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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 21573184PONE-D-10-0603710.1371/journal.pone.0019605Research ArticleBiologyMolecular Cell BiologySignal TransductionSignaling in Selected DisciplinesOncogenic SignalingA Functional Nuclear Epidermal Growth Factor Receptor, Src and Stat3 Heteromeric Complex in Pancreatic Cancer Cells A Functional Nuclear EGFR, Src and Stat3 ComplexJaganathan Soumya
1
Yue Peibin
1
Paladino David C.
1
Bogdanovic Jelena
2
3
Huo Qun
2
3
Turkson James
1
*
1
Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida, United States of America
2
NanoScience Technology Center, University of Central Florida, Orlando, Florida, United States of America
3
Department of Chemistry, University of Central Florida, Orlando, Florida, United States of America
Bonini Marcelo G. EditorUniversity of Illinois at Chicago, United States of America* E-mail: [email protected] and designed the experiments: SJ QH JT. Performed the experiments: SJ PY DCP JB. Analyzed the data: SJ QH JT. Contributed reagents/materials/analysis tools: QH JT. Wrote the paper: SJ QH JT.
2011 5 5 2011 6 5 e1960530 11 2010 12 4 2011 Jaganathan et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Evidence is presented for the nuclear presence of a functional heteromeric complex of epidermal growth factor (EGFR), Src and the Signal Transducer and Activator of Transcription (Stat)3 proteins in pancreatic cancer cells. Stat3 remains nuclear and associated with Src or EGFR, respectively, upon the siRNA knockdown of EGFR or Src, demonstrating the resistance of the complex to the modulation of EGFR or Src alone. Significantly, chromatin immunoprecipitation (ChIP) analyses reveal the nuclear EGFR, Src and Stat3 complex is bound to the c-Myc promoter. The siRNA knockdown of EGFR or Src, or the pharmacological inhibition of Stat3 activity only marginally suppressed c-Myc expression. By contrast, the concurrent modulation of Stat3 and EGFR, or Stat3 and Src, or EGFR and Src strongly suppressed c-Myc expression, demonstrating that the novel nuclear heteromeric complex intricately regulates the c-Myc gene. The prevalence of the transcriptionally functional EGFR, Src, and Stat3 nuclear complex provides an additional and novel mechanism for supporting the pancreatic cancer phenotype and explains in part the insensitivity of pancreatic cancer cells to the inhibition of EGFR, Src or Stat3 alone.
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Introduction
Many intracellular biochemical processes are triggered by the assembly of proteins into macromolecular complexes. The association between proteins or of proteins with other molecular entities modulates protein conformation, providing a means to regulate the myriad of biochemical processes that serve to efficiently manage vital biological responses. Protein dynamics and trafficking, and protein stability are also processes that can be modulated by the association of proteins with others. In the broader sense, inter-molecular associations allow specialty proteins, such as receptors, adapters, enzymes, and transcription factors to differentially modulate intracellular events, thereby creating the diversity in physiological responses and promoting context dependency.
During the induction of signal transduction, there is assembly of different proteins, each of which has specific functions important for the signal transduction and the accompanying biological response. The traditional epidermal growth factor receptor (EGFR) signal transduction pathway incorporates the activation of the mitogen-activated protein kinase kinase (MEK)-mitogen-activated protein kinase/extracellular signal-regulated kinase (ErkMAPK) and promotes mitogenic responses [1], [2]. The EGFR induction also promotes the activation of the Signal Transducer and Activator of Transcription (STAT) family of proteins, which similarly have a central role in EGF-induced biological responses [1]. The STAT proteins are latent cytoplasmic transcription factors that are activated in response to cellular stimulation by cytokines and growth factors [3] via the phosphorylation of a critical tyrosyl residue (Tyr705 for Stat3). The tyrosine phosphorylation of STATs is mediated by tyrosine kinases of growth factor receptors and by cytoplasmic tyrosine kinases, such as Src and Janus kinase (Jaks) families. Activated STATs as dimers in the nucleus bind to specific DNA response elements in the promoters of target genes to induce gene transcription. The nuclear translocation mechanism for STATs has been the subject of recent intense investigation. Stat3 nuclear translocation has been reported to be mediated by the recognition and transport by importin-α and the Ran-GTPase [4], and by mechanisms involving the chaperoning by MgcRacGAP [5], EGF receptor-mediated endocytosis [6], and by plasma membrane-associated lipid rafts trafficking [7].
The prevalence of many hyperactive signal transduction pathways that support the cancer phenotype is a major challenge to therapy. Further to the classical way of promoting crosstalks among multiple signaling pathways, macro-molecular protein assemblies provide additional unique mechanisms for inducing events that would support the malignant phenotype. Such a non-traditional signaling mechanism has been identified for the EGFR, which has been detected in the cell nucleus and observed to function as a transcription factor [8], [9]. Studies further revealed the nuclear EGFR complexes with Stat3 in breast cancer cells, and this complex induces specific genes, including the inducible nitric oxide synthase (iNOS) [10]. The additional EGFR function would compound its role as a mitogen and a promoter of cell survival, which all favor cancer. In that regard, the concurrent aberrant activation of EGFR and downstream signal mediators, including Src and Stat3, which occur with high frequencies in human cancers reflects an overall signaling complexity that supports the cancer phenotype. For example, with reference to pancreatic cancer, aberrant activation of EGFR occurs in 30–50% of cases [11], activated c-Src is noted in more than 70% of cases, and frequently accompanies EGFR overexpression [12], while aberrant Stat3 activation is also highly prevalent [13], [14], [15]. Importantly, our recent report that pancreatic cancer is more sensitive to the concurrent inhibition of aberrant Stat3 and EGFR or Src [16] shows the utilization of multiple aberrant signaling pathways for the maintenance of the cancer phenotype and how this influences the responsiveness to therapy. To extend our earlier studies [16], we sought to probe the molecular and functional interplay between Stat3, EGFR and Src and the underlying mechanisms of support of the pancreatic cancer phenotype. We herein provide evidence for a functional nuclear heteromeric EGFR, Src and Stat3 complex in pancreatic cancer cells, which promotes the induction of the c-Myc gene. Our report is the first on the identification of a nuclear EGFR, Src and Stat3 heteromeric complex that promotes the c-Myc gene induction. Understanding the dynamics of the EGFR, Src and Stat3 molecular interactions in pancreatic cancer would provide basis to design novel effective multiple-targeted therapy approaches for pancreatic cancer.
Materials and Methods
Cells and Reagents
The human pancreatic cancer, Panc-1 and Colo-357 lines have all been previously described [16], [17]. The immortalized human pancreatic duct epithelial cell (HPDEC) line was a kind gift of Dr. Tsao, OCI, UHN-PMH, Toronto) [18]. HPDEC were grown in Keratinocyte-SFM media supplemented with 0.2 ng EGF, 30 µg/mL bovine pituitary extract and containing antimycol. All other cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 5% iron-supplemented bovine calf serum and 100 units/ml penicillin-streptomycin.
Peptide synthesis
The Stat3 SH2 domain peptide inhibitor (SPI), FISKERERAILSTKPPGTFLLRFSESSK, the EGFR peptide motifs, pY1068EGFR (pY1068), PEpYINQS and the pY1086EGFR (pY1086), PVpYHNQP were purchased from Peptide 2.0 (Fairfax, VA) at >95% purity.
Nuclear Extract Preparation and Gel Shift Assays
Nuclear extract preparation from cells was carried out as previously described [17].
Sub-cellular fractionation, and SDS-PAGE/Western Blot Analysis
Western blotting analysis was performed as previously described [19], [20] on whole-cell lysates, and on cytosolic and membrane fractions, and on nuclear extracts. Sub-cellular fractions were prepared according standard protocol. Briefly, cells were washed using PBS, resuspended and lysed in low-salt buffer (20 mM HEPES (pH 7.9), 1 mM EDTA, 1 mM EGTA, 20 mM NaF, 1 mM Na3VO4, 1 mM Na4P2O7, 1 mM dithiothreitol, 1 mM TLA, 0.5 mM phenylmethylsulfonyl fluoride and 0.5% Nonidet P-40), and centrifuged (13,000 x g, 4°C, 30 s) to obtain the cytosolic fraction. The pellet was washed three times with phosphate-buffered saline (PBS), resuspended in high-salt buffer (420 mM NaCl, 20 mM HEPES (pH 7.9), 1 mM EDTA, 1 mM EGTA, 20% Glycerol, 20 mM NaF, 1 mM Na3VO4, 1 mM Na4P2O7, 1 mM dithiothreitol, 1 mM TLA and 0.5 mM phenylmethylsulfonyl fluoride), incubated at 4°C for 30 min, with rocking, and centrifuged at (13,000 x g, 4°C, 30 min) to obtain the nuclear fraction (supernatant). The pellet obtained was washed three times with PBS, resuspended in RIPA lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 0.1% SDS, 1 mM TLA, and 0.5 mM phenylmethylsulfonyl fluoride), incubated for 30 min at 4°C with rocking, and centrifuged (13,000 x g, 4°C, 20 min). The supernatant was collected as the membrane fraction. Primary antibodies used are against pY845EGFR (Upstate Biotech, Millipore, Billerica, MA), pY705Stat3, Stat3, pY1068EGFR, pY1086EGFR, pY1173EGFR, EGFR, pY416Src, Src, and β-Actin (Cell Signaling Technology, Danvers, MA), and Tata-binding protein (TBP) (Santa Cruz Biotechnology, Santa Cruz, CA). The blocking peptides were purchased from the respective companies.
Small-interfering RNA (siRNA) Transfection
siRNA sequences for EGFR and Src were ordered from Dharmacon RNAi Technologies, Thermo Scientific (Lafayette, CO). Sequences used are: EGFR sense strand, 5′-GAAGGAAACUGAAUUCAAAUU-3′; EGFR antisense strand, 5′-pUUUGAAUUCAGUUUCCUUCUU-3′; control siRNA sense strand, 5′-AGUAAUACAACGGUAAAGAUU-3′; and control siRNA antisense strand, 5′-pUCUUUACCGUUGUAUUACUUU-3′. The c-Src SMARTpool siRNA reagent (NM-005417, Catalog # M-003175-01-05) was used for Src. Transfection into cells was performed using 20 nM of EGFR siRNA or 25 nM of Src siRNA and 8 µl Lipofectamine RNAiMAX (Invitrogen Corporation, Carlsbad, CA) in OPTI-MEM culture medium (GIBCO, Invitrogen Corporation).
Immunoprecipitation (IP), and Sequential Immunoprecipitation Studies
These studies were performed as previously reported [21] using whole-cell lysates or nuclear extracts (250 µg total protein) and 5 µL of anti-EGFR or anti-Src polyclonal antibody or the monoclonal anti-Stat3 antibody (Cell Signaling Technology). For specificity, immunoblotting analysis using anti-EGFR, anti-Src and anti-Stat3 antibody was performed in the presence of the respective blocking peptide. Sequential IP studies were performed according to published procedures [10] with some modifications as follows: nuclear extracts, prepared as previously described [21], were subjected to a similar immunoprecipitation with respect to the first primary antibody, anti-EGFR antibody (Cell Signaling) or IgG (Santa Cruz) at 4°C overnight. The immunecomplex was then pelleted with 20 µL protein A/G agarose beads (Santa Cruz), washed three times using wash buffer A (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0), and then two times with wash buffer B (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl, 20 mM Tris-HCl, pH 8.0). Then the proteins were eluted with freshly prepared elution buffer (1% SDS, 100 mM NaHCO3) and subjected to the second immunoprecipitation by incubating with anti-Src antibody or IgG (Santa Cruz). The complexes were then precipitated, washed, eluted with lamelli buffer and then subjected to SDS-PAGE and Western blotting analysis probing for Stat3.
Immunostaining with laser-scanning confocal imaging
Panc-1 cells growing on coverslips in 12-well plates were treated with or without inhibitors for 1 or 24 h and subjected to immunostaining and fluorescence or laser-scanning confocal microscopy, as previously described [21]. Cells were fixed with 4% paraformaldehyde for 15 min, washed three times with PBS, permeabilized with 0.25% Triton X-100 for 10 min, and washed three times with PBS. Specimens were then blocked in 0.1% BSA in PBST for 30 min and incubated overnight at 4°C with rat monoclonal anti-EGFR (Santa Cruz), mouse monoclonal anti-Src (Cell Signaling), and rabbit polyclonal anti-Stat3 (Cell Signaling) antibodies at 1∶50 dilution (in 0.1% BSA). Subsequently, cells were rinsed three times in PBST, incubated for 1 h at room temperature in the dark with 1∶1000 dilutions of the three AlexFluor secondary antibodies, ALexaFLuor405 (goat antimouse), AlexaFluor488 (donkey anti-rabbit) and AlexaFluor546 (goat anti-rat) (Molecular Probes, Invitrogen) for EGFR, Src and Stat3 detection, respectively. Specimens were then washed three with PBST. Subsequently, coverslips were removed and mounted on slides using Fluoromount-G (Southern Biotech, Birmingham, AL) and prevented from drying by sealing the edges with nail paint. Slides were stored in the dark at 4°C until images were captured. For negative staining, secondary antibodies were added without the primary antibodies. Confocal analysis was performed by the examination of slides under Leica TCS SP5 confocal microscope (Germany) at appropriate wavelengths. Images were captured and processed using the Leica TCS SP 5 software.
Chromatin Immunoprecipitation (ChIP) and Sequential ChIP Analyses
For ChIP assay, cells in culture were treated with formaldehyde at a final concentration of 1%, for 10 min at room temperature followed by treatment with glycine at a final concentration of 0.125 M for 5 min at room temperature for cross-linking. Subsequently, cells were washed with ice-cold PBS and resuspended in and lysed with lysis buffer (20 mM HEPES, pH 7.4, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 1 mM Na3VO4, 1 mM Na4P2O7, 1 mM dithiothreitol), 1X TLA, 1 mM phenylmethylsulfonyl fluoride, 5% Nonidet P-40, and centrifuged. Then nuclear pellet was resuspended in nuclei lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS and protease inhibitors) (Roche, Indianapolis, IN). The nuclear lysates were sonicated (Omni International, Kennesaw, GA) at 30% power for 3 pulses for 10 s intervals on ice to shear DNA. The chromatin solution was pre-cleared with protein A/G agarose beads (Santa Cruz) for 1 h at 4°C with rocking. Then the pre-cleared lysates were immunoprecipitated by incubating with anti-EGFR, anti-Src, or anti-Stat3 antibodies or with IgG (for no antibody) (Santa Cruz) at 4°C overnight with rocking. The complexes were collected with 20 µL protein A/G agarose beads (Santa Cruz), washed three times using wash buffer A (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0) and two times with wash buffer B (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl, 20 mM Tris-HCl, pH 8.0). Then complexes were eluted with freshly prepared elution buffer (1% SDS, 100 mM NaHCO3). Cross-links were reversed by heating at 65°C in the presence of NaCl followed by proteinase K treatment (20 µl of a 20 mg/ml) for 6 h. The DNA was recovered and purified using DNA purification kit from Qiagen (Valencia, CA). The purified chromatin immunoprecipitated DNA was next used as a template for the polymerase chain reaction (PCR) amplification of the c-Myc promoter using the primers, Forward, 5′AAAAGGGGAAAGAGGACCTGG-3′, and Reverse, 5′-TAAAAGGGGCAAGTGGAGAGC-3′ or the TWIST promoter using the primers, Forward, 5′- AGTCTCCTCCGACCGCTTCCTG -3′.
Reverse: 5′- CTCCGTGCAGGCGGAAAGTTTGG -3′ (Invitrogen). The PCR products, 133 bp for c-Myc and the 332-bp for TWIST [22] were resolved on 2% agarose gel. The sequential ChIP studies were performed as previously reported [10] and following the sequential immunoprecipitation studies described, using the first primary antibody (anti-EGFR) and the second primary antibody (anti-Src). The recovered complexes after the secondary immunoprecipitation were eluted with the elution buffer and then subjected to ChIP assay, as described.
Gel filtration chromatography
The pre-packed Superdex 200 10/30 GL glass column was purchased from GE Healthcare Life Sciences (Piscataway, NJ). The chromatography system used in the study was the BioLogic Duoflow System (Bio-Rad, Hercules, CA). The chromatography analysis was performed following the manufacturer's instructions with a general run sequence of equilibration, load, elution, regeneration and storage. RIPA Buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Nonidet P-40) was used as the mobile phase buffer. Samples (Panc-1 cell lysate, 2 mg total protein in a volume of 250 µl) were loaded unto the column, then flow rate was adjusted to 0.25 ml/min, and then fraction (500 µl) collection was initiated right after the sample loading and monitored by the eluent absorbance at 280 nm. According to the absorbance peaks, fractions 21–34 were selected and subjected to immunoblotting analysis using antibodies against EGFR, Stat3, and Src (Cell Signaling), and against RNA helicase A (RHA) (Abcam, Cambridge, MA). For immunoprecipitation assay, 100 µl each of fractions 23–27 were pooled, from which EGFR immunecomplex was precipitated using anti-EGFR antibody (Cell Signaling) and subjected to immunoblotting analysis for Stat3, Src and EGFR.
Preparation of anti-EGFR and mouse IgG1-GNP probes
Gold nanoparticles (GNPs), 0.1 nM, with a diameter of 40 nm were purchased from Ted Pella Inc. (Redding, CA). Mouse monoclonal anti-EGFR [F4] antibody was purchased from Abcam (cat. no. ab62, conc. 1.2 mg/ml), and non-specific mouse monoclonal IgG1 was purchased from Sigma (cat. no. M9629, conc. 1 mg/ml). Polyclonal anti-Stat3 (conc. 0.2 mg/ml), polyclonal anti-Src (0.1 mg/ml), polyclonal anti-EGFR (0.2 mg/ml) and non-specific rabbit IgG (0.4 mg/ml) were purchased from Santa Cruz. All other chemicals and buffer ingredients for the assay development were purchased from Sigma. The anti-EGFR-GNP probe was prepared by adding 10 µl of mouse monoclonal anti-EGFR antibody to 1 ml of GNPs. After incubation for 15 min at room temperature, the probe was blocked with 2.5 mg BSA for 30 min. After the centrifugation at 10,000 rpm for 5 min, the supernatant was discarded and the nanoparticle residue was redispersed in 0.5 ml of 0.25% BSA in 10 mM phosphate buffer (PB). The probe was then used in the assay. The negative control mouse IgG1-GNP probe was prepared by adding 10 µl of mouse monoclonal IgG1 antibody to 1 ml GNPs, and following the procedure identical to the one used for the EGFR probe preparation. Mouse IgG1 was used here to prepare the control probe, because the anti-EGFR monoclonal antibody is a mouse IgG1 type antibody.
Detection and kinetic binding study of EGFR from nuclear extract to the GNP probes
The Panc-1 nuclear extract sample was diluted in phosphate buffer (PB) to 1 mg/ml of total protein. In a sample cell for Dynamic Light Scattering (DLS) measurement (Hellma cuvette QS 3 mm), 20 µl of the anti-EGFR-GNP probe was mixed with 2 µl of the sample, and the particle size increase was read with a DLS instrument (Zetasizer Nano ZS90 DLS system, Malvern Instruments Ltd, England) at exactly 1, 6, 11, 16, and 30 min after the mixing. The same experiment was also performed using the mouse IgG1-GNP probe. In order to confirm the specificity of the anti-EGFR-GNP probe in the detection of EGFR from nuclear extract, an inhibition experiment was conducted by treating 5 µl of the sample with 1 µl of monoclonal anti-EGFR antibody at room temperature for 7 min and 24 min prior to using the sample in the assay. After this incubation, 20 µl of the anti-EGFR-GNP probe was mixed with 2 µl of the treated sample, and the particle size was read at 1, 6 and 11 min after the mixing.
Protein complex binding partner study using polyclonal antibody
In a 1.5 ml microcentrifuge tube, 80 µl of the anti-EGFRGNP probe was mixed with 8 µl of the sample. After incubation for 30 min at room temperature, this solution was divided into four 20 µl portions. After transferring into the sample cell, the particle size of each of these portions was read with a DLS instrument. After this reading, the solution was spiked with a polyclonal antibody: either with 1 µl of anti-Stat3 or 2 µl of anti-Src or 1 µl of anti-EGFR or 0.5 µl of rabbit IgG. The particle size increase was read at exactly 5 min and 10 min after the start of the first reading.
Dynamic Light Scattering (DLS) measurements
The DLS measurements of all sample solutions were conducted using a Zetasizer Nano ZS90 DLS system equipped with a red (633 nm) laser and an Avalanche photodiode detector (APD) (quantum efficiency>50% at 633 nm) (Malvern Instruments Ltd). DTS applications 5.10 software was used to analyze the data. The average particle size (Z-average) of the solution was obtained using a Cumulant method. For each sample, ten DLS measurements were conducted with one run, and each run lasted for 10 seconds. All measurements were done at a 90° detection angle.
Results
Detection of nuclear EGFR, Src and Stat3 heterocomplex
We sought to investigate the signaling complex assembly and the dynamics of the interactions of the hyperactive Stat3, EGFR and Src [14], [16], [23] within the context of the human pancreatic cancer phenotype. Co-immunoprecipitation (co-IP) with immunoblotting analysis shows, EGFR immunecomplex from Panc-1 or Colo-357 whole-cell lysates contained both Src and Stat3 (Fig. 1A(i)), Src immunecomplex contained both EGFR and Stat3 (Fig. 1A(ii)), while Stat3 immunoprecipitate contained EGFR and Src (Fig. 1A(iii)). For specificity, the non-specific rabbit IgG in the immunoprecipitation and immunoblotting analysis for Src, Stat3 or EGFR showed no detectable protein (Fig. 1A, IgG, and data not shown), and the immunoblotting analysis performed on the immune precipitates in the presence of the respective blocking peptides (BP) showed a complete or near complete block of the immune detection of Stat3, Src or EGFR, compared to the levels detected in the absence of the blocking peptides (Fig. 1B(i)-(iii), compare + BP, lower panels to - BP, upper panels). We determined the effect of siRNA knockdown of EGFR or Src on the complex formation. Co-immunoprecipitation with immunoblotting studies of whole-cell lysates from Panc-1 cells showed that when EGFR is knockdown by siRNA (Fig.1C(i), upper band), Src immunecomplex remains associated with Stat3 (Fig. 1C(i), IP:Src), and vice-versa (Fig. 1C(i), IP:Stat3). Likewise, when Src is knockdown by siRNA (Fig. 1C(ii), upper band), EGFR immunecomplex remains associated with Stat3 (Fig. 1C(ii), IP:EGFR), and vice-versa (Fig. 1C(ii), IP:Stat3). Scrambled (con) siRNA has no effect. These findings indicate that with respect to the EGFR, Src and Stat3 heteromeric complex, Stat3 proteins remains associated with Src or EGFR, respectively, upon the siRNA knockdown of EGFR or Src.
10.1371/journal.pone.0019605.g001Figure 1 Co-immunoprecipitation with immunoblotting analysis of EGFR, Src and Stat3 association in Panc-1 and Colo-357 cells.
Immunoblotting analyses of immunecomplexes of EGFR (IP:EGFR), Src (IP:Src), and Stat3 (IP:Stat3), or of non-specific IgG non-immunoprecipitate prepared from whole-cell lysates of Panc-1 or Colo-357 cells untransfected (A and B) or transfected with EGFR siRNA, Src siRNA, or control (con) siRNA (C) and probing for Src, Stat3 and EGFR in the absence (A and C) or presence (B) of Stat3 blocking peptide (Stat3 BP), Src blocking peptide (Src BP) or EGFR blocking peptide (EGFR BP). Bands corresponding to proteins in gel are shown; input: except where indicated, represents the immunoblotting for the respective immunoprecipitated protein in the same amount of lysate used in the assay; Data are representative of 3 independent studies.
We asked the question whether the observed EGFR, Src and Stat3 heteromeric complex was present in the nucleus. Co-immunoprecipitation with immunoblotting analysis of nuclear extracts shows that EGFR immunecomplex contained both Stat3 and Src (Fig. 2A(i), IP:EGFR), Src immunecomplex contained both Stat3 and EGFR (Fig. 2A(ii), IP:Src), while Stat3 immunecomplex contained both EGFR and Src (Fig. 2A(iii), IP:Stat3). These data demonstrated the presence of the EGFR, Src and Stat3 heteromeric complex in the nucleus. For specificity of the immunoreagents, the non-specific rabbit IgG pull-down samples that were similarly immunoblotted showed no detectable EGFR, Src or Stat3 (Fig. 2A, IgG and data not shown). Immunoblotting analysis probing for the Tata-binding protein (TBP) confirmed that the extracts used in these studies are of nuclear origin (Fig. 2A(iv)). The heteromeric complex was validated by performing sequential immunoprecipitation analysis, whereby EGFR immunecomplex (IP:EGFR) was further subjected to a secondary immunoprecipitation using anti-Src antibody (IP:EGFR/IP:Src) and then immunoblotted for EGFR and Stat3. The results of these studies showed the presence of EGFR and Stat3 in the sequential immunoprecipitates (Fig. 2B, IP:EGFR/IP:Src). By contrast, IgG pull-down that was subjected to immunoblotting for EGFR or Stat3 showed no detectable levels (Fig. 2B, IgG), further confirming the specificity of the immunoreagents used.
10.1371/journal.pone.0019605.g002Figure 2 Co-immunoprecipitation with immunoblotting analysis of EGFR, Src and Stat3 complex in the nucleus and the sub-cellular distribution of EGFR, Src and Stat3.
(A and B) Immunoblotting analyses of immunecomplexes of EGFR (IP:EGFR), Src (IP:Src), Stat3 (IP:Stat3), EGFR/Src (IP:EGFR/IP:Src), or of non-specific IgG non-immuneprecpitate prepared from nuclear extracts of Panc-1 or Colo-357 cells and probing for Stat3, EGFR, Src, or the Tata-binding protein (TBP); and (C), immunoblotting analysis of membrane (mem) and cytosolic (cyto) fractions and of nuclear (nuc) extracts from Panc-1 cells probing for (i) EGFR, (ii) Stat3 and (iii) Src. Bands corresponding to proteins in gel are shown; input: except where indicated, represents the immunoblotting for the respective immunoprecipitated protein in the same amount of nuclear extract used in the assay; IP:EGFR/IP:Src, sequential immunoprecipitation with anti-EGFR and then anti-Src antibody; Data are representative of 3 independent studies.
The presence of the EGFR, Src and Stat3 complex in the nucleus of pancreatic cancer cells was further investigated by subjecting nuclear extract preparations to gel filtration column chromatography analysis (Superdex200, exclusion limit 200 kD), as described in “Materials and Methods”, in conjunction with immunoblotting analysis. The collected fractions, which showed peak absorbance at 280 nm (data not shown) were immunoprobed. Results showed that the EGFR protein first appears in the fraction 23 and is present together with Stat3 and Src, and the three proteins are concurrently detected in the fractions 23–29 (Fig. S1A). Results also showed detectable Stat3 and Src proteins in fractions 30 through 32 well after EGFR was completely eluted (Fig. S1A). Analysis of the previously-reported EGFR protein partner, RNA helicase A (RHA) [24] also showed detectable levels that were predominantly in the fractions 23 and 24 (Fig. S1A, RHA). These fractions were further subjected to co-immunoprecipitation analysis to validate the presence of the complex. Immunoblotting analysis of EGFR immunecomplex prepared from the pooled fractions 23–27 further showed the presence of Src and Stat3 (Fig. S1B). The early and the concurrent elution of EGFR, Src and Stat3 raise the possibility that each of the proteins is part of a higher molecular weight protein complex, and also that the three proteins associate as part of the same complex, as suggested by the immunecomplex formation. There has been a previous report about nuclear EGFR/Stat3 complex under conditions of cellular stimulation by EGF [10]. We therefore investigated whether the nuclear EGFR, Src, Stat3 complex was present constitutively (without ligand stimulation) in tumor cells. Immunoblotting analysis did not detect any appreciable levels of Stat3 or Src in EGFR immunecomplex or of Src or EGFR in Stat3 immunoprecipitate from the nuclear extracts prepared from human breast cancer, MDA-MB-231 and the human non-small cell lung cancer, A549 lines (Fig. S1C), suggesting a minimal possibility of the existence of a constitutive nuclear EGFR, Src, Stat3 complex in the two cancer cell types. These studies together show for the first time that EGFR, Src and Stat3 form a heteromeric complex in the nucleus of pancreatic cancer cells.
Given the detection of the EGFR, Src and Stat3 complex in whole-cell and nuclear lysates, we were interested to determine the relative levels and the sizes of EGFR, Src and Stat3 in the different sub-cellular fractions. Membrane and cytosolic fractions, and nuclear extracts were prepared from Panc-1 cells according to established protocols and which involved using 10% Nonidet P-40 lysis, and a low-salt HEPES buffer extraction for cytosolic extract, a high-salt HEPES buffer extraction for nuclear extracts (18, 22), and 0.5% SDS buffer for the membrane fraction. Immunoblotting analysis of samples of equal total protein from the sub-cellular fractions shows that with respect to each of the EGFR, Src, or Stat3 protein, the size is the same in the membrane (Mem), cytosolic (Cyto), or nuclear fraction (Nuc) (Fig. 2C). Results further show that the level of the total EGFR protein is highest in the membrane, and is higher in the cytosolic fraction than in the nuclear extract (Fig. 2C (i)). By contrast, results show that Stat3 levels are highest in the cytosolic fraction, and higher in the nuclear extract, compared to the levels associated with the cell membrane (Fig. 2C(ii)). The results for Src also showed noticeable differences, with the membrane-associated levels higher than both the cytosolic and nuclear levels, which were nearly identical (Fig. 2C(iii)). Significantly, the non-denaturing conditions (10% Nonidet P-40, low- or high-salt extraction) for preparing the cytosolic and nuclear extracts raises the potential that the EGFR protein detected in the cytosolic compartment and in the nucleus is a soluble form, and further that the Stat3 protein in the cytosolic fraction and in the nucleus is potentially a dimer. Moreover, this is the first report of the nuclear presence of c-Src tyrosine kinase in cancer cells.
The role of EGFR and Src tyrosine kinases in the nuclear EGFR, Src and Stat3 heterocomplex formation
We determined the importance of EGFR or Src in the assembly of the nuclear complex by examining the effect of siRNA knockdown. Immunoblotting analysis of immunecomplexes of Stat3 (IP:Stat3), EGFR (IP:EGFR) or Src (IP:Src) prepared from nuclear extracts showed that the siRNA knockdown of Src (Fig. 3A(i), upper panel) has little effect on the association of Stat3 with EGFR in the nucleus (Fig. 3A(i), IP:Stat3, IP:EGFR), while similarly, the knockdown of EGFR (Fig. 3A(ii), upper panel) did not alter the binding of Stat3 to Src (Fig. 3A(ii), IP:Stat3, IP:Src). Scrambled (con) siRNA has no effect. Similar to the results for the whole-cell lysates analysis in Figure 1C, these data together indicate that the knockdown of one protein does not preclude the interaction between the other two protein partners.
10.1371/journal.pone.0019605.g003Figure 3 Co-immunoprecipitation with immunoblotting analysis of the effects of modulation of EGFR, Src and Stat3 on the nuclear EGFR, Src and Stat3 complex.
(A, B, and C) Immunoblotting analyses of immunecomplexes of Stat3 (IP:Stat3), EGFR (IP:EGFR), or Src (IP:Src) prepared from nuclear extracts of Panc-1 cells untransfected or transfected with Src siRNA, EGFR siRNA, or control (con) siRNA (A), or treated with or without the EGFR inhibitor (ZD1839, ZD), Src inhibitor (Dasatinib, Das), or the Stat3 inhibitor (S3I-201) for 1 or 24 h (B), or from nuclear extracts pre-incubated for 2 h with or without 100 µM pY1068, pY1086, or SPI peptide (C) and probing for EGFR, Src, Stat3; or (D) immunoblotting analysis of nuclear extracts prepared from Panc-1 cells treated or untreated with phenylarsine oxide (PAO) and probing for Src, Stat3, EGFR. Bands corresponding to proteins in gel are shown; input: except where indicated, represents the immunoblotting for the respective immunoprecipitated protein in the same amount of lysate or nuclear extract used in the assay; Data are representative of 3 independent studies.
We next sought to determine if the heteromeric complex formation is dependent on the tyrosine kinase activities of EGFR and Src. Compared to IgG, immunoblotting of immunecomplex of EGFR showed no effect of 1 h-treatment of Panc-1 cells with the selective EGFR inhibitor, Iressa (ZD1839, ZD), the Src inhibitor, Dasatinib (Das), or the Stat3 dimerization disrupting inhibitor, S3I-201 [25] (Fig. 1G, IP:EGFR, lanes 3, 6, and 8, compared to lane 2). By contrast, immunoblotting analysis of EGFR immunecomplex showed decreased levels of associated Stat3 protein, but not Src, when cells were treated with ZD, Das, or S3I-201 for 24 h (Fig. 3B, lanes 4, 7, and 9). These findings together suggest the EGFR and Src kinase activities promote the association of Stat3 with EGFR in the nuclear heteromeric complex, while the inhibition of EGFR or Src kinase alone does not preclude the interaction between EGFR and Src. Furthermore, the Stat3 dimerization disrupting inhibitor blocks Stat3 association in the complex; however, the disruption of Stat3 dimerization, and hence its binding to EGFR does not preclude EGFR interaction with Src. The observation that the nuclear heteromeric EGFR, Src, and Stat3 complex was not completely dissociated and that EGFR/Src complex persisted by 24-h inhibition of EGFR (ZD) or Src (Das) (Fig. 3B, lanes 4, and 9) has important clinical implications in regard to the responsiveness of pancreatic cancer cells to a monotherapy targeting EGFR or Src.
During the stimulation of the EGFR signaling pathway, key phospho-tyrosine (pY) peptide motifs are induced, which recruit different signaling proteins, including Stat3 [26], [27]. The Stat3 SH2 domain binds to the receptor pY motifs through pY-SH2 domain interactions [28]. We therefore sought to probe further the observed nuclear complex of the EGFR, Src and Stat3 with respect to the peptide motifs potentially involved in the interactions by using the known Stat3-binding EGFR motifs, pY1068 peptide and pY1086 peptide, and the newly reported Stat3 peptide inhibitor, SPI, which is derived from the Stat3 SH2 domain and is capable of blocking the binding of the Stat3 SH2 domain to pY peptide motifs [29]. Immunoblotting analysis of Stat3 immunoprecipitates from nuclear extracts that have been pre-incubated with the three peptides for 2 h at room temperature showed no detectable EGFR protein (Fig. 3C(i)). These results suggest the EGFR-Stat3 interaction is promoted by pY1068EGFR and pY1086EGFR, and also that in binding to EGFR, Stat3 utilizes the key amino acid residues, 588–615 of its SH2 domain, which make up the peptide inhibitor SPI [29]. By contrast, similar probing with the SPI peptide showed no significant effect on the immuno-detection of Src protein within the Stat3 immunecomplex (Fig. 3C(ii)), suggesting the direct interaction between Src and Stat3 is independent of the amino acid residues 588–615 of the Stat3 SH2 domain.
Nuclear EGFR, Src and Stat3 heterocomplex is independent of EGFR-mediated endocytosis
We were interested to determine how the known nuclear translocation mechanisms might affect the nuclear presence of the heteromeric EGFR, Src and Stat3 complex. Several mechanisms have been reported for EGFR nuclear translocation, including receptor endocytosis, endosomal sorting machinery, importins alpha1/beta1, and exportin CRM1 [30], [31]. Previous studies have also shown that Stat3 nuclear translocation is facilitated by EGFR-mediated endocytosis [6]. However, the siRNA knockdown of EGFR did not affect the nuclear presence of Src/Stat3 (Fig. 3A(ii)), suggesting EGFR-mediated mechanisms may not be utilized for the nuclear localization of Stat3 or Src. To further determine whether endocytosis is required for the nuclear presence of the heteromeric complex, cells were treated with the pharmacological inhibitor of endocytosis, phenylarsine oxide [6]. In contrast to PAO-induced inhibition of nuclear Stat3 DNA-binding activity induced by EGF [6], immunoblotting analysis showed treatment with PAO has no effect on the nuclear presence of EGFR, Src and Stat3 heterocomplex in pancreatic cancer cells (Fig. 3D), suggesting the nuclear presence of the heteromeric complex is not dependent on endocytosis. Altogether, these findings show that EGFR, Src and Stat3 associate into a complex in a manner where all three proteins interact. However, our study does not exclude the possibility that other proteins could be present in the complex.
Detection and Analysis of the EGFR, Src and Stat3 heterocomplex through Nanoparticle Sizing (DANS) technology with Dynamic Light Scattering
The EGFR, Src and Stat3 association was further probed using DANS (Detection and Analysis through Nanoparticle Sizing) technology [32], [33], [34], [35], [36], [37]. The principle of DANS technology for protein complex detection and binding partner analysis is a single-step solution immunoassay based on gold nanoparticle (GNP) immunoprobes coupled with dynamic light scattering (DLS) detection. Details of this technology are provided in “Text S1” and illustrated in Fig. S2. Based on this general principle, the assay is designed to detect and identify protein binding partners of a protein complex. The kinetic binding assay revealed and confirmed that the anti-EGFR-GNP probe detects EGFR from the sample specifically (Fig. 4A(i)). The increase in particle size obtained with the anti-EGFR-GNP probe was much larger than the one obtained with the non-specific mouse IgG1 control probe (Fig. 4A(i)). The approximate net increase of the average nanoparticle size of the assay solution was about 70 nm, after deducting the size increase caused by non-specific interactions. For the mouse IgG1-GNP probe, the particle size increase was less than 10 nm after incubation with the sample for 30 min. Furthermore, the steepness of the kinetic curve for the EGFR probe suggested specific interactions in the assay system, while the shallow curve for the mouse IgG1 probe indicated the small size increase of the nanoparticle probes was due to non-specific interactions. To further confirm the specificity of anti-EGFR-GNP probe in the detection of EGFR from nuclear extract, an inhibition experiment was conducted in which the extract was first treated with the monoclonal anti-EGFR antibody. It is expected that the binding between the monoclonal antibody and the EGFR protein from the sample will block the subsequent binding of EGFR (in the sample) to the GNP immunoprobes, therefore, leading to decreased nanoparticle size increase during the kinetic assay. Data shown in Figure 4A(ii) confirmed the inhibitory effect of the monoclonal anti-EGFR antibody. The magnitude of this inhibition was correlated to the sample treatment time: the 24-min-treatment inhibited the particle size increase in the assay stronger than the 7 min-treatment (Fig. 4A(ii)).
10.1371/journal.pone.0019605.g004Figure 4 Studies of protein complex and protein binding partners using the Detection and Analysis through Nanoparticle Sizing technology.
(A) Kinetic binding assay of EGFR-gold nanoparticle (GNP) probe (or mouse IgG1-GNP probe as negative control) binding to (i) EGFR protein and its complex from Panc-1 nuclear extracts, and the (ii) inhibitory effect of the mouse monoclonal anti-EGFR antibody on the EGFR-GNP probe binding to the EGFR protein; and (B) Protein complex binding partner analysis whereby the polyclonal anti-Stat3, anti-Src or anti-EGFR antibody or the non-specific rabbit IgG (negative control) is added to the assay solution prepared from the (i) non-specific mouse IgG1-GNP probe (negative control), or (ii) anti-EGFR-GNP probe; Data are representative of 4 independent studies.
After using the anti-EGFR-GNP probe to “catch” the EGFR protein or protein complex from the nuclear extract sample, a second polyclonal antibody (anti-Stat3, anti-Src, or anti-EGFR antibody, or non-specific rabbit polyclonal IgG) was then added to the assay solution to identify the binding partner of the complex. In negative control studies using the mouse IgG1-GNP probe, particle size remains nearly unchanged after the addition of the four polyclonal antibodies (Fig. 4B(i)). This result confirmed the kinetic binding study that the non-specific mouse IgG1-GNP probe did not bind with the EGFR protein or protein complex from the sample. The addition of a polyclonal antibody itself to the assay solution did not cause significant nanoparticle size change. By contrast, with the anti-EGFR-GNP probe, significant particle size increase was observed when anti-Stat3 or anti-Src antibody was added to the assay solution (Fig. 4B(ii), anti-Stat3, Src). A smaller size increase was observed from anti-EGFR antibody (Fig. 4B(ii), anti-EGFR) and the rabbit IgG (Fig. 4B(ii), IgG). Per the assay principle (see Fig. S2), the substantial particle size increases observed from the addition of anti-Stat3 or anti-Src antibody to the assay solution can only be explained by the presence of EGFR in complex with Stat3 and Src in the nuclear extract. EGFR was specifically bound to the nanoparticle immunoprobes, bringing along the Stat3 and Src proteins to the nanoparticle surface, and the subsequent incubation of this assay solution with anti-Stat3 or anti-Src antibody led to further increase of the nanoparticle size. Furthermore, it appears that there is an equal amount of Stat3 and Src proteins in the EGFR, Src, and Stat3 complex. In multiple assays conducted so far, the size increase caused by anti-Src antibody is always just slightly lower than the case of anti-Stat3 antibody. Src is a smaller protein (60 KDa) than Stat3 (89–90 KDa). Therefore, with the binding of the same amount of antibody to the protein complex-GNP conjugates, the particle size should be just slightly smaller in the case of anti-Src assay solution than anti-Stat3 assay solution.
Compared to anti-Stat3 or anti-Src antibody, the particle size increase upon addition of anti-EGFR antibody to the assay solution is much smaller, only slightly higher than the non-specific rabbit IgG. This is explained by the fact that Stat3 and Src are located on the surface of the nanoparticle-bound EGFR protein, therefore, block the binding of anti-EGFR antibody to the bound EGFR proteins. The effectiveness of polyclonal anti-EGFR antibody with the EGFR protein in the sample was confirmed in a different experiment (data not shown). This result indirectly suggested that the EGFR protein detected by the nanoparticle immunoprobe from the nuclear extract exists as a complex with Stat3 and Src protein.
Detection of EGFR, Src and Stat3 heterocomplex by immunofluorescence with laser-scanning confocal microscopy
Immunofluorescence with laser-scanning confocal microscopy allowed visualization of the intracellular distribution and localization patterns of EGFR, Src and Stat3. Immunofluorescence with laser-scanning confocal microscopy confirmed the localization patterns and showed that in contrast to the negative staining (Fig. 5B(i), left panel), EGFR appearance is punctuate (red) at the plasma membrane, and in the cytoplasm and the nucleus (Fig. 5B(i), single). Similar localizations for Src (blue) and Stat3 (green) were observed, but with greater presence in the nucleus (Fig. 5B(i), single). There are stainings for colocalization of EGFR and Src (magenta, see arrows), EGFR and Stat3 (yellow, see arrows), Src and Stat3 (cyan, see arrows), and of all three entities (pale yellow/white, see white arrows) at the plasma membrane, cytoplasm, perinuclear and nuclear spaces (Fig. 5B(i), merge). These studies confirm previous reports of the association of EGFR and Stat3 in similar intracellular spaces [6], [10]. Importantly, the data show for the first time the presence of a heteromeric EGFR, Src and Stat3 complex in the nucleus, as observed by the co-IP and immunoblotting analyses, and confirmed by DANS/DLS analysis. These data contrast the results observed for the normal human pancreatic duct epithelial cells (HPDEC) (Fig. 5A). Immunofluorescence staining with laser-scanning confocal microscopy analysis of HPDEC shows a homogeneous distribution of EGFR (red), Src (blue) and Stat3 (green), all of which are strongly outside of the nucleus, with little evidence of co-localization (Fig. 5A).
10.1371/journal.pone.0019605.g005Figure 5 Immunofluorescence with laser-scanning confocal microscopy of EGFR, Src and Stat3 association in HPDEC or Panc-1 cells.
Cultured normal human pancreatic duct epithelial cells (HPDEC) (A) or pancreatic cancer, Panc-1 cells (B) were fixed, stained with primary antibodies against EGFR, Src and Stat3 and their corresponding secondary antibodies, ALexaFLuor405 (goat anti-mouse, EGFR, red), AlexaFluor488 (donkey anti-rabbit, Src, blue) and AlexaFluor546 (goat anti-rat, Stat3, green) and analyzed by laser-scanning confocal microscopy for localization (single) and colocalization (merge) studies of EGFR (red), Src (blue) and Stat3 (green) and the effects of treatment (i) without or (ii) with ZD1839 (ZD) or (iii) Dasatinib (Das) for the indicated times. Confocal images were collected using Leica TCS SP5 microscopes; Cyan, magenta, yellow and white/pale yellow arrows denote merged colors; single, one color capture, merged, three-color capture. Data are representative of 3 independent studies.
Visualization of the intracellular distribution patterns of EGFR, Src and Stat3 upon inhibition of EGFR or Src tyrosine kinase by immunofluorescence with laser-scanning confocal microscopy indicated that as with the co-IP studies (Fig. 3B), the inhibition of Src or EGFR tyrosine kinase activity alone did not completely eliminate the complex formation. However, Src or EGFR inhibition disrupted the localization patterns. Data shows EGFR, Src and Stat3 distribution in the cytoplasm and the nucleus following 1- or 24-h treatment with EGFR or Src inhibitor (Fig. 5B(ii) and (iii), 1 h, and 24 h, single). Nuclear EGFR levels are diminished, but not abolished (Fig. 5B(ii), single). Thus, EGFR, Src and Stat3 remain localized in the nucleus following tyrosine kinase inhibition. Results further showed persistent EGFR and Src (magenta), and Src and Stat3 (cyan) associations in both the nucleus and cytoplasm following treatment for 1 h with EGFR or Src inhibitor (Fig. 5B(ii) and (iii), 1 h), similar to the data in Figure 3B (1 h). Furthermore, a nuclear EGFR and Stat3 association (yellow) is detected upon 24-h treatment with the EGFR inhibitor (Fig. 5B(ii), 100 nM ZD, 24 h), while nuclear associations of EGFR and Src (magenta), and of Src and Stat3 (cyan) are detected following treatment with Src inhibitor for 24 h (Fig. 5B(iii), 100 nM Das, 24 h). The moderate differences in the observed patterns of complex formation between the co-IP (Fig. 3B) and confocal data may be due to the sensitivity differences between the two approaches. Overall, the findings are consistent with the co-IP data (Fig. 3B) in showing that the inhibition of the kinase activity of EGFR or Src alone is insufficient to completely disrupt all the proteins from the complex.
EGFR, Src and Stat3 heteromeric complex regulates the c-Myc gene expression
In our previous work, we showed that pancreatic cancer cells were insensitive to the inhibition of EGFR, Src or Stat3 activity alone, in parallel with the observation that the expression of c-Myc was also refractory to the inhibition of EGFR, Src or Stat3 alone, while the concurrent inhibition of aberrant Stat3 activity together with EGFR or Src inhibition strongly suppressed c-Myc expression and induced stronger antitumor cell effects [16]. Those findings suggest a complex regulation of c-Myc induction, which might support the cancer phenotype. Previous report identified only that the nuclear EGFR and Stat3 complex in breast cancer cells induced specific genes, including inducible nitric oxide synthase (iNOS) [10]. Studies were performed to assess the functional significance of the heteromeric complex, particularly in the context of the induction of the c-Myc gene in pancreatic cancer cells. Chromatin immunoprecipitation (ChIP) analysis was pursued to assess the association of the c-Myc promoter with the heteromeric complex. PCR amplification of DNA fragment using a primer against the c-Myc gene promoter and agarose gel electrophoresis showed that each of the anti- EGFR, Src, or Stat3 antibody-chromatin DNA immunoprecipitate contained the c-Myc gene (Fig. 6A(i), EGFR, Src and Stat3). To further confirm this finding, we pursued a modified sequential immunoprecipitation analysis similar to the one performed in Figure 2B in the context of a ChIP assay, as previously reported [10]. In the sequential ChIP assay in which EGFR chromatin immunecomplex was subjected to a second immunoprecipitation using anti-Src antibody and analyzed by PCR amplification and agarose gel electophoresis, we similarly detected the presence of the c-Myc gene (Fig. 6A(ii), EGFR/Src). By contrast, PCR analysis showed no appreciable detection of TWIST, a gene previously reported to be EGFR target [22] in the EGFR chromatin immunecomplex, while the gene was detected in the Stat3 and Src chromatin-immunoprecipitates (Fig. S3). These differences may be tumor cell-type dependent. To verify the specificity of the immunoreagents, the non-specific IgG was similarly used in the ChIP assay, and subsequent PCR analysis showed no detectable levels of the c-Myc gene (Fig. 6A, IgG). Taken together with the sequential immunoprecipitation data in Figure 2B), these studies demonstrate that EGFR, Src and Stat3 form a detectable heteromeric complex that is associated with the expression of c-myc in pancreatic cancer cells.
10.1371/journal.pone.0019605.g006Figure 6 Chromatin immunoprecipitation assay and Western blotting analysis of c-Myc, iNOS, Cyclin D1, and VEGF expression in Panc-1 and Colo-357 cells.
(A), Agarose gel electrophoresis of the Polymerase Chain Reaction (PCR)-amplified c-Myc gene fragment from the chromatin DNA precipitated with antibody against EGFR, Src, or Stat3, or with the non-specific IgG; and (B and C), Immunoblotting analysis of whole-cell lysates probing for EGFR or Src (B(i) and C(i)) or c-Myc, iNOS, Cyclin D1 or VEGF (B(ii) and C(ii)), and the effects of siRNA knockdown of EGFR (EGFR siRNA), Src (Src siRNA) or control (con) siRNA, or S3I-201 or Das). Bands corresponding to proteins or c-Myc gene in gel are shown; M, molecular weight marker, EGFR/Src, sequential immunoprecipitation with anti-EGFR and then anti-Src antibody. Data are representative of 3 independent studies, and values are mean and s.d of 3 independent studies; *p-<0.01.
To further study the potential involvement of the heteromeric EGFR, Src and Stat3 complex in the regulation of genes, we performed immunoblotting analysis of known regulated genes, including c-Myc. Results showed moderate or no significant change in the expression of c-Myc, Cyclin D1, iNOS, and VEGF upon the siRNA knockdown of EGFR or Src alone (Fig. 6B). By contrast, the concurrent knockdown of EGFR with Stat3 inhibition (by S3I-201) [25], or the concurrent knockdown of Src with Stat3 inhibition, or the concurrent EGFR knockdown with Src inhibition (by Das) resulted in a strong suppression of c-Myc expression (Fig. 6C(ii)). The bands corresponding to the expression levels of c-Myc were quantified, analyzed by ImageQuant, and represented as percent of control (Fig. 6C(ii), % numbers in parenthesis). Results show over 76% suppression of c-Myc expression (Fig. 6C(ii)) following the concurrent modulation of any two of EGFR, Src and Stat3, except for the siRNA knockdown of EGFR and Src together, which only showed 20% decrease. The moderate change in the c-Myc expression in response to the co-transfection with EGFR siRNA and c-Src siRNA may be due to the fact that we could only achieve partial knockdown of Src (Fig. 6C(i)), although EGFR is significantly suppressed by the EGFR siRNA (Fig. 6C(i)). In contrast, the use of the pharmacological inhibitor, Das, which strongly inhibits Src activity, in combination with siRNA knockdown of EGFR strongly suppressed c-Myc expression. These studies together suggest the possibility that the c-Myc gene is regulated by EGFR, Src and Stat3 complex in a manner that is susceptible to concurrent modulation of any two of the EGFR, Src and Stat3 proteins, but not to the inhibition of EGFR, Src or Stat3 alone.
Discussion
Aberrations in the EGFR, c-Src and Stat3 signaling pathways occur with a high frequency in many human cancers [14], [15], [38], [39], [40], [41] and are associated with poor prognosis. Notably, constitutively-active Stat3 induces dysregulation of gene expression, contributing to the altered gene expression profile that is a hallmark of cancer. The details of aberrantly-active Stat3-mediated dysregulation of gene expression continue to be elucidated and the initial studies indicate the mechanisms are more complex. The present studies strongly suggest that in forming a nuclear, transcriptionally-active EGFR, Src, Stat3 heteromeric complex, the EGFR and Src proteins cooperate with Stat3 to promote the altered gene expression. Such cooperation between Stat3 and other proteins for the transcriptional induction of genes has similarly been observed in other systems. Specifically, Stat3 cooperates with NF-κB to induce certain genes [42], [43]. Furthermore, there have been reports of a nuclear EGFR pathway [8], [9], in which a nuclear EGFR-Stat3 complex promotes the induction of iNOS in breast cancer cells [10]. These reports together with our present findings indicate the complicated nature of the mechanisms by which aberrantly-active Stat3 might dysregulate gene expression in cancer cells. In this context, the present studies extend our earlier report [16] in suggesting that nuclear heteromeric EGFR, Src and Stat3 complex regulates the c-Myc gene in pancreatic cancer cells. It is likely that other genes may by induced by the EGFR, Src and Stat3 nuclear complex and we note that iNOS, VEGF, and Cyclin D1 are reported to be induced by the EGFR/Stat3 complex or Stat3 [10], [44], [45] and could well be candidates for the regulation by the EGFR, Src and Stat3 complex. Taken together, these studies provide a novel mechanism for the de-regulation of gene expression in cancer cells.
The observation that the EGFR, Src and Stat3 complex is also detected in the cytoplasm raises the possibility that it is formed extra-nuclear and transported into the nuclear space. Previous reports have described inherent EGFR nuclear localization mechanisms, including facilitation via the endosomal sorting machinery and the interaction with importins α1/β1 [31], and several nuclear translocation mechanisms have also been proposed for Stat3, including EGFR-mediated endocytosis [6], [31]. Although any of these processes could facilitate the nuclear translocation of the heteromeric EGFR, Src, Stat3 complex should it be formed outside of the nucleus, the present data excludes the possibility that EGFR-mediated endocytosis is involved. Whether the other nuclear translocation pathways proposed for Stat3, including the utilization of the Ran-GTPase [4], Sec61 translocon [46], and chaperoning by MgcRacGAP [5] are involved in promoting the nuclear transport of the complex remains to be studied. Our findings also do not preclude the assembly of the complex de novo in the nucleus. Present data also suggest that only a portion of the intracellular EGFR, Src and Stat3 protein pools are utilized in the formation of the nuclear complex, raising the possibility that there may be diverse cellular pools of EGFR, Src, or Stat3 with different accessibility limitations. There also could be different pools of pre-associated complexes of the three proteins, a possibility that will be consistent with the report that cytoplasmic Stat3 exists as complexes with accessory scaffolding proteins [47]. Such pre-formed complexes would not only facilitate the signal induction [48], but may also serve to stabilize the proteins.
The incidence of signaling cross-talk has long been known, and the associations of EGFR with Src [49], Src with Stat3 [20], [50], and EGFR with Stat3 [26], [27] at the plasma membrane and the perinuclear space [6] have been reported. Specifically, Stat3 binds to pY1068 and pY1086 motifs of EGFR [26], [27], while Src binds to Y845EGFR. In cancer cells, aberrant Stat3 activation is promoted by hyperactive protein tyrosine kinases, including EGFR and Src [15], [40], [41], and evidence has indicated that c-Src phosphorylates Y845EGFR, Y1068EGFR, and Y845EGFR motifs in pancreatic cancer cells [16], [27], [51]. Present data reveals that the pY1068EGFR and pY1086EGFR motifs and the Stat3 SH2 domain amino acid residues 588–615 are essential for EGFR-Stat3 interaction within the context of the heteromeric EGFR, Src and Stat3 complex. It remains to be determined what the exact configuration is for the heteromeric complex. Also, the present data does not exclude the possibility that other accessory proteins could be present in the complex together with EGFR, Src and Stat3.
We had recently reported about the functional cooperation between EGFR, Src and Stat3 in promoting and supporting pancreatic cancer, wherein the cancer phenotype and the expression of c-Myc in the cancer cells were both insensitive to the inhibition of EGFR, Src or Stat3 alone [16]. While it is possible that the c-Myc gene may be regulated cooperatively by EGFR, Src and Stat3 through mechanisms independent of each other, our study also raises the possibility of a complex transcriptional regulation by mechanisms that involve the nuclear EGFR, Src and Stat3 heteromeric complex.
Supporting Information
Text S1
Detail description of Dynamic Light Scattering (DLS).
(DOC)
Click here for additional data file.
Figure S1
Immunoblotting analysis of EGFR, Src, Stat3, and RHA from Panc-1, MDA-MB-231, or A549 cells. Immunoblotting analysis of (A and B) fractions collected from gel filtration chromatographic analysis of Panc-1 cell lysates probing for EGFR, Src, Stat3,or RHA (A), or EGFR immunecomplex from the pooled fractions 23–27 probing for Stat3, Src or EGFR (B), or (C) immunecomplexes of EGFR or Stat3 from nuclear extracts of human breast cancer, MDA-MB-231 or non-small cell lung cancer, A549 cells probing for EGFR, Stat3 and Src. Bands corresponding to proteins in gel are shown; Data are representative of 2 independent studies.
(TIF)
Click here for additional data file.
Figure S2
The principle of DANS technology (Detection and Analysis through Nanoparticle Sizing) for protein complex detection and binding partner analysis.
(TIF)
Click here for additional data file.
Figure S3
Chromatin Immunoprecipitation assay of TWIST expression in Panc-1 cells. Agarose gel electrophoresis of the Polymerase Chain Reaction (PCR)-amplified TWIST gene fragment from the chromatin DNA precipitated with antibody against EGFR, Src, or Stat3, or with the non-specific IgG; M, molecular weight marker; Data are representative of 2 independent studies.
(TIF)
Click here for additional data file.
We thank all colleagues and members of our laboratory for the stimulating discussions and the University of Central Florida Confocal Microscopy Group for the assistance with the confocal imaging work.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by the National Cancer Institute Grants CA106439 (JT) and CA128865 (JT), Florida Department of Health Bankhead-Coley Foundation (Bridge Grant) (HQ), partial support from World Gold Council (GROW Program) (HQ), and the National Natural Science Foundation of China Overseas Young Investigator Award (20828006) (HQ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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==== Front
Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography si234910.1107/S1600536811013067ACSEBHS1600536811013067Metal-Organic Papers
cyclo-Tetrakis(μ2-3-sulfidopropyl-κ3
C
1,S:S)tetrakis[chloridocobalt(III)] [Co4Cl4(C3H6S)]4]Awan Shafique Ahmad aTahir M. Nawaz b*Muhammad Iram Khushi cAhmad Saeed cTariq Muhammad Ilyas da PAEC, PO Box # 1114, Islamabad GPO 44000, Pakistanb Department of Physics, University of Sargodha, Sargodha, Pakistanc Department of Chemistry, University of Engineering and Technology, Lahore 54890, Pakistand Department of Chemistry, University of Sargodha, Sargodha, PakistanCorrespondence e-mail: [email protected] 5 2011 13 4 2011 13 4 2011 67 Pt 5 e110500m576 m577 02 4 2011 07 4 2011 © Awan et al. 20112011This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.In the centrosymmetric title compound, [Co4Cl4(C3H6S)]4], the two independent CoIII ions are each coordinated in a distorted tetrahedral geometry by one C, one Cl and two S atoms. The molecules are stabilized by C—H⋯Cl hydrogen bonds. In the crystal, intermolecular C—H⋯Cl and C—H⋯S hydrogen bonds with R
2
2(8), R
4
2(8) and R
2
2(6) ring motifs generate a polymeric network.
==== Body
Related literature
For related background see: Shahid et al. (2009 ▶); Altaf et al. (2010 ▶). For related structures, see: Duan et al. (1997 ▶); Tremel et al. (1992 ▶). For graph-set notation, see: Bernstein et al. (1995 ▶).
Experimental
Crystal data
[Co4Cl4(C3H6S)]4]
M
r = 674.07
Monoclinic,
a = 23.6135 (12) Å
b = 7.8465 (3) Å
c = 16.8693 (9) Å
β = 130.440 (4)°
V = 2378.9 (2) Å3
Z = 4
Mo Kα radiation
μ = 3.54 mm−1
T = 296 K
0.24 × 0.16 × 0.14 mm
Data collection
Bruker Kappa APEXII CCD diffractometer
Absorption correction: multi-scan (SADABS; Bruker, 2005 ▶) T
min = 0.675, T
max = 0.683
13736 measured reflections
2152 independent reflections
1782 reflections with I > 2σ(I)
R
int = 0.058
Refinement
R[F
2 > 2σ(F
2)] = 0.041
wR(F
2) = 0.125
S = 1.04
2152 reflections
109 parameters
H-atom parameters constrained
Δρmax = 0.70 e Å−3
Δρmin = −0.62 e Å−3
Data collection: APEX2 (Bruker, 2009 ▶); cell refinement: SAINT (Bruker, 2009 ▶); data reduction: SAINT; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008 ▶); molecular graphics: ORTEPIII (Burnett & Johnson, 1996 ▶), ORTEP-3 for Windows (Farrugia, 1997 ▶) and PLATON (Spek, 2009 ▶); software used to prepare material for publication: WinGX (Farrugia, 1999 ▶) and PLATON.
Supplementary Material
Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536811013067/si2349sup1.cif
Structure factors: contains datablocks I. DOI: 10.1107/S1600536811013067/si2349Isup2.hkl
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: SI2349).
The authors acknowledge the provision of funds for the purchase of the diffractometer and encouragement by Dr Muhammad Akram Chaudhary, Vice Chancellor, University of Sargodha, Pakistan.
supplementary crystallographic
information
Comment
Recently, we have reported the crystal structure of zinc(II) and mercury(II)
complexes of pyrrolidinedithiocarbamate (PDTC) (Shahid et al.,
2009) &
(Altaf, et al., 2010). In the present study, we attempted to
prepare a
cobalt(II) complex with PDTC, but surprisingly the title compound (I, Fig. 1)
was isolated, the crystal structure of which is being presented.
The crystal structure of (II) i.e, tetranuclear molecular square[Co(HL)]44+
[H2L = tetra(2-pyridyl)thiocarbazone] (Duan et al., 1997) and
(III)
i.e., bis(tetraethylammonium)
hexakis(µ2-phenylthiolato)-tetrachloro-tetra -cobalt acetonitrile solvate
(Tremel et al., 1992) have been published which are related to
the
title compound (I).
The crystal structure of the title compound (I) is centrosymmetric. The
coordination around two independent Coiii ions is distorted tetrahedral from
one C, Cl and two S-atoms. The range of Co—C [2.038 (6)–2.051 (6) Å] is
shorter compared to Co—S [2.305 (2)–2.3648 (16) Å]. The Co—Cl bonds have
values of 2.228 (2) and 2.236 (2) Å. The important bond distances
are given in Table 1. The molecules are stabilized in the form of a
polymeric network due to C—H···Cl and C—H···S intermolecular H-bonds
(Table 2) forming R22(8), R42(8) and R22(6)
ring motifs (Bernstein et al., 1995).
Experimental
The title compound was prepared by adding two equivalents of ammonium
pyrrolidinedithiocarbamate (PDTC) in 15 ml methanol to a solution of
CoCl2.6H2O in 10 ml methanol. The addition of PDTC in the pink colored
metal ion solution resulted in the formation of green precipitates
immediately. After stirring for half an hour, the precipitates were filtered
off and dried. The blackish brown crystals of the title compound (I) were
prepared
by dissolving 0.03 g precipitates in 3 ml DMSO on heating in a vial and then
cooling the resulting solution at room temperature.
Refinement
The H-atoms were positioned geometrically (C–H = 0.97 Å) and were included in
the refinement in the riding model approximation, with Uiso(H) =
xUeq(C), where x = 1.2 for all H-atoms.
Figures
Fig. 1. View of the centrosymmetric title compound. Symmetry code i = -x + 1/2, -y - 1/2, -z + 1. The thermal ellipsoids are drawn at the 50% probability level. H-atoms are shown by small circles of arbitrary radii.
Fig. 2. The partial packing (PLATON; Spek, 2009) which shows that molecules form a polymeric network with ring motifs. H-atoms not involved in H-bondings are omitted for clarity.
Crystal data
[Co4Cl4(C3H6S)]4] F(000) = 1344
Mr = 674.07 Dx = 1.882 Mg m−3
Monoclinic, C2/c Mo Kα radiation, λ = 0.71073 Å
Hall symbol: -C 2yc Cell parameters from 1782 reflections
a = 23.6135 (12) Å θ = 2.3–25.2°
b = 7.8465 (3) Å µ = 3.54 mm−1
c = 16.8693 (9) Å T = 296 K
β = 130.440 (4)° Prisms, white
V = 2378.9 (2) Å3 0.24 × 0.16 × 0.14 mm
Z = 4
Data collection
Bruker Kappa APEXII CCD diffractometer 2152 independent reflections
Radiation source: fine-focus sealed tube 1782 reflections with I > 2σ(I)
graphite Rint = 0.058
Detector resolution: 8.10 pixels mm-1 θmax = 25.2°, θmin = 2.3°
ω scans h = −28→28
Absorption correction: multi-scan (SADABS; Bruker, 2005) k = −9→8
Tmin = 0.675, Tmax = 0.683 l = −20→20
13736 measured reflections
Refinement
Refinement on F2 Primary atom site location: structure-invariant direct methods
Least-squares matrix: full Secondary atom site location: difference Fourier map
R[F2 > 2σ(F2)] = 0.041 Hydrogen site location: inferred from neighbouring sites
wR(F2) = 0.125 H-atom parameters constrained
S = 1.04 w = 1/[σ2(Fo2) + (0.0653P)2 + 15.5359P] where P = (Fo2 + 2Fc2)/3
2152 reflections (Δ/σ)max < 0.001
109 parameters Δρmax = 0.70 e Å−3
0 restraints Δρmin = −0.62 e Å−3
Special details
Geometry. Bond distances, angles etc. have been calculated using the rounded
fractional coordinates. All su's are estimated from the variances of the
(full) variance-covariance matrix. The cell e.s.d.'s are taken into account in
the estimation of distances, angles and torsion angles
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor
wR and goodness of fit S are based on F2, conventional
R-factors R are based on F, with F set to zero for
negative F2. The threshold expression of F2 >
σ(F2) is used only for calculating R-factors(gt) etc.
and is not relevant to the choice of reflections for refinement.
R-factors based on F2 are statistically about twice as large
as those based on F, and R- factors based on ALL data will be
even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq
Co1 0.17597 (4) −0.17781 (9) 0.30876 (5) 0.0323 (2)
Co2 0.14629 (4) −0.12119 (9) 0.50575 (5) 0.0336 (2)
Cl1 0.09923 (9) −0.3988 (2) 0.25283 (12) 0.0555 (5)
Cl2 0.07469 (11) 0.0860 (3) 0.49043 (15) 0.0694 (7)
S1 0.21157 (8) −0.0272 (2) 0.45587 (11) 0.0460 (4)
S2 0.28224 (8) −0.2358 (2) 0.33225 (11) 0.0481 (5)
C1 0.1631 (4) 0.1666 (8) 0.3814 (5) 0.066 (3)
C2 0.0959 (4) 0.1316 (9) 0.2708 (5) 0.061 (2)
C3 0.1163 (3) 0.0287 (7) 0.2182 (4) 0.0362 (17)
C4 0.3356 (4) −0.0372 (9) 0.3904 (6) 0.064 (3)
C5 0.4140 (4) −0.0689 (10) 0.4862 (6) 0.065 (3)
C6 0.4164 (3) −0.1622 (7) 0.5638 (4) 0.0417 (17)
H1A 0.14799 0.22914 0.41469 0.0787*
H1B 0.19711 0.23811 0.38263 0.0787*
H2A 0.05934 0.07031 0.26878 0.0733*
H2B 0.07391 0.23855 0.23404 0.0733*
H3A 0.14572 0.09690 0.20863 0.0435*
H3B 0.07165 −0.00820 0.15024 0.0435*
H4A 0.33533 0.02242 0.33979 0.0769*
H4B 0.31166 0.03559 0.40757 0.0769*
H5A 0.43922 −0.13460 0.46849 0.0783*
H5B 0.43980 0.03894 0.51537 0.0783*
H6A 0.39644 −0.09200 0.58805 0.0502*
H6B 0.46733 −0.19168 0.62322 0.0502*
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
Co1 0.0275 (4) 0.0399 (4) 0.0300 (4) −0.0012 (3) 0.0189 (3) −0.0025 (3)
Co2 0.0338 (4) 0.0373 (4) 0.0312 (4) −0.0014 (3) 0.0217 (3) 0.0004 (3)
Cl1 0.0496 (9) 0.0537 (9) 0.0545 (9) −0.0089 (7) 0.0299 (8) −0.0081 (7)
Cl2 0.0683 (11) 0.0733 (11) 0.0725 (12) 0.0219 (9) 0.0483 (10) 0.0114 (9)
S1 0.0485 (8) 0.0520 (8) 0.0377 (7) −0.0099 (7) 0.0281 (7) −0.0045 (6)
S2 0.0407 (8) 0.0667 (10) 0.0398 (8) 0.0055 (7) 0.0274 (7) 0.0030 (7)
C1 0.091 (5) 0.045 (4) 0.054 (4) −0.010 (4) 0.044 (4) −0.004 (3)
C2 0.066 (4) 0.047 (4) 0.056 (4) 0.003 (3) 0.033 (4) 0.008 (3)
C3 0.034 (3) 0.045 (3) 0.030 (3) −0.003 (2) 0.021 (2) 0.005 (2)
C4 0.056 (4) 0.064 (4) 0.074 (5) 0.000 (3) 0.043 (4) 0.022 (4)
C5 0.056 (4) 0.067 (4) 0.074 (5) −0.010 (3) 0.043 (4) 0.003 (4)
C6 0.038 (3) 0.047 (3) 0.037 (3) −0.012 (2) 0.023 (3) −0.005 (2)
Geometric parameters (Å, °)
Co1—Cl1 2.228 (2) C5—C6 1.469 (11)
Co1—S1 2.3570 (17) C1—H1A 0.9700
Co1—S2 2.318 (2) C1—H1B 0.9700
Co1—C3 2.038 (6) C2—H2A 0.9700
Co2—Cl2 2.236 (3) C2—H2B 0.9700
Co2—S1 2.305 (2) C3—H3A 0.9700
Co2—S2i 2.3648 (16) C3—H3B 0.9700
Co2—C6i 2.051 (6) C4—H4A 0.9700
S1—C1 1.826 (7) C4—H4B 0.9700
S2—C4 1.837 (8) C5—H5A 0.9700
C1—C2 1.495 (10) C5—H5B 0.9700
C2—C3 1.492 (11) C6—H6A 0.9700
C4—C5 1.490 (13) C6—H6B 0.9700
Co1···H4Aii 3.3200 S2···H4Aii 3.0500
Cl1···C3 3.473 (6) C2···Cl2iv 3.570 (7)
Cl1···C6iii 3.343 (7) C3···Cl1 3.473 (6)
Cl1···C6i 3.379 (7) C3···S1 3.103 (5)
Cl2···C1 3.623 (11) C3···S2 3.686 (7)
Cl2···C3iv 3.512 (7) C3···C1 2.455 (9)
Cl2···C6i 3.492 (6) C3···Cl2iv 3.512 (7)
Cl2···C3v 3.412 (6) C3···Cl2viii 3.412 (6)
Cl2···S1 3.744 (4) C3···S2ix 3.539 (8)
Cl2···C2iv 3.570 (7) C6···Cl1i 3.379 (7)
Cl1···H4Aii 2.8900 C6···Cl2i 3.491 (6)
Cl1···H2Bvi 2.8800 C6···Cl1x 3.343 (7)
Cl1···H6Ai 2.6200 H1A···Cl2 2.9600
Cl1···H6Biii 2.4800 H1B···H3A 2.5900
Cl2···H1A 2.9600 H1B···S1vii 3.1000
Cl2···H3Bv 2.8100 H2A···H2Aiv 2.4400
Cl2···H3Biv 2.7400 H2B···Cl1xi 2.8800
Cl2···H5Bvii 2.9600 H3A···H1B 2.5900
S1···Cl2 3.744 (4) H3A···S2ix 2.5700
S1···S2 3.785 (3) H3B···Cl2iv 2.7400
S1···C2 2.774 (7) H3B···Cl2viii 2.8100
S1···C3 3.103 (5) H4A···Co1ix 3.3200
S1···Co1i 3.8074 (16) H4A···Cl1ix 2.8900
S1···S1i 3.773 (2) H4A···S2ix 3.0500
S2···C3ii 3.539 (8) H4B···S1 3.0000
S2···S1 3.785 (3) H4B···H6A 2.5200
S2···C3 3.686 (7) H5B···Cl2vii 2.9600
S1···H4B 3.0000 H6A···H4B 2.5200
S1···H1Bvii 3.1000 H6A···Cl1i 2.6200
S2···H3Aii 2.5700 H6B···Cl1x 2.4800
Cl1—Co1—S1 118.22 (8) C2—C1—H1B 109.00
Cl1—Co1—S2 114.37 (8) H1A—C1—H1B 108.00
Cl1—Co1—C3 108.9 (2) C1—C2—H2A 110.00
S1—Co1—S2 108.14 (7) C1—C2—H2B 109.00
S1—Co1—C3 89.52 (17) C3—C2—H2A 110.00
S2—Co1—C3 115.5 (2) C3—C2—H2B 109.00
Cl2—Co2—S1 111.09 (9) H2A—C2—H2B 108.00
Cl2—Co2—S2i 113.44 (8) Co1—C3—H3A 110.00
Cl2—Co2—C6i 109.0 (2) Co1—C3—H3B 110.00
S1—Co2—S2i 115.39 (8) C2—C3—H3A 110.00
S1—Co2—C6i 117.5 (2) C2—C3—H3B 110.00
S2i—Co2—C6i 88.68 (15) H3A—C3—H3B 108.00
Co1—S1—Co2 110.96 (8) S2—C4—H4A 109.00
Co1—S1—C1 93.3 (2) S2—C4—H4B 109.00
Co2—S1—C1 104.4 (4) C5—C4—H4A 109.00
Co1—S2—C4 102.5 (4) C5—C4—H4B 109.00
Co1—S2—Co2i 99.94 (8) H4A—C4—H4B 108.00
Co2i—S2—C4 93.3 (2) C4—C5—H5A 110.00
S1—C1—C2 113.0 (5) C4—C5—H5B 110.00
C1—C2—C3 110.6 (8) C6—C5—H5A 109.00
Co1—C3—C2 108.5 (4) C6—C5—H5B 110.00
S2—C4—C5 112.1 (5) H5A—C5—H5B 108.00
C4—C5—C6 110.6 (9) C5—C6—H6A 110.00
Co2i—C6—C5 107.9 (4) C5—C6—H6B 110.00
S1—C1—H1A 109.00 H6A—C6—H6B 108.00
S1—C1—H1B 109.00 Co2i—C6—H6A 110.00
C2—C1—H1A 109.00 Co2i—C6—H6B 110.00
Cl1—Co1—S1—Co2 −7.06 (11) S2i—Co2—S1—C1 −148.1 (2)
Cl1—Co1—S1—C1 −113.8 (3) C6i—Co2—S1—Co1 10.1 (2)
S2—Co1—S1—Co2 −139.02 (7) C6i—Co2—S1—C1 109.3 (3)
S2—Co1—S1—C1 114.2 (3) Cl2—Co2—S2i—Co1i −145.79 (9)
C3—Co1—S1—Co2 104.3 (2) Cl2—Co2—S2i—C4i 110.9 (4)
C3—Co1—S1—C1 −2.5 (4) S1—Co2—S2i—Co1i −16.01 (8)
Cl1—Co1—S2—C4 −174.3 (3) S1—Co2—S2i—C4i −119.3 (4)
Cl1—Co1—S2—Co2i −78.66 (7) Cl2—Co2—C6i—C5i −87.1 (6)
S1—Co1—S2—C4 −40.3 (3) S1—Co2—C6i—C5i 145.4 (5)
S1—Co1—S2—Co2i 55.34 (7) Co1—S1—C1—C2 30.3 (8)
C3—Co1—S2—C4 58.1 (3) Co2—S1—C1—C2 −82.3 (7)
C3—Co1—S2—Co2i 153.74 (17) Co1—S2—C4—C5 130.1 (7)
Cl1—Co1—C3—C2 95.3 (5) Co2i—S2—C4—C5 29.2 (7)
S1—Co1—C3—C2 −24.5 (5) S1—C1—C2—C3 −56.3 (9)
S2—Co1—C3—C2 −134.5 (5) C1—C2—C3—Co1 51.0 (7)
Cl2—Co2—S1—Co1 −116.41 (8) S2—C4—C5—C6 −57.7 (9)
Cl2—Co2—S1—C1 −17.1 (2) C4—C5—C6—Co2i 54.4 (7)
S2i—Co2—S1—Co1 112.67 (7)
Symmetry codes: (i) −x+1/2, −y−1/2, −z+1; (ii) −x+1/2, y−1/2, −z+1/2; (iii) x−1/2, −y−1/2, z−1/2; (iv) −x, y, −z+1/2; (v) x, −y, z+1/2; (vi) x, y−1, z; (vii) −x+1/2, −y+1/2, −z+1; (viii) x, −y, z−1/2; (ix) −x+1/2, y+1/2, −z+1/2; (x) x+1/2, −y−1/2, z+1/2; (xi) x, y+1, z.
Hydrogen-bond geometry (Å, °)
D—H···A D—H H···A D···A D—H···A
C3—H3A···S2ix 0.97 2.57 3.539 (8) 175
C3—H3B···Cl2iv 0.97 2.74 3.512 (7) 138
C3—H3B···Cl2viii 0.97 2.81 3.412 (6) 121
C6—H6A···Cl1i 0.97 2.62 3.379 (7) 135
C6—H6B···Cl1x 0.97 2.48 3.343 (7) 148
Symmetry codes: (ix) −x+1/2, y+1/2, −z+1/2; (iv) −x, y, −z+1/2; (viii) x, −y, z−1/2; (i) −x+1/2, −y−1/2, −z+1; (x) x+1/2, −y−1/2, z+1/2.
Table 1 Selected bond lengths (Å)
Co1—Cl1 2.228 (2)
Co1—S1 2.3570 (17)
Co1—S2 2.318 (2)
Co1—C3 2.038 (6)
Co2—Cl2 2.236 (3)
Co2—S1 2.305 (2)
Co2—S2i 2.3648 (16)
Co2—C6i 2.051 (6)
S1—C1 1.826 (7)
S2—C4 1.837 (8)
Symmetry code: (i) .
Table 2 Hydrogen-bond geometry (Å, °)
D—H⋯A D—H H⋯A D⋯A D—H⋯A
C3—H3A⋯S2ii 0.97 2.57 3.539 (8) 175
C3—H3B⋯Cl2iii 0.97 2.74 3.512 (7) 138
C3—H3B⋯Cl2iv 0.97 2.81 3.412 (6) 121
C6—H6A⋯Cl1i 0.97 2.62 3.379 (7) 135
C6—H6B⋯Cl1v 0.97 2.48 3.343 (7) 148
Symmetry codes: (i) ; (ii) ; (iii) ; (iv) ; (v) .
==== Refs
References
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 21572963PONE-D-11-0136210.1371/journal.pone.0019766Research ArticleBiologyMolecular Cell BiologyCellular TypesEndothelial CellsMedicineCardiovascularAtherosclerosisCardiomyopathiesCardiovascular PharmacologyCoronary Artery DiseaseHypertensionHydrogen Sulfide Attenuated Tumor Necrosis Factor-α-Induced Inflammatory Signaling and Dysfunction in Vascular Endothelial Cells Hydrogen Sulfide Attenuated InflammationPan Li-Long
1
Liu Xin-Hua
1
Gong Qi-Hai
1
Wu Dan
1
Zhu Yi-Zhun
1
2
*
1
Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai, China
2
Institute of Biomedical Sciences, Fudan University, Shanghai, China
Avraham Hava Karsenty EditorBeth Israel Deaconess Medical Center, United States of America* E-mail: [email protected] and designed the experiments: Y-ZZ L-LP X-HL. Performed the experiments: L-LP X-HL. Analyzed the data: L-LP X-HL Q-HG. Contributed reagents/materials/analysis tools: Q-HG DW. Wrote the paper: L-LP X-HL.
2011 10 5 2011 6 5 e1976616 1 2011 4 4 2011 Pan et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
Hydrogen sulfide (H2S), the third physiologically relevant gaseous molecule, is recognized increasingly as an anti-inflammatory mediator in various inflammatory conditions. Herein, we explored the effects and mechanisms of sodium hydrosulfide (NaHS, a H2S donor) on tumor necrosis factor (TNF)-α-induced human umbilical vein endothelial cells (HUVEC) dysfunction.
Methodology and Principal Findings
Application of NaHS concentration-dependently suppressed TNF-α-induced mRNA and proteins expressions of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), mRNA expression of P-selectin and E-selectin as well as U937 monocytes adhesion to HUVEC. Western blot analysis revealed that the expression of the cytoprotective enzyme, heme oxygenase-1 (HO-1), was induced and coincident with the anti-inflammatory action of NaHS. Furthermore, TNF-α-induced NF-κB activation assessed by IκBα degradation and p65 phosphorylation and nuclear translocation and ROS production were diminished in cells subjected to treatment with NaHS.
Significance
H2S can exert an anti-inflammatory effect in endothelial cells through a mechanism that involves the up-regulation of HO-1.
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Introduction
Endothelial dysfunction elicited by inflammatory cytokines is regarded as a key event in the pathogenesis of cardiovascular disorders [1], [2]. Inflammatory cytokines change the secretory activities of endothelium and causes endothelium to become dysfunctional [3]. Enhanced expression of adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), P-selectin, and E-selectin, is an early marker of endothelial activation and dysfunction in the development of early cardiovascular events [4]. Expression of adhesion molecules on the endothelium facilitates the adherence of leukocytes [2],[5], ultimately leading to the progression of numerous vascular diseases [5].
Several studies indicated expression of adhesion molecules on endothelial cells induced by tumor necrosis factor-α (TNF-α) is associated with activation of multiple signal transduction pathways, including mitogen-activated protein kinases (MAPK) and nuclear factor-κB (NF-κB) [6],[7]. In addition, the endothelial generation of reactive oxygen species (ROS) during inflammatory stimuli acts as a triggering mechanism for NF-κB activation and the elevation in adhesion molecules and chemokines expression and could ultimately contribute to endothelial dysfunction [8]–[10]. Thus, the modulation of these processes, ie, activation of NF-κB, expression of adhesion molecules, and elimination of ROS, assumes great significance in the prevention and treatment of inflammatory cardiovascular diseases.
Hydrogen sulfide (H2S), a colorless gas with a characteristic rotten-egg odor, has traditionally been considered to be a toxic environmental pollutant. More recently, H2S has been identified as the third, physiologically relevant, gaseous signaling molecule with a diverse physiological profile [11]. H2S production has been attributed to three key enzymes cystathionine γ-lyase [12], cystathionine β-synthetase, and the newest one, 3-mercaptopyruvate sulfurtransferase [13], while cystathionine γ-lyase is abundant in heart and smooth muscle and the most relevant enzyme for the cardiovascular system [12],[14]. As the third gasotransmitter H2S appears to confer cytoprotection via multiple mechanisms including anti-oxidant and anti-inflammatory effects [14]–[16]. For instance, recent reports demonstrated H2S acts as an endogenous scavenger for ROS and reactive nitrogen species [11],[17]–[19]. In addition, although H2S has been implicated to play a pro-inflammatory role in systemic inflammation [20]–22], a majority of elegant studies strongly suggest that H2S is a potent anti-inflammatory molecule in various models [16],[23]–25]. However, to the best of our knowledge, the potent anti-inflammatory mechanism of H2S in endothelial cells has not yet been clarified. Here, our present work investigated if H2S exerts anti-inflammatory and thereby potential anti-atherogenic properties in endothelial cells through inhibition of pro-inflammatory processes, such as the expression of adhesion molecules, intracellular ROS production. In addition, the underlying mechanisms and intracellular signaling pathways affected by H2S in TNF-α-stimulated endothelial cells were investigated.
Results
NaHS is non-toxic to HUVEC
The cytotoxicity experiments of NaHS in this study were performed at 10–100 µM concentration. Non-cytotoxic effect of NaHS was observed at the dosage used in this study (data not shown).
NaHS inhibited U937 cells adhesion to TNF-α-stimulated HUVEC
We first investigated the effect of NaHS on the adhesion of U937 cells to TNF-α-activated endothelial cells, a critical step in vascular inflammation. As shown in Figure 1, control-confluent HUVEC showed minimal binding of U937 cells, while the adhesion of U937 cells was remarkably increased when HUVEC were stimulated with TNF-α (10 ng/ml) for 6 h. This dose of TNF-α has been widely used to investigate the proinflammatory effects of TNF-α in cultured cells. The adhesion of U937 cells to TNF-α-stimulated HUVEC was significantly attenuated by NaHS in a concentration dependent manner. In addition, the adhesion of U937 cells to TNF-α-stimulated HUVEC was inhibited by pretreatment with NAC (5 mM) or Dex (10 µM) (Figure 1). Importantly, NaHS (100 µM) and NAC (5 mM), but not Dex (10 µM) have a similar inhibitory effect on U937 cells adhesion to TNF-α-activated endothelial cells (P>0.05). These results suggested that ROS is involved in the binding of U937 cells to TNF-α-stimulated endothelial cells.
10.1371/journal.pone.0019766.g001Figure 1 NaHS inhibited U937 cells adhesion to TNF-α-activated HUVEC.
HUVEC were incubated with indicated concentrations of NaHS, Dex (10 µM) or NAC (5 mM) for 30 min, then stimulated with TNF-α (10 ng/ml) for 6 h, U937 cells seed onto HUVEC and co-cultured for 2 h. After removing the non-adherent cells, adherent cells were detected and counted under a light microscope. (A) Pictures are representative optical fields. NaHS, Dex, and NAC concentrations were 100 µM, 10 µM, and 5 mM, respectively. (B) Quantitative analysis of the binding of U937 cells to HUVEC presented by bar graphs was counted under a light microscope. #
P<0.05 compared with unstimulated cells, *P<0.05, **P<0.01 compared with TNF-α-stimulated control. Data are the mean ± S.E.M of results from at least three independent experiments, each performed in duplicate.
NaHS inhibited TNF-α-induced mRNA levels of adhesion molecules
The effects of NaHS on TNF-α-induced mRNA levels of adhesion molecules by HUVEC were studied by quantitative real-time RT-PCR. Resting HUVEC showed a low constitutive transcription of adhesion molecules, while TNF-α caused a significant increase in mRNA levels of E-selectin, P-selectin, VCAM-1, and ICAM-1 in HUVEC after 4 h of incubation. Treatment of cells with NaHS (10-100 µM) for 30 min resulted in concentration-dependent decreases in TNF-α-induced mRNA levels of E-selectin (Figure 2A), P-selectin (Figure 2B), VCAM-1 (Figure 2C), and ICAM-1 (Figure 2D). Dex (10 µM) also significantly inhibited TNF-α-induced mRNA levels of adhesion molecules, but not significant decreased E-selectin mRNA level (Figure 2). Importantly, NaHS (100 µM) and Dex (10 µM) have a similar inhibitory effect on TNF-α-induced mRNA levels of P-selectin (Figure 2B), VCAM-1 (Figure 2C), and ICAM-1(Figure 2D) (P>0.05).
10.1371/journal.pone.0019766.g002Figure 2 NaHS inhibited TNF-α-induced mRNA levels of adhesion molecules.
HUVEC were incubated with indicated concentrations of NaHS or Dex (10 µM) for 30 min, then stimulated with TNF-α (10 ng/ml) for 4 h. mRNA levels of adhesion molecules were analyzed by real-time RT-PCR. GAPDH was used as an internal control. Bar graphs in (A), (B), (C), (D) represented the quantitative difference in mRNA levels of E-selectin, P-selectin, ICAM-1, VCAM-1, respectively, between groups. Dex concentration was 10 µM. #
P<0.05 compared with unstimulated cells, *P<0.05, **P<0.01 compared with TNF-α-stimulated cells. Data are the mean ± S.E.M of results from at least three independent experiments, each performed in duplicate.
NaHS decreased TNF-α-induced expression of ICAM-1 and VCAM-1
As the expression of adhesion molecules on endothelial cells is a prerequisite for adhesion of leukocytes, we investigated the effect of NaHS on TNF-α-induced ICAM-1 and VCAM-1 expression. Western blot analysis of cell lysates showed that levels of VCAM-1 and ICAM-1 were very low in unstimulated HUVEC, but were significantly increased by TNF-α treatment, NaHS attenuated TNF-α-induced ICAM-1 expression only at higher concentration (100 µM) (Figure 3B), while NaHS suppressed TNF-α-induced VCAM-1 expression in a dose-concentration manner at concentrations ranging from 10 to 100 µM (Figure 3C). In addition, a similar profile was also observed by pretreatment with Dex (Figure 3), as previously reported [26].
10.1371/journal.pone.0019766.g003Figure 3 NaHS inhibited TNF-α-induced expression of ICAM-1 and VCAM-1.
HUVEC were pre-treated with NaHS (50–100 µM) or Dex (10 µM) for 30 min and then stimulated with TNF-α (10 ng/ml) for 6 h. (A) Representative Western blot showed the expression of ICAM-1 and VCAM-1. Tubulin was used as loading control. Bar graphs represent the quantitative difference in expression of ICAM-1 (B) and VCAM-1 (C), respectively, in arbitrary units. #
P<0.05 compared with unstimulated cells, *P<0.05, **P<0.01 compared with TNF-α-stimulated cells. Data are the mean ± S.E.M of results from at least three independent experiments, each performed in duplicate.
NaHS up-regulated HO-1 expression in TNF-α-stimulated cells
The effect of NaHS on HO-1 expression was initially explored in cultured HUVEC, Figure 4A shows upon incubation with NaHS (10–100 µM) for 6 hours, a substantial increase in the expression of HO-1 was observed. MTT cell survival assays failed to demonstrate any cellular cytotoxicity at these concentrations (not shown). Thus, we examined the effects of NaHS on HO-1 expression in TNF-α-stimulated HUVEC. As shown in Figure 4B, treatment with TNF-α didn't reveal significant effect on HO-1 expression compared with resting cells. Contrary, NaHS concentration-dependently increased HO-1 expression in TNF-α-stimulated cells. Meanwhile, the upregulation of HO-1 was also observed in NAC-treated cells. Taken together, these results suggested that the expression of HO-1 induced by NaHS may functions as a negative regulator of TNF-α-induced inflammatory responses in HUVEC.
10.1371/journal.pone.0019766.g004Figure 4 NaHS upregulated expression of HO-1 in HUVEC.
(A) HUVEC were incubated with indicated concentrations of NaHS for 6 hours. Cells were then lysed, and HO-1 expression was analyzed by Western blot. Tubulin was used as loading control. Data represent mean ±S.E.M from 3 independent repeats. *P<0.05, **P<0.01 compared with unstimulated cells. (B) HUVEC were incubated with various concentrations of NaHS for 30 min, cells were then stimulated with TNF-α (10 ng/ml) for 6 h. HO-1 proteins were analyzed by Western blot in HUVEC. Tubulin was used as loading control. NAC concentration was 5 mM. #
P<0.05 compared with unstimulated cells, *
P<0.05, **
P<0.01 compared with TNF-α-stimulated cells. Data are the mean ±S.E.M of results from at least three independent experiments, each performed in duplicate.
NaHS reduced intracellular ROS production in TNF-α-stimulated HUVEC
To confirm whether the inhibitory effect of H2S on TNF-α-induced intracellular ROS production, HUVEC were labeled with a cell-permeable fluorescent dye H2DCF-DA and analyzed by spectrofluorometer or fluorescence microscope. Stimulation with TNF-α resulted in a great increase in the amount of intracellular ROS generation in HUVEC compared with unstimulated cells (Figure 5). However, pretreatment with NaHS (10–100 µM) significantly decreased TNF-α-induced intracellular ROS production. In addition, coincident with the reports that free radical scavenger NAC (5 mM) also abolished TNF-α-induced intracellular ROS production [27] (Figure 5). Meanwhile, NaHS (100 µM) and NAC (5 mM) also showed similar free radical scavenging capacity (P>0.05). These results strongly suggested that scavenging ROS by H2S may be responsible for inhibition in the binding of U937 cells to TNF-α-stimulated endothelial cells.
10.1371/journal.pone.0019766.g005Figure 5 NaHS inhibited TNF-α-induced intracellular ROS generation.
HUVEC were incubated with NaHS or NAC for 30 min, and then stimulated with TNF-α (10 ng/ml) for 1 h. (A) Pictures are representative fields detected by fluorescence microscope. NaHS and NAC concentration were 100 µM and 5 mM, respectively. (B) Quantitation of intracellular ROS was determined by fluorescence spectrophotometer. NAC concentration was 5 mM. #
P<0.05 compared with unstimulated cells, *P<0.05, **P<0.01 compared with TNF-α-stimulated cells. Data are the mean ±S.E.M of results from at least three independent experiments, each performed in duplicate.
NaHS inhibited TNF-α-induced p38 MAPK activation
Activation MAPK signaling pathway induced by TNF-α plays an important role in the regulation of adhesion molecules expression. In unstimulated cells, there was almost no detectable phosphorylation of p38, ERK1/2, or JNK1/2 in HUVEC. TNF-α caused a rapid phosphorylation of MAPK within 5 min, with phosphorylation peaking at 15 min and followed by dropping to normal level (Figure 6A). Based on this time course, 15 min was chosen for subsequent experiments.
10.1371/journal.pone.0019766.g006Figure 6 NaHS inhibited TNF-α-induced p38 phosphorylation.
(A) HUVEC were stimulated with TNF-α (10 ng/ml) for indicated periods. The total and phosphorylation levels of MAPK were measured by Western blot. The experiment was repeated 3 times with equal results. Cells were incubated with indicated concentrations of NaHS for 30 min, then treated with TNF-α (10 ng/ml) for another 15 min. Phosphorylation levels of p38 (B), JNK1/2(C) and ERK1/2 (D) were analyzed by Western blot. #
P<0.05 compared with unstimulated cells, *P<0.05 compared with TNF-α-stimulated cells. Data are the mean ± S.E.M of results from at least three independent experiments, each performed in duplicate.
To examine whether MAPK activation was involved in the regulation of inflammatory response by NaHS in TNF-α-stimulated HUVEC, phosphorylation of MAPK (p38, JNK1/2, and ERK1/2) were analyzed by Western blot. As shown in Figure 6, phosphorylation of p38 (Figure 6B), JNK1/2 (Figure 6C), and ERK1/2 (Figure 6D) in resting cells was significantly increased after 15 minutes treatment with TNF-α. NaHS concentration-dependently abolished the p38 phosphorylation induced by TNF-α (Figure 6B), but had little effect on JNK1/2 and ERK1/2 phosphorylation (Figure 6C, 6D).
NaHS inhibited TNF-α-induced IκBα degradation
Translocation of NF-κB from cytoplasm to the nucleus is preceded by the phosphorylation and subsequent degradation of IκBα. To determine the effect of NaHS on TNF-α-induced IκBα degradation, total cell lysate was prepared from the TNF-α with or without NaHS-treated cells. Using Western blot analysis, we demonstrated that the degradation of IκBα took place in a time dependent manner after the TNF-α induction (Figure 7A). As shown in Figure 7B, upon induction with TNF-α for 15 min the intensity of IκBα was significantly reduced. In contrast, pretreatment with NaHS inhibited TNF-α-induced IκBα degradation in a concentration dependent manner.
10.1371/journal.pone.0019766.g007Figure 7 NaHS inhibited TNF-α-induced IκBα degradation and NF-κB activation.
(A) IκBα degradation was analyzed by Western blot in HUVEC stimulated with TNF-α (10 ng/ml) for indicated periods. (B) HUVEC were incubated with indicated concentrations of NaHS for 30 min, then stimulated with TNF-α (10 ng/ml) for another 15 min. IκBα degradation was analyzed by Western blot. (C) HUVEC were incubated with indicated concentrations of NaHS for 30 min, then stimulated with TNF-α (10 ng/ml) for another 15 min. Phosphorylation levels of NF-κB p65 was analyzed by Western blot. (D) HUVEC were incubated with NaHS (100 µM) for 30 min, then stimulated with TNF-α (10 ng/ml) for another 1 h. Cytoplasmic and nuclear levels of NF-κB p65 were analyzed by Western blot. #
P<0.05 compared with unstimulated cells, *P<0.05, **P<0.01 compared with TNF-α-stimulated cells. Data are the mean ± S.E.M of results from at least three independent experiments, each performed in duplicate. (E) HUVEC were incubated with NaHS (50, 100 µM) for 30 min, then stimulated with TNF-α (10 ng/ml) for another 1 h. NF-κB p65 translocation was detected by fluorescent microscope.
NaHS inhibited TNF-α-induced phosphorylation and nuclear translocation of NF-κB p65
The phosphorylation of NF-κB p65 subunit, particular on serine residues 536 in the C-terminal transactivation domain, plays an important role in regulation transcription of adhesion molecules following exposure to inflammatory stimuli [9],[28]. To examine whether NaHS might play a role in the regulation of phosphorylation of NF-κB p65Ser536 in the TNF-α-activated HUVEC, Western blot analysis was performed. As shown in Figure 7C, TNF-α significantly induced phosphorylation of NF-κB p65Ser536 in HUVEC. NaHS markedly reduced TNF-α-induced phosphorylation of NF-κB p65Ser 536 in a concentration dependent manner.
We next asked whether NaHS might play a role in the regulation of nuclear NF-κB p65 translocation in TNF-α-stimulated HUVEC. The translocation of NF-κB p65 from the cytoplasm to the nucleus was visualized by Western blot and immunofluorescence (Figure 7D, E). TNF-α result in a significantly induction of nuclear NF-κB p65 translocation, while NaHS (100 µM) significantly suppressed TNF-α-induced nuclear NF-κB p65 translocation.
Discussion
Inflammation contributes to the pathogenesis of cardiovascular disease and elevated level of pro-inflammatory cytokine TNF-α is associated with endothelial dysfunction [1]. The outcome of the present study indicated that exogenous H2S, at the dosage used in this study, attenuated TNF-α-induced endothelial dysfunction in vitro. Our major findings showed that exogenous H2S blocked the adhesion of U937 cells to TNF-α-activated HUVEC by inhibiting expression of adhesion molecules; suppressed the TNF-α-induced activation of NF-κB by inhibiting degradation of IκBα and activation of p38 signaling pathway; eliminated TNF-α-induced intracellular ROS production; and up-regulated HO-1 expression in HUVEC.
As the third gaseous mediator H2S has multiple positive physiological functions, but the role of H2S during systemic inflammatory diseases is still a matter of debate or may be double-edged. NaHS was used as a H2S donor, because it can dissolves into Na+ and HS− in solution, HS− is released and forms H2S with H+. This provides a solution of H2S at a concentration that is about 33% of the original concentration of NaHS [29]. Several reports describe a significant decrease in plasma H2S level in cardiovascular disease [23],[30],[31]. H2S has a protective effect against atherosclerosis in apoE−/− mice and attenuated TNF-α-induced ICAM-1 expression in HUVEC [23]. Several reports mention generous plasma basal H2S levels in the 50–150 µM range [31]. So, the present study explored that in atherosclerosis-associated inflammation, H2S may function as a modulator of endothelial function at the relevant physiological concentrations (10–100 µM).
It is well known that adhesion molecules are strong predictors of atherosclerotic lesion development and future cardiovascular events [4]. TNF-α is recognized as a major risk factor in the initiation and progression of atherosclerotic lesion development and future cardiovascular events, which may promote endothelial dysfunction by increasing the production of endothelium-derived ROS and enhancing the expression of adhesion molecules on the endothelial cells [27],[32],[33]. Recent evidence suggested that H2S might exert anti-inflammatory effect via multiple mechanisms such as upregulation of antioxidant defense [17]. Exogenous H2S exert their anti-oxidative effects by inhibiting ROS production induced by cytokines or hydrogen peroxide in mouse pancreatic β-cells [25]. Consistent with the finding, we also demonstrated that NaHS treatment attenuated TNF-α-induced intracellular ROS generation in HUVEC. Meanwhile, treatment with NaHS significantly attenuated TNF-α-induced increases in the mRNA expression of ICAM-1, VCAM-1, P-selectin, and E-selectin and subsequent proteins expression of ICAM-1 and VCAM-1 as well as U937 cells adhesiveness to endothelial cells. We also demonstrated that the NAC significantly inhibited intracellular ROS production and subsequent U937 cells adherence. These observations are consistent with numerous reports that demonstrate that scavenging intracellular ROS production inhibits monocytes adhesiveness to endothelial cells by reducing the expression of various adhesion molecules [33],[34]. Thus, inhibition of TNF-α-induced ROS by NaHS may be responsible for attenuation of TNF-α-induced endothelial dysfunction. Although the effects of Dex or NaHS on adhesion molecules expression and adhesion of U937 cells were similar, their inhibitory effects on adhesion molecules may differ. Dex, a typical steroidal anti-inflammatory drug, attenuates adhesion molecules expression through direct interaction with glucocorticoid receptors [26]. Our results were consisted with the study that H2S inhibited TNF-α-induced expression of ICAM-1, as previously reported by Wang [23]. But there are some earlier reports indicated that H2S has been demonstrated to play a proinflammatory role in various disease states [20],[21]. The inconsistency between the present study and earlier studies may be a result of the dose of H2S donor used or a different inflammatory model.
HO-1 is the inducible isoform of the first and rate-controlling enzyme of heme degradation and plays a central role in the regulation of inflammatory reaction via its products bilirubin and carbon monoxide in a variety of experimental systems [33]. We reported that NaHS can dose-dependently induce HO-1 expression in endothelial cells. The persistent HO-1 induction also observed after TNF-α challenge may be due to the ability of H2S to coordinate to other thiol-containing protein, including a number of redox-sensitive transcription factors and kinases, which is a crucial modulator of the expression of antioxidant genes, including HO-1. We also suggested that HO-1 induction by NaHS might contribute to its anti-inflammatory action. Because the expression of HO-1 was induced concomitantly with the attenuation of expression of adhesion molecules and the binding of U937 cells to TNF-α-stimulated HUVEC exerted by NaHS. The result is consistent with the report that overexpression of HO-1 prevented adhesion molecules expression and leukocytes to activated endothelial cells [34],[35]. Our results suggested that the induction of HO-1 by NaHS may function in a negative feedback manner to down-regulate adhesion molecules expression, as reported by Paine A [33].
MAPK and NF-κB are key players in intracellular signaling pathways in response to inflammatory stimuli and required for adhesion molecules expression [36],[37]. Therefore, to further investigate the molecular mechanism responsible for the inhibitory effect of NaHS on expression of adhesion molecules, we examined the effect of NaHS on NF-κB and MAPK activation. Our results demonstrated that NaHS potently suppressed TNF-α-stimulated phosphorylation and nuclear translocation of NF-κB p65 in HUVEC. Consistent with previous report [38], phosphorylation and nuclear translocation of NF-κB p65 were found to be the main components of TNF-α-induced NF-κB activaton in HUVEC. The results suggested that the inhibition of NF-κB activaton by NaHS is mediated by modulation of upstream signaling pathway involved in NF-κB activation. Numerous natural components and therapeutic agents have been shown to inhibit NF-κB activation by preventing IκBα degradation [39]. Our data indicate that NaHS not only inhibited the IκBα degradation, but also attenuated the nuclear translocation of NF-κB. This provides evidence that H2S can attenuate TNF-α-induced NF-κB activation, as previously reported [23],[24]. However, the kinase responsible for IκBα degradation has not been identified. There are also studies suggesting that MAPK is involved in the regulation of NF-κB activation in TNF-α-induced endothelial cells [40]. Here, we demonstrated that NaHS inhibited TNF-α-stimulated p38 MAPK signal pathway in HUVEC, but had little effect on ERK1/2 or JNK1/2 phosphorylation. Although ERK1/2 mainly mediates cellular responses to hormones and growth factors, JNK1/2 and p38 are primarily activated by stress-related stimuli [41]. Inhibition of p38 MAPK markedly inhibited the NF-κB activation and subsequent the expression of adhesion molecules in TNF-α-stimulated endothelial cells [27]. Meanwhile, inhibition and genetic deficiency of p38 MAPK also contribute to induce HO-1 expression [42], which functions in a negative feedback manner to inhibit NF-κB activation [34].
In summary and conclusion, we demonstrated that NaHS attenuated expression of adhesion molecules and monocyte adhesion to endothelial cells, and decreased intracellular ROS production in TNF-α-stimulated endothelial cells. Remarkably, this anti-inflammatory effect is primarily achieved by the inhibition of NF-κB and p38 signaling pathways, and by the upregulation of HO-1 expression. We, thus, concluded that the inhibition of NF-κB and p38 signaling pathways, adhesion molecules, and modulation of cellular redox balance might be one of the important mechanisms of H2S that improved TNF-α-induced endothelial dysfunction. These findings suggested that H2S release agents could represent a promising approach for the treatment of inflammatory vascular diseases.
Materials and Methods
Reagents
DMEM, RPMI-1640, and fetal bovine serum (FBS) were from GIBCO-BRL (USA). Recombinant human TNF-α was purchased from Millpore (Billerica, MA, USA). 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) was from Molecular probes (Eugene, OR, USA). NaHS, N-acetyl-L-cysteine (NAC), and dexamethasone (Dex) were purchased from Sigma-Aldrich (St Louis, MO, USA). NaHS has been well established as a reliable donor of H2S in culture media [15].
Cell culture studies
HUVEC (ATCC, Manassas, VA) were grown in DMEM supplemented with 1800 mg/L NaHCO3, 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified atmosphere with 5% CO2. U937 human monocytes (ATCC, Manassas, VA) were maintained in RPMI-1640 containing 1800 mg/L NaHCO3, 4500 mg/L glucose, and 110 mg/L sodium pyruvate, supplemented with 10% FBS, 100 U/ml penicilin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere with 5% CO2.
For experiments, HUVEC were grown to confluence in 6-well plates, 24-well plates, or 60 mm dishes (Costar, Cambridge, MA). Cells were serum-starved for 12 h, to assess the effect of TNF-α or NaHS on MAPK phosphorylation, cells were incubated with TNF-α (10 ng/ml) for different periods (5, 15, 30, 45, and 90 min). In some experiments, cells were pre-incubated with or without NAC (5 mM), Dex (10 µM), or NaHS (10–100 µM) for 30 min before stimulating with TNF-α (10 ng/ml) for various periods: 15 min for measurement of MAPK (p38, JNK1/2 and ERK1/2) phosphorylation, IκBα protein, and NF-κB p65ser536 phosphorylation. 1 h for measurement of intracellular ROS production and NF-κB p65 translocation; 4 h for measurement of mRNA levels; 6 h for measurement of proteins expression of adhesion molecules, HO-1, and adhesion assay.
Cell viability assay
The cytotoxicity of NaHS was analyzed by colorimetric MTT assay as previously described [43].
Adhesion assay
Endothelial cells were starved for 12 h with serum-free medium, and then exposed to NaHS, NAC, or Dex for 30 min, TNF-α stimulated for another 6 h. An exact number of U937 monocytes was seeded on TNF-α-activated HUVEC and incubated for 2 h at 37°C in a humidified 5 % CO2 atmosphere as described previously [44]. Nonadhering U937 cells were then removed and washed with PBS for 3 times. Finally, the HUVEC were fixed with 4 % paraformaldehyde in PBS for 10 min and the number of adhered U973 monocytes to endothelial cells was calculated using a Zeiss optical microscope system. Results are expressed as means and standard deviations of number of cells counted. All experiments were repeated at least three times.
Quantitative real-time RT-PCR analysis
Total RNA was extracted from HUVEC with TRIzol Reagent (Takara, TaKaRa Biotechnology, Dalian, China) following the manufacturer's instructions. Total RNA (2 µg) of each sample was reverse-transcribed into cDNA and amplified using a PrimeScriptTM 1st Strand cDNA Synthesis Kit (Takara) according to the manufacturer's directions. real-time RT-PCR conditions include denaturation (95°C for 30 s); annealing (60°C for 30 s); number of cycles (40) and were performed using iQ5 real-time RT-PCR detection System (Bio-Rad, Richmound, USA) in a total volume of 25 µl reaction mixture containing 2 µl cDNA, 12.5 µl 2× SYBR Green 1 Master Mix (Takara), and 1 µl of each primer. GAPDH was used as an internal control to compare the amount of total mRNA of each sample. The primers used in this experiment were indicated in Table 1.
10.1371/journal.pone.0019766.t001Table 1 Primers sequences used in the present study.
Genes Sequences
ICAM-1 Sense
5′-TCACGGAGCTCCCAGTCCTAA-3′
Antisense
5′-AAAGGCAGGTTGGCCAATGA-3′
E-selectin Sense
5′-CACTCAAGGGCAGTGGACACA-3′
Antisense
5′-CAGCTGGACCCATAACGGAAAC-3′
VCAM-1 Sense
5′-CGAAAGGCCCAGTTGAAGGA-3′
Antisense
5′-GAGCACGAGAAGCTCAGGAGAAA-3′
P-selectin Sense
5′-ACCTTCAGGACAATGGACAGCAG-3′
Antisense
5′-CCCAGAGGTTGGAGCAGTTCA-3′
GAPDH Sense
5′-GCACCGTCAAGGCTGAGAAC-3′
Antisense
5′-TGGTGAAGACGCCAGTGGA-3′
Preparation of whole cell extracts and isolation of cell fractions
For whole cell extraction, cells were washed twice with ice-cold PBS and lyzed in RIPA buffer with protease & phosphatase inhibitor. After centrifugation (4°C, 10 min, 10,000 g), samples were prepared for Western blot analysis.
For preparation of cytoplasmic and nuclear fraction, HUVEC were pretreated with NaHS (100 µM), NAC (5 mM), or Dex(10 µM) for 30 min, and then stimulated with TNF-α for 1 h. Nuclear and cytoplasmic proteins of HUVEC were extracted using the NE-PER@ Nuclear and Cytoplasmic Extraction Reagents (Pierce, Inc) according to manufacturer's instructions.
Western blot analysis
Equal amounts (30 µg) of proteins were separated on 8-10 % sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinyl difluoride membrane (Millipore, USA). After being blocked with 5% nonfat dry milk, membranes were incubated overnight at 4°C with primary antibodies against ERK1/2, phosphorylated (p)-ERK1/2 (Thr202/Tyr204), JNK1/2, p-JNK1/2 (Thr183/Tyr185), p38, p-p38 (Thr180/Tyr182), NF-κB p65, p-NF-κB p65 (Ser536), IκBα (1∶1000) (all 1∶1000, Cell Signaling Technology, Beverly, MA, USA); ICAM-1 (1∶500), VCAM-1 (1∶500), β-Tubulin (1∶2000), β-actin (1∶500), GAPDH (1∶2000), or HO-1 (1∶2000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After incubation with appropriate secondary antibodies for 1 h at room temperature, proteins were visualized by enhanced chemoluminescence with a camera-based imaging system (Alpha Innotech, Santa Clara, CA, USA). The density of the signals was quantified with the AlphaEase software.
Immunofluorescence
The endothelial cells were grown on glass slides in 6-well plates. Cells were fixed in 4 % paraformaldehyde for 30 min at room temperature. Immunostained using rabbit anti-NF-κB p65 antibody (1∶25; Cell Signaling) and Alexa Fluor 488 conjugated goat anti-rabbit IgG (1∶200; Invitrogen), and counterstained for nuclei with DAPI. Immunofluorescence was visualized using a fluorescent microscope (Carl Zeiss Inc.). The results were based on three independent analyses.
Intracellular ROS production assay
The fluorescent probe, H2DCF-DA, was used to measure the intracellular generation of ROS by TNF-α [45]. Briefly, confluent HUVEC in 24-well plates were pretreated with NaHS (10-100 µM) and NAC (5 mM) for 30 min. After removing the NaHS and NAC from the wells, the cells were incubated with 20 µM H2DCF-DA for 30 min. Then stimulated with TNF-α (10 ng/ml) for 1 h, and the fluorescence intensity was measured at an excitation and emission wavelength of 485 nm and 530 nm, respectively, using a fluorescence spectrophotometer (M1000, TECAN, Austria GmbH, Austria) or a fluorescence microscope (Carl Zeiss).
Statistical analysis
Data are presented as mean ± S.E.M. Differences between mean values of multiple groups were analyzed by one-way analysis of variance with Dunnett's test for post hoc comparisons. Statistical significance was considered at P<0.05.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by the National Basic Research Program of China (973 Program, Grant No. 2010CB912600), National Science Fund for Distinguished Young Scholars (Grant No. 30888002), and Synthetic Department of Research and Development Technique of New Drugs (2009ZX09301-011). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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40 Frey RS Gao X Javaid K Siddiqui SS Rahman A 2006 Phosphatidylinositol 3-kinase gamma signaling through protein kinase C zeta induces NADPH oxidase-mediated oxidant generation and NF-kappaB activation in endothelial cells. J Biol Chem 281 16128 16138 16527821
41 Kyriakis JM Avruch J 2001 Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev 81 807 869 11274345
42 Naidu S Vijayan V Santoso S Kietzmann T Immenschuh S 2009 Inhibition and genetic deficiency of p38 MAPK up-regulates heme oxygenase-1 gene expression via Nrf2. J Immunol 182 7048 7057 19454702
43 Liu XH Pan LL Gong QH Zhu YZ 2010 Antiapoptotic effect of novel compound from herba leonuri - leonurine (SCM-198): a mechanism through inhibition of mitochondria dysfunction in H9c2 cells. Curr Pharm Biotechnol 11 895 905 20874679
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Int J Prev MedIJPVMInternational Journal of Preventive Medicine2008-78022008-8213Medknow Publications India IJPVM-2-101Brief CommunicationStomach Cancer Mortality in The Future: Where Are We Going? Amiri Masoud PhD1 Department of Epidemiology and Biostatistics, Medical Plants Research Center, Shahrekord University of Medical Sciences, Shahrekrod, Iran. Email: [email protected] 2011 2 2 101 102 12 11 2010 07 12 2010 © International Journal of Preventive Medicine2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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INTRODUCTION
Gastric cancer mortality has been fallen throughout Europe during the past decades in terms of both incidence and mortality rates.123 It is mainly as a result of remarkable improvement of life conditions in European societies.4–7 Efforts to reduce global cancer disparities begin with an understanding of geographic patterns of cancer incidence, mortality and prevalence rates, by studies such as GLOBOCAN,89 EURO-CARE,10 and Five Continents databases.11 Survival increased and mortality decreased through the combination of earlier detection, better access to care and improved treatment.12 There has also been a concomitant change in lifestyle and environmental exposures over successive generations,13 including changes in exposures to risk factors in early decades of life.14
Several studies conducted to determine the projections of future trends in gastric cancer mortality in European countries.71214–17 Mortality rates were generally expected to decline further. Based on the cancer mortality trends in the European Union until 2000, one study predicted a further fall by 11% in age-standardized cancer mortality from 2000 to 2015.15 A Dutch study also projected a substantial decline in gastric cancer mortality until 2015, based on trends until 2000.16 Similarly, an Irish study predicted mortality from gastric cancer to fall further in the near future, although with a slower rate than in the recent past.17
Determinations of future mortality trends should be based on a careful analysis of trends in the past.1819 Therefore, in a study, the analysis started with a description of trends in gastric cancer mortality over a long period, from 1980 up to 2005, in order to check whether mortality decline continued undiminished until recent years.20 In addition, it has been assessed whether the rate of decline was similar in all seven countries and both sexes, despite differences in overall rates of gastric mortality.
The noticeable decline in gastric cancer mortality was found to continue at an undiminished rate until 2005 in each of the seven European countries. If this decline were to continue in the future, gastric cancer mortality rates would decrease with about 66 percent between 2005 and 2030. A two-thirds decline would also observe in terms of the effect of gastric cancer on people’s life expectancy at birth. The absolute number of gastric cancer deaths would diminish by about 50 percent despite the ageing of national populations. Thus, by extrapolating the strong, stable and consistent mortality rate declines in recent decades, gastric cancer was projected to become increasingly less important as a cause of death in Europe in the next decades.
Empirical support for the expectation that the decline in mortality from gastric cancer will continue in the future comes from the trends that were studied in the past 25 years. First, a strong consistency exists in the recent trends in gastric cancer mortality among both sexes and among each of the seven European countries. Second, these declines have persisted up to recent years in each of these populations, including those with the lowest initial mortality rates. Furthermore, a steady decline in gastric cancer mortality rate was observed in the middle-aged and the young population as well, suggesting that they are likely to continue in the near future.3415 The latter observation is consistent with analysis of cohort-wise patterns of decline in gastric cancer mortality in European countries,11321 which may reflect the effects of life style improvement in childhood.1
It should be emphasized that, even though future declines may seem likely, in this study, we primarily aimed to explore possible future trends by extrapolating past trends. This extrapolation would provide a baseline scenario against which new studies may formulate more specific scenarios of future trends. For example, policy-based scenarios may focus on the potential effects of specific preventive policies or advancement in the treatment of gastric cancer. As the gastric cancer may become ever less important in terms of mortality, scenario studies will need to also include measures of incidence, prognosis and prevalence of gastric cancer.
Conflict of interest statement: Author has no conflict of interest.
Source of funding: None.
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1 Amiri M Kunst AE Janssen F Mackenbach JP Trends in stomach cancer mortality in relation to living conditions in childhood. A study among cohorts born between 1860 and 1939 in seven European countries Eur J Cancer 2006 42 18 3212 8 16945523
2 Kelley JR Duggan JM Gastric cancer epidemiology and risk factors J Clin Epidemiol 2003 56 1 1 9 12589864
3 Levi F Lucchini F Gonzalez JR Fernandez E Negri E La Vecchia C Monitoring falls in gastric cancer mortality in Europe Ann Oncol 2004 15 2 338 45 14760131
4 Bosetti C Bertuccio P Levi F Lucchini F Negri E La Vecchia C Cancer mortality in the European Union, 1970-2003, with a joinpoint analysis Ann Oncol 2008 19 4 631 40 18281267
5 Lepage C Remontet L Launoy G Tretarre B Grosclaude P Colonna M Trends in incidence of digestive cancers in France Eur J Cancer Prev 2008 17 1 13 7 18090905
6 Levi F Lucchini F Negri E La Vecchia C Continuing declines in cancer mortality in the European Union Ann Oncol 2007 18 3 593 5 17164227
7 Jemal A Siegel R Ward E Murray T Xu J Thun MJ Cancer statistics, 2007 CA Cancer J Clin 2007 57 1 43 66 17237035
8 Parkin DM Bray F Ferlay J Pisani P Estimating the world cancer burden: Globocan 2000 Int J Cancer 2001 94 2 153 6 11668491
9 Parkin DM Bray F Ferlay J Pisani P Global cancer statistics, 2002 CA Cancer J Clin 2005 55 2 74 108 15761078
10 Francisci S Capocaccia R Grande E Santaquilani M Simonetti A Allemani C C The cure of cancer: a European perspective Eur J Cancer 2009 45 6 1067 79 19131242
11 Kamangar F Dores GM Anderson WF Patterns of cancer incidence, mortality, and prevalence across five continents: defining priorities to reduce cancer disparities in different geographic regions of the world J Clin Oncol 2006 24 14 2137 50 16682732
12 Karim-Kos HE de Vries E Soerjomataram I Lemmens V Siesling S Coebergh JW Recent trends of cancer in Europe: a combined approach of incidence, survival and mortality for 17 cancer sites since the 1990s Eur J Cancer 2008 44 10 1345 89 18280139
13 Fitzsimmons D Osmond C George S Johnson CD Trends in stomach and pancreatic cancer incidence and mortality in England and Wales, 1951-2000 Br J Surg 2007 94 9 1162 71 17520709
14 Aragones N Pollan M Rodero I Lopez-Abente G Gastric cancer in the European Union (1968-1992): mortality trends and cohort effect Ann Epidemiol 1997 7 4 294 303 9177113
15 Quinn MJ d’Onofrio A Moller B Black R Martinez-Garcia C Moller H H Cancer mortality trends in the EU and acceding countries up to 2015 Ann Oncol 2003 14 7 1148 52 12853360
16 Coebergh JW 2004 Kanker in Nederland KWF kankerbestrijding 88 94 Avalable from http://spitswww.uvt.nl/~Fmols/kanker_in_nederland.pdf
17 O’Lorcain P Deady S Comber H Mortality predictions for esophageal, stomach, and pancreatic cancer, Ireland, up to 2015 Int J Gastrointest Cancer 2006 37 1 15 25 17290077
18 Janssen F Kunst A The choice among past trends as a basis for the prediction of future trends in old-age mortality Popul Stud (Camb) 2007 61 3 315 26 17979005
19 Wilmoth JR Demography of longevity: past, present, and future trends Exp Gerontol 2000 35 9-10 1111 29 11113596
20 Amiri M Janssen F Kunst AE The decline in stomach cancer mortality: exploration of future trends in seven European countries Eur J Epidemiol 2011 26 1 23 8 21086022
21 La Vecchia C Negri E Levi F Decarli A Boyle P Cancer mortality in Europe: effects of age, cohort of birth and period of death Eur J Cancer 1998 34 1 118 41 9624248 | 21603016 | PMC3093769 | CC BY | 2021-01-04 19:28:41 | yes | Int J Prev Med. 2011 Apr-Jun; 2(2):101-102 |
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Int J Prev MedIJPVMInternational Journal of Preventive Medicine2008-78022008-8213Medknow Publications India IJPVM-2-73Original ArticleEpigenetically Reprogramming of Human Embryonic Stem Cells by 3-Deazaneplanocin A and Sodium Butyrate Azghadi Soheila MD1Clark Amander T. PhD21 Department of Molecular Cell and Developmental Biology, University of California Los Angeles, Los Angeles, California, United States of America2 Anatomy and Cell biology, Department of Molecular Cell and Developmental Biology, University of California Los Angeles, Los Angeles, California, United States of AmericaCorrespondence to: Amander T. Clark, PhD of Anatomy and Cell biology, Department of Molecular Cell and Developmental Biology, University of California Los Angeles, Los Angeles, California, United States of America. Email: [email protected] 2011 2 2 73 78 15 11 2010 21 1 2011 © International Journal of Preventive Medicine2011This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Objectives:
Infertility affects about 6.1 million women aged 15-44 in the United States. The leading cause of infertility in women is quantitative and qualitative defects in human germ-cell development (these sentences are not mentioned in introduction so it is not correct to mention in abstract, you can omit). Human embryonic stem cell (hESC) lines are derived from the inner cell mass (ICM) of developing blastocysts and have a broad clinical potential. hESCs have been classified into three classes based on their epigenetic state. The goal of this study was to epigenetically reprogram Class II and Class III cell lines to Class I (naïve state), and to in vitro differentiation of potent hESCs to primordial germ cells (PGCs).
Methods:
Recent evidence suggests that 3-deazaneplanocin A (DZNep) is a global histone methylation inhibitor which selectively inhibits trimethylation of lysine 27 on histone H3K27, and it is an epigenetic therapeutic for cancer. The characteristics of DZNep lead us to hypothesize that it is a good candidate to epigenetically reprogram hESCs to the Class I. Additionally, we used sodium butyrate (NaBu) shown in previous studies to up-regulate the expression of germ cell specific markers (these sentences should be come in introduction).
Results:
We used these two drugs to produce epigenetically stable hESC lines. hESC lines are an appropriate system for disease modeling and understanding developmental stages, therefore producing stable stem cell lines may have an outstanding impact in different research fields such as preventive medicine.
Conclusions:
X-Chromosome inactivation has been used as a tool to follow the reprogramming process. We have used immunostaining and western blot as methods to follow this reprogramming qualitatively and quantitatively.
InfertilityStemcellModelingReprogramming
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INTRODUCTION
Human embryonic stem cells (hESCs) can be maintained in culture in a self-renewing state and differentiate into all three embryonic germ layers. Although they hold great promise for regenerative medicine, hESC-based therapy faces several challenges. Concerns have been raised regarding their genetic stability. Although not fully understood, challenges also exist over epigenetic stability. Epigenetic changes make nuclear program altered without changing the primary DNA sequence. Because epigenetic changes can substantially modify cellular behavior and are mitotically and meiotically heritable, investigation of the epigenetic properties of human hESC is desirable prior to considering their use in vivo. Epigenetic state is one of the hESCs states that enables stem cells with the unique properties to self renew or differentiate into any cell type in the body. The hESC state may be influenced by the manner in which ESCs are derived and maintained. Recent studies have showed that the efficiency of induced pluripotent stem (iPS) cells formation is enhanced upon addition of valporic acid, an inhibitor of histone deacetylases, to the culture medium. Sodium butyrate, a naturally occurring short-chain fatty acid, supports the extensive self-renewal of mouse embryonic stem cell (hESCs) and1
X-chromosome inactivation (XCI) phenomenon has been used to examine the epigenetic stability of hESC. Because XCI is one of the first measurable epigenetic changes in the early mammalian embryo and is coincident with differentiation, XCI marker serves as an excellent tool to investigate the epigenetic behavior of hESC. XCI is a mechanism to compensate gene load difference between XY males and XX females in mammals. During early embryogenesis, one of two X-chromosomes in every cell is inactivated, and stably inherited through cell division of somatic cells.2 XIST makes a noncoding RNA required to initiate silencing during XCI. Before XCI in mESCs, XIST is expressed at low level. Upon cell differentiation and the onset of XCI, XIST RNA is transcriptionally induces and forms a cloud around the inactive X (Xi). However, it is known that the two X chromosomes are active in oocytes, indicating that the inactive X chromosome must be reactivated during germ cell development. The reactivation of inactive X chromosome occurs at least twice during mammalian development, once in the epiblast cell lineage at the peri implantation stage and once in the primordial germ cells (PGCs) at the midgestation.2 The dynamics of X chromosome activity is tightly correlated with major genomic reprogramming events occurring during mammalian development.
Recently, 11 distinct hESC lines have been studied in order to investigate their epigenetic properties by using XCI markers mainly studying XIST expression. Unlike mESCs, hESCs are pre-XCI and there is variability of XIST expression among different hESC lines.3 These cell lines can be subgrouped into three classes. Class I line has the capacity to recapitulate XCI when induced to differentiate in culture. Class II cells have already undergone XCI. In class III cell lines, despite losing tirmethylation of histone H3-K27 (H3K27me3), there is a tendency to lose XIST RNA expression.
Histone lysine methylation has been shown to index silenced chromatin regions at pericentric heterochromatin or of the inactive X chromosome.4 H3K27me3 is a repressive chromatin mark. Recently, 3- deazaneplanocin A (DZNep) was discovered to selectively inhibit H3K27me3. DZNep affects multiple histone methyltransferases and can epigenetically reactivate a different cohort of genes.5
In the current study, our goal was to reprogram class II cell lines to class I by using DZNep as H3K27me3 methyltransferase inhibitor, and sodium butyrate as histone deacetylase inhibitor which enhances self-renewal status of embryonic stem cells and also up-regulates germ-cell specific markers.1 We used three Class II hESC lines: HSF6-8, HSF6-10, and HSF6-S9.
METHODS
Cells and Drug Treatment
Initial lines of hESC (HSF6-8, HSF6-10, and HSF6-S9) were cultured on a feeder layer of mouse embryonic fibroblasts (MEF). Human ESC culture medium (hESM) was consisted of DMEM/f12 supplemented with 100ml KSR, 2.5 ml L-glutamine, 500 μl BME, and 5 ml nonessential amino acids. Then cells were treated with 0.1 μM DZNep and 0.2 mM sodium butyrate.
Histone Protein Extraction
Human Embryonic stem cells were harvested and washed twice with ice-cold PBS. Cells were re-suspended in Triton Extraction Buffer (TEB: PBS containing 0.5% Triton X 100 (v/v), 2mM phenylmethylsufonyl fluoride (PMSF), 0.02% (w/v) NaN2) with a cell density of 107 cells per ml. Cells were lysed for 10 min using gentle stirring, and centrifuged at 6500× g for 10 minutes at 4 degree C to spin down the nuclei. Pellet was re-suspended in 0.2 N HCl at a density of 4x107 nuclei per ml. The extract was stored overnight at 4 degree C. Samples were centrifuged at 6500xg for 10 minutes to pellet debris. Supernatant was stored and the histone concentration was detected by Commassie Blue.
Western Blots
Cells were harvested by treatment with Try-pLE express. Cells were lysed using 1.0 ml hypotonic lysis buffer (10mM Tris-HCl, 1.5mM MgCl2 10mM KCl, 0.34M sucrose, 10% glycerol,40μl of PI stock solution, and 1.0 μl of DTT). Nuclear histones were separated on 16% SDS-polyacrylamide gels at 200v, 150mA for an hour and stained with commasie blue. Equal amounts of protein were separated in SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The blots were probed with antibodies against histone H3, H3K27me3, and H3K4me3.
RESULTS
Differential Expression of XCI Marker in Cultures of Female hESC Lines
Using immunostaining of H3K27me3, cultures of female HSF6-8, HSF6-10, and HSF6-S9 cells showed 0-50% of XCI marker. Cells were stained with DAPI (a dye binding to DNA), Oct4 (a stem cell marker), and H3K27me3. In a subset of stem cell population, H3K27me3 accompanied Xi, however, some H3K27me3 didn’t accompany Xi (Fig. 1). The absence of H3K27me3 marker is not indicative of X chromosome loss. X chromosome DNA FISH in a subset of cells which didn’t have H3K27me3 enrichment along with Xi, showed the presence of two X chromosomes (Fig. 2).
Figure 1 hESC lines have heterogeneous H3K27me3 deposition associated with Xi. A: In the stem cell population presented in panel A half of the cells don’t have H3K27me3 marker associated with XCI. DAPI is a fluorescent stain which binds to DNA. Oct4 is a stem cell marker.
Figure 2 Loss of XCI associated with H3K27me3 marker is not due to loss of X chromosome. Merge picture of Xchromosome DNA FISH and H3K27me3 marker suggests that Loss of H3K27me3 marker in a subset of cells is not due to the loss of X-Chromosome. DNA FISH shows two X-Chromosomes even in the absence of H3K27me3.
Optimization of DZNep Concentration and Colony Morphology
In cancer treatment, DZNep is used with high concentration (10μM) in order to induce apoptosis in cancerous cells, and reactivate genes which have became silent by cancer development. Therefore, in the current study the DZNep concentration should have been optimized. NaBu was added at 0.2 mM during three passages and then different concentrations of DZNep were added. The optimal concentration for growing colonies of DZNep seemed to be 0.1 μM (Fig. 3). Healthy, undifferentiated HSF6-8, HSF6-10, and HSF6-S9 colonies treated with 0.2μM NaBu, and 0.1μM DZNep seemed to have well-defined uniform borders and the individual cells within the colony appeared to be similar. More cells per colony were observed compared to the untreated colonies (Fig. 4).
Figure 3 Optimization of DZNep concentration and the colony morphology: NaBu was added at 0.2 mM during three passages and then DZNep was added at different concentrations. 0.1 μM of DZNep seemed to be the optimal concentration for growing colonies.
Figure 4 Colony Morphology. Treated cells with NaBu and DZNep appeared to have more cells per colony, and cells have typical stem cell appearance with less differentiation. p: passaging, B: NaBu.
hESCs Treated With DZNep Show Tendency to Lose XCI H3K27me3 Marker
Immunostaining analyses showed that cells tend to lose H3K27me3 enrichment associated with Xi by several passages. This effect was prominent when NaBu was added to cells for 11 passages. Cells lost H3K27me3 marker by 70%. Treating cells with DZNep for five passages caused cells to lose H3K27me3 by 90% (Fig. 5).
Figure 5 hESCs treated with DZNep show tendency to lose H3K27me3 marker. A: After several passaging hESCs tend to lose H3K27me3 marker naturally. B: hESCs treated with sodium butyrate lose H3K27me3 50% faster than the control cells. C: hESCs treated with both sodium butyrate and DZNep show dramatic effect on losing H3K27me3 marker.
X-Chromosomes are associated with Euchromatic Region in Reprogrammed Cells
HSF1 male cell line immunostained with H3K4me3 (a euchromatic region marker) showed homogenous red color with no exclusion mark which is indicative of a heterochromatic region. X-chromosome DNA FISH showed one active X chromosome (Fig. 6A). In a subpopulation of HSF6-10 cell line, there was a homogenous staining for H3K4me3 with no exclusion mark, and DNA FISH showed the presence of two active X chromosomes (Fig. 6B). In another subset of HSF6 (10) cell line, there was an exclusion mark in H3K4me3 staining, and DNA FISH showed two X chromosomes (Fig. 6C).
Figure 6 X-Chromosome is associated with Euchromatic region in reprogrammed cells. A : DNA FISH using an X chromosome specific probe was performed on HSF1 male cell line. All cells have one active X chromosome. H3K4me3 (a euchromatic region marker) is shown in red. B : H3K4me3 immunostaining and DNA FISH for X chromosome in HSF6 (10) treated cells show two active X chromosomes. C : Some portion of HSF6 (10) treated cells shows resistance to reprogramming and still has one inactive X-chromosome.
Western Blot Analyses Showed no Global Changes in Trimethylation of H3K27 and H3K4
Control cells had no drug added. NaBu and DZNep were added to treated cells with 0.2μM and 0.1μM concentrations respectively. Western analyses of HSF6-8, HSF6-10, and HSF6-S9 control cells, treated cells with NaBu, and treated cells with NaBu and DZNep showed no global changes in trimethylation of H3K27 and H3K4. Histone H3 was the loading control. The trimethylation of H3K27 demonstrated no obvious changes in control and treated cells (Fig. 7).
Figure 7 Western blot analyses show no global changes in H3K27 trimethylation. The western analyses show no global changes in H3K27 trimethylation. There are the same level of H3K27me3 and H3K4me3 in control and treated cells. Histone H3 is the loading control. * Samples with more cells per colony.
DISCUSSION
In order to mimic in vivo germ cell development pathway, hESCs should be in their native state, and keep their native state during passages. Three classes of hESC lines derived from ICM of developing blastocyst are similar in their pluripotency potential and forming teratomas when injected to mice. The different point among them is H3K27me3 enrichment deposited in Xi. Also, these three classes have different expression of XIST RNA. Class I cell lines with active X chromosomes are the ones which resembles the in vivo system. However, hESC lines are usually a kind of class II and class III, and even if they are class I, they would transform to class II or III through passaging.
Treated cells with 0.2mM NaBu and 0.1mM DZNep formed colonies with typical stem cell colony appearance which indicates that these two drugs would not interfere with self-renewal state of the cells. Additionally, treated cells had more cells per colony which suggests that the drugs even promote the self-renewal state. Less differentiation in treated cells compared the controls suggests that the differentiation pathway would stop or slow down by NaBu and DZNep.
The loss of H3K27me3 associated with Xi confirmed by immunostaining analyses suggests that NaBu and DZNep have ability to change H3K27 modification. H3K4me3 staining confirmed the existence of two active X chromosomes. In H3K4me3 staining there was no exclusion mark which suggests that both X chromosomes were active. Immunostaining results indicate that 30% of treated class II cells have been reprogrammed to Class I.
H3K27me3 is a suppressing marker. Naturally, many genes should be kept silent, such as oncogenes. Paternally or maternally expressed genes should become silent in the other parent. Therefore, the integrity maintences of necessary silent genes are crucial. Western blot analyses showed that NaBu and DZNep didn’t change the global trimethylation of H3K27 and H3K4, and they selectively change the trimethylation of H3K27 associated with Xi. Therefore, using these two drugs with mentioned concentrations is safe, and won’t have any global effect.
For future direction, we want to perform differentiation assays to confirm the proper reprogramming process. Also, gene expression assays seem to be informative for investigating gene expression profiles between control and treated cells. In order to rule out any chromosomal abnormality causing by DZNep and NaBu, cytogenetic analysis is necessary. Finally, investigating the effects of NaBu and DZNep in newly developed cell lines will further confirm the ability of the drugs to epigenetically reprogram class II cell lines to the class I.
Conflict of interest statement: All authors declare that they have no conflict of interest.
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3 Silva SS Rowntree RK Mekhoubad S Lee JT X-chromosome inactivation and epigenetic fluidity in human embryonic stem cells Proc Natl Acad Sci U S A 2008 105 12 4820 5 18339803
4 Martens JH O’Sullivan RJ Braunschweig U Opravil S Radolf M Steinlein P The profile of repeat-associated histone lysine methylation states in the mouse epigenome EMBO J 2005 24 4 800 12 15678104
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 21625550PONE-D-11-0022410.1371/journal.pone.0019652Research ArticleBiologyMolecular Cell BiologyCellular Stress ResponsesGene ExpressionSignal TransductionToxicologyToxic AgentsChemistryEnvironmental ChemistryMedicineNutritionOncologyBasic Cancer ResearchOxidative DamageLycopene Inhibits NF-kB-Mediated IL-8 Expression and Changes Redox and PPARγ Signalling in Cigarette Smoke–Stimulated Macrophages Lycopene Inhibition of CSE-Induced IL-8 ProductionSimone Rossella E.
1
Russo Marco
2
Catalano Assunta
1
Monego Giovanni
3
Froehlich Kati
2
Boehm Volker
2
Palozza Paola
1
*
1
Institute of General Pathology, Catholic University, Rome, Italy
2
Institute of Nutrition, Friedrich-Schiller-Universität, Jena, Germany
3
Institute of Anatomy, Catholic University School of Medicine, Rome, Italy
Gaetano Carlo EditorIstituto Dermopatico dell'Immacolata, Italy* E-mail: [email protected] and designed the experiments: PP. Performed the experiments: RES MR AC KF GM. Analyzed the data: PP VB. Contributed reagents/materials/analysis tools: PP VB. Wrote the paper: PP.
2011 19 5 2011 6 5 e1965220 12 2010 7 4 2011 Simone et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Increasing evidence suggests that lycopene, the major carotenoid present in tomato, may be preventive against smoke-induced cell damage. However, the mechanisms of such a prevention are still unclear. The aim of this study was to investigate the role of lycopene on the production of the pro-inflammatory cytokine IL-8 induced by cigarette smoke and the possible mechanisms implicated. Therefore, human THP-1 macrophages were exposed to cigarette smoke extract (CSE), alone and following a 6-h pre-treatment with lycopene (0.5–2 µM). CSE enhanced IL-8 production in a time- and a dose-dependent manner. Lycopene pre-treatment resulted in a significant inhibition of CSE-induced IL-8 expression at both mRNA and protein levels. NF-kB controlled the transcription of IL-8 induced by CSE, since PDTC prevented such a production. Lycopene suppressed CSE-induced NF-kB DNA binding, NF-kB/p65 nuclear translocation and phosphorylation of IKKα and IkBα. Such an inhibition was accompanied by a decrease in CSE-induced ROS production and NOX-4 expression. Lycopene further inhibited CSE-induced phosphorylation of the redox-sensitive ERK1/2, JNK and p38 MAPKs. Moreover, the carotenoid increased PPARγ levels which, in turn, enhanced PTEN expression and decreased pAKT levels in CSE-exposed cells. Such effects were abolished by the PPARγ inhibitor GW9662. Taken together, our data indicate that lycopene prevented CSE-induced IL-8 production through a mechanism involving an inactivation of NF-kB. NF-kB inactivation was accompanied by an inhibition of redox signalling and an activation of PPARγ signalling. The ability of lycopene in inhibiting IL-8 production, NF-kB/p65 nuclear translocation, and redox signalling and in increasing PPARγ expression was also found in isolated rat alveolar macrophages exposed to CSE. These findings provide novel data on new molecular mechanisms by which lycopene regulates cigarette smoke-driven inflammation in human macrophages.
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Introduction
Chronic obstructive pulmonary disease (COPD) is a syndrome characterized by progressive airflow limitation caused by chronic inflammation of the airways and lung parenchyma, which is due predominantly to chronic cigarette smoking [1]. Chronic exposure to cigarette smoke activates an inflammatory cascade in the airways resulting in the production of a number of cytokines and chemokines, with accompanying damage to the lung epithelium and increased vascular permeability and recruitment of macrophages and neutrophils [2], [3]. Macrophages are the major defence cells in the lower airways of the lung in healthy nonsmokers and appear to have an essential role in the pathogenesis of COPD by accounting for most known features of the disease [4]. Bronchoalveolar lavage (BAL) fluid from smokers compared to nonsmokers show a five-fold increase in the number of inflammatory cells in the lung, of which 85–90% are alveolar macrophages. Macrophages are predominant cells in the respiratory bronchioles of smokers; studies have shown a correlation between alveolar macrophage numbers and the extent of lung destruction in emphysema [5], [6].
The human chemokine IL-8 in particular, a member of the CXC chemokine family, activates adhesion molecules expression on endothelial cells [7] and it is an important activator and chemo-attractant for neutrophils [8] as well as T cells [9] and monocytes [10]. Increased levels of IL-8 have been found in induced sputum [11] and bronchoalveolar lavage from patients with smoking-related COPD associated with increased numbers of activated neutrophils [12] . Therefore, IL-8 has been implicated in the initiation and maintenance of chronic airway inflammation induced by cigarette smoke.
Cigarette smoke harbors a multitude of chemical compounds, including high concentrations of free radicals and other oxidant species [13] and causes direct oxidative lung damage and indirect damage through the activation of various lung cells including alveolar macrophages [14]. Therefore, reactive oxygen species (ROS) present in smoke and phagocyte-derived ROS are intimately involved in the pathogenesis of smoking-related inflammation.
Nuclear factor-kB (NF-kB) is one of the redox-sensitive transcription factors involved in the inflammatory responses to cigarette smoke in the lungs and its activity is regulated by cytoplasmic degradation of the IkB inhibitor [15]. NF-kB dimers localize to the nucleus, once IkBα is inactivated, and undergo further modification, mostly through phosphorylation of the Rel proteins [15]. In the nucleus activated NF-kB binds to promoters of its target genes and regulates the expression of genes involved in many cellular events, including inflammation [16] through the activation of the Akt/phosphoinositide 3-kinase (PI3K) and the mitogen-activated protein kinase (MAPK) cascade [17]–[20].
It is known that peroxisome proliferator activated receptor-γ (PPARγ), a member of the ligand activated nuclear receptor superfamily, is able to regulate anti-inflammatory responses in cells exposed to cigarette smoke and that ligand-activated PPARγ is able to down-regulate NF-kB transcription. Recently, reports show that upregulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), with the concomitant downregulation of PI3K-dependent signaling pathways [21]–[23], might be one of the mechanisms through which PPARγ agonists exert their anti-inflammatory actions.
Beneficial effects of tomato lycopene on the risk of smoke-related pathologies, including cancer and cardiovascular injury, have been found in epidemiological studies [24]. However, the experimental basis for such health benefits is not fully understood. One of the possible mechanisms for the protective activities of lycopene in smoke-related pathologies is by down-regulation of the cigarette smoke-stimulated inflammatory response [25], [26]. In fact, the carotenoid has been reported to inhibit pivotal pro-inflammatory mediators, including ROS [27] and cytokines [28] and to affect signal transduction pathways involved in inflammatory processes, including nicotinamide adenine dinucleotide phosphate oxidase oxidase [NADP(H)-oxidase] [28], MAPK [29], Akt/PI3K [30] and transcription factors, such as activator protein-1 (AP-1) [31] and NF-kB [31]–[35] and PPARγ cascade [28]. We recently reported that lycopene may inhibit ROS production, NOX-4 expression and cytokine release in human macrophages exposed to oxysterols [28]. Moreover, in the same model, it may enhance PPARγ expression [28].
To better understanding the role of lycopene in inflammatory processes caused by cigarette smoke, we investigated the effect of lycopene in modifying molecular pathways involved in cigarette smoke extract (CSE)-induced IL-8 production in human macrophages. Use of CSE remains a good practical model to study changes in cellular responses induced by cigarette smoke, since it mimics the in vivo soluble cigarette smoke components that are present during cigarette smoking. Here, we provide evidence of the inter-relationships between the signalling networks activated by cigarette smoke-induced stress and the anti-inflammatory effect of lycopene in macrophages.
Results
Lycopene inhibition of CSE-induced IL-8 production and NF-kB activation
Cigarette smoke induces a multitude of direct and indirect effects on lung tissue, but it is principally responsible for inflammation resulting in accumulation of macrophages and release of chemical mediators, which changes lung functions, morphology and gene expression. Since it has been reported that exposure to cigarette of macrophages stimulates release of different pro-inflammatory cytokines, including IL-8 [36] we measured the effect of different concentrations of CSE on intracellular IL-8 production in THP-1 cells incubated for 6 h and 24 h (Fig. 1A). CSE induced a dose- and a time- dependent increase in IL-8 production. A 6-h pre-treatment with lycopene at 2 µM, the maximum concentration which can be achievable in vivo after supplementation [37], down-regulated the production of IL-8 induced by CSE (Fig. 1B). Such an effect was dependent on the dose of carotenoid (Fig. 1C) and also occurred at mRNA levels (Fig. 1D). Similar results were found in the culture medium (Fig. 1E). A 6-h pre-treatment with lycopene reduced the increase of IL-8 production in culture medium induced by a 24-h treatment with CSE. These data show an anti-inflammatory potential of lycopene in CSE-exposed macrophages.
10.1371/journal.pone.0019652.g001Figure 1 Effects of Cigarette smoke extract (CSE), alone and in combination with lycopene, on IL-8 production in human THP-1 cells.
Panels A-D: intracellular IL-8 production; panel E: IL-8 production in culture medium. Panel A: effects of different CSE concentrations for 6 h and 24 h; panels B, D, E: effects of a pre-treatment for 6 h with lycopene (2 µM) followed by a 24-h CSE (0.5%) exposure; panel C: effects of a pre-treatment with different concentrations of lycopene followed by a 24-h CSE (0.5%) exposure; Panels A, B, C: representative Western Blot analyses; the values indicated represented the ratio of IL-8 and actin. Panel D: mRNA levels by reverse transcription polymerase chain reaction. Panel E: the values were the means ± SEM of three independent experiments. Panel E: the values were the means ± SEM of three independent experiments. Values not sharing the same letter were significantly different (P<0.05, Fisher's test).
Since it has been reported that IL-8 levels are modulated by NF-kB, we, then, investigated the effect of the specific NF-kB inhibitor pyrrolidine dithiocarbamate (PDTC) on CSE-induced IL-8 production in THP-1 cells. The pre-incubation of THP-1 cells with PDTC (10 µM) for 30 min prevented IL-8 protein production (Fig. 2A), demonstrating the key role of this transcription factor in CSE-stimulated IL-8 production by human macrophages. We further analysed the effects of CSE, alone and in combination with lycopene, on NF-kB DNA-binding activity in THP-1 cells. Nuclear extracts were prepared and NF-kB DNA binding activity was examined by EMSA. As shown in Figure 2B, a 3 h-treatment of the THP-1 cells with CSE resulted in an increase of the NF-kB DNA-binding activity. A 6-h pre-treatment with lycopene, at the concentration of 2 µM, inhibited CSE-mediated NF-kB DNA-binding activity. On the other hand, when compared to vehicle-control treated cells, no significant alterations in the DNA-binding activity were observed in lycopene alone-treated cells, suggesting that the NF-kB DNA-binding activity expression may not be substantially affected by physiological concentrations of the carotenoid. Moreover, CSE induced the translocation of NF-kB/p65 to the nucleus and lycopene pre-treatment suppressed it (Figure 2C). To determine whether the inhibitory action of lycopene towards NF-kB activation was due to its effect on IkBα degradation, the cytosolic levels of IKKα and IkBα were determined by Western blot analysis in cells treated with lycopene and CSE (Fig. 2D). Western blot analysis showed that CSE exposure resulted in phosphorylation of IKKα and pre-treatment of THP-1 cells with lycopene inhibited this phosphorylation (Fig. 2D). Further examining the effect of lycopene on IkBα phosphorylation by Western blot analysis, using an antibody that detects only the serine-phosphorylated form of IkBα, we found that CSE exposure resulted in increased phosphorylation of IkBα (Fig. 2D). Our data show that lycopene treatment of THP-1 cells suppressed this phosphorylation. These findings indicate that lycopene treatment of THP-1 cells resulted in inhibition of CSE-induced activation of IKKα, phosphorylation and degradation of IkBα, and subsequent activation of NF-kB.
10.1371/journal.pone.0019652.g002Figure 2 Effects of lycopene on CSE-stimulated NF-kB activation in human THP-1 cells.
Panel A: IL-8 production in culture medium after addition of the NF-kB inhibitor pyrrolidine dithiocarbamate (PDTC). THP-1 cells were pre-incubated with PDTC (10 µM) for 30 min and, then treated with CSE was added for 3 h and 24 h. Panel B: NF-kB DNA binding activity in cells pre-treated for 6 h with lycopene (2 µM) and, then exposed to CSE (0.5%) for 3 h; the specifity was demonstrated by using excess unlabeled NF-kB oligonucleotides ( = cold ssNF-kB) which competed away binding. Panel C: representative western blot analysis of nuclear NF-kB (p65)-associated proteins in cells pre-treated for 6 h with lycopene (2 µM) and, then exposed to CSE (0.5%) for 3 h; panel D: representative western blot analyses of cytosolic p-IKKα and p-IkBα in cells pre-treated for 6 h with lycopene (2 µM) and, then exposed to CSE (0.5%) for 3 h; the values indicated represented the ratio protein/actin. In panel A, values were the means ± SEM of three experiments. Values not sharing the same letter were significantly different (P<0.05, Fisher's test).
Lycopene inhibition of CSE-stimulated redox signalling
It is known that NF-kB is activated by ROS and several lines of evidence suggest that cigarette smoke is a source of ROS. Moreover, cigarette smoke constituents can directly activate vascular ROS production through induction of NAD(P)H oxidase. Therefore, we measured ROS levels in the absence and in the presence of diphenyleneiodonium (DPI), an inhibitor of cellular NADPH oxidase in THP-1 cells exposed to CSE (Fig. 3A). Exposure of THP-1 cells to CSE, caused an increase in ROS production. Such an effect was evident at 3 h, and persisted at 24 h. When cells were pre-incubated with DPI at the concentration of 10 µM for 1 h and then washed prior to the addition of CSE, CSE-induced ROS production was abolished, suggesting that, at least in our experimental conditions, ROS production was generated intracellularly and not extracellularly by smoke. Pre-treatment of THP-1 cells with lycopene, at the concentration of 2 µM was able to prevent CSE-induced ROS production (Fig. 3B). Only slight and not significant inhibitory effects on ROS production by lycopene were observed in CSE-untreated THP-1 cells. The carotenoid was also able to inhibit the increase in NAD(P)H oxidase-4 (NOX-4) expression caused by CSE (Fig. 3C). Such an effect was evident at 3 h as well as at 24 h. Generation of oxidants by cigarette smoke appears to be the primary stimulus for activation of MAPK cascades. Whether the inhibitory effect of lycopene on CSE-induced ROS production extends to MAPK signalling pathway was investigated in THP-1 cells. Employing Western blot analysis, we found that a 3 h-CSE exposure resulted in a significant increase in the phosphorylation of JNK (Fig. 4A) ERK1/2 (Fig. 4B) and p38 (Fig. 4C) MAPK proteins in THP-1 cells. The levels of p-ERK1/2 were markedly higher than those of p-p38 and p-JNK. Pre-treatment with lycopene, at the concentration of 2 µM, resulted in a marked reduction in the CSE-induced phosphorylation of p38, ERK1/2 and JNK1/2 MAPK proteins, suggesting that the carotenoid is an effective inhibitor of these pathways.
10.1371/journal.pone.0019652.g003Figure 3 Effects of lycopene on CSE-induced reactive oxygen species (ROS) production in human THP-1 cells.
Panel A: ROS levels after addition of the NAD(P)H oxidase-4 (NOX-4) inhibitor diphenyleneiodonium (DPI) in cells exposed to CSE for 3 h and 24 h. Cells were pre-incubated with DPI at the concentration of 10 µM for 1 h and then washed prior to the addition of CSE. Panel B: ROS levels in cells pre-treated for 6 h with lycopene (2 µM) and, then exposed to CSE (0.5%) for 3 h and 24 h. Panel C: representative western blot analysis of NOX-4 in cells pre-treated for 6 h with lycopene (2 µM) and, then exposed to CSE (0.5%) for 3 h and 24 h; the values indicated represented the ratio NOX-4/actin. In panels A and B, values were the means ± SEM of five experiments. Values not sharing the same letter were significantly different (P<0.005, Tukey's test).
10.1371/journal.pone.0019652.g004Figure 4 Effects of lycopene on CSE-induced MAPK phosphorylation in human THP-1 cells.
Representative western blot analyses of JNK, ERK1/2 and p38 in cells pre-treated for 6 h with lycopene (2 µM) and, then exposed to CSE (0.5%) for 3 h. The values indicated represented the ratio phosphorylated protein/total protein.
Lycopene enhancement of CSE-inhibited PPARγ signalling
PPARγ has been shown to regulate anti-inflammatory responses in CSE-exposed cells and ligand-activated PPARγ has been reported to down-regulate NF-kB transcription. Therefore, we measured the levels of PPARγ in THP-1 cells pre-treated for 6 h with lycopene and then exposed to CSE (Figs. 5A and 5B). Our results show that CSE decreased PPARγ at mRNA (Fig. 5A) and protein (Fig. 5B) levels. On the other hand, lycopene alone was able to increase the expression of this transcription factor at both mRNA and protein levels. Lycopene, in association with CSE, prevented the decrease in PPARγ levels induced by CSE, maintaining very high levels of this transcription factor.
10.1371/journal.pone.0019652.g005Figure 5 Effects of lycopene on CSE-inhibited PPARγ signalling in human THP-1 cells.
Panel A: PPARγ mRNA and panel B: representative western blot analysis of PPARγ protein cells pre-treated for 6 h with lycopene (2 µM) and, then exposed to CSE (0.5%) for 3 h. Panel C: representative western blot analysis of PTEN in cells pre-treated for 6 h with lycopene (2 µM) and/or with the PPARγ inhibitor GW9662 and, then exposed to CSE (0.5%) for 24 h. Panel D: representative western blot analysis of AKT in cells pre-treated for 6 h with lycopene (2 µM) and/or with the PPARγ inhibitor GW9662 and, then exposed to CSE (0.5%) for 24 h. GW9662 was pre-incubated at the concentration of 10 µM. The values indicated represented the ratio protein/actin (PTEN) and phosphorylated protein/total protein (AKT). In panel A, values were the means ± SEM of three experiments. Values not sharing the same letter were significantly different (P<0.05, Fisher's test).
Since PPARγ could exert its anti-inflammatory actions through an up-regulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), THP-1 cells were treated with lycopene and CSE, alone and in combination, and PTEN expression was measured in the absence and in the presence of GW9662, an irreversible antagonist of PPARγ (Fig. 5C). Lycopene alone induced a ten-fold up-regulation of PTEN in THP-1 cells (Fig. 5C). Lycopene also induced an increase in the levels of PTEN in the presence of CSE (from 0.2 to 0.9), but this increase was much lower than that observed in cells treated with lycopene alone (0.9 vs 3.2), although PPARγ expression was not significant different in the two treatments. In contrast, CSE alone did not significantly modify PTEN expression, suggesting that the pro-inflammatory effects of CSE did not involve changes in this phosphatase. (Fig. 5C). However, the strong dependency of PTEN expression by PPARγ in cells treated with lycopene alone is suggested by the results obtained in the presence of GW9662. A 6-h pre-incubation with 10 µM GW9662 completely prevented the increase in PTEN expression induced by the carotenoid. GW9662 was also able to prevent the increase in PTEN expression induced by lycopene and CSE in combination, although in a minor extent.
It has been hypothesized that the up-regulation of PTEN could result in AKT inactivation. Therefore, we measured the levels of pAKT in THP-1 cells treated with lycopene and CSE, alone and in combination, in the absence and in the presence of GW9662 (Fig. 5D). Pre-treatment of THP-1 cells with lycopene induced a marked reduction in the CSE-induced phosphorylation of AKT, which was prevented by a 6-h pre-incubation with 10 µM GW9662 (Fig. 5D). These data suggest that the anti-inflammatory effects of lycopene (increase of PTEN expression and decrease in AKT phosphorylation) are PPARγ dependent. In contrast, THP-1 cells treated with CSE alone strongly increased AKT phosphorylation, but such an increase was independent of PPARγ, since pAKT expression was not modified by GW9662.
Effects of lycopene on IL-8 expression, NF-kB/p65 nuclear translocation, ROS production and and PPARγ expression in isolated rat alveolar macrophages exposed to CSE
The ability of lycopene to modulate IL-8 production, redox and PPARγ signalling induced by CSE was also studied in rat alveolar macrophages (AMs) isolated from bronchoalveolar lavages (BALs). After the isolation of the cells and their incubation in culture conditions, the AMs were treated with lycopene and CSE, alone and in combination, in the experimental conditions indicated for THP-1 cells (Fig. 6). A 6-h pre-treatment with lycopene at the concentration of 2 µM was able to decrease CSE-induced IL-8 mRNA expression (Fig. 6A), NF-kB/p65 nuclear translocation (Fig. 6B), ROS production (Fig. 6C) and to increase PPARγ expression (Fig. 6D), confirming a key role of lycopene in the inhibition of inflammatory response induced by cigarette smoke.
10.1371/journal.pone.0019652.g006Figure 6 Effects of lycopene, alone and in combination with cigarette smoke extract (CSE), on intracellular IL-8 mRNA levels, NF-kB/p65 nuclear translocation, ROS production and PPARγ expression in isolated rat alveolar macrophages (AMs).
Panel A: IL-8 levels by reverse transcription polymerase chain reaction in cells treated for 6 h with lycopene (2 µM) followed by a 24-h CSE (0.5%) exposure. Panel B: representative western blot analysis of nuclear NF-kB (p65)-associated proteins in cells pre-treated for 6 h with lycopene (2 µM) and, then exposed to CSE (0.5%) for 3 h. Panel C: ROS levels in cells pre-treated for 6 h with lycopene (2 µM) and, then exposed to CSE (0.5%) for 3 h. Panel D: PPARγ: representative western blot analysis of PPARγ protein in cells pre-treated for 6 h with lycopene (2 µM) and, then exposed to CSE (0.5%) for 3 h. In panels A and C, values were the means ± SEM of three experiments. Values not sharing the same letter were significantly different (P<0.05, Fisher's test).
Discussion
It is widely recognized that cigarette smoke caused an inflammatory response [1]. According with this, smoking is the major risk factor for chronic obstructive pulmonary disease (COPD) and lung fibrosis, pathological processes characterized by pulmonary leukocyte infiltration, chronic inflammation and progressive tissue destruction [1]. It has been reported that exposure to cigarette smoke of cultured human blood monocytes or macrophagic cultures stimulates release of several pro-inflammatory cytokines, including IL-8 [38]–[45]. In addition, fibroblasts [46]–[48] bronchial [49], [50] and alveolar [51] epithelial cells, which are the primary target for any inhaled environmental agent, have been reported to release IL-8, when exposed to cigarette smoke. In our study, the acute exposition to CSE up-regulated IL-8 expression of human THP-1 macrophages at both mRNA and protein levels in a dose- and a time-dependent manner. In contrast, pre-treatment with lycopene, in a range of concentrations which can be achievable in vivo after supplementation [37], was able to counteract this effect, suggesting that the carotenoid may have a key role in regulating smoke-induced inflammatory processes. This finding is in agreement with previous observations showing a down-regulation of pro-inflammatory cytokine expression by lycopene in in vitro and in vivo models [28], [52]–[56]. An inhibition of pro-inflammatory cytokine levels, including IL-8, has been observed following treatment with other carotenoids, including β-carotene [57], [58]. In particular, β-carotene has been reported to arrest the increase in IL-6 and IL-8 induced by a long-term cigarette smoke exposition in serum, bronchoalveolar lavage fluid and lung tissue of rats [57]. Moreover, β-carotene had an inhibitory effect on IL-8 and TNF-α secretion in phorbol-12-myristate-13-acetate-stimulated HL-60 cells, although such an inhibition was observed at low (2 µM) but not at high (20 µM) carotenoid concentrations [58]. In agreement with the literature [49], [59]–[61] our data show that the production of the pro-inflammatory cytokine IL-8 by CSE is strongly linked to activation of NF-kB, since PDTC, a chemical that stabilizes the NF-kB/IkB-α complex [62] and inhibits the nuclear translocation of activated NF-kB, significantly inhibited it. Therefore, lycopene potential ability to influence cytokine levels may be, at least in part, explained by carotenoid localization in or within cell membrane, modulating surface molecules for primary immune response, which activate signalling pathways responsible for modulation of NF-kB. According with this hypothesis, our data also show that lycopene may counteract the effect of CSE on NF-kB activation. The inhibition of NF-κB DNA binding activity by lycopene was mediated through the downregulation of IKKα and IκB phosphorylation, and NF–κB p65 subunit translocation from cytosol to nucleus. Increasing evidence suggests that lycopene inhibited the binding activity of NF-κB [32], [33]. Moreover, tomato lycopene extract prevented lipopolysaccharide (LPS)-induced pro-inflammatory gene expression by blocking NF-kB signalling [35]. Based on data showing that one of the early events occurring in smoke-exposed cells is the increase in ROS levels, we examined the possibility that ROS were involved in NF-kB activation. We believe that the intracellularly generated ROS trigger the effects of smoke in THP-1 cells, on the basis of our experiments showing that diphenyleneiodonium (DPI), an inhibitor of cellular NADPH oxidase, was able to abolish the presence of ROS in CSE-exposed macrophages. Concomitantly, CSE was able to increase NOX-4 expression in THP-1 cells. In our study, lycopene inhibited both CSE-induced ROS production and CSE-stimulated NOX-4 expression. This is not surprising since the carotenoid has been reported to act as an intracellular redox agent, at least in vitro conditions [26]. Due to its extended system of conjugated double bonds, lycopene can effectively quench singlet oxygen [27] and free radicals [63] generated by smoking exposure. Moreover, recent data suggest that the carotenoid may also inhibit NOX-4 expression in human macrophages exposed to oxidants [28], [64].
The MAP kinases (ERK1/2, p38 and JNK) have been reported to be activated by oxidant stimuli and to mediate production of cytokines via NF-kB activation [25], [65]–[67]. Whether the inhibitory effect of lycopene on CSE-induced NF-kB activation pathway extends to MAPK signalling pathway was investigated. Employing Western blot analysis, we found that CSE exposure to THP-1 cells resulted in a significant increase in the phosphorylation of p38, ERK1/2, and JNK1/2 MAPK proteins. On the other hand, lycopene treatment resulted in a marked reduction in the CSE-induced phosphorylation of the three redox-sensitive MAPK proteins as shown in Figure 4, suggesting that lycopene is an effective inhibitor of MAPK pathways in THP-1 cells. The findings that lycopene can reduce MAPK activation in human macrophages confirm previous evidence in LPS-stimulated murine bone marrow-dendritic cells [29] and in oxysterol-exposed THP-1 cells [28] .
It has been reported that nicotine and the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, two components of cigarette smoke, induce phosphorylation of AKT (pAKT) in non-immortalized human airway epithelial cells [68]. Moreover, pAKT has been reported to activate NF-kB signalling and the consequent expression of pro-inflammatory cytokines. We therefore tested the effect of lycopene on CSE-induced activation of AKT. For this, protein lysates were analysed by Western blot using antibodies that detect the phosphorylated form of this protein. As shown in Figure 4, CSE exposure to THP-1 cells resulted in increased AKT phosphorylation, and lycopene treatment suppressed it.
A recent in vitro study also suggests that lycopene may have a key role in the modulation of AKT pathway under smoke conditions [30]. In fact, while RAT-1 fibroblasts exposed to cigarette smoke condensate exhibited high levels of phosphorylated AKT, cells exposed to a combination of tar and lycopene strongly decreased them. Moreover, in the same study, the expression of the heat shock protein (hsp)90 was increased following tar exposure [30]. Such an increase was counteracted by lycopene. This finding is particularly interesting in view of a previous report showing that hsp90 maintains Akt activity by binding to Akt and by preventing PP2A-dependent dephosphorylation of Akt [69]. Moreover, hsp90 has been reported to prevent proteasome-dependent degradation of PDK1, which is known to activate Akt.
PPARγ has been implicated in anti-inflammatory response and increasing evidence suggests that cigarette smoke strongly reduced it [70]. PPARγ activation has been shown to inhibit pro-inflammatory cytokine production by preventing activation and translocation of NF-kB [71]. In our study, lycopene was able to increase PPARγ at both mRNA and protein levels, confirming previous reports [28]. Moreover, the carotenoid, counteracted the decrease in PPARγ induced by CSE. Recently, it has been shown that upregulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), with the concomitant downregulation of PI3K-dependent signaling pathways, might be another mechanism through which PPARγ exerted its anti-inflammatory actions. Therefore, we investigated the effects of lycopene on PTEN expression. Pre-treatment of THP-1 cells with the carotenoid increased PTEN expression and decreased Akt phosphorylation. However, it should be pointed out that the increase in PTEN expression by lycopene in the presence of CSE was much lower than that observed in the presence of lycopene alone, although PPARγ expression was not significant different in the two treatments. Interestingly, GW9662, which is an irreversible antagonist of PPARγ, prevented the increase in PTEN and the decrease in pAKT induced by lycopene, suggesting that PTEN activation required an increase in PPARγ levels and antagonized the phosphorylation of Akt. It has been suggested that lycopene may have a PPARγ agonist activity [72], [73]. In THP-1 cells [72] as well as in LNCaP cells [73], the specific antagonist of PPARγ (GW9662) reverted the PPARγ-mediated effects of lycopene on cholesterol metabolism [72] and/or cell proliferation [72], [73]. In addition, Sharoni and colleagues have proposed that some carotenoids, such as lycopene, phytoene, phytofluene, and β-carotene cause the transactivation of peroxisome proliferator response element in cells co-transfected with PPARγ [74]. In contrast, CSE alone did not exert pro-inflammatory effects through a down-regulation of PPARγ expression, since CSE treatment did not significantly affect PTEN expression and the increase in CSE-induced AKT phosphorylation was unaffected by GW9662.
In conclusion, CSE exposure of human THP-1 macrophages caused an increase in the levels of the pro-inflammatory cytokine IL-8 which occurred through activation of NF-kB. Pre-treatment of cells with lycopene decreased both CSE-induced IL-8 production and NF-kB activation. Two mechanisms, could be implicated in such anti-inflammatory response of lycopene. The first one involves an interference with redox signalling, demonstrated by decrease in ROS production and in redox-sensitive MAPK activation by lycopene. The second one involves, at least in part, a PPARγ-dependent activation of PTEN which results in Akt inactivation. These findings thus represent a direct evidence that lycopene may act as an anti-inflammatory compound in cells exposed to cigarette smoke and provide novel data on important molecular mechanisms by which lycopene regulates cigarette smoke-driven NF-kB-dependent inflammation in human macrophages.
Materials and Methods
Cell culture
THP-1 (American Type Culture Collection, Rockville, MD, USA) were grown in RPMI Dutch Modified (Sigma, Milan, Italy) without antibiotics and supplemented with 10% fetal calf serum, non essential aminoacids, 2 mM glutamine, and 1 mM sodium piruvate. Cells were maintained in log phase by seeding twice a week at density of 3×108 cells/L at 37°C under 5% CO2/air atmosphere. Lycopene (LycoRed Natural Products Industries Ltd, Beer Sheva, Israel) was delivered to the cells (109 cells/L) using tetrahydrofuran (THF) as a solvent. To avoid the formation of peroxides, the solvent contained 0.025% butylated hydroxytoluene (BHT). The stock solutions of lycopene were prepared immediately before each experiment. From the stock solutions, aliquots of lycopene were rapidly added to the culture medium to give the final concentrations indicated. Control cultures were treated with THF + BHT. The amount of THF added to the cells was not greater than 0.5% (v/v) and it was the same in control cells as well as in treated ones. No differences were found between cells treated with THF and untreated cells in terms of cell viability and ROS production. After the addition of lycopene for 6 h, the medium was not further replaced throughout the experiments. Experiments were routinely carried out on triplicate cultures. At the times indicated, cells were harvested and quadruplicate haemocytometer counts were performed. The trypan blue dye exclusion method was used to evaluate the percentage of viable cells. No differences were found between vehicle control- and lycopene-treated cells, in the range of carotenoid concentration indicated, in terms of cell viability.
Isolation and culture of rat alveolar macrophages AMs
Male Wistar rats (232–305 g body weight) (Catholic University laboratories, Rome, Italy) were used for isolation of AMs. After rats were anesthetized by an intraperitoneal injection of ketamine (90 mg/kg) and xylazine (5 mg/kg), the trachea were cannulated and AMs were obtained by three bronchoalveolar lavages (BALs) using 10 ml of Ca2+− and Mg2+−free DPBS each time. Cells recovered from the pooled BAL fluid were suspended in MEM containing 0.02% BSA, 25 mM HEPES, 100 U/ml of penicillin, and 100 µg/ml of streptomycin. The cells were seeded in either 24-well culture plates or 35 mm-diameter culture dishes. After incubation for 2 h at 37°C in a 5% CO2 humidified atmosphere to allow AMs to adhere, non-adherent cells were removed by washing one time with complete medium. AMs were incubated overnight to make them quiescent. Cell viability as determined by trypan blue exclusion was >95% and the purity of AMs as determined by non-specific esterase staining (Sigma- Aldrich) was >90%. AMs from the same rat were used for the same series of experiments to reduce inter-individual variation. All animal procedures were reviewed and approved by the Ministry of Health, Veterinary Service, Rome, Italy.
Preparation of aqueous cigarette smoke extract
Research grade cigarettes (1R3F) were obtained from the Kentucky Tobacco Research and Development Center at the University of Kentucky (Lexington, KY). The total particulate matter (TPM) content of 1R3F was 17.1 mg/cigarette, tar (15 mg/cigarette) and nicotine (1.16 mg/cigarette). A 10% cigarette smoke extract (CSE) was prepared by bubbling smoke from one cigarette into 10 ml of culture medium supplemented with 1% FBS at a rate of one cigarette per minute as described previously, using a modification of the method described by Carp and Janoff [75]. The pH of the CSE was adjusted to 7.4 and was sterile filtered through a 0.45-µm filter (25-mm Acrodisc; Pall, Ann Arbor, MI). The CSE preparation was standardized by monitoring the absorbance at 320 nm (optical density of 0.74±0.05). The spectral variations observed between different CSE preparations at 320-nm wavelength were found to be within the acceptable limits. CSE was freshly prepared for each experiment and diluted with culture medium containing 1% FBS immediately before use. Control medium was prepared by bubbling air through 10 ml of culture medium supplemented with 1% FBS, adjusting pH to 7.4, and sterile filtered as described for 10% CSE. The final concentration of CSE was 0.5%. At this concentration, cell viability was always greater than 95% as measured by trypan blue exclusion.
Chemiluminescence Immunometric Assay of IL-8
IL-8 was measured by a solid-phase, two-site chemiluminescence immunometric assay (Siemens Medical Solution Diagnostics, LA, CA, USA). The intra and inter-assay CV for IL-8 was <3.8 % and 6.7%. The detection limit was 2.0 pg/ml for IL-8.
Measurement of ROS
Cells were harvested to evaluate reactive oxygen species (ROS) production using the di(acetoxymethyl ester) analog (C-2938) of 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCF) (Molecular Probes, Inc., Eugene, OR) as described [76]. Before the addition of the fluorescent probes, 2×106 cells were washed to eliminate the amount of lycopene not cell-associated. Fluorescent units were measured in each well after 30 min incubation with DCF (10 µM) by use of a Cytofluor 2300/2350 Fluorescence Measurement System (Millipore Corp., Bedford, MA). Lycopene did not alter the basal fluorescence of DCF.
Preparation of whole cell lysates, cytosolic and nuclear extracts
Cells (10×106) were harvested, washed once with ice-cold phosphate-buffered saline (PBS), and gently lysed for 30 min in ice-cold lysis buffer (1 mM MgCl2, 350 mM NaCl, 20 mM N-2-hydroxyethylpiperazine-N′-2-ethane sulfonic acid (HEPES), 0.5 mM ethylenediaminetetraacetate (EDTA), 0.1 mM ethyleneglycol-bis(β-aminoethyl ether)-N,N′-tetraacetic acid (EGTA), 1 mM dithiothreitol (DTT), 1 mM Na4P2O7, 1 mM PMSF, 1 mM aprotinin, 1.5 mM leupeptin, 1 mM Na3VO4, 20% glycerol, 1% NP40). Cell lysates were centrifuged for 10 min at 4°C (10,000 g) to obtain the supernatants (whole cell lysate). For the cytosolic extracts, the confluent cells were washed in ice-cold PBS, suspended in ice-cold hypotonic buffer (10 mM HEPES, pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.3% NP-40, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM DTT, 0.1 mM PMSF, 0.1% aprotinin), and lysed for 15 min on ice. The lysates were centrifuged for 10 min at 10 000×g. The supernatants were designated as cytosolic extracts. The nuclear pellet was resuspended in the high salt lysis buffer (10 mM HEPES, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 0.1 mM EGTA, 25% glycerol, 0.5 mM DTT, 0.1 mM PMSF, 0.1% aprotinin), and incubated at 4°C for 30 min. The resulting supernatants were reserved as nuclear extracts. The protein concentrations of samples were determined by Bio-Rad (Hercules, CA, USA) protein assay.
Western blot analysis of IL-8, p65, p-IKKα, p-IkBα, IkBα, Nox-4, p38 and p-p38, ERK1/2, pERK1/2, JNK, p-JNK, PPARγ, AKT, p-AKT and PTEN expression
The anti-IL-8 (H-60, Cat. no. SC-7922), anti-NOX-4 (N-15, Cat. No. sc-21860), anti-p38 (C-20, Cat. No. SC-535), anti-ERK1/2 (K-23, Cat. No. SC-94), , anti-JNK (C-17, Cat. No. SC-474), , anti-PPARγ (H-100, catalog. no. SC-7196), anti-AKT (C-20, Cat. no. SC-1618), anti-pAKT (anti-p-Akt1/2/3 (Thr 308-R, catalog. no. sc-16646-R), and anti-IKK (M280, Cat. no. SC-7182) polyclonal antibodies and the anti-p65 (F-6, Cat. No. SC-8008), p-p38 (D-8, Cat. No. SC-7973), anti p-ERK1/2 (E-4, Cat. No. SC-7383), anti-p-JNK (G-7, Cat. No. SC-6254) and anti-PTEN (28H6 , Cat. No. SC-56205) monoclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti- IkBα and the anti-p-IkBα (Ser32/36) monoclonal antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). The blots were washed and exposed to horseradish peroxidase-labeled secondary antibodies (Amersham Pharmacia Biotech, Arlington Heights, IL) for 45 min at room temperature. The immunocomplexes were visualized by the enhanced chemiluminescence detection system and quantified by densitometric scanning.
RT-PCR of IL-8 and PPARγ
Total RNA was extracted from cells samples using Trizol according to manufacturer's protocols (Invitrogen life technologies, Paisley U.K.) the RNA was eluted in DEPC treated water (0.01% DEPC) and stored at −80° until RT-PCR analysis. Nucleic acid concentrations were measured by spectrophotometry (Hewlett-Packard HP UV/VIS spectrophotometer 8450).
RT-PCR assay was performed using the two-step method. For the first-step of reverse transcription, we used QuantiTect Reverse® Trancription kit (Qiagen, Hilden, Germany) with 500 ηg of total RNA as template RNA, following the manufacturer's procedure. For the second step of real time PCR reactions, we employed QuantiTect SYBR® Green Kits (Qiagen) and QuantiTect® Primer Assays (Qiagen) for human and rat β-Actin, IL-8, PPARγ, according to manufacturer's protocol described for the real-time thermalcycler LightCycler (Roche). PCR data obtained by the LightCycler software were automatically analysed by the Relative Quantification Software (Roche) and expressed as target/reference ratio. Our approach was based on the calibrator-normalized relative quantification including correction for PCR efficiency.
Electrophoretic Mobility-Shift Assay
Frozen cell pellets were processed to obtain nuclear extracts. The pellet was treated as indicated in [35]. Binding reactions containing 5 µg nuclear extracts, 10 mmol/L Tris-HCl (pH 7.6), 5 % glycerol, 1 mmol/L EDTA, 1 mmol/L DTT , 50 mmol/L NaCl, and 3 mg poly(dI-dC) were incubated for 30 min with 5,000 cpm of α- 32P-end-labeled double-stranded oligonucleotide in a total volume of 20 µL. The probe was 5′-AGTTGAGGGGACTTTCCCAGGC3′. Labeling of the probe was obtained by incubating 5 pmol of oligonucleotide with 10 pmol [α-32P]ATP and 3 UT4 polynucleotide kinase for 30 min at 37°C. The probe was then purified with MicroBIO-Spin P-30 columns. Complexes were separated on 60 g/L polyacrylamide gels with 45 mmol/L Tris-borate, 1 mmol/L EDTA, pH 8 buffer. After fixation and drying, gels were exposed on phosphor screens which were then analyzed by phosphor/fluorescence imager STORM 840 (Molecular and Dynamics, Sunnyvale, CA, USA). The intensity of the revealed bands was directly quantified by Image QuaNT software (Molecular Dynamics, Sunnyvale, CA, USA).
Analysis of p65 protein
Nuclear extracts, 25–30 µg of protein, were separated by sodium dodecyl sulfate polyacrilamide gel electrophoresis with 40–120 g/L Bis-Tris gels (NOVEX, San Diego, CA, USA) and transferred to Immobilon-P membranes (Millipore Corp, Bedford, MA, USA) with the use of a semidry system. Immunoblots were blocked overnight at 4°C in 50 g/L dried milk in PBS, pH 7.4 plus 0.05% Tween 20. Blots were incubated with polyclonal primary antibodies to p65 (Santa Cruz, Biotechnology, CA, USA, clone 49.Ser 311, catalog no. SC-135769) in PBS plus 0.05% Tween 20 for 1–2 h at room temperature. The blots were visualized as described in Western blotting assay.
Statistical analysis
Three separate cultures per treatment were utilized for analysis in each experiment. Values were presented as means ± SEM. One-way ANOVA was used to determine differences between the treatments in Figs. 1D, 1E, 2A, 5A, 6A, 6C. When significant values were found (P<0.05), post hoc comparisons of means were made using Fisher's test. Multifactorial two-way analysis of variance (ANOVA) was adopted to assess any differences among the treatments and the times in Figs. 3A, 3B. When significant values were found (P<0.05), post hoc comparisons of means were made using the Tukey's Honestly Significant Differences test. Differences were analyzed using Minitab Software (Minitab, Inc., State College, PA).
We are very much grateful to Dr. Yoav Sharoni and LycoRed Natural Products Industries Ltd. for lycopene supply.
Competing Interests: We received lycopene from Dr. Yoav Sharoni and LycoRed Natural Products Industries Ltd. This does not alter our adherence to all the PLoS ONE policies and no competing interests exist.
Funding: This work was supported by LYCOCARD, European Integrated Project n° 016213. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Int J Mol SciijmsInternational Journal of Molecular Sciences1422-0067Molecular Diversity Preservation International (MDPI) 10.3390/ijms11124891ijms-11-04891ArticleKnockdown of Snail Sensitizes Pancreatic Cancer Cells to Chemotherapeutic Agents and Irradiation Zhang Kejun 1†*Jiao Xuelong 1†Liu Xiaoyi 1†Zhang Bingyuan 1Wang Jigang 2Wang Quan 3Tao Yan 4Zhang Dianliang 11 Department of General Surgery, The Affiliated Hospital of Medical College, QingDao University, Qing Dao, Shan Dong Province 266003, China; E-Mails: [email protected] (X.J.); [email protected] (X.L.); [email protected] (Q.W.); [email protected] (D.Z.)2 Department of Pathology, The Affiliated Hospital of Medical College, QingDao University, Qing Dao, Shan Dong Province 266003, China; E-Mail: [email protected] (J.W.)3 Department of Molecular Biology, The Affiliated Hospital of Medical College, QingDao University, Qing Dao, Shan Dong Province 266003, China4 Pancreatic Center, Department of General Surgery, Tongji Medical College, Huazhong University of Science and Technology, Wuhan City, Hubei Province 430000, China† These authors contributed equally to this work
* Author to whom correspondence should be addressed; E-Mail: [email protected] 26 11 2010 11 12 4891 4892 22 10 2010 7 11 2010 12 11 2010 © 2010 by the authors; licensee MDPI, Basel, Switzerland.2010This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).The prognosis of patients with pancreatic cancer remains poor; only patients with small tumors and complete resection have a chance of a complete cure. Pancreatic cancer responds poorly to conventional therapies, including chemotherapy and irradiation. Snail is a transcription factor that has been associated with anti-apoptotic and chemoresistant properties in pancreatic cancer cells. In this study, we investigated whether knockdown of Snail suppresses growth of and/or sensitizes pancreatic cancer cells to chemotherapeutic agents and irradiation through induction of apoptosis. An adeno-associated virus vector was used to deliver Snail siRNA and knockdown Snail expression in untreated pancreatic cancer cells and in pancreatic cancer cells treated with chemotherapeutic agents or γ-irradiation. Our data indicate that our adeno-associated virus vector can efficiently deliver Snail siRNA into PANC-1 cells both in vitro and in vivo, resulting in the knockdown of Snail expression at the mRNA and protein levels. We further show that knockdown of Snail expression results in potent growth suppression of pancreatic cancer cells and suppresses xenograft tumor growth in vivo through induction of apoptosis. Finally, knockdown of Snail expression significantly sensitizes pancreatic cancer cells to chemotherapeutic agents and γ-irradiation through induction of apoptosis. In conclusion, our findings indicate that Snail is an important modulator of therapeutic responses of pancreatic cancer cells and is potentially useful as a sensitizer in pancreatic cancer therapy.
pancreatic cancerSnailsensitizationchemotherapeuticsirradiation
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1. Introduction
Pancreatic cancer is a disease with a high mortality rate and short survival, as a result of the high incidence of metastatic disease at diagnosis, the fulminant clinical course and a lack of successful therapeutic strategies. In addition, the administration of chemotherapeutic agents and irradiation for the treatment of advanced disease has failed [1–3].
Cancer development is a multistage process which involves a number of genetic and epigenetic changes in the genes controlling cell survival, cell death, cell-cell communication, cell-microenvironment interactions and angiogenesis [4–7]. It has become increasingly clear that abnormalities in cell death pathways play an important role in tumorigenesis and the development of resistance to chemotherapy and radiation therapy [8,9]. The majority of the agents used in cancer therapy directly or indirectly damage DNA thereby inducing apoptosis. However, defects in the apoptotic machinery can lead to multidrug resistance [10–12]. Therapeutic manipulation of apoptotic pathways has become an attractive target to improve the clinical response of pancreatic cancer patients [13,14].
Recently, much attention has been focused on the epithelial-mesenchymal transition [EMT], a key event of embryogenesis that is observed in tumor cells and is associated with increased malignancy [15]. EMT is essential for the formation of different tissues and organs during embryonic development [16]. However, in adult tissue, EMT is inhibited to maintain epithelial integrity and homeostasis. Aberrant activation of EMT in epithelial tumors usually correlates with the development and recurrence of neoplasms [17]. Several transcription factors have been implicated in the transcriptional repression of the epithelial marker E-cadherin, including zinc-finger proteins of the Snail/Slug family, Twist, ZEB1, SIP1, and the basic helix-loop-helix factor E12/E47. Snail was the first and most important transcriptional repressor of E-cadherin to be described. Snail was identified in Drosophila as a suppressor of shotgun (an E-cadherin homolog) transcription and a regulator of embryogenesis [18,19]. Snail has a central role in morphogenesis, as it is essential for the formation of the mesoderm and neural crest, which requires large-scale cell movements in organisms ranging from flies to mammals. Absence of Snail is lethal due to severe defects at the gastrula stage during development [20]. The fundamental role of Snail in EMT and breast cancer metastasis involves suppression of E-cadherin expression. In fact, overexpression of Snail was recently found in invasive breast cancer, hepatocarcinoma and pancreatic cancer [21–23]. Furthermore, the expression of Snail in tumor cells is associated with metastasis, tumor recurrence and poor prognosis [21–24].
A few studies have focused on the role of Snail during the development of chemoresistance to anti-cancer agents in cancer cells. A recent report suggested that Snail may enhance chemoresistance of pancreatic cancer cells to 5-fluorouracil (5-FU) or gemcitabine [25]. In addition, it has found by Zhuo et al. [26] that Snail depletion sensitizes A549 lung cancer cells to cisplatin, suggesting a critical role for Snail in chemoresistance to cisplatin and raising the possibility of Snail depletion as a promising approach to lung cancer therapy.
The purpose of this study was to determine whether suppression of Snail could increase sensitivity of PANC-1 cells to the chemotherapeutic agents, 5-FU and gemcitabine, as well as γ-irradiation. RNA interference was employed to knockdown Snail expression in PANC-1 cells, and cell viability and apoptosis in response to 5-FU or gemcitabine and γ-irradiation were further assessed. Here, we identify a role for Snail in resistance to chemotherapy and γ-irradiation in pancreatic cancer cells.
2. Materials and Methods
2.1. Mice and Cells
Female Nude mice (6–8 weeks of age) were purchased from the Shanghai Animal Center and housed in our pathogen-free animal facilities. The animal experiments were carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals and the institutional guidelines. The human pancreatic cancer cell line PANC-1 was obtained from the American Type Culture Collection (Manassas, VA, U.S.) and was maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, penicillin (100 U/mL) and streptomycin (100 U/mL) at 37 °C in an atmosphere of 5% CO2.
2.2. Construction of the Adeno-Associated Virus Vectors and Production of Viral Particles
siRNA-Snail adeno-associated virus or siRNA-mock adeno-associated virus (rAAV2-siRNA-Snail or rAAV2-siRNA-mock) were generated using the pSilencer™ 2. 1-U6 neo plasmid vectors (Ambion, Austin, TX, U.S.). We generated two different siRNA vectors [26]. The following sequences were successfully constructed: siRNA-Snail sense: 5′-GAT CCG CCT AAC TAC AGC GAG CTG TTC AAG AGA CAG CTC GCT GTA GTT AGG CTT TTT TGG AAA-3′, and antisense: 5′-AGC TTT TCC AAA AAA GCC TAA CTA CAG CGA GCT GTC TCT TGA ACA GCT CGC TGT AGT TAG GCG-3′. The sequence was submitted to a BLAST search against the human genome sequence to ensure that only the Snail gene was targeted. The following unrelated nonspecific scrambled oligonucleotide was used as the control: sense: 5′-GAT CCG TAT TGC CTA GCA TTA CGT TTC AAG AGA ACG TAA TGC TAG GCA ATA CTT TTT TGG AAA-3′, and antisense: 5′-AGC TTT TCC AAA GTA TTG CCT AGC ATT ACG TTC TCT TGA AAC GTA ATG CTA GGC AAT ACG-3′. This siRNA sequence does not match any mammalian sequences currently available in online databases. The siRNA-Snail and siRNA-mock were cloned into the pGCL-GFP vector. The pGCL-GFP vectors with siRNA-Snail or siRNA-mock inserts were used as entry clone vectors and transferred into the vector pDC316-EGFP-U6 (Invitrogen, Carlsbad, CA, U.S.) using the Gateway BamHI and HindIII enzyme mix according to the manufacturer’s directions (Invitrogen) to generate pSNAV2-EGFP-siRNA-Snail-U6 or pSNAV2-EGFP-siRNA-mock-U6. The vectors were linearized with PacI enzyme and transfected into the 293A cell line using Lipofectamine 2000 reagent according to the manufacturer’s directions. The 293A cells were maintained in DMEM until a cytopathic effect was apparent 5–7 days post-transfection. Cells were collected and lysed by subjecting them to four freeze/thaw cycles. The cell debris was pelleted at 3000 rpm for 15 minutes and the supernatant was stored at −80 °C as crude viral lysate. Fifty microliters of crude viral lysate were added into each 293A cell culture dish and incubated for 2–3 days until an 80–100% cytopathic effect was observed. The rAAV2-siRNA-Snail or rAAV2-siRNA-mock were harvested and purified using the adeno-associated virus Mini Purification Kit according to the manufacturer’s directions (Clontech, Mountain View, CA, USA) and stored at −80 °C. Titers of rAAV2-siRNA-Snail or rAAV2- siRNA-mock stocks were determined using an adeno-associated virus Rapid Titer Kit according to the manufacturer’s directions (Clontech).
2.3. Evaluation of Transfection Efficiencies
After transfection of rAAV2-siRNA-mock (MOIs: 250, 100, 25, 5) for 48 h, the proportion of EGFP-expressing cells was measured by fluorescence microscope according to the manufacturer’s instruction, to give the transfection efficiency.
2.4. Drug Treatment
The anticancer drugs 5-FU and gemcitabine used in the study were purchased from Sigma (St. Louis, MO, USA). The drugs were dissolved in DMSO and diluted to appropriate concentrations with cell culture media. PANC-1 cells were exposed to γ-irradiation at 6 Gy. For combination treatments, cells were infected with rAAV2-siRNA-Snail or rAAV2-siRNA-mock for 24 hours prior to drug treatment or γ-irradiation.
2.5. In Vitro Induction of Apoptosis and Growth Assays
After treatment, attached and floating cells were harvested and analyzed for apoptosis by nuclear staining with Annexin V/propidium iodide (PI). A minimum of 300 cells were analyzed for each treatment. The PI-stained cells were analyzed by flow cytometry. Cell growth was measured using an MTT assay in 96-well plates (2,500 cells per well) using the Cell Titer 96 AQueous One Solution (Promega, Madison, WI, USA) following the manufacturer’s instructions. A490 nm was measured using a Victor III (Perkin-Elmer/Wallace, USA) plate reader. Each experiment was carried out in triplicate and repeated at least twice.
2.6. Hoechst 33342 and PI Staining
After treatment, cells were trypsinized, fixed in 1% paraformaldehyde in PBS on ice for 15 minutes, suspended in ice cold ethanol −20 °C. Microscopic detection of apoptosis and necrosis was carried out in both floating and adherent cells recovered after treatment using nuclear chromatin staining with 1 μg/mL Hoechst 33342 and 1 μg/mL propidium iodide for 15 minutes at 37 °C. Quantitative analysis of staining was obtained by counting the frequency of Hoechst-positive cells per optical field.
2.7. Western Blotting
Whole-cell proteins were isolated with the use of RIPA buffer (150 mmol/L NaCl, 0.5% deoxycholic acid, 0.1% SDS, 1% NP 40, 50 mmol/L TRIS [pH 8.0], with protease inhibitors 0.5 μg/mL aprotinin, 0.1 μg/mL leupeptin, 0.5 μg/mL pepstatin, and 1 mmol/L PMSF), sonicated for 10 seconds (Virtis; An Sp Inc, Gardiner, NY, USA), and incubated on ice for 20 minutes. Lysates were centrifuged at 16, 000 g at 4 °C for 30 minutes and the supernatant was collected and stored at −70 °C. The protein concentration was measured at 595 nm using the Bradford assay reagent (Bio-Rad Laboratories, Inc, Hercules, CA, USA). 30 μg of total protein was loaded per well, separated by 7.5% to 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc.) at 150 mA for 16 hours at 4 °C. The membranes were blocked with 5% nonfat milk in 0.1% Tween-20 in phosphate-buffered saline (PBS) and incubated with primary antibodies against Snail (rabbit polyclonal, 1:1500; Oncogene Sciences, San Diego, CA, USA) and β-actin (mouse monoclonal, 1:1000; Zymed, San Francisco, CA, USA) in 0.1% Tween-20 in PBS. The membranes were rinsed twice with 0.1% Tween 20 in PBS for 15 minutes, then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (1:5000, Oncogene Sciences, San Diego, CA, USA) and HRP-conjugated donkey anti-rabbit IgG antibody (1:5000) for 1 hour at room temperature. Membranes were then washed 3 times with 0.1% Tween-20 in PBS for 15 minutes. Immunoblots were detected using an electrochemiluminescence kit (Amersham, Piscataway, NJ, USA) and exposed to X-OMAT AR film (Kodak, Rochester, NY, USA).
2.8. RNA Isolation and RT-PCR
Total RNA was extracted from cells using the TRIzol reagent (Life Technologies, San Diego, CA, U.S.) following the manufacturer’s instructions. 2 μg of total RNA was used for synthesis of cDNAs by reverse transcription (cDNA Synthesis Kit, Invitrogen) following the manufacturer’s instructions. cDNA was amplified using 1 μL of the reaction products in 25 μL with 10 pmol of each primer for 35 cycles. Each cycle consisted of 30 seconds of denaturation at 94 °C, 30 seconds of annealing at 65 °C and 60 seconds of extension at 72 °C. The primers used for cDNA amplification included: Snail forward, 5′-TTC CAG CAG CCC TAC GAC CAG-3′ and reverse, 5′-CGG ACT CTT GGT GCT TGT GGA-3′; and β-actin forward, 5′-CAA CTG GGA CGA CAT GGA GA-3′ and reverse 5′-CAG GCA GCT CGT AGC TCT TC-3′. β-actin was used as the internal control in all reactions.
2.9. In Vivo Tumor Study
All animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Qingdao. Xenograft tumors were established by s.c. injection of 4 × 106 PANC-1 cells into the flanks of 6- to 8-week-old female Nude mice. Tumor treatment was initiated by injecting each tumor (50–100 mm3) with rAAV2-siRNA-Snail or rAAV2-siRNA-mock (1 × 109 plaque-forming units in 50 μL of PBS). Each treatment was repeated twice. Tumor growth was monitored three times per week using calipers to calculate tumor volumes according to the formula [length × width2]/2.
2.10. In Vivo Apoptosis Detection
Apoptosis was evaluated by terminal transferase dUTP nick end labeling (TUNEL) staining using the Apoptag Peroxidase In Situ Detection Kit S7100 (Chemicon) according to the manufacturer’s instructions. Briefly, histological sections were deparaffinized, hydrated in deionized water, then rinsed with PBS. The sections were treated with 20 μg/mL of proteinase K for 15 minutes to digest protein, and with 3% H2O2 for 5 minutes to quench endogenous peroxidase activity. After washing with PBS, the equilibration buffer was added. The slides were then treated with working strength TdT enzyme for 60 minutes at 37 °C. Subsequently, the sections were incubated with preheated working strength Stop solution for 10 minutes, followed by peroxidase conjugated anti-digoxigenin for 30 minutes. The signal was detected with Pierce Metal Enhanced 3,3′-diaminobenzidine (DAB) and the sections were counterstained with methyl green (Vector stock solution) or Mayer’s hematoxylin and then mounted. Control slides were ordered from Serologicals Corporation. The results were obtained using an optical microscope. The percentage of apoptotic cells was calculated as the number of apoptotic cells per number of total cells × 100%.
2.11. In Vivo Immunohistochemistry
Frozen sections from Xenograft tumors were consecutively cut into 4–6-μm thick sections for H&E staining for routine histological analysis and for immunohistochemical studies. The immunohistochemical staining was carried out using the Histostain-SP kit (Zymed). The primary antibody was polyclonal anti-Snail antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (dilution 1:100). Briefly, after deparaffinization and rehydration, the sections were boiled in 0.01 mol/L citrate buffer pH 6.0 for 15 minutes in a microwave oven. Then sections were immersed in 3% hydrogen peroxide in methanol for 10 minutes to block the endogenous peroxidase activity and incubated with serum blocking solution to block nonspecific binding. The sections were incubated with primary antibody at 4 °C overnight, followed by incubation with biotinylated secondary antibody and enzyme conjugate. Staining was developed by addition of DAB chromogen. The sections were counterstained with hematoxylin. The results of the immunostaining were assessed microscopically by two pathologists. Snail-expressing cells within the tumors were counted and the proportion of Snail-positive cells to total tumor cells was calculated.
2.12. Statistical Analysis
P values were calculated by Student’s t test or two-way ANOVA using SPSS10.0 software. The mean ± SD are shown in the figures. P < 0.05 was considered to be statistically significant.
3. Results
3.1. Adeno-Associated Virus Vector Effectively Delivers Snail-siRNA into Cultured PANC-1 Cells
Two adeno-associated virus vectors rAAV2-siRNA-Snail and rAAV2-siRNA-mock that contained the GFP gene as a control were constructed. PANC-1 cells were infected with rAAV2-siRNA-Snail viral particles with different multiplicity of infections (MOIs). The infection efficiencies at the following MOIs: 250, 100, 25, 5 and 0 for 48 hours were 86%, 75%, 26%, 18% and 0%, respectively (Figure 1). Snail expression was detected by RT-PCR and Western blotting analysis. Both assays demonstrated not only a reduction in Snail gene expression in the infected cells, but also showed a clear dose-dependent repression of Snail protein expression (Tables 1–2).
3.2. Knockdown of Snail Significantly Suppresses Growth of PANC-1 Cancer Cells through Induction of Apoptosis
The anti-apoptotic function of Snail (25) prompted us to investigate whether knockdown of Snail suppresses pancreatic cancer cell growth. PANC-1 cells were infected with rAAV2-siRNA-Snail or rAAV2-siRNA-mock resulting in at least 70% infection as indicated by the GFP signal. After 48 hours, cells were analyzed using the MTT assay. rAAV2-siRNA-Snail was found to cause significant growth suppression in PANC-1 cells (P = 0.0025), whereas rAAV2-siRNA-mock had virtually no effect on cell growth compared to the untreated cells (P = 0.337; Figure 2A). An increased frequency of cells infected by rAAV2-siRNA-Snail, but not by rAAV2-siRNA-mock, underwent apoptosis as demonstrated by Hoechst 33342 and PI staining and Annexin V/PI expression (Figures 2B, C). These data show that Snail knockdown leads to growth suppression and induction of apoptosis in PANC-1 cancer cells.
3.3. Knockdown of Snail Sensitizes PANC-1 Cancer Cells to Chemotherapeutic Agents and γ-Irradiation
Abnormalities in the regulation of apoptosis have been shown to contribute to the development of resistance to chemotherapy and radiation therapy in cancer cells [27,28]. The important anti-apoptotic function of Snail suggests that downregulated Snail expression may restore sensitivity of cancer cells to anticancer agents. To test this hypothesis, PANC-1 cells were treated with a low dose of rAAV2 siRNA-Snail (5 MOI) alone or in combination with γ-irradiation or chemotherapeutic agents, including 5-FU and gemcitabine. rAAV2-siRNA-Snail was found to significantly enhance the growth inhibitory effects of these chemotherapeutic drugs and γ-irradiation (Figures 3A, C and D). For example, up to a 6–7-fold increase in growth suppression was achieved when rAAV2-siRNA-Snail was combined with 5-FU (Figure 3B). We also determined the IC50 values of the chemotherapeutic agents in PANC-1 cells with or without rAAV2-siRNA-Snail and found that rAAV2-siRNA-Snail significantly lowered the IC50s of these agents by 4-fold (gemcitabine) to more than 10-fold (5-FU; Table 3). These results show that knockdown of Snail significantly sensitizes PANC-1 cells to chemotherapeutic agents.
We next determined whether knockdown of Snail sensitizes PANC-1 cells to these anticancer agents through induction of apoptosis. PANC-1 cells are resistant to apoptosis induced by 5-FU (up to 5 μg/mL) and γ-irradiation (up to 6 Gy, data not shown). rAAV2-siRNA-Snail (10 MOI) infected cells and 5-FU (0.5 μg/mL) treated cells alone did not induce significant apoptosis in PANC-1 cells (Figure 4A). However, almost 70% of cells underwent apoptosis following 72 hours of the combination treatment (Figure 3A). Similarly, a combination of γ-irradiation (6 Gy) with rAAV2-siRNA-Snail led to a markedly enhanced apoptotic response in PANC-1 cells (Figure 3E). In contrast, the control rAAV2-siRNA-mock had no effect on apoptosis when combined with 5-FU or γ-irradiation (Figures 3E,F). Analysis of apoptosis levels using Hoechst 33342 and PI staining confirmed these results (data not shown). These results show that knockdown of Snail promoted apoptosis induced by 5-FU or γ-irradiation.
3.4. Knockdown of Snail Suppresses Tumor Growth in Vivo
To determine whether the knockdown of Snail confers antitumor activity in vivo, PANC-1 xenograft tumors (∼50–100 mm3) were treated with three injections of rAAV2-siRNA-Snail and the control rAAV2-siRNA-mock at 1 × 109 plaque-forming units (Figure 4A). rAAV2-siRNA-mock did not have any effect on tumor growth compared with PBS alone, with tumors reaching eight-times their initial volume in 21 days (Figures 4 A). In contrast, tumors subjected to rAAV2-siRNA-Snail treatment grew much slower and reached less than twice their initial volume, with approximately 80% growth suppression compared to tumors treated with rAAV2-siRNA-mock (P < 0.01; Figures 4A). We analyzed tumor histology by H&E staining, Snail protein expression by immunohistochemistry and in situ apoptosis by TUNEL. We found that Snail protein expression was inhibited significantly in rAAV2-siRNA-Snail treated tumors (Figure 4B). These data show that knockdown of Snail can effectively inhibit growth of established tumors in vivo through induction of apoptosis.
4. Discussion
In addition to its role as an inducer of EMT and cell migration, Snail is considered to be a critical factor in cell survival [29]. When stably expressed in epithelial cells, Snail confers resistance to cell death induced by withdrawal of growth factors and by pro-apoptotic signals [30]. Aberrant expression of Snail may also promote resistance to programmed cell death elicited by DNA damage [31]. Pancreatic cancer is a malignancy that resists nearly all present chemotherapeutic strategies. Yin et al. [25] reported that expression of Snail not only confers the invasive phenotype to pancreatic cancer cells, but also promotes chemoresistance. Therefore, in this study, we have used a pancreatic cell line, PANC-1 to examine whether knockdown of Snail suppresses growth of pancreatic cancer cells and/or sensitizes pancreatic cancer cells to chemotherapeutic agents and irradiation.
We first examined the efficiency of adeno-associated virus transduction and the effect of MOI on the expression level of Snail in pancreatic cancer cells in vitro using RT-PCR and Western blotting. We found that the expression of Snail in PANC-1 cells after knockdown by adeno-associated virus vectors is clearly viral particle dose-dependent. Higher MOIs resulted in a marked decrease in the expression level of Snail protein and mRNA.
We also demonstrated that knockdown of Snail via adeno-associated viruses resulted in apoptosis and enhanced sensitivity to chemotherapeutic agents and γ-irradiation, suggesting that Snail knockdown is crucial for triggering apoptotic responses to these agents. Interestingly, knockdown of Snail seems to be the most effective in enhancing growth suppression and apoptosis when combined with 5-FU, gemcitabine or γ-irradiation, consistent with the notion that Snail plays a critical role in DNA damage–induced apoptosis [26]. These observations suggest that knockdown of Snail sensitizes pancreatic cancer cells to chemotherapeutic agents and irradiation, and warrants further evaluation.
We also investigated the in vivo therapeutic effect of adeno-associated virus-mediated Snail siRNA knockdown by intratumoral injections of viruses. Our results show that on average, tumors treated with rAAv-siRNA-Snail were 42% smaller than mock-treated tumors. Tumor volumes of the rAAv-siRNA-Snail treated group were significantly smaller on day 21 (P < 0.01) than the other groups. This reduction in tumor size can be attributed to increased apoptosis in tumors treated with rAAv-siRNA-Snail.
5. Conclusion
We successfully down-regulated Snail expression in PANC-1 cells using adeno-associated Snail siRNA vectors, resulting in increased 5-FU, gemcitabine or γ-irradiation-induced PANC-1 cell apoptosis. These results suggest that appropriate chemotherapeutic or radiation therapies concomitant with Snail depletion might be a promising approach to treat pancreatic cancer.
We thank Dong Zhu for helpful technical support. We take this opportunity to specifically thank the reviewers and editors for their kind instructions that may be helpful for our further studies.
Competing interests
The authors promised there were not any possible conflicts of interest in this research.
Figure 1. EGFP expression detection by fluorescence microscope. Images of EGFP-expressing cells at 25 MOI (top left), 100 MOI (top right), 250 MOI (bottom left), and plot of transfection efficiencies: the infection efficiencies at the following MOIs: 250, 100, 25 and 5 for 48 hours were 86%, 75%, 26% and 18%respectively.
Figure 2. Knockdown of Snail suppresses the growth of pancreatic cancer cells through induction of apoptosis. (A) Cell growth was measured by MTT assay 48 hours after rAAV2-siRNA-Snail (or mock) infection (100 MOI), as described in Materials and Methods. Growth of untreated cells was defined as 100%. (B) Following the indicated treatments for 48 hours, levels of apoptosis were analyzed using annexin-V staining. (C) Following the indicated treatments for 48 hours, levels of apoptosis were examined using Hoechst 33324 and PI staining.
Figure 3. Knockdown of Snail sensitizes pancreatic cancer cells to chemotherapeutic agents and γ-irradiation. PANC-1 cells were treated with chemotherapeutic agents and γ-irradiation, alone or in combination with rAAV2- siRNA-Snail or rAAV2-siRNA-mock (5 MOI), as described in Materials and Methods. Cell growth was measured by MTT assay. (A, B) Growth inhibition of PANC-1 cells after treatment with chemotherapeutic drugs or irradiation alone. 5-FU, 0.5 μg/mL; gemcitabine 0.5 μg/mL; IR, γ-irradiation, 6 Gy. (C, D) Effects of different concentrations of the chemotherapeutic drugs on rAAV2-siRNA-Snail or rAAV2-siRNA-mock infected cells on cell growth. (E, F) The sensitizing effects of Snail knockdown are mediated by enhanced induction of apoptosis. PANC-1 cells were subjected to the indicated treatments and analyzed for apoptosis, as described in Materials and Methods. rAAv was used at 10 MOI. Apoptosis was analyzed by Annexin V/PI assays. 5-FU, 0.5 μg/mL; γ-irradiation, 6 Gy.
Figure 4. Knockdown of Snail suppresses the growth of xenograft pancreatic tumors. (A) PANC-1 tumors (n = 6 per group) were subjected to rAAV2-siRNA-Snail or rAAV2-siRNA-mock treatment for 21 days, as described in Materials and Methods. (B) Tumor histology was analyzed by H&E staining (top panel). Frozen sections of the PANC-1 tumors 48 hours after the second injection were analyzed by TUNEL to determine the level of apoptosis (middle). Snail protein expression levels were analyzed by immunohistochemistry (bottom). Magnification ×200.
Table 1. RT-PCR analysis for Snail mRNA (n = 3, mean ± SD).
Snail mRNA/β-actin mRNA
Group 0 MOI 5 MOI 25 MOI 100 MOI 250 MOI
siRNA-Snail 0.896 ± 0.057 0.506 ± 0.046 0.36 ± 0.028 0.21 ± 0.014 0.12 ± 0.006
siRNA-mock 0.824 ± 0.053 0.873 ± 0.066 0.904 ± 0.062 0.849 ± 0.067 0.856 ± 0.072
siRNA-Snail vs. siRNA-mock, P = 0.0023.
Table 2. Western blotting analysis for Snail protein (n = 3, mean ± SD).
Snail/β-actin value
Group 0 MOI 5 MOI 25 MOI 100 MOI 250 MOI
siRNA-Snail 0.658 ± 0.034 0.465 ± 0.027 0.321 ± 0.023 0.214 ± 0.020 0.10 ± 0.012
siRNA-mock 0.721 ± 0.042 0.657 ± 0.036 0.684 ± 0.046 0.703 ± 0.056 0.674 ± 0.049
siRNA-Snail vs. siRNA-mock; P = 0.0057.
Table 3. IC50 of the chemotherapeutics in PANC-1 cells with or without rAAV2- siRNA-Snail.
IC50
rAAV2- siRNA-mock rAAV2- siRNA-Snail
Drug gemcitabine 5-FU
37.4 μg/mL 9.16 μg/mL
68.57 μg/mL 7.46 μg/mL
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 21647453PONE-D-11-0467010.1371/journal.pone.0020152Research ArticleBiologyBiochemistryNucleic acidsDNADNA recombinationDNA repairGeneticsGene FunctionGenetics and GenomicsBiochemistryBiology and Life SciencesBiochemistryProteinsPost-Translational ModificationPhosphorylationBiology and Life SciencesCell BiologyChromosome BiologyChromosomesChromatidsBiology and Life SciencesGeneticsGenetic LociBiology and life sciencesGeneticsDNADNA damageBiology and life sciencesBiochemistryNucleic acidsDNADNA damageBiology and life sciencesGeneticsDNADNA repairBiology and life sciencesBiochemistryNucleic acidsDNADNA repairBiology and Life SciencesComputational BiologyGenome AnalysisChromatin ImmunoprecipitationBiology and Life SciencesGeneticsGenomicsGenome AnalysisChromatin ImmunoprecipitationPhysical SciencesChemistryChemical CompoundsOrganic CompoundsCarbohydratesMonosaccharidesGalactosePhysical SciencesChemistryOrganic ChemistryOrganic CompoundsCarbohydratesMonosaccharidesGalactoseBiology and life sciencesGeneticsDNADNA recombinationBiology and life sciencesBiochemistryNucleic acidsDNADNA recombinationRtt107 Phosphorylation Promotes Localisation to DNA Double-Stranded Breaks (DSBs) and Recombinational Repair between Sister Chromatids Rtt107 Phosphorylation and DSBsUllal Pranav Vilella-Mitjana Felipe Jarmuz Adam Aragón Luis
*
Cell Cycle Group, Medical Research Council, Clinical Sciences Centre, Imperial College, London, United Kingdom
Lustig Arthur J. EditorTulane University Health Sciences Center, United States of America* E-mail: [email protected] and designed the experiments: LA. Performed the experiments: PU. Analyzed the data: PU. Contributed reagents/materials/analysis tools: FV-M AJ. Wrote the paper: LA.
Competing Interests: The authors have declared that no competing interests exist.
2011 25 5 2011 12 4 2017 6 5 e2015210 3 2011 19 4 2011 © 2011 Ullal et al2011Ullal et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Efficient repair of DNA double-stranded breaks (DSB) requires a coordinated response at the site of lesion. Nucleolytic resection commits repair towards homologous recombination, which preferentially occurs between sister chromatids. DSB resection promotes recruitment of the Mec1 checkpoint kinase to the break. Rtt107 is a target of Mec1 and serves as a scaffold during repair. Rtt107 plays an important role during rescue of damaged replication forks, however whether Rtt107 contributes to the repair of DSBs is unknown. Here we show that Rtt107 is recruited to DSBs induced by the HO endonuclease. Rtt107 phosphorylation by Mec1 and its interaction with the Smc5–Smc6 complex are both required for Rtt107 loading to breaks, while Rtt107 regulators Slx4 and Rtt101 are not. We demonstrate that Rtt107 has an effect on the efficiency of sister chromatid recombination (SCR) and propose that its recruitment to DSBs, together with the Smc5–Smc6 complex is important for repair through the SCR pathway.
This work was funded by European Research Council ERC starting grant 202337 (LA) and by the Medical Research Council UK (LA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Double-stranded breaks (DSB) can arise from exposure to a variety of DNA damaging agents, but also as a consequence of cellular processes, for instance during intermediate stages in the repair of various DNA lesions or during DNA replication. DSBs trigger a cellular response that involves checkpoint, repair, chromatin and structural proteins many of which are recruited to the site of damage. This response is highly conserved, highlighting the importance of ensuring correct DSB repair to prevent genomic instability.
Cells can repair DSBs using a variety of pathways, including non-homologous end-joining (NHEJ) [1] and homologous recombination (HR) [2]. HR involves the use of similar sequences as a template to repair the break, whereas during NHEJ the broken ends of DNA are directly rejoined. Because of its nature, HR requires an intact donor DNA molecule. Sister-chromatids are the ideal templates to repair DSBs via HR because they contain an exact copy of the broken site. Therefore it is not surprising that DSB repair by sister chromatid recombination (SCR) is the preferred choice in eukaryotic cells during the S and G2/M periods of the cell cycle [3]. Upon DSB induction a response that culminates in the enforcement of cohesion around the break site is triggered, which involves the recruitment of cohesin, the protein complex that maintains sister chromatids paired [4], [5] to the DSB [6], [7]. Increased cohesion between the broken chromatid and the intact one is thought to promote post-replicative DSB repair between them. The Smc5–Smc6 complex, related to cohesin, is also recruited to DSB sites [8], [9], [10] to mediate repair via SCR [8], [9]. Although the exact role of Smc5–Smc6 during SCR repair is not well understood, the fact that cohesin loading is altered in the absence of Smc5–Smc6 raises the possibility that Smc5–Smc6 mediates SCR by promoting cohesin loading to DSB sites.
In budding yeast, the checkpoint kinases Mec1 (ATM) and Tel1 (ATR) are recruited early to break sites [11]. Mec1p/Tel1p-dependent phosphorylation of a variety of proteins at the site promotes a cascade of events important to detect, signal and repair the lesion [12]. H2AX is a Mec1 target, whose phosphorylation is required for cohesin loading [7]. Rtt107, also a target of Mec1, is a phosphoprotein thought to function as a scaffold necessary for the assembly of repair proteins onto sites of damage [13], [14], [15], [16]. Rtt107 promotes restart of DNA replication forks after DNA damage [13], [14], [15], [17] and interacts with a number of DNA repair proteins including the Rad51 paralogs, Rad55 and Rad57 [15] as well as the Smc5–Smc6 complex [18].
Previously we demonstrated that yeast Smc5–Smc6 is recruited to DSBs to mediate SCR [8]. Here, we report that the Smc5–Smc6 interacting factor Rtt107 is also recruited to DSBs and affects SCR repair. We show that phosphorylation of Rtt107, which depends on Mec1p and Smc5–Smc6 is sufficient for its recruitment to HO-induced breaks.
Results
Rtt107 localises to HO-induced DSBs
On the basis of the interaction between Rtt107 and the Smc5–Smc6 complex [18], we investigated whether Rtt107 localises to an HO endonuclease catalyzed DSB formed at a unique site in the MAT locus on chromosome III [19]. Transcriptional regulation of the HO endonuclease using the galactose inducible promoter allowed us to control the timing of DSB induction [20]. DSBs at the MAT locus are repaired by homologous recombination with HML and HMR loci [21]. To prevent repair of HO-induced DSBs, both HM loci were deleted in our strains, thus maximising the persistence of the break to facilitate possible detection of Rtt107 at this site.
Cells were engineered to express an epitope tagged version of Rtt107 (RTT107-9xMYC). Chromatin binding of this protein to sites around the DSB were assayed by chromatin immunoprecipitation (ChIP). We used different primer pairs covering at least 30 kb on either side of the DSB (Fig. 1A). DNA sequences were amplified from the input chromatin and chromatin immunoprecipitated to calculate the relative percentage of immunoprecipiated material. To control for DSB-independent effects on protein occupancy we also used a primer pair specific for sequences located in a different chromosome (chromosome VI). First we tested the efficiency of DSB induction and intact DNA damage checkpoint activation. We used the checkpoint protein Ddc2 fused to GFP as an in vivo marker of DSB formation in our strains [22]. Two hours after galactose-mediated HO induction ∼80% of the cells arrested as dumbbells with a single Ddc2 focus (data not shown) demonstrating that the HO break at the MAT locus is efficiently induced and not repaired, thus causing G2/M arrest in our experimental system.
10.1371/journal.pone.0020152.g001Figure 1 Rtt107 is enriched on regions flanking the HO-induced DSB.
(A) A DSB was induced at the MAT locus in strains expressing Rtt107-9myc. A strain containing a Myc-tagged version of Rtt107, and a galactose-inducible HO endonuclease (CCG6983) were grown at 30°C in YP raffinose. Cells were then split and one half transferred to galactose (cut), while the other half were grown in the absence of galactose (uncut). Chromatin immunoprecipitation was performed around the HO cleavage site in the MAT locus of chromosome III. The binding of Rtt107 around the locus was evaluated 2 and 4 hours post-induction (2 hrs, 4 hrs) or in the absence of the DSB (uncut). Input DNA and DNA immunoprecipitated were amplified with primers at the indicated distances from the HO site. The average of two independent experiments with the corresponding standard deviation is shown. A locus on chromosome VI was used as a control. (B) Epitope tagged Rtt107 levels were compared with an isogenic untagged strain (CCG2781) before and after HO mediated DSB.
Next we evaluated the binding of Rtt107 around the HO site. In the absence of a DSB, we found low Rtt107 binding across the region (Fig. 1A–B; uncut). After 2 hours of HO induction, we detected a general increase in binding around the regions flanking the break (Fig. 1A; 2 hrs). The maximum DSB-induced increase was ∼7-fold and localised to regions 0.5–10 kb away from the DNA break on either side (Fig. 1A–B; 2–4 hrs). The relative binding decreased with distances greater than 20 kb from the break, however the regions immediately adjacent to the DSB site were found to be also high in binding (Fig. 1A–B; 2–4 hrs). Thus the presence of a DSB induces a significant increase in Rtt107 binding in a domain around the DSB.
Smc5–Smc6 complex contribute to Rtt107 phosphorylation
Rtt107 is extensively phosphorylated by Mec1 in response to DNA damage [13]. Consistent with this, we found that exposure to MMS, hydroxyurea, camptothecin (CPT) or an HO-induced DSB in wildtype cells expressing RTT107-9MYC led to substantial mobility changes of Rtt107 on SDS-PAGE gels. The mobility change was reversed by treatment of the extracts with λ-phosphatase (Fig. 2A and Fig. S1). Previous studies of Rtt107 phosphorylation used western blot analysis with antibodies reported to recognise phosphorylated SQ/TQ motifs, which are sites of Mec1/Tel1 phosphorylation [23]. We have been able to resolve Rtt107 phosphorylation through changes in protein mobility on SDS-PAGE gels using a 30∶1 ratio of acrylamide:bisacrylamide (Fig. S1). We therefore tested whether in addition to Mec1 other factors contribute to Rtt107 phosphorylation. Rtt107 was tagged in different checkpoint kinase mutant backgrounds, including the effector kinases Rad53 and Chk1 and the adaptor kinases Rad9 and Mrc1, and cells were exposed to different genotoxic agents (Fig. 2A). We found that none of these kinases contributes to Rtt107 phosphorylation (Fig. 2A). In fission yeast, the Rtt107 homologue Brc1 was identified as a multi-copy suppressor of the UV hypersensitivity associated with loss of Rad18 [24], the homologue of Smc6. Recently, a physical interaction between budding yeast Smc5–Smc6 complex and Rtt107 was described [18]. We therefore investigated whether Smc5–Smc6 function contributes to Rtt107 phosphorylation in response to DNA damage. To this aim, Rtt107 was tagged in cells carrying the conditional allele for SMC6, smc6–9
[25]. Rtt107 was phosphorylated upon exposure to MMS in wildtype and smc6-9 cells at permissive temperatures (Fig. 2B). In contrast, we observed Rtt107 phosphorylation only in wildtype cells under non-permissive temperatures (Fig. 2B). These results demonstrate that Smc5–Smc6 function is required for complete phosphorylation of Rtt107 by the Mec1 kinase.
10.1371/journal.pone.0020152.g002Figure 2 Rtt107 phosphorylation upon DNA damage requires Mec1 kinase and the Smc5–Smc6 complex.
(A) Phosphorylation of Rtt107 upon DNA damage is dependent on the ATR kinase, Mec1. Logarithmically growing cultures of Wt (CCG6886), mec1Δ (CCG6887), rad53Δ (CCG6888) rad9Δ (CCG6683), chk1Δ (CGG6890) and mrc1Δ (CCG6720) carrying myc tagged Rtt107 were treated with 0.3% MMS, 50 µM HU or 50 µg CPT for 90 minutes. An untreated control was also used. Samples were then fixed with RIPA buffer. Indicated extracts were then run on SDS-PAGE gels (as in Fig. S1). Immunoblots were probed with anti-Myc antibodies. Logarithmically growing cultures of Wt (CCG6983) containing a Myc-tagged version of Rtt107, and a galactose-inducible HO endonuclease were grown at 30°C in YP raffinose. Cells were then split and half transferred to galactose (cut), while the other half were grown in the absence of galactose (uncut). Samples were then fixed with RIPA buffer at the indicated timepoints. Extracts were then fractionated on SDS-PAGE gels (as in Fig. S1). Immunoblots were probed with anti-Myc antibodies. (B) Logarithmically growing cultures of smc6–9 (CCG6365) carrying myc tagged Rtt107 were treated with 0.3% MMS for 90 minutes. The experiment was conducted at 25°C and 37° in order to detect phosphorylation at permissive and non-permissive temperatures. To inactivate the smc6–9 allele, cultures were grown overnight at 25°C and then transferred to 37°C for 2 hours, prior to the addition of MMS. Samples were processed as in A.
Mec1 and Smc5–Smc6 complex are necessary for Rtt107 localisation to HO-induced DSBs
Based on the observed phosphorylation of Rtt107 in response to DNA damage by Mec1 and Smc5–Smc6 [13], we investigated whether these factors are required for the localization to HO-catalyzed DSBs (Fig. 1). We examined Rtt107 levels around the MAT DSB in the temperature sensitive allele smc6–9 and the mec1Δ mutant. We focused our analysis in the regions immediately flanking the break (5 kb; Fig. 3). Deletion of the Mec1 kinase completely abolished the recruitment of Rtt107 to the MAT DSB (Fig. 3A), demonstrating that Mec1 is indeed required for Rtt107 recruitment. Next, we analysed the contribution of the Smc5–Smc6 complex. We observed that even at permissive temperatures for the smc6–9 allele (25°C), the recruitment of Rtt107 to the MAT DSB was partially compromised (Fig. 3B–C). The effect was more dramatic under non-permissive temperatures (37°C) where, like in mec1Δ cells, Rtt107 recruitment to sites flanking the MAT DSB was not observed (Fig. 3C). These results demonstrate that Mec1 and Smc5–Smc6 function are important for Rtt107 recruitment to DSBs. Furthermore, we found that deletion of the Tel1 or Rad53 kinases, which do not prevent Rtt107 phosphorylation (Fig. 2A) do not affect its recruitment to DSBs (Fig. S2), demonstrating a correlation between Rtt107 phosphorylation and its ability to be recruited to DSBs.
10.1371/journal.pone.0020152.g003Figure 3 Mec1 kinase and the Smc5–Smc6 complex are required for Rtt107 recruitment to induced MAT DSB.
(A) Phosphorylation of Rtt107 upon DNA damage is dependent on the ATR kinase, Mec1. A DSB was induced at the MAT locus in a wildtype strain expressing Ddc2-GFP (CCG6983) and Ddc1-GFP in a mec1Δ mutant (CCG7143) both also expressing Rtt107-9myc. Cells were collected at indicated times after HO induction and scored for formation of Ddc2 and Ddc1 foci. The binding of Rtt107 around the MAT DSB locus on chromosome III was assayed by chromatin immunoprecipitation at the indicated time points, after (cut- 2 hours) and before (uncut) DSB induction. The averages of two independent experiments with the corresponding standard deviation are shown. A locus on chromosome VI was used as a control. (B) Rtt107 enrichment to regions flanking the HO-induced DSB is dependent on the function of the Smc5–Smc6 complex. A DSB was induced at the MAT locus in wildtype (CCG6983) and smc6–9 (CCG6985) strains expressing Ddc2-GFP and Rtt107-9myc tagged at permissive (25°C) temperature. The binding of Rtt107 around the MAT DSB locus on chromosome III was assayed by chromatin immunoprecipitation at the indicated time points, after (cut- 2 hours) and before (uncut) DSB induction in wildtype and smc6–9 cells at 25°C. The averages of two independent experiments with corresponding standard deviations are shown. A locus on chromosome VI was used as a control. (C) Rtt107 enrichment to regions flanking the HO-induced DSB is dependent on the function of the Smc5–Smc6 complex. A DSB was induced at the MAT locus in wildtype (CCG6983) and smc6–9 (CCG6985) strains expressing Ddc2-GFP and Rtt107-9myc tagged at non-permissive (37°C) temperature. The binding of Rtt107 around the MAT DSB locus on chromosome III was assayed by chromatin immunoprecipitation at the indicated time points, after (cut- 2 hours) and before (uncut) DSB induction in wildtype and smc6–9 cells at 37°C. The averages of two independent experiments with corresponding standard deviations are shown. A locus on chromosome VI was used as a control.
Rtt107 phosphorylation is sufficient for DSB recruitment
Mec1 phosphorylation occurs in Ser/Thr–Gln (SQ/TQ) motifs [23]. A number of SQ/TQ motifs are found scattered within the Rtt107 aminoacid sequence, however a cluster of SQ/TQ motifs at the C-terminal region of Rtt107 have been previously identified as critical for Mec1 phosphorylation [13]. In order to investigate whether phosphorylation in these motifs is required for Rtt107 recruitment to DSBs, we mutated the Ser743, Thr758, Thr773 and Ser806 to alanine (‘A’) residues to generate the Rtt107-AQ allele (Fig. 4A). We expressed the Rtt107-AQ allele tagged with 6 copies of the HA epitope in the C-terminal region under the control of the GAL1-10 promoter in rtt107Δ cells carrying the HO MAT DSB system. The resulting strain was sensitive to a variety of DNA damage agents (data not shown) [13]. We then evaluated the binding of Rtt107-AQ around the HO site. In the absence of a DSB at MAT, we found no Rtt107 binding across the region (Fig. 4A; uncut). After 4 hours of HO induction, we did not find evidence for Rtt107-AQ recruitment to the regions flanking the break (Fig. 4A). We therefore conclude that phosphorylation on the C-terminal SQ/TQ cluster is required for Rtt107 recruitment to the MAT DSB.
10.1371/journal.pone.0020152.g004Figure 4 Rtt107 phosphorylation is required for its recruitment to the MAT DSB.
(A) The phospho-mutant allele of Rtt107, Rtt107-AQ is not recruited to HO-induced DSBs. A DSB was induced at the MAT locus in a strain expressing wildtype Rtt107-6HA from the GAL1-10 promoter (CGG7290) or the phospho-mutant version Rtt107-AQ-6HA (CCG7291) is shown. The binding of Rtt107 and Rtt107-AQ around the MAT DSB locus on chromosome III was assayed by chromatin immunoprecipitation at the indicated time points, after (cut- 4 hours) and before (uncut) DSB induction. The averages of two independent experiments with corresponding standard deviation are shown. A locus on chromosome VI was used as a control. Western blot analysis of Rtt107-6HA and Rtt107-AQ-6HA upon DSB induction is also shown. No band-shift was observed for Rtt107-AQ, suggesting that this protein is not phosphorylated upon DSB induction. (B) The phospho-mimmicking allele of Rtt107, Rtt107-DQ is recruited to HO-induced DSB in the absence of the Mec1 kinase. A DSB was induced at the MAT locus in a mec1Δ strain expressing wildtype Rtt107-6HA from the GAL1-10 promoter (CGG7558) or the phospho-mimmicking version Rtt107-DQ-6HA (CCG7556). The binding of Rtt107 and Rtt107-DQ around the MAT DSB locus on chromosome III was assayed by chromatin immunoprecipitation at the indicated time points, after (cut- 4 hours) and before (uncut) DSB induction. The averages of two independent experiments with corresponding standard deviations are shown. A locus on chromosome VI was used as a control.
To investigate whether phosphorylation on the Rtt107 C-terminal SQ/TQ cluster is sufficient for DSB recruitment, we mutated Ser743, Thr758, Thr773 and Ser806 to phospho-mimicking aspartic acid (‘D’) residues to generate the Rtt107-DQ allele (Fig. 4B). We expressed the tagged Rtt107-DQ in mec1Δ cells, where wildtype Rtt107 is neither phosphorylated in response to DNA damage [13] (Fig. 2A) nor recruited to HO-induced DSBs (Figs. 3A and 4B). Unlike wildtype Rtt107, Rtt107-DQ was recruited to the regions flanking the break following DSB induction at MAT in the absence of Mec1 (Fig. 4B). These results show that phosphorylation on the C-terminal SQ/TQ cluster of Rtt107 is sufficient for its recruitment to DSBs.
Rtt107 regulators are dispensable for DSB recruitment
The N-terminal region of Rtt107 contains four tandem BRCT domains, which are characteristic of proteins involved in cell cycle checkpoint functions related to DNA damage [26]. These domains have been shown to interact with a number of proteins, including the Slx4/Slx1 nuclease complex [14]. In addition, Rtt107 is recruited to stalled replication forks via interaction with the cullin Rtt101 protein [17]. We therefore wished to address whether these interactions are important for the observed recruitment of Rtt107 to DSBs. First, we investigated whether Rtt107 is recruited to DSBs in the absence of Slx4. Slx4Δ largely prevents Rtt107 SQ/TQ phosphorylation [14], a requirement for Rtt107 DSB recruitment (Fig. 4). Surprisingly, we found no significant defects in Rtt107 recruitment to MAT DSBs in the absence of Slx4 (Fig. 5A). Next we tested whether the cullin Rtt101 is required for the localization of Rtt107 to breaks. We found Rtt107 recruitment to HO-induced DSBs to be normal in rtt101Δ cells (Fig. 5B). Therefore, we conclude that while the Slx4/Slx1 and Rtt101 interactions might be important for Rtt107 functions at damaged forks [14], [17] they are not essential for its recruitment to DSBs induced by HO.
10.1371/journal.pone.0020152.g005Figure 5 Slx4 and Rtt101 are not required for Rtt107 recruitment to the MAT DSB.
(A) Slx4 deletion does not prevent Rtt107 recruitment to HO-induced DSB. A DSB was induced at the MAT locus in wildtype (CCG6983) and slx4Δ (CGG7970) cells expressing Rtt107-9myc. The binding of Rtt107 around the MAT DSB locus on chromosome III was assayed by chromatin immunoprecipitation at the indicated time points, after (cut- 4 hours) and before (uncut) DSB induction. The averages of two independent experiments with corresponding standard deviations are shown. A locus on chromosome VI was used as a control. (B) Rtt101 deletion does not prevent Rtt107 recruitment to HO-induced DSBs. A DSB was induced at the MAT locus in wildtype (CCG6983) and rtt101Δ (CGG7970) cells expressing Rtt107-9myc. The binding of Rtt107 around the MAT DSB locus on chromosome III was assayed by chromatin immunoprecipitation at the indicated time points, after (cut- 4 hours) and before (uncut) DSB induction. The averages of two independent experiments with corresponding standard deviations are shown. A locus on chromosome VI was used as a control.
Rtt107 contributes to sister chromatid recombination
The role of Smc5–Smc6 complex at DSBs is to promote repair of the lesion by sister chromatid recombination [8], [9]. Deletion of Rtt107 has been shown to have a small effect on sister chromatid exchange (SCE) rates in S-phase cells exposed to DNA damage [13]. To investigate whether phosphorylation is important for Rtt107 function in SCR, we used an assay that can measure spontaneous recombination between sister chromatids in the absence of DNA damage [27]. The assay is based on two ade2 marker genes separated by a TRP1 gene. Recombination between the ade2 alleles that generates a wildtype ADE2 gene and retains the TRP1 gene is indicative of gene conversion events between sister chromatids [27]. Deletion of the Rad51 paralogs, Rad57 and Rad55, has been shown to significantly decrease spontaneous recombination events between sister chromatids in this assay [27]. First, we investigated whether the smc6–9 conditional mutant, known to be important for SCR, shows defects in the formation of Ade2+Trp1+ recombinants. We measured the rate of Ade+ Trp1+ recombinants in wildtype and smc6–9 cells growing at 30°C, which is semi-permissive for the smc6–9 allele [25], and found a 6-fold reduction of recombinant formation in the smc6–9 mutants (Fig. 6A). Consistent with previous reports, rad55Δ caused a 1000-fold reduction in gene conversion events between sister chromatids [27] (Fig. 6A). The reduction in Ade2+Trp1+ recombinants in rtt107Δ was found to be similar to that observed for smc6–9 cells (Fig. 6A).
10.1371/journal.pone.0020152.g006Figure 6 Rtt107 contributes to spontaneous recombination events between sister chromatids.
(A) The smc6–9 alele and rtt107Δ mutant show decreased recombination between sister chromatids. Wildtype (CCG7802), rad55Δ (CCG7804), rtt107Δ (CCG7855) and smc6–9 (CCG7856) strains carrying the ade2-TRP1-ade2 recombination assay [27] were grown on YPD plates at 30°C. Five independent colonies were inoculated into 5 ml of YPD and grown overnight at 30°C. Cells were pelleted and re-suspended in 1 ml of sterile water. Serial dilutions were then plated on SC medium minus adenine and tryptophan, and incubated for 3–4 days, after which colonies were counted. Ade+Trp+ recombination frequencies are plotted on the y-axis. (B) Rtt107 phosphorylation is required for its role in spontaneous sister chromatid recombination. Wt (CCG7802), rtt107Δ and rtt107Δ containing wildtype RTT107 (CCG8214), phospho-mutant RTT107-AQ (CCG8215) and phospho-mimmetic mutant RTT107-DQ (CCG8216) (under the galactose inducible promoter GAL1-10) strains with the engineered ade2-TRP1-ade2 recombination assay were grown on YPD plates at 30°C. Five independent colonies were inoculated into 5 ml of YPD and grown overnight at 30°C. Cells were then transferred to YP galactose and expression of constructs was induced for 4 hours. Cells were pelleted and re-suspended in 1 ml of sterile water. Serial dilutions were then plated on SC galactose medium minus adenine and tryptophan, and incubated for 3–4 days, after which colonies were counted. Ade+Trp+ recombination frequencies are plotted on the y-axis.
To evaluate whether Rtt107 phosphorylation promotes gene conversion events between sister chromatids, we used the Rtt107-AQ and Rtt107-DQ alleles in the genetic assay. Expression of wildtype Rtt107 from the GAL1-10 promoter in rtt107Δ cells yielded similar levels of Ade2+Trp1+ recombinants to wildtype cells (Fig. 6B). Recombinant levels in cells expressing the phospho-defective mutant Rtt107-AQ were comparable to those found for rtt107Δ cells (Fig. 6B). In contrast, we observed partial rescue of Ade2+Trp1+ recombinant formation in rtt107Δ cells expressing the phospho-mimmicking Rtt107 allele, Rtt107-DQ (Fig. 6B). These results demonstrate that phosphorylation of Rtt107 is involved in the function of this protein promoting sister chromatid recombination events.
Discussion
Genomes are constantly being challenged by lesions on their DNA that are either induced as a consequence of the action of exogenous agents, such as different drugs causing DNA damage, or as a consequence of the cell's own metabolism, for instance during DNA replication. DSBs are one of the most serious lesions in DNA and can be lethal if not repaired or can generate deleterious effects to the genome if repaired improperly. Therefore, accurate mechanisms for DNA double-strand break repair (DSBR) are important for all living organisms. Repair generally occurs in a stepwise manner and begins with the recruitment of different factors to the break site to orchestrate a coordinated response that involves signalling and repair activities. It is therefore important to understand the order of events at DSBs as well as the dependencies between the factors that are recruited.
The checkpoint kinase Mec1 is recruited to break sites early during the repair response [11]. Mec1 phosphorylation then acts on a variety of proteins at the site [12]. One of Mec1 targets is the Rtt107 scaffold protein [13], however, prior to this study Mec1-dependent phosphoylation of Rtt107 had only been studied in the context of damaged replication forks [13]. Previous investigation of Rtt107 homologues in fission yeast [24] and mammalian cells, raised a possible function for Rtt107 at DSBs; PTIP (mammalian Rtt107 homologue) is indeed recruited to DNA damage sites formed by ionizing radiation [28]. Here, we have investigated the role of Rtt107, and its phosphorylation, at DNA double-stranded breaks. We observed that following the induction of an irreparable break by the HO endonuclease, Rtt107 is recruited to regions surrounding the break. We demonstrated that the Mec1 kinase is required for both Rtt107 phosphorylation in response to a single DSB as well as its recruitment, as both events are absent in mec1Δ mutants. This result indicates that the recruitment of Rtt107 to DSBs is controlled by Mec1 phosphorylation. We confirmed this hypothesis showing that the phospho-mutant allele of Rtt107 (Rtt107-AQ) is unable to be recruited to breaks in the presence of Mec1 while a phospho-mimicking allele (Rtt107-DQ) is indeed recruited even when Mec1 is non-functional. The dependency of Rtt107 recruitment to DSB on phosphorylation by the Mec1 kinase is in contrast to Rtt107 recruitment to stalled forks, which is independent of Mec1 [17], and thus phosphorylation.
A number of protein interactions have been described for Rtt107 [14], [15], [17], [18]. We investigated whether some of these interactions are important for Rtt107 recruitment to DSBs. We found that recruitment was drastically reduced in the smc6–9 mutant allele, suggesting that intact Smc5–Smc6 function is a requirement for Rtt107 DSB-loading. Furthermore, we observed that Rtt107 phosphorylation is impaired in smc6–9 mutants, confirming that Rtt107 phosphorylation is important for recruitment. Surprisingly, deletion of Slx4 did not prevent Rtt107 recruitment to DSBs, despite the fact that Slx4 is required for Mec1-dependent phosphorylation [14]. Interestingly, Slx4 is also not essential for Rtt107 binding to stalled replication forks [17]. It is possible that low-level Rtt107 phosphorylation is retained in slx4Δ cells and that this might be sufficient to promote detectable Rtt107 recruitment to damaged forks and/or DSBs.
Unlike bacterial models, the role of recombination at stalled forks is poorly understood in eukaryotes. It is presently unclear why recombination at collapsed forks can, under some circumstances, rescue replication while in other cases it might generate genomic rearrangements. Prompted by the positive role of the Smc5–Smc6 complex in sister chromatid recombination [8], [9] and its interaction with Rtt107 [18], we explored a potential role of Rtt107 in promoting repair by the SCR pathway. Importantly, we found that rtt107Δ cells exhibit a defect in the formation of recombinant products between sister chromatids (an assay measuring unequal exchange between sister chromatids). The defect was similar to that observed for smc6–9 mutants in the same assay. Furthermore, we showed that Rtt107 phosphorylation contributes to its role in SCR since the Rtt107-DQ but not the Rtt107-AQ allele could partially restore the formation of recombinant products between sister chromatids in rtt107Δ cells.
A future question is the role of the Rtt107 scaffold protein at DSBs in the recruitment of downstream repair factors. The Rad55-Rad57 complex is known to play a role in the stabilization of the Rad51 nucleoprotein filament, and rad55-rad57Δ have strong defects in SCR [27]. In fission yeast, Rhp55/Rhp57 (homologues of Rad55/Rad57) are required for Brc1 (Rtt107 homologue) suppression of smc6–74 mutants [29]. In budding yeast, the Rad55/Rad57 heterodimer interacts with Rtt107 [15] and Rad55 is a known target of the Mec1 kinase [30]. Interestingly, Rad55 phospho-mutants show similar phenotypes to both Rtt107 and smc6–9 mutants, i.e. inability to complete replication and failure to re-enter pulsed-field gels after treatment with the DNA damage agent MMS [13], [30], [31], [32]. It is tempting to speculate that the phosphorylation of the BRCT-domains of Rtt107 could attract phosphorylated Rad55 and other repair factors to mediate repair of DNA lesions by the error-free sister recombination pathway.
Methods
Yeast, Media and Cell growth conditions
Yeast strains used are listed in supplementary materials (Table S1). Media for yeast growth, both complete YP (1% yeast extract, 2% peptone) and synthetic drop out media lacking various amino acids were prepared according to standard protocols. Media were mixed with different carbon sources, depending on the experiment: glucose (D), galactose or raffinose all at 2% final concentration. Carbon sources were sterilised by filtration. To detect Rtt107 phosphorylation for the various experiments, liquid cultures were grown overnight to a final concentration of 0.7<OD595<0.4 and were then incubated in the presence of different DNA damage agents; methyl methanesulfonate (MMS), camptothecin (CPT) or hydroxyurea (HU) and grown for 2 hours more before processing. Final concentration of MMS was 0.03%, final concentration of CPT was 5 µg/ml and the final concentration of HU was 50 µM. The drugs were added to the cultures as in [13]. Cultures growing in liquid media were incubated at 25°C, 30°C or 37°C in flasks (the volume of the flask was 5 times the volume of the culture), shaken at high speed [250 rotations per minute (rpm) in a New Brunswick G25 shaking incubator (GMI Inc.), with 4 cm rotation diameter, or at 150 rpm in a SM1003 shaking incubator (Kuhner), with 44 cm rotation diameter]. For growth of Rtt107 alleles under the GAL1–10 promoter, cells were grown in 20 ml YP media with raffinose (supplemented with 0.01% glucose) overnight to 0.3<OD595<0.5 at 25°C. Expression of the constructs was subsequently induced by addition of galactose at a final concentration of 2% w/v. The cultures were then grown for 2–4 hours, before processing for western blotting or immunoprecipitation.To detect Rtt107 in smc6–9 temperature sensitive strains, cells were grown overnight at 25°C in YP media with raffinose overnight to 690.3<OD595<0.5 and then transferred to 37°C for 1 hour to inactivate the smc6–9 allele, before the addition of MMS.
Culture conditions for HO endonuclease induction
The HO endonuclease was induced in strains carrying a stably integrated GAL10::HO construct [33]. Cells were grown in YP media with raffinose (supplemented with 0.01% glucose) overnight to 0.3<OD595<0.5 at 25°C. HO expression was subsequently induced by addition of galactose at a final concentration of 2% w/v. The cultures were then grown for 2–4 hours and samples were taken to check Ddc2-GFP foci formation to detect the efficiency of the DSB induction in the population. To induce the HO endonuclease in smc6–9 temperature sensitive alleles, strains were grown overnight at 25°C in YP media with raffinose to 0.3<OD595<0.5 and then transferred to 37°C for 1 hour to inactivate the smc6–9 allele, before HO induction at 35°C.
Construction of Rtt107 phosphorylation mutants
The Rtt107 constructs cloned for this study were made using gene synthesis (GeneCust). Three constructs were synthesized; Rtt107, Rtt107-AQ and Rtt107-DQ. All contructs contained 6HA epitopes in the C-terminal region and were expressed using the GAL1–10 promoter. In Rtt107-AQ, Ser743, Thr758, Thr773 and Ser806 were changed to alanine, while in Rtt107-DQ these sites were changed to aspartic acid. All constructs were cloned in the integrative yeast plasmid pRS406 containing the URA3 selectable marker. For transformation, constructs were digested by Stu I and integrated into the genome at the endogenous ura3 locus.
SDS-PAGE and Western blot
To detect phosphorylation shifts of Rtt107, gels with different ratios of acrylamide: bisacrylamide were prepared. Gels were run at 100 V in Tris-glycine-SDS running buffer (National Diagnostics) and were transferred to polyvinylidene fluoride transfer membrane (Hybond-P, Amersham Biosciences) in a Biorad blotting system in Tris- glycine transfer buffer (National Diagnostics) containing 20% methanol. Transfer was for 1.5 hours at 285 mA or overnight at 30 V. Membranes were blocked in 5% skimmed milk powder in PBS with 0.1% Tween 20 (PBST) for at least 30 minutes, then incubated with mouse monoclonal anti-Myc IgG1 antibody 9E10 (Roche), at a 1/5000 dilution (from stock 5 mg/ml) in blocking solution for 1 hour at room temperature or overnight at 4°C in 2.5% milk powder in PBST. Following several washes in PBST, membranes were incubated with sheep anti-mouse IgG Horseradish-Peroxidase-linked antibody (GE Healthcare) at 1/10000 in 2.5% skimmed milk powder in PBST. After several further washes in PBST, the ECL Plus Western Blotting Detection System (GE Healthcare) was used to detect the secondary antibody.
Chromatin Immunoprecipitation
Chromatin immunoprecipitation experiments were conducted as in [34]. Primers sites used were as in [8]. Real-time PCR was used to quantify enrichment. PCR reactions were performed using the SensiMix NoRef Kit (Quatance). Reactions were carried out according to the manufacturer's instructions in a total volume of 20 µl, containing 1 µl of immunoprecipitated or input DNA and 1.5 µl of 10 µM oligonucleotide primer pairs. Reactions were amplified using a DNA Engine Opticon2 thermal cycler and were analysed using Opticon software (MJ Research). Each PCR reaction was reproduced in duplicate: reactions in which the difference between the two duplicates was bigger than 0.5 cycles were not considered. PCR amplifications were analysed for the melting curve profile to confirm the absence of contaminant PCR products. Mean “threshold cycle number” (or Ct value) was calculated for each PCR, selected in the window of exponential amplification phase. Enrichment was calculated using the following formula: enrichment = 2(Ct IP DNA – Ct input DNA), where Ct IP is the Ct value for the immunoprecipitated sample, and Ct input is the Ct value for the input DNA. The specificity of the enrichment was tested by comparing the values from tagged and untagged strains.
Recombination assays for sister chromatid exchanges
The sister chromatid exchange used has been described before [27]. In brief, yeast strains were grown on YPD plates for 2–3 days at 30°C. 5 independent colonies were inoculated into 5 ml of YPD, and cultures were grown overnight at 30°C. For the Rtt107 phosphorylation mutants, strains were grown on YPD plates for 2–3 days at 30°C. Colonies were inoculated into 5 ml of YPD, and cultures were grown overnight at 30°C. Cells were then washed and transferred to medium containing galactose, and the constructs were expressed for 2 hours, before washing and aliquoting into dilutions. Cells were then plated on SC medium, and SC medium minus adenine and tryptophan supplemented with galactose. Cells were pelleted and resuspended in 1 ml of sterile water. Aliquots of appropriate dilutions were plated onto SC medium to determine the number of viable cells in each culture and onto SC medium minus adenine and tryptophan for the recombination assay, to determine the total number of recombinants in each culture. Plates were incubated for 3–5 days, after which the colonies were counted. For each strain, recombination rates were measured three times on independent isolates and the mean values are shown.
Supporting Information
Figure S1
Western blot analysis showing different conditions to detect Rtt107 phosphophorylation upon DNA damage. Logarithmically growing cultures were treated with 0.3% MMS, 50 µM HU and 50 µg CPT for 90 minutes. Samples were then fixed with RIPA buffer. Extracts were run on SDS-PAGE gels with different concentrations of acrylamide:bis- acrylamide as shown. (A) SDS-PAGE gels with a 37∶1 ratio of acrylamide:bisacrylamide. Immunoblots were probed with anti-Myc antibodies. (B) SDS-PAGE gels with a 30∶1 ratio of acrylamide:bisacrylamide. Immunoblots were probed with anti-Myc antibodies.
(TIF)
Click here for additional data file.
Figure S2
Tel1 and Rad53 kinases are not required for Rtt107 recruitment to MAT DSBs. (A) Tel1 deletion does not prevent Rtt107 recruitment to HO-induced DSB. A DSB was induced at the MAT locus in wildtype (CCG6983) and tel1Δ (CGG7967) cells expressing Rtt107-9myc. The binding of Rtt107 around the MAT DSB locus on chromosome III was assayed by chromatin immunoprecipitation at the indicated time points, after (cut- 4 hours) and before (uncut) DSB induction. The averages of two independent experiments with corresponding standard deviations are shown. A locus on chromosome VI was used as a control. (B) Rad53 deletion does not prevent Rtt107 recruitment to HO-induced DSB. A DSB was induced at the MAT locus in wildtype (CCG6983) and rad53Δ (CGG7968) cells expressing Rtt107-9myc. The binding of Rtt107 around the MAT DSB locus on chromosome III was assayed by chromatin immunoprecipitation at the indicated time points, after (cut- 4 hours) and before (uncut) DSB induction. The averages of two independent experiments with corresponding standard deviations are shown. A locus on chromosome VI was used as a control.
(TIF)
Click here for additional data file.
Table S1 Yeast strains used in this study.
(DOC)
Click here for additional data file.
We thank members of the Aragon laboratory for discussions and critical reading of the manuscript. We also thank Jim Haber and Patrick Sung for providing materials.
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10.1371/journal.pone.0019945
Research Article
Biology
Molecular Cell Biology
Signal Transduction
Medicine
Obstetrics and Gynecology
Gynecologic Cancers
Oncology
Basic Cancer Research
Tumor Physiology
Cancers and Neoplasms
Gynecological Tumors
Endometrial Carcinoma
Cancer Risk Factors
Molecular Biology
Oncology
Epithelial Membrane Protein-2 Promotes Endometrial Tumor Formation through Activation of FAK and Src
EMP2 Activates FAK and Src in Endometrial Cancer
Fu Maoyong 1
Rao Rajiv 1
Sudhakar Deepthi 1
Hogue Claire P. 1
Rutta Zach 1
Morales Shawn 2
Gordon Lynn K. 2
Braun Jonathan 1 3
Goodglick Lee 1 3
Wadehra Madhuri 1 *
1 Department of Pathology and Laboratory Medicine, University of California Los Angeles, Los Angeles, California, United States of America
2 Jules Stein Eye Institute, University of California Los Angeles, Los Angeles, California, United States of America
3 Jonsson Comprehensive Cancer Center, University of California Los Angeles, Los Angeles, California, United States of America
Zhang Lin Editor
University of Pennsylvania, United States of America
* E-mail: [email protected]
Conceived and designed the experiments: SM MF LKG JB LG MW. Performed the experiments: MF RR DS CPH ZR MW. Analyzed the data: MF RR DS SM LKG JB LG MW. Contributed reagents/materials/analysis tools: LKG JB LG MW. Wrote the paper: LKG JB LG MW.
Competing Interests: The authors have declared that no competing interests exist.
2011
27 5 2011
6 5 e1994513 1 2011
6 4 2011
© 2011 Fu et al
2011
Fu et al
https://creativecommons.org/licenses/by/4.0/ Except for the EMP2 and β-actin panels of Figure 3A, this is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Endometrial cancer is the most common gynecologic malignancy diagnosed among women in developed countries. One recent biomarker strongly associated with disease progression and survival is epithelial membrane protein-2 (EMP2), a tetraspan protein known to associate with and modify surface expression of certain integrin isoforms. In this study, we show using a xenograft model system that EMP2 expression is necessary for efficient endometrial tumor formation, and we have started to characterize the mechanism by which EMP2 contributes to this malignant phenotype. In endometrial cancer cells, the focal adhesion kinase (FAK)/Src pathway appears to regulate migration as measured through wound healing assays. Manipulation of EMP2 levels in endometrial cancer cells regulates the phosphorylation of FAK and Src, and promotes their distribution into lipid raft domains. Notably, cells with low levels of EMP2 fail to migrate and poorly form tumors in vivo. These findings reveal the pivotal role of EMP2 in endometrial cancer carcinogenesis, and suggest that the association of elevated EMP2 levels with endometrial cancer prognosis may be causally linked to its effect on integrin-mediated signaling.
The authors acknowledge the following agencies who have helped support this work: National Institutes of Health (NIH) grants HD48540 (J. Braun), R21 CA131756 (M. Wadehra), CA016042 (University of California at Los Angeles Jonsson Comprehensive Cancer Center flow cytometry core); Early Detection Research Network NCI CA86366 (L. Goodglick); American Cancer Society # RSG-03-160-01-LIB (L. Goodglick). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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pmcIntroduction
Endometrial cancer is a significant disease in women with nearly one out of every thirty-five women developing the disease over her lifetime [1], [2]. In the United States endometrial cancer is the most common malignancy of the female genital tract. It is generally accepted to be an endocrine-related neoplasm with a pronounced impact of sex hormone status, and an increased incidence with age [3].
One protein associated with the development of endometrial cancer is epithelial membrane protein-2 (EMP2). EMP2 is a member of the GAS-3/PMP22 subfamily, which together with tetraspanins and connexins, comprise the three subfamilies of the large 4-transmembrane family. In the endometrium, EMP2 functions as a prognostic marker which can help predict patients who will progress from endometrial hyperplasia to cancer [4]. In patients with endometrial cancer, EMP2 positive tumors have been shown to be more aggressive and invasive, and its expression within tumors correlates with poor prognosis and survival [4], [5].
Although the exact role of EMP2 in endometrial cancer remains poorly defined, EMP2 may function as a trafficking molecule for a variety of proteins and glycolipids to efficiently transfer from a post-Golgi endosomal compartment to the plasma membrane. Accordingly, modulation of EMP2 expression and localization causes pleiotrophic changes on the plasma membrane of several classes of molecules, including integrins, MHC class I, and GPI-linked proteins [6], [7], [8], and EMP2 appears to mediate trafficking of these molecules to detergent-insoluble glycosphingolipid-enriched (DIG) membrane domains. DIG domains are thought to be important for receptor complexing and resultant signal transduction (reviewed in [9], [10], [11]).
In this study, we examine the mechanism by which EMP2 contributes to the etiology of endometrial cancer. Previous studies have shown that EMP2 selectively interacts with integrins in endometrial cells [6]. As EMP2 expression also correlates with tumor invasiveness in patients with endometrial cancer, we examine if modulation of EMP2 alters integrin-associated signaling proteins. Specifically, we focus on focal adhesion kinase (FAK), one of the critical focal adhesion signaling proteins and a protein that EMP2 has been shown to associate with in other cell systems [12], [13]. In endometrial cancer cells, EMP2 promotes FAK and Src phosphorylation, and contributes to their localization within lipid raft domains. As cell adhesion and spreading is a crucial step in the formation and invasion cascade of cancer cells, our results suggest that EMP2 is a critical regulator of metastatic potential of endometrial cancer cells.
Materials and Methods
Cell lines
The human endometrial adenocarcinoma cell lines HEC-1A (HTB112, ATCC, Manassas, VA) and Ishikawa (generous gift from Dr. Carmen Williams, NIH) cells were cultured in McCoys or DMEM media supplemented with 10% fetal calf serum at 37°C in a humidified 5% CO2. Cell lines were used within 2 months after resuscitation of frozen aliquots and were authenticated based on viability, recovery, growth, morphology, and isoenzymology. Both cell lines were also tested for murine pathogens including mycoplasma by the Division of Laboratory Animal Medicine at the University of California, Los Angeles. Cells were passaged every 3–4 days. Stably transfected HEC-1A cells containing a human EMP2-GFP fusion protein (48 kD), control GFP, or EMP2 specific ribozyme have been previously described [6]. Endogenous EMP2 is expressed at 20 kD. Ishikawa cells were infected with a MSCV-EMP2 or control retrovirus, creating the cell lines Ishikawa/EMP2 and Ishikawa/V [14]. Infected cells were sorted by GFP using flow cytometry.
Preparation of Xenografts
Ethical Treatment of Animals Statement
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The procedures used were approved by the Animal Research Committee at the University of California, Los Angeles under protocol #2004-182. All efforts were made to minimize animal suffering.
Four to six-week-old nude BALB/c female mice were obtained from Charles River Laboratories (Wilmington, MA) and maintained at the University of California, Los Angeles. Animals were inoculated subcutaneously (s.c.) with 1×106 HEC-1A/OE, HEC-1A/V, or HEC-1A/RIBO cells into the right and left shoulder flanks, respectively. Six mice were used per group. Tumors were measured twice a week, and tumor volumes were calculated by the formula π/6 x larger diameter x smaller diameter [15], and data expressed as mean ± standard error of the mean (SEM). Two-way ANOVA was used to evaluate overall differences in the means between different groups as a function of time, and significance was calculated using p<0.05. At day 30, tumors were excised, fixed in formalin, and then processed for hematoxylin and eosin staining by the Tissue Procurement Laboratory at UCLA.
Immunohistochemistry
Tumor samples were stained with human EMP2 antisera or a preimmune control. Briefly, for antigen retrieval, sections were incubated at 95°C for 20 minutes in 0.1 M citrate, pH 6.0. EMP2 expression was detected using rabbit human EMP2 antisera at a dilution of 1∶400 as previously described [5] followed by visualization using the Vector ABC kit (Vector Labs, Burlingame, CA) according to the manufacturer's instructions. DAB staining was quantified using Photoshop 7 [16].
Proliferation Assays
Cellular Proliferation was monitored using two independent assays. First, a standard static growth assay was performed as previously described [6], [17]. Briefly, 1×103 cells were plated in triplicates in a 96-well plates from 0–7 days. At each indicated day, cells were stained with toluidine blue, lysed with 2% SDS (Biowhittaker, Walkersville, MD), and absorbance measured at 595 nm. Each experiment was repeated at least three times, and groups were compared using an unpaired Student's t test.
Second, the BrdU Cell Proliferation Assay (EMD Chemicals, Gibbstown, NJ) was performed as per manufacturer's instructions. Briefly, triplicate cultures of 104 cells were cultured in a 96-well plate. Cells were then incubated in DMEM +0.5% FCS overnight to arrest the cells. Cells were then released in complete media containing BrdU for 2 or 24 hrs. Cells were then fixed, permeabilized and the DNA denatured. A detector anti-BrdU monoclonal antibody was then added and ultimately detected using a horseradish peroxidase-conjugated goat anti-mouse IgG1. To determine the amount of incorporated BrdU, a fluorogenic substrate was added and the absorbance quantitated at both 450 and 595 nm using a spectrophotometer.
Wound healing
105 HEC-1A and Ishikawa cells with modulated EMP2 expression were seeded in 35-mm tissue culture dishes. When cells were confluent, a “wound” was created using a 100-µl “yellow” pipette tip as described [13], [18]. Wound healing was monitored over 48 hrs with a 10x phase contrast objective, and images were collected using a Power Shot S80 camera (Canon, Lake Success, NY). Manual measurements were made to quantitate the wound healing process. Three independent experiments were performed, and the results averaged.
In some experiments, inhibitors were added to determine the contribution of specific pathways to wound healing. Wildtype HEC-1A or Ishikawa cells were treated with 10 µM of the FAK-Src small molecule inhibitor PP2 [19] or 5 µM of Akt inhibitor VIII ([20]; Calbiochem, San Diego, CA), the EGFR inhibitor Erlotinib ([21]; 10 µM, Genentech), or the Src family tyrosine kinase inhibitor Dasatinib ([22]; 10 nM, Bristol-Myers Squibb). Efficacy of the inhibitors was tested at the manufacturer's recommended dosage, and potential toxicity was measured using trypan-blue exclusion.
Immunoprecipitations
Cells were washed two times with PBS, placed in lysis solution (1% Nonidet P-40 containing 2 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 2 µg/ml, pepstatin, 10 mM iodoacetamide, 0.1 mM EDTA, 0.1 mM EGTA, 10 mM HEPES, and 10 mM KCl), and solubilized for 30 min at 4°C. Lysates were sonicated for 15 s, and the insoluble materials were pelleted at 10,000 rpm for 10 min. The cell lysates were precleared by incubation with protein A/G-agarose beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Precleared, equivalent lysates were incubated overnight with agarose beads bound to either anti-FAK rabbit polyclonal antisera (Santa Cruz Biotech), EMP2 antisera, or isotype rabbit sera. The beads were washed three times in lysis solution and finally in 50 mM Tris buffer (pH 8). Immune complexes were eluted from the beads using Laemmli sample buffer (62.5 mM Tris-Cl, pH 6.8, 10% glycerol, 2% SDS, 0.01% bromphenol blue, 2% β-mercaptoethanol). As EMP2 contains multiple glycosylation sites (13), N-linked glycans were cleaved using peptide N-glycanase (PNGase; New England Biolabs, Beverly, MA). Eluates were treated as per the manufacturer's instructions at 37°C for 2 h and analysed by Western blot analysis as described below. Blots were digitized using a flatbed scanner and the band density measured using NIH program Image J. EMP2:FAK stoichiometry was calculated by dividing the volume intensities of immunoprecipitation with anti-EMP2 sera over that with anti-FAK antisera. Experiments were repeated three times, averaged, and the SEM calculated.
Western blot analysis
Cells were lysed in Laemmli buffer. Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane (Amersham Biosciences), and stained with Ponceau S (Sigma-Aldrich, St. Louis, MO) to determine transfer efficiency. Membranes were blocked with 10% low fat milk in PBS containing 0.1% Tween 20 and probed with EMP2 antisera (1∶1000), anti- 576/577p-FAK (Santa Cruz Biotechnology), anti- total FAK (BD Biosciences), anti-416 p-Src (Cell Signaling, Danvers, MA), anti-total Src (Cell Signaling) or β-actin (Sigma-Aldrich). In some experiments, blots were probed with anti-Flottillin-2 (BD Biosciences) or anti-EEA1 antibodies (BD Biosciences). Protein bands were visualized using a horseradish peroxidase-labeled secondary antibody (BD Biosciences; Southern Biotechnology Associates, Birmingham, AL) followed by chemiluminescence (ECL; Amersham Biosciences). Band intensities were quantified using the NIH program Image J as above. To account for loading variability, β-actin was used to normalize each sample. At least three independent experiments were performed and, where indicated, the results were evaluated for statistical significance using a Student's t-test (unpaired, one-tail). A level of p<0.05 was considered to be statistically significant.
Immunofluorescence
HEC-1A or Ishikawa cells were plated onto glass coverslips (Fisher Scientific, Pittsburgh, PA). Cells were fixed in cold methanol, blocked in 1% normal goat serum, and then incubated overnight at 4°C with combinations of EMP2, anti-397p-FAK (BD Biosciences, 1∶250) or a total FAK antibody (BD Biosciences, 1∶250) in a humidified chamber. Cells were rinsed with PBS +0.01% Triton X-100, then incubated (2–4 hours. at RT) with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (1∶50) or rhodamine-conjugated donkey anti-mouse IgG (1∶500; Jackson ImmunoResearch Laboratories, West Grove, PA). Negative controls included incubation of cells with secondary antibody alone. Cells were washed in PBS +0.01% Triton, rinsed briefly with double deionized H20, and mounted in a 3.5% n-propyl gallate-glycerol solution.
Cell images were captured using a Zeiss LSM 510 confocal microscope at 600X magnification. The colocalization coefficient between EMP2 and 397p-FAK or total FAK was determined using Zeiss LSM 5 PASCAL software (Carl Zeiss MicroImaging GmbH, Germany), and values represent the relative number of colocalizing pixels compared to the total number of pixels above threshold. The mean colocalization coefficient was averaged from at least 3 independent images.
Lipid Raft Fractionation
Cells (5×107) were harvested and washed in PBS. Cells were resuspended in Tris-buffered saline (50 mM Tris, pH 7.5, 20 mM EDTA, complete protease inhibitors and PhosSTOP (both from Roche Diagnostics, Indianapolis, IN). Cells were lysed by sonication (Moran and Miceli, 1998; Lusa et al., 2001) and then dissolved in 1% Triton X-100 or 1% Brij-58 and incubated on ice for 60 min. The extract was mixed 1∶1 with 80% sucrose (40% final), followed by step overlays with 35 and 5% sucrose, and centrifuged at 46,000 rpm for 18 h with a Sorvall SW55 rotor (Global Medical Instrumentation, Albertville, MN). Fractions (500 µl) were collected from the top of the gradient, and analyzed by SDS-PAGE. In some cases, samples were treated with PNGase prior to protein resolution by SDS-PAGE.
Cholesterol depletion was performed as described previously (Claas et al., 2001). Briefly, cells were washed in PBS to remove serum. Cells were incubated in DMEM containing 20 mM methyl-β cyclodextrin (MβCD; Sigma-Aldrich) for 60 min at 37°C. Cells were analyzed by trypan blue exclusion to insure the lack of toxicity by MβCD before being fractionated as described above.
Statistical Analysis
All values in the text are mean + SEM. Differences between means were evaluated using a two-tailed Student's t test or ANOVA as indicted. Significant differences were taken at the p<0.05 level.
Results
EMP2 expression promotes endometrial tumor development
Endometrial tumors with high levels of EMP2 have been shown to be more aggressive and correlate with poor clinical outcome [5]. In order to determine if EMP2 expression directly contributed to tumor formation, we engineered endometrial carcinoma cells to express modulated levels of the protein (see Methods and Materials; [6]). 106 HEC-1A/EMP2, HEC-1A/V, or HEC-1A/RIBO were injected s.c. into nude mice, and tumor volume was then measured over 30 days (Figure 1A). EMP2 overexpression promoted a two fold increase in tumor growth over HEC-1A/V cells (Figure 1B) while tumors with reduced EMP2 expression exhibited a three fold reduction in growth compared to HEC-1A/EMP2 cells (Figure 1C). Two-way ANOVA indicated that a statistically significant difference existed in the effect of EMP2 on tumor growth (p = 0.019).
10.1371/journal.pone.0019945.g001 Figure 1 Tumor volume is increased in HEC-1A/EMP2 tumors.
(A) HEC-1A/EMP2, HEC-1A/V, and HEC-1A/RIBO cells were injected s.c. into nude mice. Tumor volume was determined using calipers. HEC-1A/EMP2 tumors were larger than tumors injected with HEC-1a/V and HEC-1a/RIBO cells. Values are averages (±SEM, n = 6). Comparison by ANOVA, p<0.05 (B) Representative nude Balb/c mouse displaying HEC-1A/EMP2 and HEC-1A/V subcutaneous tumors. (C) Representative Balb/c nude mouse displaying HEC-1A/V and HEC-1A/RIBO subcutaneous tumors. At day 30, HEC-1A/EMP2 (D), HEC-1A/V (E), and HEC-1A/RIBO (F) tumors were excised, fixed, and stained by hematoxylin and eosin or EMP2. H/E Magnification, 40X; EMP2 Staining Magnification, 400X. Scale bar = 10 µM.
After 30 days, tumors were excised, fixed, and stained by hemotoxylin/eosin or for EMP2 expression by immunohistochemistry (Figure 1D–F). Hemotoxylin and eosin staining confirmed the size of the tumors, where HEC-1A/RIBO tumors were significantly smaller than HEC-1A/EMP2 and HEC-1A/V tumors. To correlate the change in tumor size with EMP2 expression in vivo, immunohistochemistry was performed. As expected, two and four fold higher levels of EMP2 were observed in HEC-1A/EMP2 tumors compared to HEC-1A/V and HEC-1A/RIBO tumors, respectively (Figure 1D–F).
EMP2 expression does not alter cellular proliferation
One possible mechanism by which EMP2 could contribute to the malignant phenotype of endometrial cancer cells would be through alterations in cell proliferation. In order to test this, we first characterized the growth kinetics of HEC-1A/EMP2, HEC-1A/V, and HEC-1A/RIBO cells. Equivalent numbers of cells were plated on day 1 and then manually counted over the next 7 days. No significant differences in total cell numbers were observed between the three cell lines (Figure 2A). In order to validate this result, a BrdU cell proliferation assay was performed. Cells were incubated from 0–24 hours with BrdU to monitor its incorporation in proliferating cells (Figure 2B). No statistical differences were found in the kinetics of proliferation between the three cell lines.
10.1371/journal.pone.0019945.g002 Figure 2 EMP2 expression and FAK/Src signaling promote wound healing.
(A) HEC-1A/EMP2, HEC-1A/V, and HEC-1A/RIBO cells were analyzed quantitatively for total cell numbers from 0–7 days or (B) BrdU incorporation after 2 or 24 hours. No significant differences in cellular proliferation or total cell numbers were observed between the three cell lines. The experiment was repeated at least 3 times with similar results. (C) HEC-1A/EMP2, HEC-1A/V, or HEC-1A/RIBO cells were grown in a monolayer and then a “wound” created. After 24 h, wound closure was measured. Experiments were performed at least three times, and the results averaged. Comparison by Student's t test, * p = 0.03; ** p = 0.05. (D) The same experiment was also performed on Ishikawa/EMP2 and Ishikawa/V. Comparison by Student's t test, * p = 0.02. (E) HEC-1A cells were grown to reach a confluent monolayer. Cells were incubated with the PP2, Dasatinib, Erlotinib, AKTi VIII, or a vehicle control, and a wound created. After 36 h, plates were imaged, and the percentage of wound closure was calculated. Values are averages (±SEM, n = 3). Comparison by Student's t test, * p<0.05. (F) The same experiment was also performed on Ishikawa cells. Values are averages (±SEM, n = 3). Comparison by Student's t test, * p<0.05.
EMP2 expression promotes cellular migration
In previous studies examining its expression in clinical endometrial cancer specimens, EMP2 correlated with invasive and more aggressive tumors [5]. To determine the contribution of EMP2 expression to endometrial cancer cell migration, HEC-1A/EMP2, HEC-1A/V, or HEC-1A/RIBO were grown to confluence. The cell layer was then wounded by using a sterile pipette tip, and images of the wound healing response were recorded over 48 hours. As shown in Figure 2C, HEC-1A/EMP2 cells exhibited a 33.5% increase in wound healing compared to HEC-1A/V (p = 0.03) and a 47.5% increase compared to HEC-1A/RIBO cells (p = 0.01). To confirm that this effect was not specific to HEC-1A cells, another endometrial cancer cell line was tested. Ishikawa cells were engineered to over express EMP2, and consistent with the previous results, Ishikawa/EMP2 exhibited a significant increase in wound healing over Ishikawa/V cells (Figure 2D; p = 0.02).
To determine the contribution of select signaling pathways on endometrial cancer cell migration, inhibitors were added to HEC-1A (Figure 2E) or Ishikawa cells (Figure 2F). Cells were then assayed for their response to migrate and close the wound. Both inhibition of Akt phosphorylation and EGFR using erlotinib had no significant effect on both HEC-1A and Ishikawa cell migration. In contrast, both the FAK-Src small molecule inhibitor PP2 and the Src family inhibitor dasatinib significantly inhibited migration. Importantly, minimal to no toxicity was observed for any of these inhibitors at the dosages indicated as determined by trypan blue exclusion (data not shown). These results suggested that activation of FAK and Src promoted migration in these cells.
EMP2 correlates with increased FAK and Src phosphorylation
Given the requirement for the FAK/Src pathway and EMP2 for migration of endometrial cancer cells, we investigated the relationship of these proteins in HEC-1A and Ishikawa cells. HEC-1A cells with modulated EMP2 levels were analyzed for EMP2, FAK, and Src phosphorylation. Total levels of FAK, Src, and β-Actin were used as controls. HEC-1A/EMP2 cells express a EMP2-GFP fusion protein at 48 kD and the endogenous protein at 20 kD. As expected, HEC-1A/RIBO cells reduced EMP2 expression by two-fold compared with the vector control. Increased expression of EMP2 resulted in an increase of activated FAK (phosphorylation at Y-576/577) as well as activated Src (phosphorylation at Y-416). Conversely, a reduction of EMP2 expression significantly reduced the expression of activated FAK and Src (Figure 3A). Similarly, Ishikawa cells with modulated EMP2 levels were analyzed for activated FAK and Src expression, and concordantly, upregulation of EMP2 promoted a two fold increase in the activation of FAK and Src (Figure 3B).
10.1371/journal.pone.0019945.g003 Figure 3 EMP2 promotes activated FAK and Src expression.
Expression of EMP2, 576/577 P-FAK, and 416 P-Src were assessed in (A) HEC-1A/EMP2, HEC-1A/V and HEC-1A/RIBO cells or (B) Ishikawa/EMP2 and Ishikawa/V cells. Semi-quantitative analysis of 576/577 P-FAK after correction for total FAK in both HEC-1A (C) and Ishikawa cells (D) from three independent experiments, respectively. β-actin expression was used as an additional loading control. Comparison by Student's t test, * p<0.05. EMP2 and β-actin panels of Figure 3A are excluded from this article's CC-BY license. See the accompanying retraction notice for more information.
EMP2 associates with FAK
In order to understand the relationship of FAK and EMP2 in endometrial cells, confocal microscopy was utilized to compare the localization of EMP2 and total FAK. In both HEC-1A and Ishikawa cells, EMP2 was expressed within the cytoplasm and at the plasma membrane (Figure 4A) [6], [23]. FAK displayed a similar localization pattern within cells, with intense cytoplasmic and membrane staining, and significant colocalization between EMP2 and FAK was observed. In HEC-1A cells, 34.5±1.3% of EMP2 colocalized with FAK (Figure 4A), and in Ishikawa cells, 42.6±8% colocalization was observed (data not shown).
10.1371/journal.pone.0019945.g004 Figure 4 Total FAK and EMP2 associate with each other.
(A) Cellular images of HEC-1A cells. Cells were stained for EMP2 (FITC) and total FAK (Rhodamine) expression and imaged using confocal microscopy. EMP2 and FAK colocalize (yellow) in the cytoplasm and on the membrane of HEC-1A cells. Scale bar, 20 µM. (B) In order to assess if EMP2 and FAK immunoprecipitate together, HEC-1A cells were lysed in 1% NP-40 and an interaction assessed using immunoprecipitation/SDS-PAGE analysis. Both α-FAK antibodies and EMP2 antisera pulled down EMP2, FAK, and 576/577 p-FAK. Normal rabbit antisera served as the negative isotype control. Experiments were repeated three times with similar results; a representative image is displayed.
As microscopy experiments suggested that EMP2 and FAK resided within similar cellular compartments, we next determined if the two proteins associated with each other. Immunoprecipitations were preformed in wildtype HEC-1A cells using 1% Nonidet P-40 to prevent nonspecific hydrophobic interactions [24], [25]. Anti-EMP2 antisera pulled down both EMP2 and FAK (Figure 4B). Conversely, pull-down with anti-FAK antibodies resulted in detectable FAK and EMP2 (Figure 4B). We next determined the proportion of activated FAK that associated with EMP2. Approximately 35±3% of activated FAK immunoprecipitated with EMP2.
EMP2 colocalizes with activated FAK
Biochemical studies suggested that a subset of EMP2 immunoprecipitated with activated FAK. To confirm and extend this observation, the association of EMP2 and activated FAK was examined by confocal microscopy. Cells were stained for EMP2 (FITC) and activated FAK (Rhodamine). Approximately 30% of EMP2 in HEC-1A/EMP2 and 40% of EMP2 in Ishikawa/EMP2 colocalized with activated FAK.(Figure 5A, C). In HEC-1A/V and Ishikawa/V cells, a two fold reduction in colocalization was observed. As summarized in Figures 5B and D respectively, approximately 15 or 20% of EMP2 and activated FAK colocalized with HEC-1A/V and Ishikwawa/V cells, respectively. Finally, little colocalization (<5%) was observed between EMP2 and activated FAK in HEC-1A/RIBO cells.
10.1371/journal.pone.0019945.g005 Figure 5 EMP2 and activated FAK colocalize.
(A) Cellular images of HEC-1A/EMP2 cells stained for confocal microscopy using EMP2 antisera (FITC) and FAK (activated at tyrosine 397; Rhodamine). (B) Data from at least four separate samples were quantitated using Pascal software to calculate pixel intensity, and the resultant data was evaluated by Student's unpaired t-test. Overexpression of EMP2 demonstrated a two-fold increase in colocalization with p-FAK compared to vector control cells. Less than 5% colocalization was observed in HEC-1A/RIBO cells. Student's t test, * p = 0.01; ** p = 0.0001. Scale bar, 25 µM. (C) Ishikawa/EMP2 cells were stained using EMP2 antisera (FITC) and FAK (activated at tyrosine 397; Rhodamine) and analyzed by confocal microscopy. (D) Data from at least four separate samples were quantitated and analyzed as above. Student's t test, * p = 0.01. Scale bar, 25 µM.
As elevated EMP2 expression correlated with increased FAK activation, we next characterized the distribution of p-FAK within endometrial carcinoma cells. Increased EMP2 levels promoted activated FAK expression on the plasma membrane, and in contrast, the EMP2 specific ribozyme appeared to disrupt the organization of activated FAK (data not shown). HEC-1A/RIBO cells expressed little activated FAK on the plasma membrane, and instead, p-FAK was localized within intracellular compartments (data not shown).
EMP2 resides within lipid raft domains
Previous studies have shown that in NIH3T3 fibroblast cells, EMP2 localized within DIG membrane domains [7]. We thus hypothesized that EMP2 may create a functional signaling complex with activated FAK and Src within a DIG lipid raft membrane domain that regulated the migratory potential of endometrial cancer cells. To test this hypothesis, we initially confirmed the localization of EMP2 to lipid raft domains in wildtype HEC-1A cells. Using both 1% Brij-96 (Figure 6A) or 1% Triton X-100 (Figure 6B), EMP2 localized to the lipid fractions identified by a common raft protein ganglioside GM1 [26]. Cholesterol depletion of rafts using methyl-β cyclodextrin (MβCD) redistributed EMP2 into the non-raft soluble fractions.
10.1371/journal.pone.0019945.g006 Figure 6 EMP2 forms a complex with FAK and Src in DIG lipid raft membrane domains.
(A) EMP2 expression in HEC-1A lipid raft membrane domains was verified by Brij-58 insolubility. Cells were lysed in 1% Brij-58 and centrifuged in a sucrose density gradient. Nine fractions (500 µl each) were collected from the top of the gradient and tested for GM1 by a cholera toxin-HRP dot blot and for EMP2 (∼Mr 20 kDa) by using SDS-PAGE and Western blot analysis. (B) To verify the cholesterol dependence of EMP2 expression within the lipid raft. HEC-1A cells were preincubated in the presence (+) or absence (–) of MβCB. Cells were lysed in 1% Triton X-100, gradient fractionated, and EMP2 expression detected by Western blot analysis. (C) HEC-1A/EMP2, HEC-1A/V, and HEC-1A/RIBO cells were lysed in 1% Triton X-100 and centrifuged as above. Samples were probed by Western blot analysis for EMP2, activated FAK, activated Src, total FAK, total Src, Flotillin-2, and EEA1. Experiments were performed independently three times with similar results.
We next determined if modulation of EMP2 expression altered the distribution of Src, FAK, or their activated forms into lipid raft domains (Figure 6C). In both HEC-1A/EMP2 and HEC-1A/V, the distribution of total FAK and Src was similar, with a significant localization within lipid raft fractions (fractions 2–5) as well as in the 1% Triton X-100 detergent soluble fractions (fractions 6–9). In contrast, decreased levels of total FAK and Src were present in lipid raft fractions in HEC-1A/RIBO cells. Similar results were observed with the activated forms of these proteins. Upregulation of EMP2 expression resulted in increased localization of activated FAK and Src in lipid rafts. In contrast, reduced EMP2 expression decreased reduced the levels of activated FAK and eliminated all activated Src within lipid domains. The redistribution of the FAK-Src signaling complex appeared to be specific as EMP2 did not alter the distribution of a known raft protein Flotillin-2 or the detergent soluble endosome protein EEA1 [27], [28].
Discussion
The tetraspan protein EMP2 has been implicated as a prognostic and survival marker for endometrial cancer [4], [5]. In this study, we examine the tumor growth of endometrial carcinoma cells with increased or reduced EMP2 expression to determine the contribution of EMP2 to endometrial cancer tumorigenicity. Dramatically, HEC-1A tumors with increased EMP2 expression (HEC-1A/EMP2) exhibit a two fold increase in tumor volume compared to HEC-1A/V and a three fold increase compared to cells with reduced EMP2 expression (HEC-1A/RIBO). Mechanistically, we have started to characterize how EMP2 expression affects endometrial tumor progression. We demonstrate that EMP2 is sufficient to activate FAK and its signal transduction cascade. This effect may be direct as EMP2 and FAK associate with each other, and a significant proportion of activated FAK and Src reside within an EMP2-lipid raft complex.
Intact lipid structures have been shown to play critical roles for proper signal transduction and migration [29], [30]. DIG membrane microdomains are enriched in cholesterol and sphingolipids and make up two types of membrane compartments. The first are flask-shaped invaginations called caveolae which contain caveolin-1 [31], and the second type are flat domains which lack caveolins called lipid rafts [32]. Endometrial epithelial cells do not have caveolae as endometrial epithelial cells lack expression of both caveolin-1 and caveolin-2 (Wadehra and Braun, unpublished results). Our data supports the conclusion that EMP2 complexes with FAK and Src leading to enhanced protein phosphorylation, and that this association occurs within lipid raft microdomains. We predict that EMP2 expression is necessary to traffic and/or stabilize FAK and Src phosphorylation within a lipid raft domain with the functional consequence of this interaction being improved cell migration.
FAK acts as a receptor-proximal protein that bridges the growth-factor-receptor and integrin signaling pathways [33]. A previous study has shown that in endometrial epithelial cells, EMP2 regulates αvβ3 integrin expression [6], and it is known that in epithelial cells β3 integrin activation directly leads to Src and FAK phosphorylation resulting in stable focal adhesions [34], [35]. Analysis of EMP2-β3 integrin-FAK association data suggest that in the endometrium EMP2 may act as a molecular adaptor for efficient integrin-mediated FAK activation. In this way, we predict that EMP2 works to link select integrin isoforms and their associated signaling modules within DIG lipid raft membrane domains [25], [36].
This is similar to the role proposed for other members of the tetraspan superfamily as several groups have hypothesized that tetraspanins create a scaffold to regulate signaling, trafficking, and structural characteristics of their membrane protein constituents [37], [38]. Tetraspanins which have been shown to participate in the formation of a variety of signaling complexes important for integrin signaling, B-cell receptor signaling, and fertilization [24], [25]. With regard to integrin signaling, for example, it has been shown that the interaction of CD151 with integrin α3β1 helps modulate cell adhesion and motility on the extracellular matrix laminin [39], [40], suggesting that the GAS-3 and tetraspanins families of proteins may have more in common than was previously appreciated. Interestingly, tetraspanins are not found in a lipid raft domains [41]. Thus, it may be that GAS-3 proteins such as EMP2 help organize lipid raft domain signaling while the tetraspanins organize non-raft domain signaling.
EMP2 is an early prognostic biomarker in endometrial hyperplasia, and high expression of EMP2 in endometrial cancer is associated with a poor prognosis. Similarly, we would predict that in endometrial cancer, β3 integrin expression will be upregulated with a concomitant increase in FAK and Src activation. Clinical data supports this hypothesis. First, β3 integrin expression has been shown to be upregulated in a number of cancers, including endometrial [42]. Second, activation of FAK and Src kinase has been documented in the progression of benign to malignant endometrium [42], [43]. We predict that a better understanding of the mechanism of EMP2 may help predict endometrial cancer incidence, prognosis and treatment.
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Patholog Res IntPatholog Res IntPRIPathology Research International2090-80912042-003XSAGE-Hindawi Access to Research 2166026610.4061/2011/431246Review ArticleOral Carcinogenesis and Oral Cancer Chemoprevention: A Review Tanaka Takuji
1, 2
*Tanaka Mayu
3
Tanaka Takahiro
4
1Director TCI-CaRP, 5-1-2 Minami-Uzura, Gifu City, Gifu 500-8285, Japan2Department of Oncologic Pathology, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Shikawa 920-0293, Japan3Department of Pharmacy, Kinjo Gakuin University of Pharmacy, Moriyama-Ku, Nagoya, Aichi 463-8521, Japan4Department of Physical Therapy, Kansai University of Health Sciences, Kumatori-Machi, Sennan-Gun, Osaka 590-0482, Japan*Takuji Tanaka: [email protected] Editor: Stefan E. Pambuccian
2011 22 5 2011 2011 43124627 10 2010 19 3 2011 Copyright © 2011 Takuji Tanaka et al.2011This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Oral cancer is one of the major global threats to public health. The development of oral cancer is a tobacco-related multistep and multifocal process involving field cancerization and carcinogenesis. The rationale for molecular-targeted prevention of oral cancer is promising. Biomarkers of genomic instability, including aneuploidy and allelic imbalance, are possible to measure the cancer risk of oral premalignancies. Understanding of the biology of oral carcinogenesis will yield important advances for detecting high-risk patients, monitoring preventive interventions, and assessing cancer risk and pharmacogenomics. In addition, novel chemopreventive agents based on molecular mechanisms and targets against oral cancers will be derived from studies using appropriate animal carcinogenesis models. New approaches, such as molecular-targeted agents and agent combinations in high-risk oral individuals, are undoubtedly needed to reduce the devastating worldwide consequences of oral malignancy.
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1. Introduction
Head and neck cancer is the sixth most common human cancer [1], representing 3% of all types of cancer. They are located in the oral cavity in 48% of cases, and 90% of these are oral squamous cell carcinoma [2]. They are sometimes preceded by precancerous lesions, such as leukoplakia and erythroplakia. More than 300,000 new cases of oral squamous cell carcinoma are diagnosed annually [3]. Approximately 35,000 new cases are recorded annually in the US [2], 40,000 new cases are recorded in the EU and 10915 new cases in Japan [4]. The most common site for intraoral carcinoma is the tongue, which accounts for around 40% of all cases in the oral cavity proper. Tongue cancers most frequently occur on the posterior-lateral border and ventral surfaces of the tongue. The floor of the mouth is the second most common intraoral location. Less common sites include the gingival, buccal mucosa, labial mucosa, and hard plate.
The incidence of oral cancer has significant local variation. Oral and pharyngeal carcinomas account for up to half of all malignancies in India and other Asian countries, and this particularly high prevalence is attributed to the influence of carcinogens and region-specific epidemiological factors, especially tobacco and chewing betel quid. An increase in the prevalence of oral cancer among young adults is a cause of special concern. There has been a 60% increase in the number of under 40 years olds with tongue cancer over past 30 years. However, little has been published on the etiology and natural history of this increase [5]. Oral malignancy, including tongue cancer, is associated with severe morbidity and long-term survival of less than 50% despite advances in the treatment (surgery, radiation, and chemotherapy) of oral cancer. The survival of the patients remains very low, mainly due to their high risk of developing a second primary cancer. Therefore, the early detection and prevention of oral cancer and premalignancy are quite important [6–10]. This article will focus on the current understanding of oral carcinogenesis for the early detection and prevention of oral malignancy.
2. Oral Carcinogenesis
Oral carcinogenesis is a highly complex multifocal process that takes place when squamous epithelium is affected by several genetic alterations. The use of several molecular biology techniques to diagnose oral precancerous lesions and cancer may markedly improve the early detection of alterations that are invisible under the microscope. This would identify patients at a high risk of developing oral cancer [11]. The natural history of oral cancer and sequence of genetic alterations are illustrated in Figure 1. There are several approaches to understanding the molecular basis of oral cancer [12–14]. They include microarray technology, methylation microarrays, gene expression microarrays, array comparative genomic hybridization, proteomics, mitochondrial arrays, and micro-RNA arrays [15]. High-throughput approaches are currently being used to search for oral cancer biomarkers in biofluids, such as saliva and serum [15].
Field cancerization' refers to the potential development of cancer at multiple sites [16, 17]. This has been observed during the development of cancer in the tissues covered with squamous epithelium (head and neck tumor) and transitional epithelium (urothelial carcinoma). It is evident that oral cancer, like carcinomas in other tissues, develops over many years, and during this period, there are multiple sites of neoplastic transformation occurring throughout the oral cavity. “Field cancerization” may also be defined by the expression of mutations in the exons of tumor suppressor genes. One such tumor suppressor gene is p53, and mutations of this gene have been observed in various sites of premalignant leukoplakia and carcinoma in the same oral cavity [18]. A reduction in tumor suppressor activity by the gene and the development of mutations in p53 are associated with smoking and an increased risk for oral carcinoma development [19]. Therefore, multifocal presentations and mutational expressions of tumor suppressor genes may be the consequence of long-term (e.g., 20 ~ 40 years) exposure to various environmental and exogenous factors. The continual presence of mutations may also signify changes in DNA repair and apoptosis, thereby increasing the susceptibility to future transformation. Mutational adaptations that modify the survivability of particular clones of transforming cells may also further enhance the level of resistance to therapeutic control. A recent genetic analysis revealed that cancers developing at distant sites within the oral cavity often are derived from the same initial clone [20]. The multiplicity of the oral carcinogenesis process makes it difficult to interrupt the progression to cancer through the surgical removal of a premalignant lesion.
3. Risk Factors of Oral Cancer
The most important risk factor for the development of oral cancer in the Western countries is the consumption of tobacco [21] and alcohol [22]. Although drinking and smoking are independent risk factors, they have a synergistic effect and greatly increase the risk together. The use of smokeless tobacco products such as gutkha and betel quid in Asian countries [5, 23] is responsible for a considerable percentage of oral cancer cases.
3.1. Genetic
Several studies have reported a significant familial component in the development of oral cancer. The estimates of risk in the first degree relatives of oral cancer patients vary widely and range from 1.1 [24] to 3.8 [25], although some of these cancers refer to head and neck cancer in general. Familial aggregation of oral cancer, possibly with an autosomal dominant mode of inheritance, is observed in a very small percentage of oral cancer patients [26]. Polymorphic variation of genes in the xenobiotic metabolism pathways such as in CYP1A1 or the genes coding for glutathione S-transferase-M1 [27, 28] and N-acetyltransferase-2 [29] may be implicated. Individuals that carry the fast-metabolizing alcohol dehydrogenase type 3 (ADH3) allele [30] may be particularly vulnerable to the effects of chronic alcohol consumption and could be at increased risk to develop oral cancer [31]. The single nucleotide polymorphism A/G870 in the CCND1 gene that encodes Cyclin D is associated with susceptibility to oral cancer. The AA genotype [32] or the GG wild-type genotype [33] may increase risk for oral cancer.
3.2. Inflammation
Cytokines, including interleukins (ILs), tumor necrosis factors (TNFs), and certain growth factors, are an important group of proteins that regulate and mediate inflammation and angiogenesis. Tumor growth, invasion and metastasis are facilitated when there is a deregulation in their production. Genetic association studies suggest a putative correlation between functional DNA polymorphisms in cytokine genes and oral cancer [34]. Increased serum levels of proinflammatory cytokines, interleukin (IL)-1β, IL-6, IL-8, and TNF-α as well as the anti-inflammatory cytokine, IL-10, are seen in patients with oral cancer in comparison to healthy controls. The anti-inflammatory cytokine IL-4 inhibits oral cancer invasion by the downregulation of matrix metalloproteinase-9.
3.3. Infection
Human papillomavirus (HPV), particularly HPV type 16, may be an etiologic factor, especially among persons who do not smoke or drink alcohol [35, 36]. Ang et al. [37] reported that tumor HPV status is a strong and independent prognostic factor for survival among patients with oropharyngeal cancer. They also noted that the risk of death significantly increased with each additional pack-year of tobacco smoking. Although the idea that bacterial infections could lead to oral cancer has been generally discounted, there is an increasing body of evidence to suggest a possible relationship between micro-organisms and the development of oral cancer. The mostnotable example is that of the common pathogenic bacterium Helicobacter pylori and its association with gastric cancer. The mouth contains a variety of different surfaces that are home to a huge diversity of micro-organisms, including more than 750 distinct taxa of bacteria, thus suggesting that the oral squamous epithelium is constantly exposed to a variety of microbial challenges, on both cellular and molecular levels. It is therefore important to consider how such factors may be related to oral cancer development [38, 39].
3.4. Preneoplasia
There are clinically apparent oral premalignant lesions of oral cancer. They include leukoplakia, erythroplakia, nicotine stomatitis and tobacco pouch keratosis, lichen planus, and submucous fibrosis, [40]. The term “leukoplakia” was first used by Schwimmer in 1877 [41] to describe a white lesion of the tongue that probably represented a syphilitic glossitis. The definition of leukoplakia has often been confusing and controversial. Some clinicians now avoid using this term. The World Health Organization defines leukoplakia as ‘a white patch or plaque that cannot be characterized clinically or pathologically as any other disease [42]. Therefore, leukoplakia should be used only as a clinical term. The term has no specific histopathological connotation and should never be used as a microscopic diagnosis. Leukoplakia is a clinical diagnosis of exclusion. Sometimes a white patch is initially believed to represent leukoplakia, but the biopsy reveals another specific diagnosis. These lesions should no longer be categorized as a leukoplakia. Leukoplakia is seen most frequently in middle-aged and older males, with an increasing prevalence with age [43]. Fewer than 1% of males below the age of 30 have leukoplakia, but the prevalence increases to an alarming 8% in men over the age of 70 [43]. The prevalence in females past the age of 70 is approximately 2%t. The most common sites are the buccal mucosa, alveolar mucosa, and lower lip. However, lesions occurring on the floor of mouth, lateral tongue, and lower lip are most likely to show either dysplastic or malignant changes [44].
The term “erythroplasia” originally used by Queyrat [45] to describe a red, precancerous lesion of the penis is used for a clinically and histopathologically similar process that occurs on the oral mucosa. Similar to the definition for leukoplakia, erythroplakia is a clinical term that refers to a red patch that cannot be defined clinically or pathologically as any other condition [42]. This definition excludes inflammatory conditions that may result in a red clinical appearance. Oral erythroplakia occurs most frequently in older males and appears as a red macule or plaque with a soft, velvety texture. The floor of mouth, lateral tongue, retromolar pad, and soft palate are the most common sites of involvement. Often the lesion is well demarcated, but some examples may gradually blend into the surrounding mucosa. Some lesions may be intermixed with white areas (erythroleukoplakia). Erythroplakia is often asymptomatic, although some patients may complain of a sore, burning sensation.
3.5. Tobacco
Nicotine stomatitis is a thickened, hyperkeratotic alteration of the palatal mucosa that is most frequently related to pipe smoking, but milder examples can also develop secondary to cigar smoking or, rarely, from cigarette smoking [42]. The palatal mucosa becomes thickened and hyperkeratotic, sometimes developing a fissured surface. The surface often develops numerous elevations with red centers, which represent the inflamed openings of the minor salivary gland ducts.
Another specific tobacco-related oral mucosal alteration occurs in association with smokeless tobacco use, such as either snuff or chewing tobacco [40]. Such lesions typically occur in the buccal or labial vestibule where the tobacco is held, but they can also extend onto the adjacent gingiva and buccal mucosa. Early lesions show slight wrinkling that disappears when the tissues are stretched. Other lesions may appear as hyperkeratotic, granular patches. Advanced lesions exhibit greatly thickened zones of grayish white mucosa with well-developed folds and fissures. The degree of clinical alteration depends on the type and quantity of tobacco, the duration of tobacco usage, and host susceptibility. Smokeless tobacco keratosis shows microscopic hyperkeratosis and acanthosis of the mucosal epithelium. True epithelial dysplasia is uncommon, and when dysplasia is found, it tends to be mild [46].
3.6. Mutations
Genetic mutations often produce early phenotypic changes that may present as clinically apparent, recognizable lesions. An oral premalignant lesion is an area of morphologically or genetically altered tissue that is more likely than normal tissue to develop cancer. The reported rates of malignant transformation of leukoplakia range from less than 1% to 18% [47, 48]. There is no accepted method to predict the risk of malignant progression of an individual oral premalignant lesions, but various factors, such as the location within the oral cavity, clinical appearance (homogeneous versus heterogeneous), and the presence of dysplasia are correlated with the risk of progression. The histological finding of dysplasia is strongly associated with an increased rate of invasive cancer development [47]. A velvety reddish mucosal lesion, known as erythroplakia, is associated with a higher rate of cancer development, occurs much less frequently, and is more difficult to detect clinically than oral leukoplakia. Virtually all erythroplakic lesions contain severe dysplasia, carcinoma in situ, or early invasive carcinoma at the time of presentation [49]. Formalized classification and staging systems for oral preneoplastic lesions have been proposed [50, 51], and their use is important to facilitate uniform reporting and comparisons of data.
Detection and diagnosis of oral neoplasia has traditionally relied heavily on the clinical experience of the examiners and their ability to recognize often subtle morphologic changes. However, some early malignant lesions are clinically indistinguishable from benign lesions, and some patients develop carcinomas in the absence of clinically identifiable oral premalignant lesions. Furthermore, it can be difficult, even for experts, to determine which oral premalignant lesions are at significant risk to progress to invasive carcinoma. Therefore, an accurate, objective, and noninvasive method to help identify premalignant lesions and to distinguish those at risk of malignant conversion is needed.
4. Biomarkers of Oral Cancer
Biomarkers help in evaluating the preventive measures or therapies and the detection of the earliest stages of oral mucosal malignant transformation. Biomarkers reveal the genetic and molecular changes related to early, intermediate, and late end-points in the process of oral carcinogenesis. These biomarkers will refine the ability to enhance the prognosis, diagnosis, and treatment of oral carcinomas [52]. Genetic and molecular biomarkers will also determine the efficacy and safety of chemopreventive agents. Chemopreventive agents are chemicals of natural or synthetic origin. Unlike other drugs, which do not prevent disease, chemopreventive agents reduce the incidence of diseases such as cancer before clinical symptoms occur. This development is critical for the understanding of early oral mucosal transformation. Biomarkers will also reduce the number of patients and the time for long-term follow-up required to define a significant clinical response to a chemopreventive agent [53, 54]. The markers may therefore clarify the types, doses, frequencies, and regimens to achieve the maximum level of benefit from chemopreventive agents. Decreasing the cost of the clinical trials is another factor that drives the development of biomarkers.
Biomarkers have been categorized following the recommendation by the Committee on Biological Markers of the National Research Council/National Academy of Sciences [55]. They fall into broad groups that detect exposure, progression, susceptibility to carcinogens, and/or the responses by the target cellular populations [54].
Oral cancer studies have a distinct advantage due the anatomical access to the developing premalignant and malignant lesions. One could readily analyze biopsies of the primary lesion as well as apparently normal mucosal sites to determine the levels of DNA adducts and oral cancer risk. DNA adduct studies and cytogenetic analyses may also provide evidence for altered structure and function of susceptibility sites in the DNA following DNA binding studies of nuclear proteins such as p53. Some studies have focused on microscopic cytogenetic and somatic mutation changes as early biologic markers. One of the markers used to define chromosomal aberrations is the staining for micronuclei in exfoliated buccal mucosal cells [56]. Micronuclei have also been used to evaluate the reversal of leukoplakia and the effectiveness of retinoids, carotenoids, and vitamin E [57, 58]. Other methods include the determination of aneuploidy and the assessment of losses and gains of genetic material particularly associated with somatic and sex chromosomes. Other sites of chromosomal aberrations are found in sister chromatid exchanges, and allele typic variations designated by losses on chromosomes 3, 4, 5, 6, 8, 9, 11, 13, 17, and 19.
Some molecular biomarkers with potential diagnostic relevance include DNA content and chromosome polysomy, loss of heterozygosity, nucleolar organizer regions, histo-blood group antigens, proliferation markers, increased epidermal growth factor receptor (EGFR), and decreased expression of retinoic acid receptor-β, p16, and p53 [59, 60]. Although a reliable, validated marker panel for providing clinically useful prognostic information in oral premalignant lesions patients has not yet been established, the advent of high-throughput genomic and proteomic analysis techniques may soon yield major advances toward a prognostically relevant molecular classification system (Table 1).
5. Animal Models for Oral Carcinogenesis
A variety of animals have been used for the study of tumor growth, the process of carcinogenesis, and the prevention/treatment research [8, 61–64]. The continual development of transgenic or knockout mice has improved our understanding of the role of specific genes in tumor growth. The most widely used animal models for oral carcinogenesis are the hamster cheek pouch model [62, 65] and the 4-nitroquinoline 1-oxide- (4-NQO-) induced oral (tongue) carcinogenesis model [8, 61, 66, 67].
DMBA is one of the widely used carcinogens in experimental oral carcinogenesis. Induction of SCC in cheek pouch of hamsters was first described with the aid of three polycyclic aromatic hydrocarbons, such as 7,12-dimethylbenz(a)anthracene (DMBA), 20-methyleholanthrene (20-MC), and 3,4-benzpyrene [68]. A complete carcinogen, DMBA (0.5%), is applied to the hamster cheek pouch three times a week for 16 weeks. All animals exhibit invasive oral squamous cell carcinoma by week 16. Many studies have been conducted using the hamster buccal pouch model and thus elucidated an array of changes that are analogous to those observed in human invasive oral carcinoma [62, 65]. These include a mutation in codon 61 of Ha-ras, which manifested in an A→T transversion in the second position of codon 61, thus resulting in an amino acid change from glycine to leucine. The expression of c-Ki-ras in malignant tumors of the pouch, but not in the normal oral mucosa, is also observed at the very early stages of tumor development [65]. Although the hamster oral tumor model appears to parallel several changes observed in human oral cancer, the hamster still has several areas of uniqueness which must be considered in any evaluation of results from oral carcinogenesis studies. The hamster cheek pouch provides a relatively large surface area of oral mucosa for the development of invasive carcinoma, while the human does not possess this type of mucosal structure. In contrast to humans, mice, or rats, the hamster cheek pouch lacks lymphatic drainage, which thus allows various drugs or molecules to accumulate in the pouch. The Syrian hamster population was also derived from a small breeding pair that resulted in a restricted polymorphism for the antigen recognition region (Ia region) and some of the major histocompatibility K and D regions [69]. In addition, the number of T-cells in the hamster spleen exhibits a lower number/gram weight of the organ in comparison to the mouse or human [69]. The hamster may also respond to antigenic tumor sources with a natural killer macrophage or granulocyte cytotoxicity rather than a T cell response [69]. DMBA and its solvent vehicle (acetone or benzene) are significant local irritants that cause severe inflammatory response, necrosis, and sloughing. Therefore, it is difficult to examine early squamous cell lesions [66, 70, 71]. Neoplasms induced by DMBA in the hamster cheek pouch possess many differences in histological features of differentiated SCC and do not closely resemble the lesions observed in human [72, 73].
The latter animal models for the study of oral carcinogenesis include those in rats and mice using the water soluble carcinogen, 4-NQO. The carcinogen is supplied either in the water (20 ppm) for the rats [66, 71, 74–86] or by painting for the mice [87]. The administration of 4-NQO in drinking water (20 ppm) for 8 weeks in rats and mice produces tongue lesions including squamous cell neoplasms within 32 weeks [83], while topical application of the carcinogen to the mouse palates for up to 16 weeks just like the hamster model develops palate tumors within 49 weeks [87]. The 4-NQO-induced tongue carcinogenesis model is quite useful for investigating oral carcinogenesis and identifying cancer chemopreventive agents, because the most common site for intraoral carcinoma is the tongue and the administration drinking water containing of 4-NQO is a simple and easy method [66, 71, 74–86, 88–96]. Increased levels of polyamine synthesis, as well as nucleolar organizer regions (NORs) with the progression of oral carcinogenesis, have been noted in the rat model [66]. The mouse model with 4-NQO has demonstrated some molecular mimicry of human oral cancers, as is true of the hamster model [87]. A number of chemical carcinogens, including coal tar, 20-MC, DMBA, and 4-NQO, have been used in experimental oral carcinogenesis. However, 4-NQO is the preferred carcinogen apart from DMBA in the development of experimental oral carcinogenesis. 4-NQO is a water soluble carcinogen, which induces tumors predominantly in the oral cavity. It produces all the stages of oral carcinogenesis and several lines of evidences suggest that similar histological as well as molecular changes are observed in the human system. There are several review articles that collate the available information on the mechanisms of action of 4-NQO. In addition, studies have been conducted for the development of biomarkers and chemopreventive agents using 4-NQO animal models [8–10, 61, 66, 67, 74–86].
The complexity and variety of biochemical changes that can increase tumor development is demonstrated in the p53−/− mice [97]. Unfortunately, this model and other genetic mouse models have not been exploited for studying the relationships among chemical oral carcinogenesis, specific genetic defects, and chemoprevention. Genetically altered mouse and rat models have been developed to evaluate molecular-targeted prevention and treatment of oral carcinoma [64]. The rasH2 transgenic mouse carcinogenesis model [98] and human c-Ha-ras proto-oncogene transgenic rat model [99] have been developed for chemoprevention studies on oral (tongue) carcinogenesis.
6. Chemoprevention
Chemoprevention is the use of natural or synthetic substances to halt, delay, or reverse malignant progression in tissues at risk for the development of invasive cancer [8–10]. Retinoids are the most extensively studied agents for chemoprevention of oral cancer [100]. Administration of 13-cis-retinoic acid for only 3 months yields a clinical response rate of 67% versus 10% for placebo. However, the toxicity is considerable, and there is a very high rate of relapse within 3 months of stopping treatment. Subsequent studies with retinoids in patients with oral premalignant lesions have confirmed clinical and pathologic response rates, though toxicities remain a concern [101]. However, translational studies show that molecular abnormalities persist in some patients with a complete clinical and pathologic response to retinoid therapy [102], suggesting that cancer development may be delayed rather than prevented by these agents. Other agents that have been assessed in clinical trials to evaluate the chemoprevention activity in oral leukoplakia patients include vitamin E [52], Bowman-Birk inhibitor concentrate (BBIC) derived from soybeans [103], curcumin [104], and green tea polyphenol epigallocatechin-3-gallate. Small clinical trials using oral BBIC have revealed no significant toxicity and a 32% response rate [103].
Attention is currently focused on the development of agents targeted to specific steps in the molecular progression from normal to oral premalignancy and to invasive carcinoma. Examples of molecularly targeted agents that have shown promise in vitro, in animal models, or in early clinical trials include cyclooxygenase- (COX-) 2 inhibitors and epidermal growth factor receptor (EGFR) inhibitors [105–107]. Data from several sources suggest that the cyclooxygenase pathway is a good target for oral cancer prevention. COX-2 is overexpressed in head and neck squamous carcinoma [108], and COX-2 inhibitors prevent oral cancer development in animal models [109]. A randomized placebo-controlled trial of the COX-2 inhibitor ketorolac administered as an oral rinse in oral leukoplakia patients revealed that the treatment is well tolerated but does not result in a greater clinical response than placebo [110]. However, an analysis of the results of this trial is somewhat confounded by the high response rate (32%) in the placebo arm and difficulty in determining whether topical delivery of the agent allowed penetration to the damaged cells. The future of COX-2 inhibitors as chemoprevention agents will also depend on determining the extent of risk for cardiac toxicities associated with this class of agents. The EGFR is also a promising molecular target for intervention in oral malignant progression [105–107]. EGFR is a receptor tyrosine kinase that is overexpressed in oral dysplasia and invasive cancer and associated with poor prognosis in patients with head and neck squamous carcinoma [111, 112]. EGFR inhibitors, alone or in combination with chemotherapy and radiotherapy, show activity against head and neck squamous carcinoma in clinical trials and are generally well tolerated [113]. Evidence suggests that combination therapy targeting COX-2 and EGFR may be efficacious [107, 114]. Although chemoprevention appears to be a promising approach to managing oral premalignancy, prospective clinical trials using specific agents, and strong corollary translational and laboratory investigations, are needed to evaluate clinical, histological, and molecular efficacy. It may be possible and necessary to individualize medical therapy to specific genetic abnormalities detected within the oral mucosa.
7. Conclusion
Human oral cancer is the sixth largest group of malignancies worldwide. Seventy percent of oral cancers appear from premalignant lesions. The process of formation of oral cancer results from multiple sites of premalignant change in the oral cavity (field cancerization). Animal models are now being widely used for the development of diagnostic and prognostic markers. The appearance of these premalignant lesions is one distinct feature of human oral cancer. There is currently a dearth of biomarkers to identify which of these lesions will turn into malignancy. Regional lymph node metastasis and locoregional recurrence are the major factors responsible for the limited survival of patients with oral cancer. The paucity of early diagnostic and prognostic markers strongly contributes to the higher mortality rates. Determining high- and low-risk populations by measuring reliable biomarkers is expected to contribute to achieving a better understanding the dynamics and prevention of oral cancer development. The quantitation of genetic and molecular changes and the use of these changes as markers for the detection and prevention of early premalignant change require the harvesting of tissues and cells. Promising technologies are being rapidly developed to assist in the identification of an abnormal oral mucosa, noninvasive and objective diagnosis and the characterization of identified mucosal lesions, and in the therapies for patients with oral cancer. Undoubtedly, the prevention or reduction in the use of tobacco products and alcohol consumption would have a profound influence on the incidence of oral cancer. Chemoprevention also has an impact on the development of malignant changes in the oral mucosa. Prevention through chemoprevention and/or the use of systemic medications is an extensively studied strategy and continues to hold promise as a way of diminishing the morbidity and mortality associated with this malignancy.
Conflict of Interests
The authors declared that there is no conflict of interest.
Acknowledgments
This review was based on studies supported in part by a Grant-in-Aid for the 3rd Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labour and Welfare of Japan; the Grant-in-Aid for Cancer Research from the Ministry of Health, Labour and Welfare of Japan; the Grants-in-Aid for Scientific Research (nos. 18592076, 17015016, and 18880030) from the Ministry of Education, Culture, Sports, Science and Technology of Japan; and the Grant (H2010-12) for the Project Research from High-Technology Center of Kanazawa Medical University.
Abbreviations
BBIC:Bowman-Birk inhibitor concentrate
COX:Cyclooxygenase
DMBA:7,12-dimethylbenz(a)anthracene
EGFR:Epidermal growth factor receptor
HPV:Human papillomavirus
IL:Interleukin
20-MC:20-methylcholanthrene
NORs:Nucleolar organizer regions
4-NQO:4-nitroquinoline 1-oxide
RAR:Retinoid acid receptor
TNF:Tumor necrosis factor.
Figure 1 The natural history of oral carcinogenesis.
Table 1 Potential biomarkers for oral carcinogenesis.
Category of biomarkers Measurements
Genomic Micronuclei, DNA adduct, DNA content, Chromosomal aberration
Oncogenic Oncogenic expression, Modified tumor suppressor genes, Src genes
Proliferation Nuclear and cyclin related antigens, Mitotic frequency, Ornithine decarboxylase (ODC), Polyamines
Differentiation Cytokeratins, Transglutaminase Type I, Transcription factor (AP)-1
Oxidative stress Glutathione S-transferase, Stress proteins (HSPs), Superoxide dismutase
Apoptosis Bcl-2 family, Chromatin condensation factors, Caspases, Mitochondrial pathway
Immunologic Various cytokines
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 21673986PONE-D-11-0648910.1371/journal.pone.0020537Research ArticleMedicineGastroenterology and HepatologyGastrointestinal CancersOncologyCancers and NeoplasmsGastrointestinal TumorsPancreatic CancerActivated K-ras and INK4a/Arf Deficiency Cooperate During the Development of Pancreatic Cancer by Activation of Notch and NF-κB Signaling Pathways Activated Notch and NF-kB in Compound KCI MiceWang Zhiwei
1
Banerjee Sanjeev
1
Ahmad Aamir
1
Li Yiwei
1
Azmi Asfar S.
1
Gunn Jason R.
5
6
Kong Dejuan
1
Bao Bin
1
Ali Shadan
2
Gao Jiankun
2
3
Mohammad Ramzi M.
2
Miele Lucio
4
Korc Murray
5
6
Sarkar Fazlul H.
1
*
1
Department of Pathology, Karmanos Cancer Institute, School of Medicine, Wayne State University, Detroit, Michigan, United States of America
2
Division of Hematology and Oncology, Department of Internal Medicine, School of Medicine, Wayne State University, Detroit, Michigan, United States of America
3
Sichuan College of Tranditional Chinese Medicine, Mianyang, Sichuan, People's Republic of China
4
University of Mississippi Cancer Institute, Jackson, Mississippi, United States of America
5
Department of Medicine and Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire, United States of America
6
Norris Cotton Comprehsive Cancer Center, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire, United States of America
Batra S. K. EditorUniversity of Nebraska Medical Center, United States of America* E-mail: [email protected] and designed the experiments: ZW SB A. Ahmad YL A. S. Azmi J. Gunn DK BB SA J. Gao RM LM MK FHS. Performed the experiments: ZW SB A. Ahmad YL A. S. Azmi J. Gunn DK BB SA J. Gao RM LM MK FHS. Analyzed the data: ZW SB A. Ahmad YL A. S. Azmi J. Gunn DK BB SA RM LM MK FHS. Contributed reagents/materials/analysis tools: ZW SB A. Ahmad YL A. S. Azmi J. Gunn DK BB SA RM LM MK FHS. Wrote the paper: ZW SB FHS.
2011 3 6 2011 6 6 e2053712 4 2011 2 5 2011 Wang et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the United States, suggesting that novel strategies for the prevention and treatment of PDAC are urgently needed. K-ras mutations are observed in >90% of pancreatic cancer, suggesting its role in the initiation and early developmental stages of PDAC. In order to gain mechanistic insight as to the role of mutated K-ras, several mouse models have been developed by targeting a conditionally mutated K-rasG12D for recapitulating PDAC. A significant co-operativity has been shown in tumor development and metastasis in a compound mouse model with activated K-ras and Ink4a/Arf deficiency. However, the molecular mechanism(s) by which K-ras and Ink4a/Arf deficiency contribute to PDAC has not been fully elucidated.
Methodology/Principal Findings
To assess the molecular mechanism(s) that are involved in the development of PDAC in the compound transgenic mice with activated K-ras and Ink4a/Arf deficiency, we used multiple methods, such as Real-time RT-PCR, western blotting assay, immunohistochemistry, MTT assay, invasion, EMSA and ELISA. We found that the deletion of Ink4a/Arf in K-rasG12D expressing mice leads to PDAC, which is in part mediated through the activation of Notch and NF-κB signaling pathways. Moreover, we found down-regulation of miR-200 family, which could also play important roles in tumor development and progression of PDAC in the compound transgenic mice.
Conclusions/Significance
Our results suggest that the activation of Notch and NF-κB together with the loss of miR-200 family is mechanistically linked with the development and progression of PDAC in the compound K-rasG12D and Ink4a/Arf deficient transgenic mice.
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Introduction
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignant disease, which is ranked as the fourth leading cause of cancer-related death with a median survival of 6 months, and with an estimated 43,140 newly diagnosed cases and an approximately 36,800 deaths in the United States in 2010 [1]. It has been accepted that the development of PDAC occurs through the progression of precursor lesions such as pancreatic intraepithelial neoplasia (PanIN), ranging from low-grade PanINs (PanIN-1A, PanIN-1B) to high-grade PanINs (PanIN-2, PanIN-3) [2], [3]. PDAC has been shown to have multi-step molecular progression including high frequency of activating K-ras mutations and subsequent inactivation of p16INK4a, p53, SMAD4, p14ARF tumor suppressors and other additional genetic abnormalities in mouse models [4]–[6] and in human [7]. The most common K-ras mutation in human PDAC is on codon 12 (KrasG12D), which is related to the activation of GTPase activity. Therefore, several mouse models of PDAC have been generated by targeting a conditionally mutated K-rasG12D to recapitulate the progression of PDAC [8]–[12]. One compound mouse model containing activated K-ras and Ink4a/Arf deficiency showed to cooperate in producing metastatic PDAC [13], [14]. However, the molecular mechanism(s) by which activated K-ras and Ink4a/Arf deficiency contribute to PDAC aggressiveness has not been fully elucidated.
In recent years, many signaling pathways including Notch pathway have been investigated and found to play important roles in PDAC [15]. Notch signaling has critical functions on the control of cell growth, differentiation, apoptosis, migration, invasion, and metastasis in PDAC [16]. Notch genes encode proteins which can be activated by interacting with a family of its ligands. To date, four Notch receptors (Notch1–4) and five ligands (Dll-1, Dll-3, Dll-4, Jagged-1, Jagged-2) have been identified [17]. Interestingly, it has been reported that the function of Notch signaling in tumorigenesis can be either oncogenic or oncosuppressive, and the function is also context dependent in PDAC [18], [19]. Notch signaling is frequently deregulated with up-regulated expression of Notch receptors and their ligands in PDAC [20]. We have shown that down-regulation of Notch-1 using specific siRNA was correlated with decreased proliferative rates, increased apoptosis, reduced migration, and decreased invasive properties of pancreatic cancer cells [21], [22]. Recently, it has been found that active Notch signaling can synergize with K-ras in PanIN initiation and progression to invasive adenocarcinoma [8], [23]. Inhibition of Notch signaling pathway resulted in the inhibition of tumor progression in a mouse model (K-ras, p53 L/+ mice) of PDAC [24]. More recently, Mazur et al. found that deficiency of Notch-2 stops PanIN progression, prolong survival through inhibition of Myc signaling in K-ras-driven pancreatic carcinogenesis [25]. Surprisingly, Notch-1 was recently found as a tumor suppressor in a model of K-ras-induced PDAC [18], suggesting that additional studies are required to determine the role of Notch signaling in PDAC.
Notch pathway has been reported to cross-talk with NF-κB, one of the major transcription factor associated with cell growth and apoptotic regulatory pathways in pancreatic cancer. Studies from our group revealed that Notch signaling could induce NF-κB activity in pancreatic cancer [21]. Recently, it was shown that NF-κB pathway is required for the development of tumors in a mouse model (K-ras, p53 L/L mice) of lung adenocarcinoma [26]. Moreover, it was found that genetic deletion of the NF-κB subunit p65 in a K-ras-induced lung cancer mouse model reduced lung tumorigenesis in the presence and in the absence of the tumor suppressor p53 [27]. However, it is largely uncertain whether NF-κB is necessary for K-ras-induced PDAC progression.
In the present study, we assessed the molecular alterations in mouse tumors developed in the compound transgenic mice with activated K-ras and Ink4a/Arf deficiency. Here, we show, for the first time, that deletion of Ink4a/Arf in K-ras expressing mice leads to PDAC, which is in part mediated through the activation of Notch and NF-κB signaling pathways. Moreover, we found alterations in the expression of miR-200 family, which could also play important roles in tumor development and progression of PDAC in the compound transgenic mice with activated K-ras and Ink4a/Arf deficiency.
Results
Notch signaling pathway is highly expressed in Pdx1-Cre;LSL-K-rasG12D;Ink4a/Arf mouse
To delineate the mechanistic role of mutated K-ras in the development and progression of PDAC, we assessed the expression of Notch pathway in the murine model. In this model, oncogenic K-ras (KrasG12D) is knocked-in into its own locus and transcriptionally silenced due to the insertion of a LoxP-Stop-LoxP element (LSL). When LSL- KrasG12D mice are bred with transgenic mice which express Cre recombinase under the control of the Pdx1 promoter, expression of Cre recombinase in pancreatic progenitor cells allows the removal of the floxed transcriptional STOP cassette, leading to the activation of the oncogenic K-ras allele. In this model, there was no tumors readily found up to 30 weeks of age in LSL- K-rasG12D; Pdx1-Cre (we called KC in this manuscript) mice, consistent with previous study [13]. We also found no evidence of PDAC in the Ink4a/Arf; Pdx1-Cre (we called IC in this manuscript) animals up to an age of 24 weeks, similar to the observation documented by other groups [13]. However, all 25 mice from LSL- K-rasG12D; Pdx1-Cre; Ink4a/Arf (we called KCI for this manuscript) group were found to have pancreatic tumors ranging in diameter from 4 to 10 mm between 45 to 80 days (Fig. 1A). The compound KCI mice with tumors became moribund (Fig. 1A, survival curve). The tumors were confirmed by histopathologic examination (Fig. 1B). The Ki-67, a known proliferation marker, was highly expressed in KCI pancreatic tumors (Fig. 1B). It has been reported that Notch signaling pathway has critical roles in the development and progression of pancreatic cancer. Therefore, we assessed the expression of Notch genes in these transgenic mice tissues. It is important to note that we focused our studies on the cleaved Notch because it is the active functional form of Notch. Therefore, Notch in our all figure legends means active cleaved Notch. We found that Notch signaling was activated in the tumors of KCI mice when compared with the pancreata of KC and IC mice, respectively (Fig. 1C, D). The expression of Notch-2 and Notch-4 was up-regulated both at the mRNA and protein levels in KCI mice. However, Notch-1 expression showed no change and Notch-3 expression was increased only at the mRNA level in the tumors of KCI mice, suggesting that roles of Notch-2, and Notch-4 could be more important in progression of pancreatic cancer. We also found that all five Notch ligands were up-regulated in the tumors derived from the KCI mice (Fig. 2A, B). To confirm these results, we evaluated the expression of Notch downstream genes such as Hes-1 and Hey-1. We found that the expression of Hes-1 and Hey-1 was increased in the tumors of KCI mice (Fig. 2A, B), which was expected based on up-regulated expression of Notch-2, Notch-4 and their ligands.
10.1371/journal.pone.0020537.g001Figure 1 Notch receptors are highly expressed in KCI mice.
A, Top left panel: Tumor incidence in KCI mice (N>25). Control mice: KC and IC mice. Top right panel: Average length of tumors in KCI mice (N>20). Bottom panel: Kaplan-Meier pancreatic tumor-free survival curve for KCI mice and control animals. Control mice: combinations of KC and IC mice. B, Top panel: Microscopic examination of tumors derived from the KCI mice composed of cells which are forming ducts at places (red arrows). Focally tumor cells are in sheets. Cells are large, highly atypical, with large, pleomorphic nuclei and prominent 2–3 nucleoli (yellow arrows) per cell. Cytoplasm is eosinophilic with pale eosinophilic inclusions (green arrows) in few cells giving a rhabdoid feature to the cells. Surrounding stroma shows spindled cells with elongated nuclei (black arrows) and scattered inflammatory infiltrate comprising of neutrophils, lymphocytes and few plasma cells. Bottom panel: Ki-67 was highly expressed in tumors obtained from the KCI mice as assessed by immunohistochemistry. C, Notch signaling pathway was up-regulated at mRNA level as assessed by Real-time RT-PCR in tumors derived from the KCI mice. D, Notch pathway was highly expressed in tumors derived from the KCI mice as assessed by western blotting analysis and immunohistochemistry, respectively.
10.1371/journal.pone.0020537.g002Figure 2 The expression of Notch ligands and NF-κB is upregulated in KCI mice.
A, The expression of Notch ligands and Notch downstream genes was increased at mRNA level as assessed by Real-time RT-PCR in tumors derived from the KCI mice. B, The expression of Notch ligands and Notch downstream genes was highly expressed in tumors derived from the KCI mice as assessed by western blotting analysis. C, NF-κB p65 activity was increased in tumors derived from the KCI mice by ELISA. D, Left panel, NF-κB p65 DNA-binding activity is increased in tumors derived from the KCI mice as assessed by EMSA. Right panel, phospho-p65 was highly expressed in tumors obtained from the KCI mice as assessed by immunohistochemistry.
NF-κB DNA binding activity was activated in KCI mice tissue
NF-κB has been reported to cross-talk with Notch pathway [28]. We have reported earlier that Notch-1 can up-regulate NF-κB DNA binding activity in pancreatic cancer [22]. Therefore, we investigated whether the downstream effect of Notch up-regulation could be mechanistically associated with the activation of NF-κB pathway in the tumors of these animals. It is well known that the NF-κB family is composed of homo- and heterodimers of Rel proteins; NF-κB1 (p50); NF-κB (p52), RelA (p65), RelB, and c-Rel (Rel). NF-κB (p50/p65) is a ubiquitous, constitutive and inducible heterodimer. In general, the DNA binding activity of NF-κB traditionally refers to the p50/p65 (p50/RelA) heterodimer-mediated binding to the DNA, and it is a known regulator of cell survival and anti-apoptosis signaling. In the nucleus, the p65 NF-κB subunit is a strong activator of a wide variety of genes; therefore, we assessed the nuclear expression of p65 protein by immunohistochemistry. Nuclear proteins from the tumors obtained from the transgenic mice were also subjected to NF-κB p65 ELISA, and NF-κB p65 DNA-binding activity as measured by EMSA. The results showed that the NF-κB p65 activity as assessed by ELISA, and the NF-κB p65 DNA binding activity was activated in tumors derived from the compound KCI mice (Fig. 2C, D). These results showed an increased nuclear accumulation of the p65 in the tumors of KCI mice, suggesting that both activation of K-ras and Ink4a/Arf deficiency are required for enhanced NF-κB activation. Moreover, immunostaining also showed that phospho-p65 was highly expressed in the nuclear compartment in the tumor tissues of KCI mice (Fig. 2D).
NF-κB downstream genes are activated in KCI mice
It has been reported that Notch pathway stimulates NF-κB activity in cervical cancer cells by associating with the IKK signalosome through IKKα [29]. Previous study has shown that Notch pathway regulates the IKKα expression in pancreatic cancer [30]. Thus, we investigated the expression of IKK protein in the tumors of KCI mice. We found that all IKK family members such as IKKα, IKKβ and IKKγ were activated in the tumors of KCI mice (Fig. 3A). To further explore the effects of NF-κB activation, we examined the expression levels of certain NF-κB target genes including COX-2, cyclin D1, MMP-9, MMP-2, Bcl-2, c-myc, and survivin by real-time RT-PCR and western blotting, respectively, using the tumor tissues obtained from the compound KCI transgenic animals. Real-time RT-PCR and western blot analysis showed that the expression of these genes was activated in the tumors of KCI mice (Fig. 3A, B). We also found that the expression of Stat3 was activated in the tumors form KCI mice (data not shown). It is well known that these genes play critical roles in cell growth, invasion and metastasis. Therefore, these results further support the role of NF-κB in tumor growth and progression in the compound KCI mice.
10.1371/journal.pone.0020537.g003Figure 3 The expression of Notch target genes is increased in KCI mice.
A, Western blot analysis showing the up-regulated expression of IKK, p65, and NF-κB downstream genes in tumors derived from KCI mice. B, Real-time RT-PCR showing increased expression of NF-κB downstream genes such as survivin, cyclin D1, Bcl-2, C-myc, MMP-2, and MMP-9 in the tumors derived from the KCI mice. C, The expression of miR-200 family was down-regulated in the tumors of the KCI mice as assessed by real-time RT-PCR. D, Real-time RT-PCR showing decreased expression of E-cadherin, and increased expression of vimentin, and a modest increase in the expression of ZEB1 whereas a 30-fold increased expression of ZEB2 in tumors derived from the KCI mice.
The miRNA-200 family was down-regulated in KCI mice
The miR-200 family have been found to regulate Notch signaling pathway [31]. The miR-200 family has five members: miR-200a, miR-200b, miR-200c, miR-141 and miR-429. We found that Notch-1 could be one of the target genes of miR-200 family (miR-200b, miR-200c) because over-expression of these miRNAs significantly inhibited Notch-1 expression in prostate cancer [31] and pancreatic cancer (unpublished data). To address whether miR-200 family is involved in the tumors of KCI mice which showed high expression of Notch (Notch-2 and 4) signaling pathway, we investigated the expression of miR-200 family. As expected, the expression of miR-200a, miR-200b and miR-200c was significantly decreased in the tumors of KCI mice (Fig. 3C). These results suggest that the tumors developed in the compound mice could show aggressive behavior such the acquisition of epithelial-to-mesenchymal transition (EMT) phenotype, and thus we have further investigated the molecular make-up of the tumors derived from the compound KCI transgenic mice as detailed below.
Evidence of EMT phenotype in the tumors derived from the compound KCI transgenic mice
Recently many studies have shown that the miR-200 family regulates EMT by targeting zinc-finger E-box binding homeobox 1 (ZEB1) and ZEB2. EMT is a process by which epithelial cells undergo remarkable morphological changes characterized by a transition from epithelial cobblestone phenotype to elongated fibroblastic phenotype. Our previous studies have shown that miR-200a, miR-200b, and miR-200c were down-regulated in gemcitabine-resistant pancreatic cancer cells, consistent with the observed EMT phenotype [32], [33]. Furthermore, we have shown that miR-200 family regulates the expression of ZEB1, slug, E-cadherin, and vimentin, and thus suggested the re-expression of miR-200 could be useful for the reversal of EMT phenotype to mesenchymal-to-epithelial transition (MET), which has been partly documented in our recent publication [34]. Since we found low expression of miR-200 family in the tumors of KCI mice, we assessed the EMT markers to investigate whether the tumors in the KCI mice underwent EMT or not. We found loss of E-cadherin expression and elevated expression of vimentin and ZEB2 in the tumors of KCI mice although the expression of ZEB1 showed modest increase (Fig. 3D), suggesting that the expression of these factors may be important to induce EMT phenotype in the tumors of the KCI mice, which appears to be consistent with the aggressive behavior of the tumors developed in the compound KCI transgenic mice.
Inhibition of Notch pathway caused reduced cancer cell growth in a mouse PDAC cell line
To further assess the potential role of Notch pathway in pancreatic cancer, we used Rink-1 cell line which was derived from the KCI pancreatic tissues and studied the effects of inhibitors of Notch pathway. Previous studies have shown that Rink-1 cells exhibited rapid growth in vitro and formed tumors in nude mice [14]. Since Notch signaling is activated via the activity of γ-secretase, several forms of γ -secretase inhibitors including DAPT and L-685,458 have been used to inactivate Notch pathway. Therefore, we determined the cell viability of Rink-1 cells treated with GSI by the MTT assay, and the data are presented in Figure 4A. The treatment of Rink-1 cells for 72 hours with DAPT, and L-685,458 resulted in cell growth inhibition. To determine which Notch receptor could be an effective therapeutic target for pancreatic cancer, the effect of Notch 1–4 siRNA on cell growth of the pancreatic cancer cells was examined. The efficacy of GSI and Notch siRNA for knockdown of Notch protein was confirmed through western blotting. We observed that Notch protein level was barely detectable in GSI treated or Notch siRNA transfected cells (Fig. 4B). Very interestingly, only inactivation of single Notch receptor did not significantly inhibit cell growth (Fig. 4A). These results suggest that inactivation of multiple Notch receptors by GSI are good way to treat PDAC. To confirm this conclusion, we tested the expression of Notch target genes in Rink-1 cells treated with GSI or Notch siRNA. Because only Notch-2 and Notch-4 siRNA slightly inhibited cell growth, we detected the expression of Notch target genes in Rink-1 cells treated with these two siRNAs. As we expected, we found that GSI inhibited the expression of Notch target genes including Hes-1, Survivin, Bcl-2, c-myc, uPA to more degree, compared to Notch-2 siRNA or Notch-4 siRNA transfection (Fig. 4C). Therefore, we used GSI in the following experiments. Next, we tested the effects of treatment on cell viability by clonogenic assay. GSI treatment resulted in a significant inhibition of colony formation of Rink-1 cells when compared with control (Fig. 5A). Overall, the results from clonogenic assay were consistent with the MTT data, suggesting that the inactivation of Notch pathway could inhibit cell growth of Rink-1 cells.
10.1371/journal.pone.0020537.g004Figure 4 Inhibition of Notch pathway by Notch siRNA or GSI inhibited Rink-1 cell growth.
A, Left panel, Inhibition of Rink-1 cell growth by Notch 1–4 siRNA tested by MTT assay. The results were plotted as means ± SD of three separate experiments having six determinations per experiment for each experimental condition. Middle and Right panel: L-685,458 and DAPT were γ-secretase inhibitors (GSI), which prevent the cleavage of the Notch receptor, blocking Notch signal transduction. GSI significantly inhibited Rink-1 cell growth. Cells were seeded in 96-well plates at 5,000 cells per well and treated with GSI for 72 hours. After treatment, cell densities were determined by MTT assay. Each value represents the mean ± SD (n = 6) of three independent experiments. *P<0.05, compared to the control. B, The expression of Notch pathway was down-regulated in Rink-1 cells treated with GSI or transfected with Notch 1–4 siRNA as assessed by western blotting analysis. C, The expression of Notch target genes was down-regulated in Rink-1 cells treated with GSI or transfected with Notch-2 siRNA or Notch-4 siRNA as assessed by as assessed by real-time RT-PCR.
10.1371/journal.pone.0020537.g005Figure 5 GSI induced apoptosis, inhibited migration and invasion in Rink-1 cells.
A, Top, Left panel: Cell survival of Rink-1 cells treated with GSI. Cells treated with GSI for 72 hours were evaluated by the clonogenic assay. Photomicrographic difference in colony formation in cells untreated and treated with GSI. Right panel: There was a significant reduction in the colony formation in Rink-1 cells treated with GSI compared with control cells. P values represent comparisons between cells treated with GSI and control using the paired t test. Bottom, Left panel: Characterization of apoptosis was carried out after propidium iodide (PI) and Annexin V-FITC staining with apoptosis detection kit followed by flow cytometric analysis after 48 h of GSI treatment of Rink-1 cells. The percentage of apoptotic cells increased from 10% in the control to 28–33% in GSI treated cells. Right panel: GSI induced apoptosis in Rink-1 cells. Rink-1 cells were exposed to GSI for 72 hours. Apoptosis was measured by Histone DNA ELISA. Values are reported as mean ± SD. *P<0.05, compared to the control. B, Top, Left panel, Invasion assay using GSI treated cells showing low penetration of cells through the Matrigel-coated membrane, compared with control cells. Right panel: The graphs showing the value of fluorescence from the invaded Rink-1 cells. The values indicate the comparative amount of invaded Rink-1 cells. Bottom, Wound healing assay was conducted to assess the capacity of cell migration. GSI treatment decreased the cell migration in Rink-1 cells. C, GSI inhibited the NF-κB DNA binding activity in Rink-1 cells as assessed by EMSA. D, Real-time RT-PCR and western blot analysis showed that L-685,458 inhibited the expression of Survivin, c-myc, Bcl-2, and uPA genes.
Inhibition of Notch pathway caused apoptotic cell death in Rink-1 cell line
Next, we investigated whether the overall growth inhibitory effects of GSI are in part due to induction of apoptosis, which was examined by using an ELISA-based assay. These results provided convincing data that GSI induced apoptosis in Rink-1 cell line (Fig. 5A). To confirm these results, we also used annexin V-FITC method to detect the apoptosis induced by GSI for which Rink-1 cells were treated with GSI for 48 hours. By staining cells with annexin V-FITC and PI, FACS analysis was used to distinguish and quantitatively determine the percentage of dead, viable and apoptotic cells after treatment. We found that the percentage of apoptotic cells increased from 10% in the control to 28–33% in Rink-1 cells after GSI treatment (Fig. 5A). These results provided convincing data showing that inactivation of Notch pathway could induce apoptosis in Rink-1 pancreatic cancer cells.
Inhibition of Notch pathway decreased cancer cell migration and invasion
Notch pathway is believed to be critically involved with the processes of tumor cell invasion and metastasis. Previous studies have shown that pancreatic tumors arising in the compound KCI mice have extensive invasion of adjacent organs, including the duodenum, stomach, liver, and spleen [13]. In order to better understand whether Notch pathway has a critical role in invasion, we tested the effects of inactivation of Notch pathway on cancer cell invasion. We found that GSI treated cells showed a lower level of penetration through the matrigel-coated membrane compared with the control cells. The value of fluorescence from the invaded Rink-1 cancer cells was decreased about 5–7 fold compared with that of control cells (Fig. 5B). In order to further examine the effect of GSI on cell migration and invasion, we conducted wound healing assay in Rink-1 cells. The results show that GSI treatment inhibited the capacity of wound healing in Rink-1 cells (Fig. 5B), suggesting that GSI can inhibit cell migration and invasion. These results suggest a direct role of Notch signaling in Rink-1 cancer cell migration and invasion, and these results are consistent with aggressiveness of tumors developed in the compound KCI transgenic animal.
Inhibition of Notch pathway decreased NF-κB DNA-binding activity
We investigated whether the downstream effect of Notch-1 down-regulation was mechanistically associated with the NF-κB pathway. Nuclear proteins from GSI treated cells were subjected to analysis for NF-κB p65 DNA-binding activity as measured by EMSA. The results showed that GSI significantly inhibited NF-κB p65 DNA-binding activity compared to control (Fig. 5C). These results provided evidence in support of a mechanistic crosstalk between Notch and NF-κB in pancreatic cancer. Furthermore, we also found that GSI inhibited NF-κB downstream gene expression, such as Survivin, Bcl-2, c-myc, and uPA (Fig. 5D).
Over-expression of miR-200b inhibited the cell growth through Jagged ligands
Recently, it has been reported that miR-200 family members target Notch pathway components, such as Jagged-1 [35], [36]. In order to examine whether miR-200 family regulate Notch pathway, we transfected miR-200b precursor into Rink-1 cells. We confirmed that the transfection of miR-200b precursor increased the relative level of miR-200b in Rink-1 cells (Fig. 6A). Over-expression of miR-200b decreased the relative mRNA levels of Jagged-1, Jagged-2 and their target genes by real time RT-PCR assay (Fig. 6A). The data from western blot analysis demonstrated that over-expression of miR-200b decreased the relative protein levels of Jagged-1 and its target gene such as Hes-1, Hey-1, and Bcl-2 (Fig. 6B). Moreover, we found that over-expression of miR-200b inhibited cell growth in Rink-1 cells (Fig. 6B). Next, we detected whether inhibition of Jagged-1 could inhibit cell growth. Jagged-1 siRNA significantly decreased the expression of Jagged-1 and its target Hes-1 and Hey-1 at mRNA and protein levels (Fig. 6C). Furthermore, we found that inhibition of Jagged-1 by Jagged-1 siRNA inhibited the Rink-1 cell growth (Fig. 6C), suggesting that Jagged-1 could be a potential target for pancreatic cancer.
10.1371/journal.pone.0020537.g006Figure 6 The miR-200b inhibited Rink-1 cell growth and Jagged-1 expression.
A, Left panel, Re-expression of miR-200b was established in Rink-1 cells by transfection with its precursor. Middle panel, Re-expression of miR-200b did not regulate the expression of Notch receptors in Rink-1 cells. Right panel, Re-expression of miR-200b regulated the expression of Jagged-1 and Jagged-2 mRNAs in Rink-1 cells. B, Left and middle panel, Re-expression of miR-200b inhibited the expression of Jagged-1 target genes at mRNA and protein levels in Rink-1 cells. Right panel, Re-expression of miR-200b inhibited Rink-1 cell growth test by MTT assay. C, Left and middle panel, Jagged-1 siRNA inhibited the expression of Jagged-1 target gene Hes-1 and Hey-1 at mRNA and protein levels in Rink-1 cells. Right panel, Jagged-1 siRNA inhibited Rink-1 cell growth test by MTT assay.
Discussion
PDAC is the fourth leading cause of cancer-related deaths in the United States [1]. Although some progress in chemotherapy, radiation therapy, and surgical technique, the overall survival rate for five years is less 4% of all patients diagnosed with PDAC [1]. These disappointing outcomes suggest that new and alternative approaches to the understanding the mechanisms of PDAC progression is critically needed. Transgenic mice are good models to identify the pathogenic role of specific gene mutations and core signaling pathways associated with pancreatic cancer.
It has been known that K-ras mutations are observed in 80%–90% of pancreatic cancer. Oncogenic K-ras is involved in the initiation or early stages in the development of PDAC. Therefore, the conditional KC mice are considered good tools for mechanistic studies of pancreatic cancer progression. Since KC mice mimic slow progression from PanIN to invasive cancer in around 12–15 months [6], [37], but the KC mice bred with many other transgenic mice showed rapid development the PDAC. For example, Smad4/Dpc4 haploinsufficiency shortened the life span of KC mice to median survival of approximately 8 months [10]. LSL- K-rasG12D; Pdx1-Cre; Trp53R mice have a dramatically shortened median survival of approximately 5 months [6]. The median survival time of KC mice with LKB1 heterozygosity was 4.5 months [11]. PTEN haploinsufficiency significantly shortened the life span of KC mice to a median survival of around 3.5 months [9]. The p21 heterozygosity made the KC mice with a median survival of 2.5 months [11]. One mouse model having activated K-ras and Ink4a/Arf deficiency had median survival of 2 months [13]. Therefore, for the present study, we used the compound KCI mice (activated K-ras and Ink4a/Arf deficiency) to investigate the mechanisms of pancreatic cancer progression.
Pancreatic cancer has been shown to have deregulated Notch signaling pathway. Although Notch pathway has been reported to have a tumor suppressive role in certain specific condition [18], the majority of studies show that the activated Notch pathway contributes to PDAC tumorigenesis [19], [25], [38]–[40]. The high level expression of Notch receptors, Notch ligands and Notch target genes have also been observed in human pancreatic cancer [19]–[21], [38]–[40]. Notch activity is required for TGF-α-induced acinar-to-ductal transition and prevention of Notch activation by GSI prevents acinor-to-ductal metaplasia in TGF-α-treated cells [41]. It has been reported that GSI inhibited tumor progression in LSL-K-rasG12D; Pdx1-Cre; Trp53R mouse model of PDAC [24]. Moreover, it has been found that Notch signaling was downstream of K-ras gene in pancreatic cancer [23], [42]–[44]. In this study, we used the compound KCI mice, which recapitulated most features of human pancreatic cancer to determine whether Notch signaling could be required for the development of PDAC. Indeed, we found over-expression of Notch signaling pathway in the tumors of KCI mice. The molecular explanation for the high expression of Notch in the tumors of KCI mice could be due to Ink4a/Arf deficiency. Furthermore, inhibition of Notch pathway by GSI in murine pancreatic cancer cell line Rink-1 inhibited cell growth, migration, and invasion, suggesting that Notch signaling pathway appears to be a viable therapeutic target for PDAC, which has been an active area of drug development.
The cross-talk between Notch and NF-κB in PDAC has been found in human cancer including pancreatic cancer [22], [28], [45]. It was found that Notch pathway stimulated NF-κB activity in cervical cancer cells by associating with the IKK signalosome through IKKα [29], [46]. We have reported that Notch pathway can regulate NF-κB activity in pancreatic cancer [21], [22]. In the present study, we found that NF-κB was activated in the tumors of KCI mice, suggesting that the downstream effect of Notch pathway up-regulation was mechanistically associated with the activation of NF-κB signaling pathway in the tumors developed in the compound KCI transgenic mice. Moreover, activated NF-κB-regulated genes which are involved in cell growth, apoptosis, migration, and invasion are also activated. Furthermore, GSI inhibited NF-κB activity and its downstream genes in Rink-1 cells. These results demonstrate the importance of NF-κB signaling and provide a basis to consider the pharmacological inhibition of the NF-κB for the treatment of PDAC, which has also been an active area of drug development.
In recent years, microRNAs (miRNAs) have been reported to participate in Notch pathway regulation in pancreatic cancer [47]. One important miRNA is miR-200 family, which is involved in the regulation of EMT, stem cells and the regulation of Notch pathway [35], [36]. The miR-200 family has five members: miR-200a, miR-200b, miR-200c, miR-141 and miR-429. Our previous study has shown that Notch pathway could be one of the target of miR-200b [31]. Consistent with this notion, we found loss of miR-200a, miR-200b, and miR-200c expression in the tumors of KCI mice, suggesting that activated Notch pathway could also be due to the loss of expression of miR-200 family. Over-expression of miR-200b decreased the expression of Jagged ligands and Notch target gene such as Hes-1, Hey-1, and Bcl-2, leading to cell growth inhibition. Moreover, we found that over-expression of miR-200b inhibited cell growth in Rink-1 cells (Fig. 6B). Most interestingly, recent studies have shown that the miR-200 family regulates EMT by targeting ZEB expression. We have reported earlier that miR-200a, miR-200b, and miR-200c are down-regulated in gemcitabine-resistant pancreatic cancer cells, which have high expression of Notch pathway and contributed to the acquisition of EMT phenotype [33], [34]. The acquisition of EMT has been documented to be involved with invasion and metastasis, and thus our data on the loss of miR-200 suggest that the EMT phenotypic tumors in our compound mice, and that the tumors in these animals are invasive and metastatic compared to pancreata with K-ras activation or Ink4a/Arf loss alone.
Based on our results, we conclude that one possible mechanism by which the tumors developed in the compound KCI transgenic mice with activated K-ras and Ink4a/Arf deficiency is in part due to the loss of miR-200 family, which leads to the activation of Jagged/Notch and NF-κB signaling pathway, resulting in the up-regulation of NF-κB target genes, such as MMP-9, c-myc, survivin, Bcl-2, cyclin D1, and COX-2 as summarized in the cartoon diagram (Fig. 7) and contributes to tumor aggressiveness. Although we have demonstrated the loss of miR-200, and the activation of Notch and NF-κB signaling pathway in the current animal model; however, there maybe other genetic alterations causing tumor aggressiveness in this compound mice with activated K-ras and Ink4a/Arf deficiency, suggesting that further in-depth studies are needed to investigate the precise molecular mechanism of tumor progression in this mouse model. Moreover, novel strategies for the re-expression of miR-200 and its consequence could be tested in this animal model, which would help in the rational drug design in addition to Notch and NF-κB targeted drugs for the treatment of human PDAC for improving the overall survival of patients diagnosed with this devastating disease.
10.1371/journal.pone.0020537.g007Figure 7 The schematic representation of our proposed molecular mechanism involved in the development and progression of tumors in the compound KCI transgenic mice.
Materials and Methods
Ethics Statement
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Any animal found unhealthy or sick were promptly euthanized. The protocol was approved by the Committee on the Ethics of Animal Experiments of Wayne State University institutional Users Animal Care Committee (Permit Number: A-10-03-08).
Mouse Model
The LSL-K-rasG12D strain was bred to the following strains: Pdx1-Cre, INK4a/Arf lox/lox as previously described [14], [48]. Pancreata were collected and processed for further analysis.
Genotyping
For genotyping, genomic DNA was extracted from tail cuttings using the REDExtract-N-Amp Tissue PCR kit (Sigma-Aldrich, St. Louis, Missouri). Three PCR reactions were carried out for each animal to investigate the presence of the oncogenic K-ras, p16 and Pdx1-Cre transgenes, respectively.
Cell lines
Rink-1 murine pancreatic tumor cell line was generated from the pancreatic tissue obtained from LSL- KrasG12D; Pdx1-Cre; Ink4a/Arf mice as previously described [13], [14].
Histopathology and immunohistochemistry
Histopathologic analysis of pancreata was carried out. The expression of Ki-67, Notch, and phospho-p65 was assessed in histological sections of tumors as described before [49].
Real-time reverse transcription-PCR analysis for gene expression studies
The total RNA from animal tissues was isolated by Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer's protocols. The primers used in the PCR reaction were described earlier [21], [22], [31], [50]. Real-time PCR amplifications were performed as described earlier [21].
Western blot analysis
The animal tissues were homogenized and sonicated in 62 mM Tris-HCl and 2% SDS. In another set of experiments, cytoplasmic and nuclear proteins were also extracted. The proteins were used for western blotting as described earlier [21].
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared from animal tissues and electrophoretic mobility shift assay was done by incubating 10 µg nuclear extract with IRDye-700-labeled NF-κB oligonucleotide as described earlier [22].
NF-κB p65 DNA-binding activity assay
Nuclear extracts (5 µg) was used to determine p65 DNA-binding activity using an enzyme-linked immunosorbent assay (ELISA)-based assay according to the manufacture's instructions (Active Motif TransAM).
TaqMan miRNA real-time reverse transcription–PCR
To determine the expression of miRNAs in transgenic mice tissues, we used TaqMan miRNA assay kit (Applied Biosystems) following manufacturer's protocol. Total RNA was extracted, and 5 ng from each sample were reverse transcribed as described earlier [32]. Real-time PCR reactions were then carried out in a total volume of 25 µL reaction mixture using Smart Cycler II (Cepheid) as described earlier [32].
Cell invasion assay
The invasive activity of the cells was tested using the BD BioCoat Tumor Invasion Assay System (BD Biosciences, Bedford, MA) as described earlier [22].
Wound healing assay
Wound healing assay was conducted to examine the capacity of cell migration. Briefly, the wound was generated in the cells with 90–95% confluent by scratching the surface of the plates with a pipette tip. The cells were then incubated in the absence and presence of GSI for 24 h, and then photographed with a Nikon microscope.
siRNA, miRNA and transfection experiments
Cells were transfected with 100 nmol/L of Notch-1, Notch-2, Notch-3, Notch-4, Jagged-1 siRNA or control siRNA (Santa Cruz) as well as 20 nmol/L of miR-200b (Ambion, Austin, TX) using DharmaFECT3 siRNA transfection reagent (DHARMACON, Lafayette, CO) as previously described [31].
Densitometric and statistical analysis
The statistical significance of differential findings between experimental groups and control groups was statistically evaluated using GraphPad StatMate software (GraphPad Software, Inc., San Diego, CA). P values lower than 0.05 were considered statistically significant.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by the National Cancer Institute, National Institutes of Health grants 5R01CA131151, 5R01CA131151-S02, and 5R01CA132794 (F.H. Sarkar) and CA-075059 to M. Korc. The authors thank Puschelberg and Guido foundations for their generous financial contribution. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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BMC Vet ResBMC Veterinary Research1746-6148BioMed Central 1746-6148-7-172152934910.1186/1746-6148-7-17Research ArticleDetection and differentiation of Borrelia burgdorferi sensu lato in ticks collected from sheep and cattle in China Niu Qingli [email protected] Guiquan [email protected] Jifei [email protected] Yuguang [email protected] Zongke [email protected] Youquan [email protected] Miling [email protected] Zhijie [email protected] Junlong [email protected] Aihong [email protected] Qiaoyun [email protected] Wayne [email protected] Jianxun [email protected] Hong [email protected] State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Key Laboratory of Grazing Animal Diseases MOA, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China2 Centre of Animal Science, QAAFI, University of Queensland 4072 Australia2011 29 4 2011 7 17 17 25 10 2010 29 4 2011 Copyright ©2011 Niu et al; licensee BioMed Central Ltd.2011Niu et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
Lyme disease caused by Borrelia burgdorferi sensu lato complex is an important endemic zoonosis whose distribution is closely related to the main ixodid tick vectors. In China, isolated cases of Lyme disease infection of humans have been reported in 29 provinces. Ticks, especially ixodid ticks are abundant and a wide arrange of Borrelia natural reservoirs are present. In this study, we developed a reverse line blot (RLB) to identify Borrelia spp. in ticks collected from sheep and cattle in 7 Provinces covering the main extensive livestock regions in China.
Results
Four species-specific RLB oligonucleotide probes were deduced from the spacer region between the 5S-23S rRNA gene, along with an oligonucleotide probe which was common to all. The species specific probes were shown to discriminate between four genomic groups of B. burgdorferi sensu lato i.e. B. burgdorferi sensu stricto, B. garinii, B. afzelii, and B. valaisiana, and to bind only to their respective target sequences, with no cross reaction to non target DNA. Furthermore, the RLB could detect between 0.1 pg and 1 pg of Borrelia DNA.
A total of 723 tick samples (Haemaphysalis, Boophilus, Rhipicephalus and Dermacentor) from sheep and cattle were examined with RLB, and a subset of 667 corresponding samples were examined with PCR as a comparison. The overall infection rate detected with RLB was higher than that of the PCR test.
The infection rate of B. burgdoreri sensu stricto was 40% in south areas; while the B. garinii infection rate was 40% in north areas. The highest detection rates of B. afzelii and B. valaisiana were 28% and 22%, respectively. Mixed infections were also found in 7% of the ticks analyzed, mainly in the North. The proportion of B. garinii genotype in ticks was overall highest at 34% in the whole investigation area.
Conclusion
In this study, the RLB assay was used to detect B. burgdorferi sensu lato in ticks collected from sheep and cattle in China. The results showed that B. burdorferi senso stricto and B. afzelii were mainly distributed in the South; while B. garinii and B. valaisiana were dominant in the North. Borrelia spirochaetes were detected in Rhipicephalus spp for the first time. It is suggested that the Rhipicephalus spps might play a role in transmitting Borrelia spirochaetes.
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Background
Lyme borreliosis has been recognized as the one of most common vector-borne diseases in the world. The disease was first reported in the USA by Steere in 1975. Subsequently its clinical manifestations were described by a scholar in Yale University in 1977, and named Lyme disease in 1980 [1]. Burgdorfer and his colleagues isolated a spirochete and confirmed it as the causative agent of Lyme disease [2]. The spirochaete was formally named Borrelia burgdorferi in 1984 [3].
Lyme disease is distributed in over 30 countries and regions of Asia, Europe, America, Africa and Oceania, with more than 0.3 million clinical cases per year [4]. In the USA, it is particularly severe with approximately 20-100 cases per 100,000 [5]. The number of cases is increasing, and the disease was listed as a key target for prevention and control by WHO in 1992 [6].
In China, the disease was first identified in the forest regions of Hailin county in Heilongjiang province in 1986 [7]. Liang and Zhang latter confirmed that three B. burgdorferi isolates from the region caused Lyme disease [8]. To date, serological investigations have confirmed that Lyme disease is present in more than 29 provinces and autonomous regions, and more than 130 isolates of B. burgdorferi have been recovered from patients, ticks or animals in 19 provinces and autonomous regions [9,10], especially in the northeast forest areas. The prevalence of Lyme disease correlates with the geographic distribution and activity of vector ticks [11]. Of the 109 species of ticks identified in China, Lyme disease pathogens have been isolated from 9 Ixodid ticks, including Ixodes persulcatus, I. granulatus, I. acutitarsus, H. longicornis, H. bispinosa, H. concinna, H. formosensis, Boophilus microplus and Dermacentor silvarum[12].
The B. burgdorferi sensu lato complex comprises at least 13 species, B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. lusitaniae, B. valaisiana, B. bissettii, B. andersonii, B. japonica, B. tanukii, B. turdi, B. sinica, B. spielmanii and B. californiensis. The following five species of B. burgdorferi have been isolated from ticks: B. burgdorferi sensu stricto (I. scapularis, I. dammini, I. ricinus and I. pacificus), B. garinii (I. ricinus and I. persulcatus), B. afzelii (I. ricinus and I. persulcatus), B. lusitaniae (I. ricinus) and B. valaisiana (I. ricinus and I. columnae) [13-17].
In this study, we developed a reverse line blot (RLB) to detect and differentiate 4 B. burgdorferi sensu lato species on the basis of the variable spacer region regions between 5S and 23S rRNA genes sequences. We then used the RLB to investigate the distribution and prevalence of B. burgdorferi sensu lato in China.
Results
Specificity of the RLB
Amplification of the 5S-23S rRNA gene internal transcribed spacer was performed by nested PCR on four isolates (Figure 1.). The primers specifically amplified the spacer region between 5S-23SrRNA genes sequence from Borrelia and no cross-reaction occurred with DNAs from M. pneumoniae, C. psittasi, Anaplasma. marginale and A. ovis. All probes bound to their respective target sequence only; no cross reaction was observed at dilutions used, resulting in the clear recognition of individual strains, species, or groups. Each of the 4 B. burgdorferi species was identified by one of the oligonucleotide probes (Table 1). Isolate BO23 was identified by two oligonucleotide probes: Sl (453-430), and a specific probe recognizing Af (305-278), isolate B31 was identified by two oligonucleotides probes: the Sl (453-430), and a specific probe recognizing Ss (322-299). Isolates SZ, T25, PBr, 20047, IP90 and TN were recognized by two oligonucleotides as well: Sl (453-430), and a specific probe recognizing Ga (322-298). Isolate VS116 was identified by two oligonucleotide probes: Sl (453-430), and a specific probe recognizing Vs (303-278), No signal was detected from genomic DNA of M. pneumoniae, C. psittasi, A. marginale and A. ovis, or water (Figure 2).
Figure 1 The second PCR amplification of the 5S-23S intergenic spacer region. Lanes 1-14:BO23: Borrelia. afzelii; B31: Borrelia.burgdorferi sensu stricto; SZ, T25, PBr, 20047, IP90, TN: Borrelia. garinii;VS116: Borrelia. valaisiana; Mycoplasma ovipneumoniane,, Chlamydia psittacii, Anaplasma marginale, Anaplasma ovis and pathogen-free tick DNA as negative controls.
Table 1 Species and their recognition pattern by oligonuclotide probes
Isolate designation Species Oligonucleotide*
Species-specific Group-specific
BO23 B. afzelii Af
B31 B. burgdorferi s.s. Ss
T25 B. garinii
PBr B. garinii
SZ B. garinii Ga Sl
20047 B. garinii
IP90 B. garinii
TN B. garinii
VS116 B. valaisiana Vs
*See Table 3 for oligonucleotide sequence data.
Figure 2 Reverse line blot (RLB) assay specificity test for Borrelia burgdorferi. Sl: Probe of Borrelia burgdorferi sensu lato; Ga: Probe of Borrelia.garinii; Ss: Probe of Borrelia.burgdorferi sensu stricto; Af: Probe of Borrelia. afzelii;Vs: Probe of Borrelia. valaisiana; row 1-14: BO23: Borrelia. afzelii; B31: Borrelia.burgdorferi sensu stricto; SZ, T25, PBr, 20047, IP90, TN: Borrelia. garinii;VS116: Borrelia. valaisiana; Mycoplasma ovipneumoniane, Chlamydia psittacii infections, Anaplasma marginale, Anaplasma ovis and pathogen-free tick DNA as negative control.
Comparison of sensitivity of nested PCR and RLB
Sensitivity of the RLB was assessed using 10 fold diluted genomic DNA (10-1~10-12) of B. burgdorferi sensu stricto, B. garinii, B. afzelii, and B. valaisiana. The RLB system was capable of detecting 1pg of B. burgdorferi sensu stricto, B. afzelii, DNA and 0.1pg of B. garinii, B. valaisiana DNA. The detection level of the nested PCR carried out in parallel was also restricted to about 1pg~0.1pg (Figure 3). Unlike the nested PCR, the RLB was able to identify which species B. burgdorferi sensu stricto, B. garinii or B. afzelii, the tick was infected with.
Figure 3 The sensitivity of the RLB assay for B. burgdorferi sensu lato. Plate 1 shows the sensitivity of the nested-PCR using Ba and Bb probes; Plate 2 shows the sensitivity of RLB using Ba and Bb probes; Plate 3 shows the sensitivity of the nest-PCR using Bg and Bv probes; Plate 4 shows the sensitivity of the RLB of using Bg and Bv probes. Lane1-12: 100-11 10 × fold dilution DNA of Genome for B.burgdorferi sensu lato Row 1-6: oligonucleotide probe of B. burgdorferi sensu lato (Sl); oligonucleotide probe of B. burgdorferi sensustricto; (Ss); oligonucleotide probe of B. garinii( Ga); oligonucleotide probe of B. afzelii (Af); oligonucleotide probe of B. valaisiana (Vs); oligonucleotide probe of B. burgdorferi sensu lato (Sl).
Detection of B. burgdorferi sensu lato in ticks collected from sheep and cattle in China
The prevalence of each B. burgdorferi species was identified by RLB and PCR (Table 2). The number of samples in which B. burgdorferi sensu stricto, B. garinii, B. afzelii, or B. valaisiana alone were detected were 201 (28%), 245 (34%), 160 (22%), 85 (12%) respectively (Table 2), and 7% (50/723) were mixed infections and mainly in northern China. For the positive samples from southern China, it was found that the majority (40%) of them were infected with B. burgdorfei sensu stricto, while B. garinii was dominant (40%) in the positive samples from northern China. The results have revealed that most of the positive samples were infected by B. burgdorfei sensu stricto, B. garinii and/or B. afzelii. Sixty-nine samples (Shangzhi and Huichun), 15 samples (Lintan) and 1 sample (Huaihua) were detected from Dermacentor, Haemaphysalis and Boophilus, which belonged to B. valaisiana.
Table 2 Comparison of the results of examination of field tick samples by RLB and PCR
Origin Genra of tick No. of tick infected/examined
RLB PCR
BB BG BA BV Mixa
Huaihua Boophilus 62/146(42) 54/146(37) 33/146(23) 1/146(0.7) 44/146(30)
Nanping Haemaphysali 3/39(7.6) 16/39(41) 1/39(2.6) 1/39(2.6)
Huizhou Rhipicephalus 4/48(8.3) 7/48(14.6) 3/48(6.3) 14/45(31)
Laibin Boophilus 41/42(98) 3/42(7) 41/42(98) 15/39(38)
Total in south 110/275(40) 80/275(29) 78/275(28) 1/275(0.4) 74/269(27)
Shangzhi Dermacentor 44/154(29) 91/154(59) 44/154(29) 54/154(35) 33/154(21) 7/134(5)
Huichun Dermacentor 24/160(15) 34/160(21) 16/160(10) 15/160(9.4) 9/160(5.6) 3/130(2.3)
Total in Northeast 68/314(22) 125/314(40) 60/314(19) 69/314(22) 42/314(13) 10/264(4)
Lintan Haemaphysalis 23/134(17) 43/134(32) 22/134(16) 15/134(11) 8/134(6) 10/134(7)
Total 201/723(28) 248/723(34) 160/723(22) 85/723(12) 50/723(7) 94/667(14)
aBB: B. burgdorferi sensu stricto, BG: B. garinii, BA: B. afzelii, BV: B. valaisiana
Mix: a tick is infected by more than one species of B. burgdorferi.
Discussion
Lyme disease is a tick-borne disease caused by Borrelia. Animals such as cattle, sheep, horses, dogs and rats can be infected by the pathogen, and in most cases, play a role as reservoirs with varied clinical symptoms [18-20]. Lyme disease spirochetes have been isolated and detected from the following tick vectors: Ixodes persulcatus, I. crenulatus, Haemaphysalis longicornis, H. japonica, Boophilus microplus, Dermacentor silvarum and D. nuttalli [21]. The main mode of transmission is via the salivary gland when infected ticks feed on mammalian hosts. The infection rate of I. persulcatus, I. granulates and H. bispinosa were 40%-50%, 16%-40% and 24% respectively in north of China, suggesting that I. persulcatus is the principal vector of Lyme disease spirochete in northern China, while I. granulates and H. bispinosa are the main vectors in the south of China [22].
Although Lyme disease was found more than 20 years ago in China, knowledge of the epidemiology of the causative organism in the tick vectors is limited. We therefore considered that it would be very useful to develop a validated method to detect all four Lyme disease pathogens associated with ticks to better understand the epidemiology of the disease in different regions of China.
The RLB assay was here developed into a useful diagnostic tool to simultaneously detect and differentiate B. burgdorferi subspecies in ticks. Each species can be identified by a species-specific oligonucleotide probe using a line-blotter apparatus which is quickly becoming a standard molecular tool for diagnostic and epidemiological studies in an increasing number of laboratories all over the world. In this study, a RLB assay was established based on the spacer region between 5S and 23S rRNA genes of B. burgdorferi senso lato. B. burgdorferi sensu stricto, B garinii, B. afzelii, and B. valaisiana as decribed Barandika et al [23]. All four Borrelia species could be differentiated with 4 species-specific oligonucleotides while the oligonucleotide probe Sl designed specifically for the genera of B. burgdorferi hybridized with all 4 species. Any new species or genotype that may present would therefore be detected by the SI probe but not by the species-specific probes. Meanwhile, in order to confirm whether there was false positives, the sequence analysis of 120 samples was conducted for amplicons (PCR product) from these samples by random sampling. In all cases, it was found that the fragment was about 400 bp and 250bp, and the homology was 99.5% and 99.2% with the 5S-23S rRNA intergenic spacer region gene of B. burgdorferi by DNAstar analysis, suggesting that the ticks were infected by B. burgdorferi sensu lato. The RLB was a reliable diagnostic tool and when PCR amplification steps were included, the specificity was further improved. Different strains can be detected concurrently, and coinfection of different strains of B. burgdorferi sensu lato could be distinguished.
We have shown here that the infection rate of Borrelia spirochaetes in ticks varies considerably between different geographical regions. B. burgdorferi sensu stricto is found more frequently in the South (Laibin and Huaihua) and B. garinii more frequently in the Northeast (Shangzhi and Huichun) and Northwest (Lintan), this is not accordance with the recent reports [24]. Our results do not agree with previous findings which suggest that B. afzelii is the dominant species in northeast China. However, in the present study, the B. afzelii infection rate of samples from the north was lower than those samples infected with B. garinii and B. burgdorferi sensu stricto. Although the infection rate of ticks with B. afzelii was 19%, which was much higher than that previously reported [25], the low infection rate of B. afzelii might be associated with the limited number of ticks collected from field. Another explanation could be that the tick infection rate of each of the four Borrelia varies among samples from different regions, and it is possible that samples in this study were collected from areas where the tick infection rate of B. afzelii is lower.
To the best of our knowledge, this is the first study in which Lyme disease species of B. burgdorferi sensu lato were detected by RLB in field ticks in China. The prevalence and distribution of Borrelia spirochaetes in ticks are key factors for risk assessment of Lyme borreliosis. From our results, it could be seen that B. garinii is the dominant species and widely distributed in China, which agrees with the previous data [26-28]. B. afzelii and B. burgdorferi sensu stricto have been found only in a limited number of ticks, however, the tick infection rate with B. burgdorferi sensu stricto was higher than that of B. afzelii, which is different from previously reported.
B. valaisiana has only been isolated from rats from Guizhou Province in China and it has been assumed that distribution of B. valaisiana is confined to the southwest of China [29]. In this study B. valaisiana was detected in relatively high levels in the samples from northeast China (Table 2). Masuzawa et al [30] have confirmed that B. valaisiana is present in Korea which is geographically very close to northeast China. The prevalence of B. valaisiana varied among our study sites. It could be inferred that the distribution of B. valaisiana is much wider than originally thought. Previous reports suggested that B. valaisiana was more difficult to isolate from clinically diagnosed cases of Lyme Borreliosis than genomospecies B. burgdorferi sensu stricto, B. afzelii and B. garinii [14,31]. As most of the current prevalence data on Lyme disease are from human medical institutions and identification of Borrelia is based on the isolation, it is reasonable to believe that in northeast China, B. valaisiana is more prevalent but may be misdiagnosed due to the difficulty of isolation of the pathogen.
We collected samples of four genera of ticks from sheep and cattle, which were Haemaphysalis (n = 173), Boophilus (n = 188), Rhipicephalus (n = 48) and Dermacentor (n = 314) from seven regions, where the primary forests are preserved. The infection rates of the four genera of ticks with the four species of Borrelia burgdorferi l were 15%, 34%, 13% and 0.8% (Haemaphysalis), 54%, 28%, 39% and 0.05% (Boophilus), 0.8%, 1.4%, 0.6% and 0% (Rhipicephalus), and 21%, 39%, 19% and 22% (Dermacentor); while the infection rates detected by PCR were 6% (Haemaphysalis), 32% (Boophilus), 31% (Rhipicephalus) and 4% (Dermacentor) [31]. Recently, Richter and Matuschka (2010) have found that although about a quarter of I. ricinus ticks questing on the pasture were infected by spirochetes, the positive rate of the ticks collected from goats and cattle was much lower, and no molted ticks that had previously engorged or repletion on ruminant harbored Lyme disease spirochetes [32]. The authors also concluded that the spirochetes are cleared from I. ricinus ticks during feeding on ruminants. To confirm this hypothesis, more engorged ticks of these four species should be collected to test the status of spirochete infection of their offspring. In the present study, the results indicated that the positive detection rates of the RLB technique were higher than that by PCR. We further confirmed the sensitivity of the RLB, and the detection results of the infection state of three genera of ticks are also consistent with two methods on the whole, except for the results with Rhipicephalus. It has been still unknown yet why the positive rate by PCR was higher than by RLB with the Rhipicephalus samples. Our data suggests that the infection rate of different genera of ticks is also variable. However, the differences in infection rates among tick species were not statistically significant, and these differences could be due to geographical and seasonal variations of tick and host populations rather than tick vector competence. Because the ticks were collected from animals which could have been already infected with Borrelia burgdorferi, we can therefore only say that these ticks are potential vectors. The data showed the infection rate of Boophilus with Borrelia was relatively high and this species might play a role in transmission of Lyme diseases [21,33]. As Boophilus does not readily attach to humans, it could transmit the pathogen between cattle and perhaps sheep, and these infected animals would become reservoirs for other tick species which will attach to humans. These results are consistent with previous studies and confirm that Boophilus plays an important role in Lyme disease transmission amongst livestock. It has been reported that Haemaphysalis and Dermacentor are the vectors of Lyme disease [27,34] yet Rhipicephalus spp. have not previously been confirmed as vectors. In this study, we detected Borrelia burgdorferi in Rhipicephalus spp suggesting that Rhipicephalus spp. might transmit Borrelia burgdorferi. In the present study, it was not possible to confirm that these four genera of ticks were capable of transmitting Borrelia sporochaetes, and the competence of the ticks as vector will need to be determined by further experimental study.
Conclusion
This study reports development of a RLB assay that is able to detect 4 species of B. burgdorferi sensu lato in field ticks simultaneously. The genomic DNA of B. burdorferi senso stricto and B. afzelii, B. garinii and B. valaisiana have been detected in samples prepared from 4 genera of ticks collected from seven different areas in China. These findings extend the knowledge of the epidemiology of Borrelia burgdorferi and its possible transmission by adult ticks in China. RLB is a powerful tool for such epidemiological studies and for further investigation of the association between tick vectors B. burgdorferi sensu lato species and clinical manifestations of Lyme disease. It can also be used for screening ticks and can easily be expanded to include additional Borrelia species. We also demonstrated that Rhipicephalus spps are the potential vectors of Borrelia burgdorferi. Several tick species may be involved in the transmission of Borrelia in China and the high infection rate suggests that there is a high risk of human infection. Little is known of the epidemiology, diagnosis and control of Lyme borreliosis in livestock, and more research is needed.
Methods
Borrelia strains and culture
Three B. burgdorferi sensu lato strains, B. burgdorferi sensu stricto (B31), B. afzelii (BO23) were purchased from American Type Culture Collection. B. garinii (SZ) was isolated from Dermacentor ticks as described by Niu et al. [35]. The spirochaetes were cultured in BSKII medium at 33°C as described previously [18]. DNA of standard Borrelia genotypes used in the study was provided by Dr. Fingerle (Nationales Referenzzentrum für Borrelien Max von Pettenkofer-Institut, LMU München) in Germany. The designations and origins of the strains are given in Table 1[34,13,36].
Control samples
DNA is from four species of bacteria, Mycoplasma ovipneumoniane, Chlamydia psittacii infections, Anaplasma marginale and Anaplasma ovis, were provided by colleagues in Lanzhou Veterinary Research Institute, CAAS and used as negative controls.
DNA from adult Haemaphysalis qinghaiensis was also used a control. The ticks were originally collected from sheep, goats and cattle in Lintan county, Gansu province. Prior to DNA extraction, the final generation of ticks was fed on splenectomised sheep to confirm absence of any tick borne pathogens. The examination of the sheep by microscopy and PCR proved that the sheep has not been infected by Borrelia. Furthermore, the same DNA was tested by both PCR and RLB, and the results were negative. Therefore, the remaining unfed adult ticks of H. qinghaiensis were designated a Borrelia-free strain of H. qinghaiensis.
Field samples
From March to August in 2009 (Figure 4), ticks (n = 723) were collected on cattle and sheep from Nanping in Fujian province, Huizhou in Guangdong province, Laibin in Guangxi province, Huaihua in Hunan province, Shangzhi in Heilongjiang province, Huichun in Jilin province and Lintan in Gansu province. Ticks were identified to genus using keys previously described by Deng and Jiang 1991 [37].
Figure 4 Map of China showing the number of ticks examined in each of the seven regions in this study.
Tick DNA extraction
Each tick was soaked in 70% ethanol in 15 min, dried, and ground in a separate 1.5 ml Eppendorf tube to avoid cross contamination. The sample was incubated with proteinase K for 2h at 56°C, and then boiled at 100°C for 10 min to inactivate proteinase K. After centrifugation, the supernatant was transferred to a fresh sterile microtube, and DNA was extracted using a Genomic DNA Purifcation Kit (Gentra, USA) according to the manufacturer's instructions.
Primers and probes
Two pairs of primers were designed from 5S-23S rRNA intergenic spacer region [38,39]. They are: 23SN1 (5'-ACCATAGACTCTTATTACTTTGAC-3' 469-446), 23SC (5'-TAAGCTGACTAATACTAATTACCC-3' 92-115) and 5SCB (5'-biotin-GAGAGTAGGTTATTGCCAGGG-3' 243-263), 23SN2 (5'-ACCATAGACTCTTATTACTTTGACCA-3' 469-444).
Species-specific RLB oligonucleotide probes were deduced from the hypervariable 5S-23S rRNA gene intergenic spacer region (Table 3). All the specific oligonucleotide probes contained a N-(trifluoroacetamidohexyl-cyanoethyl, N, N-diisopropyl phosphoramidite [TFA])-C6 amino linker and were diluted to 10-1,200 pmol/150 μl in 500 mM NaHCO3 (pH 8.4) to provide optimal sensitivity and specificity (summarized in Table 4).
Table 3 Sequence, concentration and position of specific oligonucleotide probes for B. burgdorferi sensu lato
Type of primers and isolate Oligonucleotide Sequence(5'-3') Concentration used
(pmol) Position on 5S-23S
intergenic spacer region
23SN1 ACCATAGACTCTTATTACTTTGAC 50 469-446
23SC TAAGCTGACTAATACTAATTACCC 50 92-115
5SCB biotin-GAGAGTAGGTTATTGCCAGGG 50 243-263
23SN2 ACCATAGACTCTTATTACTTTGACCA 50 469-444
B.burgdorfei sensu lato (S1) a-CTTTGACCATATTTTTATCTTCCA 800 453-430
B. burgdorferi sensu stricto (Ss) a-AACACCAATATTTAAAAAACATAA 20 322-299
B. garinii (Ga) a-AACATGAACATCTAAAAACATAAA 10 322-298
B. afzelii (Af) a-AACATTTAAAAAATAAATTCAAGG 200 305-278
B. valaisiana (Vs) a-CATTAAAAAAATATAAAAAATAAATTTAAGG 10 303-278
Table 4 Strain designation, species, and geographic origin of isolates used in this study
Strain Species Origin Geographic origin Reference
BO23 B. afzelii Skin Germany 13
B31 B. burgdorferi s.s. Ixodes dammini United States 13
SZ B. garinii Dermacentor China 35
T25 B. garinii Ixodes ricinus Germany 13
PBr B. garinii Human (CSF) Germany 13
20047 B. garinii I. ricinus France 36
IP90 B. garinii I. persulcatus Russia 36
TN B. garinii Ixodes ricinus Germany 13
VS116 B. valaisiana Ixodes ricinus Switzerland 17
Nested PCR amplification
All DNA samples of ticks and B. burgdorferi sensu lato isolates were amplified in duplicate by nested PCR. Genomic DNA of M. pneumoniae, Chlamydia psittacii, A. marginale and A. ovis, and water were used as controls. Primers, Taq DNA polymerase and buffers were obtained from TakaRa (Dalian, China). The first PCR was performed in a reaction volume of 50 μl. For each sample, the PCR mixture was prepared as follows: H2O 38.7 μl, 10 × reaction buffer (200 mM Tris-HCl (pH 8.55), 160 mM (NH4)2SO4 and 20 mM MgCl2) 5 μl, 10 mM dNTP 4 μl, 50 pmol each of primers 23 SN1 and 23 SC, Taq polymerase 1.8 U and 1 μl of DNA sample or water. PCR amplification was performed in an automatic DNA thermocycler (Eppendorf). The program for the first PCR consisted of an initial denaturation at 94°C for 3 min followed by the thermal cycle reaction program of 1 min at 94°C, 90 s at 50°C, and 90 s at 72°C for 40 cycles with a final extension step at 72°C for 5 min. Samples were held at 4°C until analysis. 1 μl of the product from the first PCRs was added to the second PCR tubes containing reaction mixture which were then were briefly vortexed, centrifuged and then transferred to a thermal cycler. After a denaturation step, (1 min at 94°C), 40 rounds of temperature cycling (94°C for 30 s, 55°C for 30 s, and 72°C for 1 min) were performed.
Analysis of amplified products and sequence analysis
All PCR products were separated electrophoretically in 1.5% agarose and visualized under UV light after ethidium bromide staining.
Nine independent positive clones for each B. burgdorferi sensu lato isolates were sequenced using the BigDye Terminator Mix (TaKaRa Company, China). The sequences of the first PCR was size of approximately 362-392 bp, and the second PCR product was 218-235 bp, respectively. The homology was 96.8-98.7% amongst the other isolates in GenBank by DNAstar analysis, confirming that they were from genomic DNA of Borrelia species.
Reverse line blot hybridization
Protocols for preparation of RLB membrane and hybridization were carried out as previously described [40] with the following modifications: Biodyne C membrane was cut into a 14.5 cm square and activated by 10 min incubation in 10 ml freshly prepared 16% (w/v) 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) in demineralized water, in a rolling bottle at room temperature. The membrane was then washed with agitation in demineralized water for 2 min and placed on a supporting cushion in a clean miniblotter system. Each probe was diluted in with water to give a range of concentrations (800 pmol, 400 pmol, 200 pmol, 100 pmol, 50 pmol, 20 pmol and 10 pmol), aliquoted into the miniblotter slot and incubated for 1 min. The solutions were aspirated and the membrane incubated in 100 mM NaOH for 10 min, and then washed at 42°C for 5 min in 2 × SSPE, 0.1% (SDS). Subsequently, the membrane was placed into the miniblotter, perpendicular to its previous orientation. The denatured PCR samples were aliquoted into the slots of the miniblotter for 10 min at 42°C, then aspirated and the membrane washed at 42°C for 10 min in 2 × SSPE, 0.1% SDS. Subsequently, the membrane was treated at 42°C for 45~60 min with peroxidase-labeled streptavidin diluted 1:4,000 in 2 × SSPE/0.1% SDS, washed twice at 42°C for 10 min, and washing twice at room temperature for 5 min in 2 × SSPE, 0.1% SDS. Finally detection of binding was by chemiluminescence performed according to manufacturer's recommendations (Santa Cruz Biotechology ).
Specificity and sensitivity of the RLB
The RLB was performed by 20 μl of 100ng amplified product of B. burgdorferi sensu stricto, B. garinii, B. afzelii, and B. valaisiana extracted. Meanwhile, Mycoplasma ovipneumoniane, Chlamydia psittacii, Anaplasma marginale, Anaplasma ovis and pathogen-free tick DNA as negative control were used as negative controls. The sensitivity of the RLB was tested by hybridizing 20 μl aliquots of 10-fold serially diluted PCR product. The genomic DNA of the four pathogens was serially diluted from 100ng/μl to 0.000001pg/μl by 10-fold dilution as templates for the PCR, and then hybridized by RLB.
Epidemiological study
A total of 723 ticks were examined by the established RLB assay and the positive and negative samples were recorded to assess the Borrelia spirochaetes infection rate in the four genera of ticks. Furthermore, 667 of these same samples were tested with the PCR.
Authors' contributions
QLN carried out the samples detection, RLB analysis and drafted the manuscript. HY, JXL participated in the design of the study. GQG, JFY, YGF, ZKX, YQL, MLM, JLL, AHL and QYR participated in sampling. WJ reviewed and commented on the manuscript. All authors read and approved the final manuscript.
Acknowledgements
This study was financially supported by the 973 Program (2010CB530206), NSFC(№30800820; №30972182, № 31072130, №31001061;), "948"(2010-S04), Key Project of Gansu Province (1002NKDA035 and 0801NKDA033), National Beef and YakIndustrial Technology System Programme, MOA, Specific Fund for Sino-Europe Cooperation, MOST, China, State Key Laboratory of Veterinary Etiological Biology Project (SKLVEB2008ZZKT019); The research was also facilitated by the following projects of the European Commission, Brussels, Belgium: EPIZONE (FOOD-CT-2006-016236), ASFRISK(№211691), ARBOZOONET (№211757) and PIROVAC (KBBE-3-245145).
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PLoS PathogPLoS PathogplosplospathPLoS Pathogens1553-73661553-7374Public Library of Science San Francisco, USA 21695245PPATHOGENS-D-10-0023310.1371/journal.ppat.1002062Research ArticleBiologyImmunologyBacteria-Induced Dscam Isoforms of the Crustacean, Pacifastacus leniusculus
PlDscam Isoforms Induced by BacteriaWatthanasurorot Apiruck
1
Jiravanichpaisal Pikul
1
2
Liu Haipeng
1
3
Söderhäll Irene
1
Söderhäll Kenneth
1
*
1
Department of Comparative Physiology, Uppsala University, Uppsala, Sweden
2
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani, Thailand
3
State Key Laboratory of Marine Environmental Science, College of Oceanography and Environmental Science, Xiamen University, Xiamen, Fujian, People's Republic of China
Schneider David S. EditorStanford University, United States of America* E-mail: [email protected] and designed the experiments: AW PJ IS KS. Performed the experiments: AW HL. Wrote the paper: AW PJ IS KS.
6 2011 9 6 2011 7 6 e100206228 10 2010 25 3 2011 Watthanasurorot et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.The Down syndrome cell adhesion molecule, also known as Dscam, is a member of the immunoglobulin super family. Dscam plays an essential function in neuronal wiring and appears to be involved in innate immune reactions in insects. The deduced amino acid sequence of Dscam in the crustacean Pacifastacus leniusculus (PlDscam), encodes 9(Ig)-4(FNIII)-(Ig)-2(FNIII)-TM and it has variable regions in the N-terminal half of Ig2 and Ig3 and the complete Ig7 and in the transmembrane domain. The cytoplasmic tail can generate multiple isoforms. PlDscam can generate more than 22,000 different unique isoforms. Bacteria and LPS injection enhanced the expression of PlDscam, but no response in expression occurred after a white spot syndrome virus (WSSV) infection or injection with peptidoglycans. Furthermore, PlDscam silencing did not have any effect on the replication of the WSSV. Bacterial specific isoforms of PlDscam were shown to have a specific binding property to each tested bacteria, E. coli or S. aureus. The bacteria specific isoforms of PlDscam were shown to be associated with bacterial clearance and phagocytosis in crayfish.
Author Summary
Invertebrate animals lack an adaptive immune system and have no antibodies. Vertebrate antibodies belong to the immunoglobulin super family of proteins, and one other member of this large family is the Down syndrome cell adhesion molecule or Dscam. Of specific interest is that Dscam proteins in invertebrates show a great diversity of isoforms, and its gene structure in Drosophila melanogaster and other insect species allow for more than 30,000 different isoforms. Dscam proteins are important for the interaction between neurons in insects, but recently a role for this hypervariable protein in immune defense has been shown. Here, we show that Dscam proteins with similar highly variable structures are present in a crustacean, the freshwater crayfish Pacifastacus leniusculus. We also found that specific isoforms could be induced in the animal after injection of different bacteria. The Dscam isoforms induced by Escherichia coli were found to cluster together in a phylogenetic analysis. Furthermore we produced recombinant proteins of the different isoforms that were induced by E. coli and Staphylococcus aureus and we could demonstrate that these proteins can bind specifically to their corresponding bacteria. The bacteria specific isoforms of Dscam were also shown to be associated with bacterial clearance and phagocytosis in crayfish. Our study therefore provides new insights into the function of invertebrate Dscams in immunity.
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Introduction
The immunoglobulin super family (IgSF) is composed of proteins that contain at least one immunoglobulin domain [1]. Several members of IgSF are expressed on the cell surface and there serve as receptors for diverse ligands, and contribute to a variety of cellular activities [2]. In vertebrates, many IgSF members play essential roles as immune molecules (also known as antibodies) by recognizing non-self entities and then promoting their elimination [3]. Although it appears as if all invertebrates lack true antibodies, diversified IgSF molecules have been shown to be involved in immune defense of several invertebrates [4], [5]. However, this does not imply that the diversification of IgSF in invertebrates have any relation to the antibody diversification in vertebrates [5]. Recently, one IgSF member, the Down syndrome cell adhesion molecule gene or Dscam, that can generate hypervariable isoforms through alternative splicing was shown to act as an opsonin and to enhance phagocytosis in insects [4], [6].
Dscam was first detected on the human chromosome 21q22, a region associated with Down Syndrome [7]. Then, orthologues of Dscam were identified in various species and the typical domain structure of the Dscam gene is highly conserved. The Dscam molecules are widely expressed in the nervous system and play an essential role in neural circuit formation [8], [9]. Moreover, transcripts of fly Dscam was detected in fat body cells and hemocytes, which both are important components of the insect immune system [4], [10]. The Dscam in hemocytes of Drosophila melanogaster and Anopheles gambiae can bind to Escherichia coli and potentially acts as both a phagocytic receptor and as an opsonin. In the mosquito, Dscam can generate pathogen-specific spliced forms upon immune challenge [6]. These findings provide some evidences that Dscam may have functions not only in neuronal wiring, but also in innate immunity in insects.
In the present study, the full-length cDNA and variable regions of P. leniusculus Dscam (PlDscam) were identified and characterized. We also present results showing that different isoforms of PlDscam can be induced by immune challenge, that they can bind bacteria and that they are important in bacterial clearance.
Results
Isolation and characterization of the PlDscam
A large open reading frame of PlDscam (6,009 bp) was identified that encodes a polypeptide of 2,002 aa (Figure 1A). The closest sequence matching that of PlDscam was Dscam of L. vannamei (identity = 85%). Domain homology analysis using SMART showed that the deduced amino acid sequence contains a signal peptide at amino acids 1–24, ten tandem repeated immunoglobulin domains (Ig), six fibronectin type III domains (FNIII) and a transmembrane domain (TM). The domain organization of PlDscam is 9(Ig)-4(FNIII)-(Ig)-2(FNIII)-TM (Figure 1B). It also has a conserved cell attachment sequence (Arg-Gly-Asp: RGD motif) between Ig6 and Ig7 (Figure 1A). The sequence in the 3′ UTR contains a polyadenylation signal (AATAA) and is located at 55 bp upstream of the poly A tail (Figure 1C).
10.1371/journal.ppat.1002062.g001Figure 1 Identification and characterization of Dscam in P. leniusculus.
A) Amino acid sequence of PlDscam; double underlining represents signal peptide, while the Immunoglobulin (Ig) and Fibronectin type III (FNIII) domains are indicated by light and dark gray shading, respectively. A conserved RGD motif between Ig6 and Ig7 is indicated by single underlining. In addition, the single transmembrane domain is shown in a box. B) Schematic diagram showing the domain organization of the PlDscam protein. C) 3′ UTR containing the polyadenylation signal (AATAA) is indicated by double underlining and a stop codon is bold and underlined.
Expression of PlDscam transcript diversity
To identify the variable regions of PlDscam, several regions of the PlDscam were amplified using different primer pairs and the location of each pair of primers is shown in Figure S1. Fifty clones of each of the six amplified regions were selected and sequenced, translated and aligned using ClustalW. Alternatively spliced mRNA segments of the PlDscam were detected in the N-terminal of Ig2 and Ig3, in the entire length of the Ig7 domain, in the transmembrane domain and in the cytoplasmic tail (Figure 2B–E and Figure S1A). In total 12, 29, 32 and 2 alternative spliced forms of the exons encoding Ig2, Ig3, Ig7 and the transmembrane domains, respectively were detected and therefore at least 22,000 different unique isoforms could in theory be generated (Figure 2A–2E). The other domains were highly conserved, especially Ig8-FNIII4. The cytoplasmic tail of PlDscam contains some highly conserved motifs similar to the corresponding area in Dscams of other species (Figure S1B).
10.1371/journal.ppat.1002062.g002Figure 2 High diversity regions of Dscam in freshwater crayfish.
A) Diagram showing the locations of putative alternatively spliced exons of PlDscam. The variable regions are indicated at the N-terminal parts of Ig2 (red) and Ig3 (pink) and complete Ig7 (blue) and transmembrane (green) domains in crayfish. The number of alternative exons is presented by bars under the diagram. B–E) Multiple amino acid sequence alignment of PlDscam variable regions of Ig2(B), Ig3(C), Ig7(D) and transmembrane domain (E) aligned by ClustalW, Dark shading indicates the boundary of each domain.
Tissue expression and phylogenetic analysis of PlDscam
In crayfish, PlDscam was abundantly expressed in the heart, moderately expressed in the testis, the hematopoietic tissue (HPT), brain and nerve, whereas low expression was detected in the stomach, gill, muscle and hemocytes. The PlDscam was not detected in hepatopancreas or in intestine (Figure 3A).
10.1371/journal.ppat.1002062.g003Figure 3 Tissue distribution and phylogenetic analysis.
A) The expression of PlDscam was studied in various tissues, such as HP = Hepatopancreas, ST = Stomach, IN = Intestine, G = Gill, TT = Testis, H = Heart, M = Muscle, B = Brain, HC = Hemocyte and N = abdominal nerve. A 40S ribosomal gene was used as an internal control. B) A phylogram based on multiple alignments between PlDscam and other Dscam proteins; Drosophila melanogaster (AAF71926), Apis mellifera (AAT96374), Homo sapiens (AF217525), Mus musculus (NP_112451), Danio rerio (AAT36313), Daphnia pulex (ACC65888), Xenopus tropicalis (NP_001136135), Gallus gallus (XP_416734), Bos taurus (XP_002693048), Canis familiaris (XP_546506) Rattus norvegicus (AAL57167), Macaca mulatta (XP_002803170), Callithrix jacchus (XP_002761477), Tribolium castaneum (NP_001107841), Nasonia vitripennis (XP_001599258), Acyrthosiphon pisum (XP_001949262), Equus caballus (XP_001491675), Aedes aegypti (EAT37388), Aplysia californica (ABS30432) and Litopenaeus vannamei (ACZ26466). A human junctional adhesion molecule A or JAMA was used as outgroup (NP_058642).
A phylogenetic analysis clearly separated the Dscam proteins of vertebrates and invertebrates in two different groups and PlDscam clustered with other members of invertebrate Dscams (Figure 3B).
LPS and bacteria injection induce PlDscam transcription
To investigate whether immune challenge induces higher expression of PlDscam in crayfish hemocytes, LPS, peptidoglycan (PG), E. coli, S. aureus or WSSV was used as immune elicitors. The results of quantitative RT-PCR revealed that the PlDscam mRNA expression was significantly induced by LPS-, E. coli- and S. aureus at 6 h to 24 h post injection compared to controls (Figure 4A, 4C, 4D). In contrast, the expression profile of PlDscam in PG- and WSSV- injected animals was not changed (Figure 4B and 4E).
10.1371/journal.ppat.1002062.g004Figure 4 Expression profiles of PlDscam in response to immune challenge.
PlDscam expression was significantly higher than the control from 6 h post-injection with LPS (A), E. coli (C) and S. aureus (D). In contrast, PG (B) and WSSV (E)-injection did not have any effect on the PlDscam expression level. The asterisk indicates that the expression levels are significantly different (*P<0.05, **P<0.01).
Clustering of PlDscam isoforms in hemocytes during a bacterial infection
In order to characterize the major isoforms of PlDscam in hemocytes of normal and bacteria injected crayfish, PlDscam cDNA fragments encompassing the signal peptide to Ig3 from each group were amplified and cloned (Figure 5A). A total of 50 clones of each cDNA fragment were sequenced. Ten isoforms were detected in all groups, i.e. normal, E. coli injected and S. aureus injected crayfish. These isoforms were named as Normal isoform (N) (Table S2 and Table S3). Furthermore ten abundant isoforms were found only in the E. coli or S. aureus injected group and were named E. coli induced isoform (E) or S. aureus induced isoform (S), respectively (Table S2 and Table S3). All isoforms were subjected to multiple sequence alignment using clustalW. The similarity of each PlDscam isoform was then clustered by the maximum likelihood (ML) and Bayesian inference (BI) methods. As shown in Figure 5B, the clustering tree contained two major branches. The E. coli induced isoforms were separated into one branch, whereas the other branch consisted of two sub-branches of normal isoform and S. aureus induced isoform (Figure 5B).
10.1371/journal.ppat.1002062.g005Figure 5 Bacteria-induced PlDscam isoforms.
A) Different isoforms of PlDscam were amplified from hemocyte cDNA of normal crayfish, at 12 h post injection with E. coli or S. aureus. The PCR products were detected on 1.2% agarose gel. The PCR-products from normal crayfish (N) are shown in lanes 1–3, while PCR-products from E. coli (E) and S. aureus (S) injected animals are shown in lanes 4–6 and 7–9 respectively. B) Clustering of all PlDscam isoforms is based on the similarity of their amino acid sequence. The gray shaded boxes represent the chosen isoforms. C) Multiple amino acid sequence alignment of the isoforms E2, N6 and S9 aligned by ClustalW. Dark shading indicates the boundary of each domain.
The E isoforms of PlDscam isoforms had a VNKEYIIRGDSA(F/I)LKCSIPSFVA(D/N) motif and a EIGSPATFTCRAQAHPVPQY motif present at the N-terminal of the Ig2 and Ig3 domains, respectively. As shown in Figure 5B and 5C the isoforms N6, E2 and S9 are different, and therefore we used these isoforms as representative alternative spliced forms of normal, E. coli- and S. aureus-induced isoforms for a bacterial binding assay.
Binding of rPlDscam to E. coli and S. aureus
Recombinant proteins covering the Ig1-Ig3 domains of the PlDscam isoforms of N6, E2 and S9 were produced and tested whether these domains have any putative function in binding to E. coli or S. aureus. These proteins were expressed in bacterial systems and the size of all soluble recombinant proteins was ∼67 kDa (Figure 6A). All recombinant proteins were fused with GST at the N-terminus and contained the Ig1-Ig3 domains. We used these rPlDscams in bacterial binding assays to reveal whether different isoforms are capable of direct binding to bacteria. The GST protein was used as a non-specific binding control in these experiments. The in vitro bacterial binding assays showed that all isoforms of PlDscam had different binding ability to the two tested bacteria. The rPlDscam of isoform E2 clearly bound to E. coli and had significantly higher binding than the N6 and S9 isoforms. In contrast, binding of rPlDscam S9 to S. aureus was significantly higher (P<0.05) than that of N6 and E2 (Figure 6B).
10.1371/journal.ppat.1002062.g006Figure 6 Bacteria-induced PlDscam isoforms have different binding ability to bacteria.
A) Different PlDscam isoforms were amplified from N6, E2 and S9 subclones and recombinant proteins were produced. The recombinant isoforms were subjected to 12% SDS and were confirmed by western blotting. Isoforms from non-injected animals in lanes 1 and 4; E. coli- induced isoform in lanes 2 and 5; and S. aureus-induced isoform in lanes 3 and 6. B) In vitro binding assays of rPlDscam isoforms of E. coli (left) and S. aureus (right), respectively. Binding of bacteria to different rPlDscam isoforms, consisting of N6 = isoforms from non-induced animal, E2 = E. coli induced isoforms, S9 = S. aureus induced isoforms or GST = control recombinant protein was determined using ELISA by measuring the absorbance of samples at 450 nm (OD450). C and D) The effect of bacteria induced isoforms on the clearance process in vivo was tested by pre-incubation of the bacteria E. coli (C) and S. aureus (D) with each recombinant isoform prior to injection into crayfish. The bacterial numbers in crayfish were determined as CFU/ml of crayfish in the hemolymph at 40 min (black) and 3 h (grey) after the bacterial challenge. This experiment was repeated three times and data represent means of these experiments. E) Phagocytosis and nodule formation of FITC-conjugated heat-killed bacteria by crayfish hemocytes. Black arrows show nodule formation under normal light microscopy. White arrows show the ingested FITC-conjugated heat-killed bacteria by crayfish hemocytes under UV light microscope. Red arrows show nodules that contain several ingested bacterial particles under UV light microscope. F) The percentage of crayfish hemocytes ingesting FITC-conjugated heat-killed E. coli (left) and S. aureus (right) that had been pre-incubated with each isoform of the recombinant proteins and the phagocytic hemocytes was determined at 1.5 h after challenge. Significant difference compared to control is marked by * = P<0.05 and ** = P<0.01.
The effect of specific bacteria induced isoforms on bacterial clearance and phagocytosis
Recombinant proteins of isoforms E2 and S9 that specifically interacted with E. coli and S. aureus, respectively, were found to interfere with bacterial clearance and phagocytosis in crayfish. Pre-incubation of E. coli with E2 followed by injection into the animals did increase the number of bacteria in circulation (Figure 6C). Similarly with S. aureus, the number of bacteria was the highest in the S9 isoform of rPlDscam pre-incubated group (Figure 6D). This result clearly indicates that the specific rPlDscam could interfere with bacterial binding to the hemocytes. These results were in agreement with the phagocytosis assay, since if E. coli and S. aureus were coated with the E2 and S9 isoforms respectively, this resulted in a significant decrease in the phagocytic activity (Figure 6F).
In vitro effect of PlDscam gene silencing on WSSV replication
The role of PlDscam during a WSSV infection was investigated using PlDscam RNAi to suppress the PlDscam expression. The PlDscam gene was completely knocked down in an HPTcell culture (Figure 7A) whereas the 40S ribosomal gene was unaffected. However, PlDscam silencing did not have any effect on WSSV replication as shown with no changes in transcription level of WSSV structural protein transcript VP28 between control and PlDscam silenced groups (Figure 7B and 7C). Moreover, this result also indicates that the expression of PlDscam was not affected by WSSV infection. This agrees with our previous experiment where we injected WSSV to live crayfish and the transcript level of Dscams was not affected (Figure 4E).
10.1371/journal.ppat.1002062.g007Figure 7 Effect of PlDscam silencing on viral replication of WSSV in vitro.
A) Silencing of PlDscam using dsRNA in cultured HPT cells was confirmed by RT-PCR before WSSV inoculation. B) WSSV VP28 expression in PlDscam silenced animals (lane 7–9) and the control (lane 4–6). UV-killed WSSV was served as control (lane 1–3), analyzed by semi-quantitative RT-PCR. C) Statistical analysis after comparing different band intensity of the results shown in (B). Significant differences are indicated by asterisks (P<0.05).
Discussion
The typical domain structure of Dscam with an extracellular domain, a single transmembrane domain and a C-terminal cytoplasmic tail, is highly conserved within arthropods and vertebrates [7], [11]. This domain architecture was also found in PlDscam. Diversity in Dscam is generated through alternative splicing and variable alternative exons were found in the N-terminal half of Ig2 and Ig3, in the entire Ig7 domain and in the complete transmembrane domain [12]. These four variable domains are highly conserved within arthropods, including the PlDscam. It is noticeable that the alternative splicing of these exons clearly contributes to separate Dscam of vertebrates from invertebrates in our phylogenetic analysis [11].
The Dscam was initially identified for its essential roles in neuronal wiring, so its transcript is present in high quantity in neuronal organs [13]. The PlDscam was also detected in the neural system of crayfish. However, recently a putative role of Dscam in host defense was shown in D. melanogaster and A. gambiae
[4], [6]. Both D. melanogaster and A. gambiae Dscams were required for host resistance and phagocytosis of bacteria [14]. Dscams of the mosquito respond to pathogen infection by generating specific isoforms and these pathogen-specific isoforms of Dscam can bind directly to pathogens [15]. The response of IgSF molecules to pathogens does not only generate specific isoforms, but also an increase in the number of these isoforms, such as is the case with the fibrinogen-related proteins (FREPs) in snails [5]. FREP production is enhanced following parasitic invasion, and these proteins can bind to parasitic invaders or their products. In crayfish challenge with both Gram-negative and Gram-positive bacteria induced higher transcription of PlDscam. A high transcription of PlDscam was obtained after LPS injection, whereas PG or viral injection of WSSV had no such effect. Most Gram-positive bacterial cell walls or cell membranes contain several components, including PG, lipoteichoic acid (LTA), and lipoproteins [16]. The high transcription of PlDscam achieved as a response to S. aureus injection but no response to PG may indicate that the PlDscam respond to other components of the S. aureus cell wall, such as LTA which has similar physiochemical properties to LPS from Gram-negative bacteria [17].
Previous results from D. melanogaster and L. vannamei showed that the hemocytes of immune challenged animals exhibited higher variability of the Ig2 and Ig3 domains but only a few Ig7 variants compared to normal animal [4], [18]. This is the reason why we studied bacteria specific induced isoforms and produced recombinant proteins covering only the Ig1-Ig3 region. Interestingly, PlDscam isoforms of E. coli injected crayfish mainly encoded “VNKEYIIRGDSA(F/I)LKCSIPSFVA(D/N)” and “EIGSPATFTCRAQAHPVPQY” motifs at the N-terminal part of the Ig2 and Ig3 domains, respectively. This implies that these two motifs might be important parts of the specific PlDscam isoforms in E. coli-infected crayfish. This is consistent with results from our bacterial binding assay, which showed that the recombinant proteins containing these two motifs (E2), could bind to E. coli better than the other isoforms (N6 and S9). In addition, binding of S. aureus-induced isoforms (S9) to S. aureus was also higher than E2 and N6. These results indicate that the pathogen induced isoforms of PlDscam have a specific binding property to each type of challenged bacteria. This specific interaction may be associated with some immune defense reaction as shown with mosquito Dscam [6]. To address this question, recombinant proteins (N6, E2 and S9) were pre-incubated with bacteria or FITC conjugated heat killed bacteria to study bacteria clearance and phagocytosis in vivo. When, the bacteria-induced PlDscam isoforms were coated on E. coli or S. aureus and then injected into live animals, this resulted in lowered clearing rates of bacteria in the hemolymph. This implies that the specific PlDscam fragments covered the binding sites of the bacteria so they could not bind to the membrane bound PlDscam on the hemocytes and hence the bacterial number increases since the clearance of bacteria by phagocytes is inhibited.
In the case of mosquito, AgDscam is not only a determinant of resistance to bacteria but also affects the resistance towards the malaria parasite Plasmodium
[6]. This implies that Dscam might be involved in other host pathogens reactions in crayfish. The Dscam belongs to a subfamily of the Immunoglobulin super family (IgSF). Indeed, many members of IgSF proteins have been reported to interact with and promote entry of numerous virus, including for example the junctional adhesion molecule A (JAM A), that could bind with and facilitate entry of reovirus [19], [20]. So, it is possible that PlDscam could bind to virus like the other members of IgSF and maybe facilitate entry of virus into crayfish hemocytes. WSSV is a virulent pathogen that causes death in many species of crustaceans such as crayfish [21] and therefore we tested a possible relationship between PlDscam and this important arthropod virus. We performed experiments to reveal whether WSSV challenge could increase transcription of PlDscam and whether PlDscam RNAi had any effect on WSSV replication. However, we could not detect any increase in PlDscam mRNA expression after WSSV infection, and more important, if the PlDscam gene was completely silenced this could not affect WSSV infection or replication.
Materials and Methods
Crayfish
Healthy intermolt freshwater crayfish (P. leniusculus) were obtained from Lake Hjälmaren, Sweden and maintained in aerated tap water at 10°C.
Cloning of full length PlDscam cDNA
Total RNA (at least 1 µg) was extracted from the heart and converted into cDNA using ThermoScript (Invitrogen). The degenerate primers were designed from the conserved region of insect Dscam including Drosophila, Apis, Aedes and Tribolium (DSCAM-e5 F: 5′-AARCAYMGIYTIACIGGIGARAC-3′; DSCAM-e7 R: 5′-GTI ARIACIGTYTCIACI SWYTC-3′). The resulting PCR product was purified and cloned into a TOPO vector (Invitrogen) and sequenced. A partial sequence was used for the further step. Gene specific primers for Rapid amplification of cDNA ends (RACE) technique (Table S1) were designed from the partial sequence and 5′ or 3′ RACE-PCR was performed with a SMART universal primer A mix (SMARTer RACE cDNA Amplification Kit user manual, Clontech). Thermal cycling was as follows: 25 cycles of 94°C 30 s, 68°C 30 s, and 72°C 3 min. The 5′ and 3′ RACE PCR products were cloned into TOP10 vector and sequenced.
PlDscam sequence analysis and phylogenetic analysis
The nucleotide sequence of PlDscam was compared to others in Genbank using BlastX. Multiple sequence alignment was done by ClustalW (http://www.ebi.ac.uk/Tools/clustalw/index.html). The deduced amino acid domain was predicted with SMART (http://smart.embl-heidelberg.de/). A phylogenetic tree representing the relationship between PlDscam and other proteins was analyzed by the maximum likelihood (ML) and Bayesian inference (BI) methods. A PhyML program (under the Whelan and Goldman (WAG) and gamma model with four categories) was used in ML analysis [22]. For the BI method, we used MrBayes program [23] with CAT model (3,000 cycles, first 1,000 cycles removed as burn-in, and the analysis was repeated three times with identical results). Internal blanch support values was from analysis of 1,000 ML bootstrap replicates. This evolutionary phylogram was based on the conserved region of Dscam from Ig8 to FNIII4, whereas phylogram for clustering of all PlDscam isoforms was based on the similarity of their amino acid sequence and were used with the same methods as described above.
Tissue distribution of PlDscam mRNA
RNA from various tissues, including hepatopancreas, stomach, intestine, heart, hematopoietic tissue (Hpt), muscle, brain, hemocytes and nerves, was extracted following the instruction of GenElute Mammalian Total RNA Miniprep kit (Sigma) followed by treatment with RNase-Free DNase I (Ambion, Austin, TX). Complementary DNA was synthesized using ThermoScript. PlDscam gene specific primers (GSP-PlDscam-F, 5′- TGGGAAGTGATGCCAGGTTAGA-3′; GSP-PlDscam-R, 5′-TTGAATCAGCAGACATAACCAAAGC-3′) were designed from full-length cDNA of PlDscam and its PCR product covered the conserved Dscam region (from Ig8 to FNIII3). A 40S ribosomal gene was used as internal control in all PCR experiments and its specific primers (40S-F, 5′-CCAGGACCCCCAAACTTCTTAG-3′; 40S-R, 5′-GAAAACTGCCACAGCCGTTG-3′) were designed from P. leniusculus Lamda Zap Express library Hpt cDNA (Genbank accession no. CF542417). PCR conditions were as follows: 94°C 2 min, followed by 30 cycles of 94°C 20 s, 58°C 20 s, and 72°C 1 min for the PlDscam gene and 25 cycles for 40S ribosomal gene. The PCR products were analyzed on 1.2% agarose gel stained with ethidium bromide.
Identification of alternatively expressed regions in PlDscam
Due to the large size of full length of PlDscam, it was necessary to use several pairs of primers for identification of the different variable regions. PCR of each region was performed with gene specific primers (Table S1) and thermal cycling was as follows: 94°C 2 min, followed by 30 cycles of 94°C 20 s, 60°C 20 s, and 72°C 1.30 min. The PCR products from four different tissues, such as heart, brain, hemocytes and HPT, were cloned into TOP10 vector and 25 individual clones from each tissue were sequenced.
Immune challenge, sample collection and PlDscam transcription analysis
E. coli and S. aureus were cultured in LB broth at 37°C until OD 600 was ca 0.5. The bacteria were washed three times with 0.85% NaCl by centrifugation at 900 g for 10 min at room temperature. The pellets were resuspended in sterile 0.85% NaCl and adjusted to an approximate concentration of 2×108 CFU/ml. Two groups of three crayfish were injected in the base of the forth walking leg with 100 µl of E. coli and S. aureus, respectively (approximately 2×107 CFU/crayfish). The control group was injected with 100 µl of 0.85% NaCl.
WSSV was purified with a method described by Xie et al.[24]. The purified virus was resuspended in sterile crayfish saline buffer (CFS: 0.2 M NaCl, 5.4 mM KCl, 10 µM CaCl2, and 10 mM MgCl2, 2 mM NaHCO3, pH 6.8) at a concentration of 2×107 copies/ml. One hundred microliter of WSSV (equivalent to 2×106 copies) or CFS (as control group) was injected as previously described. The experimental setup was made in triplicates.
Two groups of three crayfish received injections with 100 µl lipopolysaccharides (LPS: Sigma, from E. coli) or peptidoglycan (PG: Sigma, from S. aureus) in a sterile CFS with a concentration of 0.2 mg/ml. One hundred microliter of CFS was used for the controls.
Hemolymph of crayfish from all experiments was collected at 0, 6, 12 and 24 h post injection and the hemocytes were separately isolated for RNA extraction. The transcript levels of PlDscam were detected by quantitative RT-PCR using the QuantiTect SYBR green PCR kit (QIAGEN). The expression of PlDscam was normalized to the expression of the mRNA encoding the crayfish ribosomal protein gene (R40s) for each sample. The primers used are shown in Table S1. The qPCR reactions contained 5 µl of 1∶10 diluted cDNA template, 1× QuantiTect SYBR Green PCR master mix (QIAGEN) and 5 µM forward and reverse primers in a 25-µl reaction volume. The following amplification profile was used: 95°C for 15 min, followed by 45 cycles of 94°C for 15 s, 58°C for 30 s, and 72°C for 30 s. All qPCR reactions were performed in duplicate. The hemocytes from a least three crayfish were used for each time point.
Transcription analysis of PlDscam isoforms in response to E. coli and S. aureus
The variable region, from the signal peptide to the Ig3 domain region, was amplified from hemocyte cDNA templates from groups of crayfish injected with E. coli or S. aureus, respectively, and the control group at 12 h post injection with F1 and R2 primers (Table S1). The PCR products from each of these groups were subcloned into TOP10 vector and fifty colonies of each group were sampled and subsequently sequenced. Multiple sequence alignment was done by ClustalW and clustering tree of the different isoforms was constructed as described above.
Recombinant protein of the PlDscam isoforms
The ORFs without the signal peptide encoding different PlDscam isoforms were amplified from the original templates N4, E1 and S7 with Dscam-expression-BamHI-Forward (5′-TTTTGGATCCAACCCGACAACCGTGTGGACTTCA-3′) and Dscam-expression-XhoI-Reverse (5′- TTTCTCGAGCCAGGTAACAGACTTGACGGGGTTG-3′) primers. The resulting insert was cloned into pGEX-4T-1(GE healthcare) at BamHI and XhoI and transformed into BL21 E. coli. Single colonies were grown in LB medium containing 100 µg/ml ampicillin to OD600 = 0.6 and induced with 1 µM IPTG for 5 h at 37°C. The protein was expressed as a fusion product with a glutathione S-transferase part at the N-terminus of rPlDscam. After purifying this GST-fusion protein on a GST-trap FF column (GE healthcare), the presence of the recombinant protein was confirmed by western blot. The protein samples were subjected to 12% SDS-PAGE and then transferred electrophoretically to PDVF membranes. The membrane was blocked by immersion in 10% skimmed milk in TBST for 1 h and washed three times in 1 x TBST (10 mM Tris-HCl, pH 7.5, containing 150 mM NaCl and 0.1% Tween 20). The membrane was then incubated with 1: 2,000 dilution of a primary antibody for GST (Sigma) in TBST for 1 h. Then, the previous washing procedure was repeated before the membrane was incubated with anti-mouse-IgG peroxidase-linked species-specific whole antibody from sheep (GE Healthcare) at 1: 3,000 in 1xTBST for 1 h and washed with TBST for 3×10 min. For detection, the ECL Western blotting reagent kit (Amersham Biosciences) was used according to the manufacturer's instructions.
Bacterial binding assay
A bacterial binding assay was modified from the method described by Yu et al.[25] Briefly, E. coli or S. aureus was prepared as previously described. An aliquot 100 µl of the bacteria was immobilized on 96 well plates at approximately 108 CFU/well by incubating for 30 min at room temperature and then shifted to incubation at 4°C overnight. The bacteria were then blocked with 3% (w/v) BSA in TBS buffer at room temperature for 1 hour. To access the binding of the proteins to the bacteria, 20 µg of each PlDscam isoform (with GST fusion tag) or GST diluted in 1% BSA in TBS were added to the wells and incubated at room temperature for 2 h. Following three careful washes with TBS, the bound PlDscam protein was detected with the GST antibody (1∶2,000) followed by rabbit antimouse HRP-conjugated secondary antibody (1∶3000). After adding 100 µl tetramethylbenzidine or TMB substrate (Sigma) for 20 min and 100 µl of stop solution (0.5 M sulfuric acid), the absorbance of the resulting color was measured at 450 nm. All binding assays were performed in triplicates and experiment was repeated three times.
Bacteria clearance assay
E. coli and S. aureus were washed six times in 0.9% NaCl at 1,200× g for 10 min and then incubated with 10 µg recombinant proteins of each PlDscam isoforms for 1 h at 4°C, followed by washing six times with 0.9% NaCl. One hundred microliter of E. coli or S. aureus at concentrations of 6×108 and 3×109 CFU/ml, respectively, in CFS were injected into crayfish. The bacteria count was carried out in hemolymph collected 40 min and 3 h after the bacterial injection. The homonym was serial diluted serially and was then dotted onto LB agar (10 µl for each dot) and then incubated at 37°C overnight followed by counting the bacterial colony forming units (CFU).
Phagocytosis assay
Both, heat killed E. coli and S. aureus were conjugated with fluoresce in isothiocyanate (FITC) using a method previously described by Hed [26]. Briefly, heat-killed E. coli and S. aureus were washed six times in 0.9% NaCl at 1,200× g for 10 min and then incubated at concentration of 109 particles/ml in 0.1 M Na2CO3 containing 0.1 mg/ml FITC (Sigma), pH 9.5 for 30 min at 37°C, followed by washing six times with 0.9% NaCl as above. The FITC-conjugated heat-killed bacteria were incubated with 10 µg recombinant proteins of each PlDscam isoforms for 1 h at 4°C. The bacteria were washed five times as described above and resuspended at a concentration of 108 particles/ml in CFS. Two hundred microliter of FITC-conjugated heat-killed bacteria coated with recombinant PlDscam isoform (4×106 particles/ml in CFS) was injected into the animals via the base of the fourth walking leg. Hemocytes were bled for phagocytosis detection at 1.5 h post FITC-conjugated heat-killed bacteria injection. The fluorescence of FITC-conjugated heat-killed bacteria particles was quenched by adding 20 µl of 0.04% trypan blue (Pfaltz & Bauer, Waterbury, CT). The ingested bacteria were easily detected under the UV light microscope due to their fluorescence. The percentage of phagocytosing cells was determined by counting 10 individual fields and dividing the number of cells with ingested fluorescent bacteria with the total number of counted cells. Each experiment was performed in triplicates.
Crayfish Hpt cell culture and maintenance
The hematopoietic tissue was dissected according to Söderhäll et al.[27]. The Hpt was washed with CPBS (crayfish phosphate buffered saline: 10 mM Na2HPO4, 10 mM KH2PO4, 150 mM NaCl, 10 µM CaCl2, and 10 µM MnCl2, pH 6.8) and incubated in 600 µl of 0.1% collagenase (type I and IV) (Sigma) in CPBS at room temperature for 45 min to separate the Hpt cells. The separated cells were washed twice with CPBS by spinning down at 800× g for 5 min at room temperature. The cell pellet was re-suspended in modified L-15 medium and subsequently cells were seeded at a density of 2.5×106 cells/150 µl in 96-well plates. The Hpt cells were supplemented with partially purified plasma as a source of astakine [28] after 1 h of attachment at room temperature and the culture plates were incubated at 16°C, and 1/3 of the medium was changed at 48 h intervals.
Generation of dsRNA
dsRNA of PlDscam was designed from the conserved region during Ig8-FNIII3. Gene specific primers for PlDscam and GFP was incorporated with a T7 promoter sequence (italic letters) at 5′ ends (DscamRNAi-F, 5′- TAATACGACTCACTATAGGGGCATCAAGCTGAGTGGACAA-3′; DscamRNAi-R, 5′- TAATACGACTCACTATAGGG GAAGCCAGGTAGGGGAAATC -3′ and GFP 63+, TAATACGACTCACTATAGGG
CGACGTAAACGGCCACAAGT; GFP 719-, TAATACGACTCACTATAGGG
TTCTTGTACAGCTCGTCCATG) and used to amplify PCR products as template for dsRNA synthesis. A GFP transcript was amplified with the pd2EGFP-1 vector (Clontech) as template and used as control. The amplified products were then purified using GenElute Gel extraction kit (Sigma) followed by in vitro transcription using the MegaScript kit (Ambion). The dsRNA was purified with the Trizol LS reagent (Invitrogen).
RNAi in vitro study
The Hpt cells were divided into three groups with four replicates in each group. The Hpt cells received different treatments as follows: group 1: GFP dsRNA plus UV-killed WSSV, group 2: GFP dsRNA plus WSSV and group 3: PlDscam dsRNA plus WSSV. The dsRNA transfection and WSSV infection into Hpt cell cultures were performed as described by Liu et al.[29]. Briefly, 4 µl of dsRNA (250 ng/µl) was mixed with 3 µl of histone H2A (1 mg/ml) and with 20 µl of modified L15 and added to one well of 1-day-old Hpt cell cultures. The cells were then incubated at 16°C. At day 3, one replicate of group 2 and 3 were subjected to RNA extraction to determine RNAi efficiencies. For remaining replicates, the medium was replaced with 150 µl of L15 medium together with 5 µl of WSSV stock suspension and 5 µl crude astakine preparation and were further incubated for 36 h at 20°C followed by isolation of RNA. Total HPT RNA was extracted to determine PlDscam and WSSV VP28 transcripts by semi-quantitative RT-PCR. PCR was performed with three oligonucleotide primers (WSSV VP28 (Genbank accession no. AF502435):-F: 5′-TCACTCTTTCGGTCGTGTCG-3′ and -R: 5′- CCACACACAAAGGTGCCAAC-3′; previous primer for 40s ribosomal and PlDscam gene). The PCR conditions were as follows: 94°C 2 min, followed by 30 cycles of 94°C 20 s, 60°C 20 s, and 72°C 30 s for PlDscam and VP28, while 25 cycles for 40S ribosomal gene.
Statistic analysis
The relative expression levels of different time groups were examined by One-way ANOVA followed by Duncan's new multiple range test and Tukey test. Differences were considered statistically significant at P<0.05. Results are expressed as the mean ± SE.
Supporting Information
Figure S1 Variable regions in the cytoplasmic tail of PlDscam. A) Multiple alignment of amino acid sequence of seven different cytoplasmic tails of PlDscam. B) Amino acid sequence alignment of the cytoplasmic tail of Pacifastacus leniusculus (PlDscam), Drosophila melanogaster (DmDscam: AAF71926), Apis mellifera (AmDscam: AAT96374), Aedes aegypti (AaDscam: EAT37388), Tribolium castaneum (TcDscam: NP_001107841), Daphnia pulex (DpDscam: ACC65888). The squares highlight homologous regions and some conserved motifs are indicated by different colors, including SH3-binding motif (violet), endocytosis motif (yellow), SH2-binding motif (green), Polyproline motif (blue), PDZ motif (red) and ITIM motif (in a box).
(TIF)
Click here for additional data file.
Figure S2 Diagram showing the binding locations of primers that were used in this study. The locations of primers that were used for RACE-PCR (A), identification of alternatively expressed regions in PlDscam (B) and quantification of PlDscam expression (C).
(TIF)
Click here for additional data file.
Figure S3 Dscam is important for phagocytosis of crayfish hemocytes. Phagocytosis of FITC-conjugated heat killed E. coli or S. aureus particles were determined using normal or UV light microscopy. A) phagocytosis of FITC conjugated heat-killed E. coli after pre-incubation with different isoforms (N2, E6 or S9) of recombinant proteins of PlDscam. B) phagocytosis of FITC conjugated heat-killed S. aureus after pre-incubation with different isoforms (N2, E6 or S9) of recombinant proteins of PlDscam. The white, black and red arrows represent ingested FITC-conjugated heat-killed bacterial particles, nodule formation and nodules that contain several ingested bacterial particles, respectively.
(TIF)
Click here for additional data file.
Table S1 Primer pairs used in this study. All primer sequences are given in the format 5′-sequence-3′.
(DOC)
Click here for additional data file.
Table S2 Comparison of Dscam Ig2 and Ig3 combinations in normal, E. coli injected and S. aureus injected crayfish. Fifty individual cDNAs, expressed in each group, were sent to sequence.
(DOC)
Click here for additional data file.
Table S3 The chosen clones were used for construction of the clustering phylogram and the clones in bold letters were expressed as recombinant proteins.
(DOC)
Click here for additional data file.
The authors have declared that no competing interests exist.
This work has been done under financial support from the Swedish Science Research Council. (621-2009-5715). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Int J Mol SciijmsInternational Journal of Molecular Sciences1422-0067Molecular Diversity Preservation International (MDPI) 10.3390/ijms12031464ijms-12-01464ArticlepH Dependent Molecular Self-Assembly of Octaphosphonate Porphyrin of Nanoscale Dimensions: Nanosphere and Nanorod Aggregates Bhosale Sheshanath V. 1*Kalyankar Mohan B. 12Nalage Santosh V. 2Lalander Cecilia H. 1Bhosale Sidhanath V. 2*Langford Steven J. 1Oliver Ruth F. 11 School of Chemistry, Monash University, Wellington road, Clayton Victoria 3800, Australia; E-Mails: [email protected] (M.B.K.); [email protected] (C.H.L.); [email protected] (S.J.L.); [email protected] (R.F.O.)2 Department of Organic Chemistry, North Maharashtra University, Jalgaon 425 001, India; E-Mail: [email protected] (S.V.N.)* Authors to whom correspondence should be addressed; E-Mails: [email protected] (S.V.B.); [email protected] (S.V.B.); Tel.: +61-3-9905-5980; Fax: +61-3-9905-4597.24 2 2011 2011 12 3 1464 1473 27 1 2011 17 2 2011 22 2 2011 © 2011 by the authors; licensee MDPI, Basel, Switzerland.2011This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Self-assembled nanostructures of zwitterionic octaphosphanatoporphyrin 1, of either nanoparticles or nanorods, depending on small changes in the pH, is demonstrated based on the J-aggregates. Porphyrin 1 self-assembled into nanosphere aggregates with a diameter of about 70–80 nm in the pH range 5–7, and nanorod aggregates were observed at pH 8.5. Hydrogen bonding, π-π stacking and hydrophilic interactions play important roles in the formation of this nanostructure morphology. Nanostructures were characterized by UV/Vis absorbance, fluorescence, atomic force microscopy (AFM) and transmission electron microscopy (TEM). This interesting pH dependent self-assembly phenomenon could provide a basis for development of novel biomaterials.
molecular self-assemblyporphyrinaggregationAFMTEM
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1. Introduction
Porphyrins, particularly natural i.e., chlorophylls and the protoporphyrin IX, contain hydrophobic, rigid, apolar and polar parts [1]. This characteristic prevents crystallization and favors self-aggregation [2]. Such supramolecular self-assemblies are of interest for polymeric porphyrin wires for long range energy and electron transport [3]. In contrast, symmetric tetraphenylporphyrins (TPPs) lack this diversity of functional groups and have a high tendency to crystallize. However, water-soluble symmetric TPP derivatives [4,5] form nanostructures or form well-defined Langmuir-Blodgett monolayers on smooth solid surfaces [6], and TPP derivatives carrying long side chains with water-soluble groups produces fibrile structures at low pH [7,8]. Nevertheless, recently we have shown that substituted protoporphyrin (IX) with triethylene glycol [9], which shows good solubility in organic solvents, produces well defined tunable nanostructures from varying solvent mixes.
Phosphonatoporphyrin were first developed as building blocks for porphyrin towers on silicon or gold electrodes for electrochemical investigations, in such a case zirconium-(IV) was first applied as a cement between phosphonate groups and led to broad rocks, instead of ill-defined monomolecular aggregation [10]. Self-assembly of phosphonatoporphyrin with more flexible chains was proven to result in formation of ill-defined monomolecular aggregates [11]. There are only few reports of porphyrin vesicles from charged and/or amphiphilic porphyrins [12–19]. Nanoscaled three-dimensional aggregates of supramolecular porphyrin arrays have been explored and their non-specific intermolecular interactions have been described. A porphyrin derivative substituted by four cartenoid molecules (“bixin”) spontaneously formed vesicles in water, as observed by Fuhrhop and co-workers [18]. Vesicles of meso-tetrakis-[(bixinylamino)-o-phenyl] porphyrin formed in water at pH = 9 can remain essentially intact even on dry solid surfaces [18,19]. Systematic studies on the anionic substituent are desired to further develop the research scope of the zwitterionic self-assembly of porphyrins. For the J-aggregate formation, the electrostatic interactions between the positively charged porphyrin core and the negatively charged groups play an important role. However, studies along these lines are still limited [4,5]. Nevertheless, in our earlier work, we have demonstrated synthesis of novel octaphosphanatoporphyrin (OctaPhosPor) 1 [20], and its cofacial reversible self-assembly with cyclam, which yields micrometer long monomolecular nanowires. We speculated that self-assembly was modulated by intermolecular hydrogen bonding of the amino groups of cyclam with phosphonato moieties of 1. In this present study, we report pH dependent self-assembly of zwitterionic OctaPhosPor 1 in water, and the displacement of peripheral phosphanato (anionic) groups of porphyrins is shown to have a major effect on the aggregation behavior.
2. Results and Discussion
Porphyrin 1 is equipped with two phosphonate moieties on the meta-positions of each phenyl group with methylene bridges (Figure 1). Short flexible spacers, i.e., methyl, were found to be necessary to achieve good solubility of the phosphonate esters in organic solvents and phosphonate salts in water. By manipulating pH, we show that supramolecular adducts form in solution and further stabilize by H-bonding to form nanospheres and nanorods in water (Figure 1). To the best of our knowledge, phosphonate moieties on porphyrin forming nanoscale aggregates have not yet been reported.
We hypothesize that pH dependent assemblies of OctaPhosPor 1 were obtained via three protonation steps (see Figure 1b): (1) first protonation of the phosphonate groups (pKa = 8.0), (2) protonation of the core nitrogen atoms (pKa = 5.6), and (3) second protonation of the phosphonate groups (pKa = 3.0). It is expected that the second protonation step, leading to a zwitterionic form, might induce aggregation. On the other hand, the first protonation step produces a nonzwitterionic species that is able to aggregate. In the latter case, in addition to π-π interactions, hydrogen bonds involving the phosphonic groups might also foster self-assembly. In this case, dimers of porphyrins interact axially through π-π stacking and laterally by means of strong edge-to-edge hydrophobic contacts. The whole structure may further stabilize by hydrogen bonding of phosphonato groups. Our experimental results agree with previous literature assignments [11]: OctaPhosPor 1 is (i) a monomer at pH > 9.0, (ii) a spherical particle aggregate at pH range 5–7, and (iii) higher aggregates are observed in the pH range 7–9. The stability of the particles and tubular aggregates made of porphyrins is presumably caused by an ordering of the porphyrins by π-π interactions and by hydrogen bridges between partly protonated phosphonate groups in water at higher pH. The aggregation behavior of 1 in aqueous solution, as a function of pH, has been studied by means of UV/Vis, fluorescence emission spectroscopy, atomic force microscopy (AFM) and transmission electron microscopy (TEM) techniques.
2.1. UV/Vis Absorption and Fluorescence Spectroscopy
Typically OctaPhosPor 1 is readily soluble in water at pH 9.0 and the corresponding UV/Vis spectrum exhibits an intense Soret band at 418 nm (ɛ = 2.56 × 105 L M−1 cm−1), together with four weaker Q-bands at 516, 558, 584, and 639 nm (Figure 2, broken curve). In fact, at pH 5.5, the most prominent band in the absorption spectrum is red-shifted with respect to that of the species existing at basic pH 9.0. At pH 5, absorption of 1 shows a red-shifted Soret band at 440 nm with vanishing of three Q-bands and appearance of a red-shifted Q-band at 656 nm (Figure 2, solid curve). The appearances of such characteristic absorption bands indicate the formation of J-aggregates. The “lack” of the Soret band of the protonated form (418 nm, monomer porphyrin band) can be attributed to the aggregation phenomena [12–18]. The effect of changing pH has been investigated in the pH range 3–11 (Table 1). The absence of the Soret band of the protonated form strongly suggests that the higher aggregate presiding over the hierarchy is that formed at a pH close to 6–8.
Furthermore, fluorescence emission of OctaPhosPor 1 shows two bands at 651 and 718 nm at pH 8.5 upon excitation at λmax = 418 nm. In sharp contrast, the fluorescence decreases with a decrease in pH with a slight bathochromic shift, which also indicates aggregation behavior of 1 (Figure 3). At pH 9–11, porphyrin appear to be in monomeric form, and in acidic pH (in the range 5–9) fluorescence quenching was observed. Such fluorescence quenching may be due to the self-aggregation of porphyrins at such pHs [12–18].
To support this hypothesis, we have characterized self-assembly of 1 at different pHs from 5–9, by employing atomic force microscopy (AFM) and transmission electron microscopy (TEM) techniques.
2.2. Atomic Force Microscopy and Transmission Electron Microscopy
Porphyrin 1 (10−4 M) gives nanospheres with a mean diameter of ca. 70–80 nm and a height of ca. 30 nm at pH 7.0 in water (Figure 4). The mean diameter of the particles (75 nm) is significantly larger than the molecular dimension of 1 (ca. 2.2 nm), hence nanoaggregates should be vesicular aggregates rather than micellar aggregates. Furthermore, transmission electron microscopy (TEM) gave clear evidence of formation of vesicular aggregates of porphyrin 1. TEM shows well-defined completely regular and isolated pattern of aggregated nanospheres of 1 with a mean diameter of 70–100 nm (Figure 4d). The TEM images are in excellent agreement with the results obtained from AFM, with respect to the size and diameter of these nanospheres measured. In addition, it can be seen from the TEM images that the spherical particles are completely dispersed and do not tend to associate with each other.
At pH 8.5, 1 produces not only the common particular aggregates but also regular parallel-lying self-organized nanorods (Figure 5). The AFM images showed that most of the tubules were surrounded by disordered and mobile patches of flat-lying porphyrins (Figure 5a). Occasionally these tubules showed some crystalline order at one end and the other end narrow. TEM examination of the same sample prepared from a dilute solution of 1 at pH 8.5 revealed the presence of well-defined rod like nanoaggregates with a diameter of ∼15 ± 3 nm and of several nanometers in length (600–900 nm) as shown in Figure 5b. Interestingly, the change to pH < 8.0 reduces the average size of the aggregates and leads to the formation of assemblies with short nanorods.
That self-assembly only occurs in pH 5–9 was confirmed by fluorescence spectroscopy, as in the pH range 3–5, the emission of 1 was diminished, which may be due to the protonation of phosphonate end groups and the two nitrogen atoms of the core. AFM clearly shows formation of larger aggregates i.e., nanosheets at pH values above 10 and below pH 5 as shown in Figure S1 and S3, respectively.
3. Experimental Section
3.1. Octaphosphanato Porphyrin 1 Synthesis
Synthesis and characterization of OctaPhosPor 1 was described previously [1].
3.2. Standard Solution of Octaphosphanato Porphyrin 1
Stock solutions (concentration 1 × 10−3 M) of 1 were made in water at pH 7.0. For spectral measurements, this solution was injected each time with 2 mL of water (varying pH) in a cuvette using a micropipette.
3.3. UV-Vis Absorption Spectroscopy
Stock solutions (concentration 1 × 10−3 M) of 1 were made in water (pH 7.0). A 0.2 mL aliquot of the stock solution was transferred to several different volumetric flasks in water at varying pH 3–11, each of 2 mL volume. The solutions were allowed to equilibrate for 2 h prior to the spectroscopic measurements. The most prominent features are a reduction in the peak intensity along with a significant red shift of the absorption maximum and a loss of the fine structure.
3.4. Atomic Force Microscopy (AFM) of 1
The samples were characterized using an Atomic Force Microscope (AFM) from Agilent Technologies (5500 AFM). Micromach Ultrasharp probes with silica wafer coating for enhanced reflectivity (NSC15/AIBS), with a typical resonance frequency of 325 kHz and a force constant of 40 N/m, were used for imaging. Sample of OctaPhosPor 1 were prepared by spin-coating the freshly prepared solution (1 × 10−4 M in water, at varying pH 3 to 11) onto silica coating at 2000 rpm. The particle diameter and height determination was performed by measuring the mean horizontal distance and height of particles.
3.5. Transmission Electron Microscopy (TEM) of OctaPhosPor 1
TEM measurements were performed on an electron microscopy Igor 1200EX, operating at an accelerating voltage of 80 kV. 0.5 μL freshly prepared sample solution (1 × 10−4 M in water at different pH values) was dropped onto a TEM grid (400-mesh copper grid coated with carbon) and the solvent was allowed to evaporate before introduction into the vacuum system. Negative staining was performed by addition of a drop of uranyl acetate onto the carbon grid. After few minutes, remaining solvent was removed by tapping with filter paper and images were collected.
4. Conclusions
In this paper, we have shown that by tuning the pH systems through the formation of complementary hydrogen bonds it is possible to promote the formation of nanospheres and nanorods of phosphonato porphyrin in water. The formed self-assembled nanostructures, i.e., nanospheres and nanorods, were stable enough to be investigated in solution via UV/vis, fluorescence spectroscopy and visualized through AFM and TEM microscopy. We are also currently producing porphyrin phosphonate nanoscale aggregates from zinc and tin(IV) complexes in order to measure electric conductivities of cation and anion π-radical stacks [21].
Shesh.V.B and S.J.L gratefully acknowledge the Australian Research Council for support under their Discovery program (DP0878756 and DP0878220). Sid. V.B wishes to thanks the CSIR, India for financial support from grant no. 01(2283)/08/EMR-II.
Figure 1. (a) Schematic representation of the self-assembly of the OctaPhosPor 1 into: nanosphere aggregates at pH 5–7, and further aggregation of 1 into nanorods at pH 7–9 in water; (b) protonation and aggregation mode of OctaPhosPor 1.
Figure 2. Absorption spectrum of OctaPhosPor 1 (1 × 10−4 M) at pH 9.0 (broken curve) and 5.0 (solid curve), inset figure shows as an expansion of Q-bands (490–690 nm).
Figure 3. OctaPhosPor 1 shows a pH dependent (10.4–3.0) change in fluorescence spectrum at a concentration of 1 × 10−4 M, λex at 418 nm.
Figure 4. Atomic force microscopy (AFM) height images of OctaPhosPor 1 (10−4 M) upon spin-cast on silicon wafer plate after the solvent has evaporated, gives nanospheres in water at pH 7.0: (a) height image (scale bar = 1 μm); (b) magnified view of height image a (scale bar = 400 nm); (c) height image (scale bar = 1 μm); (d) Transmission electron microscopy (TEM) micrograph of 1 on holey, carbon-coated copper grids (scale bar = 100 nm); (e) high magnification of d; and (f) cross-section analysis magnified region from image c, provides a mean diameter of ca. 75 nm and a height of 30 nm.
Figure 5. (a) Atomic force microscopy (AFM) height images of 1 (10−4 M) upon spin-cast on silicon wafer plate after the solvent has evaporated; Inset = high magnification of AFM image (b) Transmission electron microscopy (TEM) micrograph of 1 on holey, carbon-coated copper grids (scale bar = 500 nm), shows nanorods in water at pH 8.5. Inset = high magnification of TEM image.
Table 1. UV/vis Absorption and Fluorescence Emission Data for OctaPhosPor 1 under Different pH Conditions in Water.
pH B-band (λ, nm; 105 ɛ, M−1 cm−1) Q-bands (λ, nm; 103 ɛ, M−1 cm−1) emission (λ, nm)
3 442 (1.4) 516 (9.6), 656 (36) no[a]
5 440 (2.19) 534 (21.2), 594 (14.8), 644 (12.8) 657, 721[b]
8 420 (2.38) 516 (13.8), 559 (12.8), 583 (12.0), 636 (10.6) 651, 718
10 418 (2.56) 510 (13.8), 545 (12.8), 578 (12.0), 626 (10.6) 651, 718
[a] very weak emission;
[b] weak emission.
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J Cardiothorac SurgJournal of Cardiothoracic Surgery1749-8090BioMed Central 1749-8090-6-692156941210.1186/1749-8090-6-69Research ArticleRapamycin-loaded nanoparticles for inhibition of neointimal hyperplasia in experimental vein grafts Zou Junjie [email protected] Xiwei [email protected] Hongyu [email protected] Yi [email protected] Hao [email protected] Shui [email protected] Department of General Surgery, the First Affiliated Hospital of Nanjing Medical University, Nanjng, Jiangsu province, China2 Division of Vascular Surgery, Department of General Surgery, the First Affiliated Hospital of Nanjing Medical University, Nanjng, Jiangsu province, China2011 12 5 2011 6 69 69 18 10 2010 12 5 2011 Copyright ©2011 Zou et al; licensee BioMed Central Ltd.2011Zou et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
Nanoparticles possess several advantages as a carrier system for intracellular delivery of therapeutic agents. Rapamycin is an immunosuppressive agent which also exhibits marked antiproliferative properties. We investigated whether rapamycin-loaded nanoparticles(NPs) can reduce neointima formation in a rat model of vein graft disease.
Methods
Poly(lactic-co-glycolic acid) (PLGA) NPs containing rapamycin was prepared using an oil/water solvent evaporation technique. Nanoparticle size and morphology were determined by dynamic light scattering methodology and electron microscopy. In vitro cytotoxicity of blank, rapamycin-loaded PLGA (RPLGA) NPs was studied using MTT Assay. Excised rat jugular vein was treated ex vivo with blank-NPs, or rapamycin-loaded NPs, then interposed back into the carotid artery position using a cuff technique. Grafts were harvested at 21 days and underwent morphometric analysis as well as immunohistochemical analysis.
Results
Rapamycin was efficiently loaded in PLGA nanoparticles with an encapsulation efficiency was 87.6%. The average diameter of NPs was 180.3 nm. The NPs-containing rapamycin at 1 ng/ml significantly inhibited vascular smooth muscular cells proliferation. Measurement of rapamycin levels in vein grafts shown that the concentration of rapamycin in vein grafts at 3 weeks after grafting were 0.9 ± 0.1 μg/g. In grafted veins without treatment intima-media thickness was 300.4 ±181.5 μm after grafting 21 days. Whereas, Veins treated with rapamycin-loaded NPs showed a reduction of intimal-media thickness of 150.2 ± 62.5 μm (p = 0.001). CD-31 staining was used to measure luminal endothelial coverage in grafts and indicated a high level of endothelialization in 21 days vein grafts with no significant effect of blank or rapamycin-loaded NPs group.
Conclusions
We conclude that sustained-release rapamycin from rapymycin loaded NPs inhibits vein graft thickening without affecting the reendothelialization in rat carotid vein-to-artery interposition grafts and this may be a promising therapy for the treatment of vein graft disease.
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Background
Surgical bypass via autologous vein remains an evidence-based treatment of choice for selected patients with coronary occlusive disease or infra-inguinal lower extremity. However, contemporary data shows that almost 60% of lower extremity vein bypass grafts develop occlusive lesions or fail within a year [1], and almost half of cardiac bypass patients will lose (≥ 75% stenosis) a vein graft within a year [2]. Neointimal hyperplasia, which develops immediately after grafting, is the most important early change of the grafted vein. Although not solely responsible for the graft failure, this neointimal process is to be regarded as the guiding track easing the development of fatal sclerotic changes [3] With the exception of aggressive lipid lowering, [4] no therapy has been shown to improve long-term vein graft patency in clinical studies. Earlier experimental studies such as placement of external porous dacron stents [5] or perivascular application of decoy oligonucleotides [1,2] have failed to translate into clinical benefits because of early graft thrombosis and poor efficacy, respectively. Gavin and colleagues [6] have recently shown that immersion of vein grafts in rapamycin solution immediately before grafting inhibits neointima formation in porcine vein grafts; however, this effect is not sustained.
Nanoparticles possess several advantages as a carrier system for intracellular delivery of therapeutic agents. These advantages include their subcellular size, good suspensibility, an easy penetration into the vessel wall without causing trauma, and the capacity for sustained intracytoplasmic release [7,8]. Therefore, we hypothesized that rapamycin-loaded nanoparticles (NPs) could be an innovative therapeutic strategy for preventing vein graft failure. In this study, we have explored the efficacy of NPs as an intracellular ex vivo delivery system to the excised vein, and whether these NPs suppressed vein graft neointima formation in vivo. To our knowledge, however, no prior studies have examined whether rapamycin-loaded PLGA (RPLGA) NPs are useful as therapeutic strategy for preventing vein graft failure.
Methods
Materials for nanoparticle preparation
Rapamycin was obtained from Sigma, St. Louis, MO, USA. Polyvinyl alcohol (PVA) 87-89% hydrolyzed, MW 31,000-50,000 was purchased from Advanced Technology & Industrial Co. Ltd, Hong Kong, China. Poly(lactic-co-glycolic acid) (PLGA) copolymer, monomer ratio 50:50, MW 20000 was purchased from Dai Gang Biology, Shandong, China.
Preparation of PLGA NPs
PLGA NPs containing rapamycin was prepared using an oil/water solvent evaporation technique based on a single emulsion method [9]. Briefly, rapamycin was added to PLGA solution in Dichloromethane. The resulting solution was emulsified in 20 mL of 1% w/v PVA solution in PBS using a magnetic stirrer (Model 50-HI190M-1 Sciencelab. Inc. Houston, Texas. USA) and ultrasound probe 250W for 4 min, then the organic solvent was removed from the final emulsion by evaporation. The nanoparticle suspension was filtered through a 0.45 μm microporous membrane. Blank nanoparticles were also prepared by the same method without adding rapamycin at any stage of the preparation. The PLGA nanoparticles were stored at -4°C for further studies.
Characterization of RPLGA NPs
Nanoparticle size and size distribution were determined by Dynamic Light Scattering methodology using Zetasizer (Model 3000 Malvern Instruments Worcestershire, United Kingdom). The analysis was performed at a scattering angle of 90 and at a temperature of 25°C using sample appropriately diluted 20 times with filtered distilled water (0.22 μm GV, Millipore, Ireland). The morphology of the nanoparticles was examined using transmission electron microscopy(TEM). A drop of the nanoparticle suspension was transferred onto a small metal cylinder. After drying, the sample was transferred in the sample holder of a Jeol JEM-1010 electron microscope(Tokyo, Japan).
Measurement of Encapsulation Efficiency
Encapsulation efficiency was performed using dialysis bag diffusion technique. RPLGA NP samples (6 ml), enclosed in dialysis bags (cellulose membrane, mw cut-off 12400, Sigma), were incubated in 120 ml 5% sodium lauryl sulphate (SLS) at 37°C under 300 r/min agitation in a water bath for 8 h in order to release free dissolved drug. The amount of rapamycin in incubation medium was estimated by spectrophotometry based on the UV absorbance at 277 nm, and the entrapment efficiency was calculated by the equation:
Study of the in vitro release of the rapamycin from the RPLGA NPs
In vitro release studies were also carried out using dialysis bag diffusion technique [10]. At predetermined time intervals, 500 μl samples from SLS were withdrawn from the incubation medium and analyzed for rapamycin with spectrophotometry as described above. After sampling, the incubation medium was replaced by fresh 5% SLS. A control experiment to determine the release behavior of the free drug was also performed. Rapamycin was dissolved in 5% SLS (13.95 μg/ml) and 5 ml of this solution was enclosed in a dialysis bag and was immersed in 100 ml 5% SLS, 37°C. Then, the procedure described above for the nanoparticle samples was followed.
Cellular Uptake and Antiproliferative Effect of RPLGA NPs
The vascular smooth muscular cells (VSMCs) were incubated in the growth medium containing RPLGA NPs (the rapamycin concentration: 50 ug/ml) for 1 hour. The cells were then washed with PBS and centrifuged for 8 min with 3000 rpm. The centrifugal cell were fixed in 2% glutaraldehyde dissolved in 0.15M phosphate buffer at pH 7.2 for 1 h, followed by post-fixation in 1% osmium tetroxide dissolved in 0.9% sodium chloride for 1 h. Fixed material was stained in bloc in 0.5% aqueous uranyl acetate overnight. After this procedure, the samples were dehydrated in graded acetone series, and embedded in Araldite resin. Ultrathin sections (70 nm) were obtained using a diamond knife in an LKB ultramicrotome, placed on 200-mesh copper grids and double-stained by uranyl acetate and lead citrate. The grids were studied and micrographed with transmission electron microscope, operating at 80 kV.
In vitro cytotoxicity of blank, RPLGA NPs, and free rapamycin were investigated using the primary VSMCs culture. NPs were prepared under aseptic conditions. Rapamycin was dissolved in 96% ethanol. To obtain different test concentrations, several dilutions of nanoparticle suspensions were prepared with DMEM culture medium. Ethanol amounts used for dilution showed no influence on the cell viability during the experiments. VSMCs were seeded into 96-well microtiter plates (Nunclon™, Nunc, Germany) at a density of 4000 cells/well. After an incubation period of 24 h the culture medium was replaced with fresh, rapamycin, blank NPs, and RPLGA NPs culture medium. After an additional incubation time of 24 h the culture medium was replaced with fresh culture medium. After an additional incubation time of 48 h the viability of the cells was evaluated by the MTT assay (n = 7). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Invitrogen, USA) was dissolved in phosphate buffered saline at 5 mg/ml and 20 μl were added to each well reaching a final concentration of 0.5 mg/ml. After an incubation time of 4 h unreacted dye was removed by aspiration, the purple formazan product was dissolved in 200 μl/well dimethyl sulfoxide and quantitated by a plate reader (Titertek Plus MS 212, ICN, Germany) at wavelengths of 490 nm.
Experimental Animal Models
Male Sprague-Dawley rats (n = 25) (350 to 400 g; Slac Laboratory Animal Shanghai China) were anesthetized with an intraperitoneal injection of a 10% Chloral Hydrate (0.05 mg/kg), then a midline neck incision was made, and an approximately 1-cm segment of the lateral branches of external jugular vein was dissected free; all side branches were ligated. The vein segments were gently flushed, and placed in 1 ml solution containing either blank-NPs (n = 5), or RPLGA NPs containing rapamycin at 50 μg/ml (n = 5) for 30 minutes at room temperature. The operation was performed as described previously [11,12]. In brief the treated vein segments were interposed into ipsilateral carotid arteries (everted over a cuff). Animals were killed with a lethal dose of anesthesia on days 21. The vein grafts were harvested, flushed with saline, and used for histopathologic studies.
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996)
Histopathologic Analysis
For histological analysis the animals underwent autopsy on days 21 (n = 5 each). The grafts were perfusion fixed with 4% phosphate-buffered formaldehyde via puncture of the left ventricle as previously described [12]. The interposed vein segments were cut out at the cuff ends and fixed with 4% phosphate-buffered formaldehyde. Consecutively the veins were formalin fixed and embedded in paraffin. Sections of 4 μm thickness were stained by Hematoxylin and Eosin for measurement of the intimal thickness. The measurements were done using Digimizer 3.1.1.0 image analysis software (MedCalc Software, Belgium). For achieving reproducible results, the cross sections of the veins were divided into 4 quadrants. In each quadrant 3 measurements were performed. The median value of all measurements was regarded as representative for the intimal-median thickness.
Evaluation of endothelial coverage within grafts was achieved using an immunohistochemistry stain for CD-31 as previously described. CD-31 antibody was obtained from Santa Cruz Biotechnology, USA. Reendothelialization was calculated as the percentage of luminal surface covered by CD31-positive cells 3 weeks after operation. Endothelial coverage was calculated as an average score over 4 sections per graft.
Measurement of Rapamycin Levels
To determine vein graft drug uptake and retention, vein grafts were removed at different time points after administration of NPs. Rapamycin levels were measured by high-performance liquid chromatography as previously described [13]. Briefly, whole-blood samples (1 mL) or homogenized vein graft tissue were treated with precipitation reagent (methyl tert-butyl ether:I-chloroethane:methanol, 45:45:10) plus internal standard solution (32-0-desmethoxysirolimus) followed by C18 solid-phase extraction. UV detection of the absorbance peaks was performed at 278 nm, and values were expressed as micrograms per gram of dry tissue.
Statistical analysis
The SPSS™ for Windows statistical software package (SPSS 17 for Windows, SPSS Inc., Chicago, IL, USA) was used for analysis. The value of MTT and intimal-median thickness is given as median ± 2SE. Comparisons of reendothelialization and histological measurements of the intimal-median thickness were made by the two tailed, unpaired Student's t test. Results were considered statistically significant at p values of less than 0.05.
Results
Characterization of RPLGA nanoparticles
Rapamycin was efficiently loaded in PLGA nanoparticles reaching a loading capacity of 23 ug of drug to 1 mg of polymer and an encapsulation efficiency of 87.6%. Dynamic light scattering analysis revealed an average diameter of 180.3 nm (particle size range of 100-450 nm) for nanoparticles(Figure 1A). TEM showed a spherical shape for RPLGA nanoparticles (Figure 1B). The diameter of nanoparticles measured from TEM pictures was in the range of 100-300 nm, which was in agreement with the results of light scattering measurements.
Figure 1 Characterization and in vitro release of the rapamycin from the RPLGA nanoparticles. A, Dynamic light scattering analysis for nanoparticles. B, Transmission electron micrograh RPLGA NPs. Bar represents 1 μm. C, Transmission electron microscopic picture of cross-section of VSMCs incubated with NP for 1 hour. Arrows indicate NP in the cytoplasm. Scale bar = 1 μm. D, In vitro release profile of rapamycin from RPLGA NPs. The percentage of rapamycin released was plotted against time.
In vitro release of the rapamycin from the RPLGA nanoparticles
An analysis of the in vitro drug release kinetics from rapamycin loaded PLGA nanoparticles showed an early burst of drug release, approximately 27% of the total amount of drug, was observed on day 1, followed by sustained release that lasted for 21 days. During this period 80.2% of the drug was released (Figure 1D).
In Vitro Cell Uptake and Antiproliferative Effect of RPLGA NPs
TEM of the cellular cross sections revealed the intracellular localization of NPs after 1 h of incubation (Figure 1C).
The MTT test served as an assay for proliferation and cell viability by measuring the mitochondrial activity of cells. The RPLGA NPs at 1 ng/ml significantly inhibited VSMCs proliferation (78.5 ± 5.74 vs 100 ± 3.01; P < 0.01). The effect of inhibiton was similar to that of free drug at 10 ng/ml(78.5 ± 5.74 vs. 79.1 ± 4.82; P = 0.85 Figure 2). There was non-antiproliferative character of unloaded nanoparticles prepared from PLGA(97 ± 6.79 vs. 100 ± 3.01; P = 0.35).
Figure 2 In vitro effects of RPLGA NPs on proliferation of SMCs. Data represents mean ± 1SEM (n = 6 each) and percentage changes from control (100%). *P < 0.01 vs control.
Rapamycin Pharmacokinetics
Measurement of rapamycin levels in vein grafts demonstrated the presence of drug in vein grafts at 0 days(after the vein being immersed in rapamycin-loaded NPs solution), 1 weeks and 3 weeks after grafting (n = 5) (Figure 3). The concentrations of rapamycin in vein grafts at 0 days, 1 weeks and 3 weeks after grafting were 5.4 ± 0.9 μg/g, 1.3 ± 0.2 μg/g and 0.9 ± 0.1 μg/g respectively. Rapamycin was not detected with high-performance liquid chromatography in whole-blood samples of any experimental animal, 1 week after grafting.
Figure 3 Rapamycin Pharmacokinetics. Graph showing vein graft rapamycin levels at 0, 7 and 21 days after grafting. Bars represent mean ± 1SEM.
Effects of RPLGA NPs on Vein Graft Failure in rats
The autologous interposition jugular vein graft was explanted and examined 21 days. Figure 4 demonstrates intima-media thickening at 21 days after implantation of the vein into the arterial circulation. The intima and media are measured together as the rat jugular vein has no well-defined boundary between the two. In grafted veins without treatment (controls, n = 5 at each point of time) intima-media thickness was 300.4 ±181.5 μm after 21 days. Veins treated with rapamycin loaded NPs (n = 5 at each point of time) showed a reduction of intimal-media thickness with 150.2 ±62.5 μm 21 days (p < 0.001 vs controls). Figure 1D demonstrates quantitative analysis of remodeling in this model, with increased thickness of the vein graft intima-media at 21 days.
Figure 4 Effects of ex vivo treatment with rapamycin-loaded NPs on vein graft neointima formation and reendothelializaton in rats at 21 days. A and B, Vein graft sections of animals treated with PLGA NPs (left, control) and with RPLGA-NPs (right, treated), stained with Hematoxylin and Eosin. D, Bar graphs represent the morphometric analysis of vein graft sections about the inhibition of hyperplasia (intima-media thickness) in different treatment groups. Quantitative data derived from 3 vein graft sections at different levels from each animal in each group. *P < 0.001 for treated (n = 5) versus control (n = 5) groups. C, Endothelial coverage within grafts was achieved using an immunohistochemistry stain for CD-31. E, Bar graphs represent no significant effect of control or RPLGA-NPs group P = 0.8. Bars represent mean ± 1SEM.
CD-31 staining (Figure 1C) was used to measure luminal endothelial coverage in grafts and indicated a high level of endothelialization in 21 days with no significant difference between control and RPLGA NPs group(88.2% ± 7.1% vs. 86.4% ± 7.5%; P = 0.8) (Figure 1E).
Discussion
In this study, we have demonstrated for the first time that PLGA NPs is an excellent system for intracellular delivery of rapamycin in excised veins. This NPs system is bioabsorbable polymer with a long history of safe use in medical applications. The RPLGA NPs was endocytosed rapidly by VSMCs. After cellular uptake of NPs, they slowly release encapsulated drugs into the cytoplasm whereas PLGA gets hydrolyzed, resulting in an intracellular drug delivery and thereby significantly inhibiting VSMCs proliferation. An important finding was, long-term release of rapamycin in the vein until day 21 after grafting which suggests that RPLGA NPs delivery system may be applied to human beings clinically. The bio-absorption time of PLGA in the body can be controlled by changing material make-up of PLGA, thus the function of the intracellular drug delivery system can be modified. Therefore, this NPs-mediated drug delivery system works as an excellent ex vivo delivery of rapamycin for the excised vein to prevent vein graft failure.
Rapamycin is a macrocyclic, lipophilic lactone with immunosuppressive antibiotic activity derived from the actinomycete streptomyces hygroscopicus. The well-established effect of rapamycin are the inhibition of the progression of the cells from the G1 to the S phase within the mitotic cycle and VSMCs migration [14-16]. The drug is clinically used in transplantation medicine and as a coating of coronary artery stents. Rapamycin-eluting stents significantly reduce the incidence of restenosis and late loss of arterial luminal diameter [17]. Schachner and colleagues [18] have shown in an experimental study that rapamycin could inhibit the development of neointimal hyperplasia in a mouse autologous graft transplantation model (vein to artery), with a peri-paravascular application of rapamycin applied locally in pluronic gel. With the use of pluronic gel some prolonging of drug contact to the graft could be reached but one disadvantage of this method, however, is the limited release time of the gel. As an example, Fulton and colleagues [19] found that after 5 days, 80% of antisense oligonucleotides to proliferating cell nuclear antigen (PCNA) were released from pluronic gel.
In a recently published study Walpotha and colleagues shown a reduction of neointimal hyperplasia with a high dose of systemic rapamycin treatment (3 mg/Kg) either after 30 or 60 days in a rat infrarenal graft model. This dosage was similar to the one used in the transplantation model. However, they observed drug related side effects as well as a catabolic metabolism with lack of weight increase, which would limit the clinic use of this method [20].
In our experiment we found that the proliferation of VSMCs was inhibited significantly by RPLGA NPs when the concentration of rapamycin was 1 ng/ml. The concentration was one-tenth of free drug. This shows that the PLGA nanopariticle increases the effects of rapamycin, which may be related to nanoparticles ability to increase the drug solubility and cell membrane penetration. At the same time, the rapamycin-loaded NPs can penetrate into the vessel wall easily ex vivo and sustained release the drug at least 3 weeks, so the early stage intimal hyperplasia of vein graft was inhibited significently.
The study of Thirumaran and colleagues [21] have shown that periadventitial application of rapamycin-eluting polyvinyl alcohol microspheres to porcine saphenous vein-to-carotid artery interposition grafts inhibited vascular smooth muscle cell proliferation in 1-week grafts. The inhibition of vein graft thickening was not sustained; however, there was no therapeutic benefit evident in 12-week grafts. It may be related to the shorter time of drug retention (rapamycin was not detected at 28 days). Kawatsu et al [22] reported rapamycin-eluting biodegradable PLA-CL film applied externally can inhibit neointimal hyperplasia of anastomotic sites of arterial and vein grafts in a canine model. By the end of fourth week, the film had been completely absorbed; relatively drug retention time was also less. However, in our study the rapamycin was detected after 21 days, and the concentration was relatively higher, similar observations were made by Reddy et al wherein they have used cross-linked gel-like polymeric NPs for delivery system of rapamycin in injured artery [23]. Because RPLGA NPs can easily penetrate into the vessel wall and their sustained release of rapamycin into cell cytoplasm, the inhibition to the intimal hyperplasia of vein graft would maintain for a longer durations. In our study, the rapamycin level in the vein graft was 5.4 μg/g after being immersed, and the level decreased during the first week but remained constant thereafter for the next 2 weeks. It is possible that the NPs localized in the intimal layer might have washed away with blood flow and the NPs localized in the adventitia diffused into the surrounding tissue after the initial localization, resulting in the initial decrease in drug level. However, the NPs localized in the vein graft were retained and able to maintain the drug level.
Because rapamycin has been reported to suppress the adhesion of endothelial progenitor cells and their differentiation into endothelium [24]. The biggest drawback of coated stent is adversely influencing the process of reendothelialization, thereby causing acute thrombosis. However, we have observed that the RPLGA NPs did not affect reendothelialization of the vein graft. One of the main reasons that stented arteries do not undergo reendothelialization very well is because of their direct contact with the intimal layer of the drug used to coat the stent, consequently either inhibiting the proliferation of endothelial cells or preventing adhesion and differentiation of circulating endothelial progenitor cells to the injured endothelium [25]. The drug localized in the vein graft was probably not interfering in the process, allowing vascular repair to occur by the natural mechanism.
Conclusions
In conclusion, sustained-release of rapamycin from RPLGA NPs inhibits vein graft thickening in rat carotid vein-to-artery interposition grafts 3 weeks, this does not affect the reendothelialization of the vein graft. Therefore, this NPs-mediated drug delivery system works as an excellent ex-vivo delivery of rapamycin for the excised vein to prevent vein graft failure.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
JZ designed study, analyzed and interpreted the data, and prepared manuscript. XZ contributed to study design, data analysis and interpretation, and preparation of manuscript. HY contributed to data collection, and preparation of manuscript. HM contributed to interpretation of data and manuscript preparation. YZ contributed to study design, and preparation of manuscript. SW contributed to study design, analyzed and interpretation of data and manuscript preparation. All authors read and approved the final manuscript.
Acknowledgements
The authors wish to express their appreciation to Liu Jianping, PhD, for her superb technical assistance during the preparation and characterization of RPLGA NPs.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 2169823310-PONE-RA-1974210.1371/journal.pone.0020614Research ArticleBiologyMolecular cell biologyGene expressionRNA interferenceMedicineOncologyCancer TreatmentGene TherapyRadiation TherapyMMP-2 siRNA Inhibits Radiation-Enhanced Invasiveness in Glioma Cells Inhibition of Glioma InvasionBadiga Aruna Venkata
1
Chetty Chandramu
1
Kesanakurti Divya
1
Are Deepthi
1
Gujrati Meena
2
Klopfenstein Jeffrey D.
3
Dinh Dzung H.
3
Rao Jasti S.
1
3
*
1
Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine, Peoria, Illinois, United States of America
2
Department of Pathology, University of Illinois College of Medicine, Peoria, Illinois, United States of America
3
Department of Neurosurgery, University of Illinois College of Medicine, Peoria, Illinois, United States of America
Lesniak Maciej S. EditorThe University of Chicago, United States of America* E-mail: [email protected] and designed the experiments: AVB JSR. Performed the experiments: AVB CC DK DA. Analyzed the data: AVB MG JDK DHD JSR. Contributed reagents/materials/analysis tools: JSR. Wrote the paper: AVB CC. Provided discussion and revision of critically important intellectual content: JSR.
2011 16 6 2011 6 6 e2061411 6 2010 9 5 2011 Badiga et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
Our previous work and that of others strongly suggests a relationship between the infiltrative phenotype of gliomas and the expression of MMP-2. Radiation therapy, which represents one of the mainstays of glioma treatment, is known to increase cell invasion by inducing MMP-2. Thus, inhibition of MMP-2 provides a potential means for improving the efficacy of radiotherapy for malignant glioma.
Methodology/Principal Findings
We have tested the ability of a plasmid vector-mediated MMP-2 siRNA (p-MMP-2) to modulate ionizing radiation-induced invasive phenotype in the human glioma cell lines U251 and U87. Cells that were transfected with p-MMP-2 with and without radiation showed a marked reduction of MMP-2 compared to controls and pSV-transfected cells. A significant reduction of proliferation, migration, invasion and angiogenesis of cells transfected with p-MMP-2 and in combination with radiation was observed compared to controls. Western blot analysis revealed that radiation-enhanced levels of VEGF, VEGFR-2, pVEGFR-2, p-FAK, and p-p38 were inhibited with p-MMP-2-transfected cells. TUNEL staining showed that radiation did not induce apoptosis in U87 and U251 cells while a significant increase in TUNEL-positive cells was observed when irradiated cells were simultaneously transfected with p-MMP-2 as compared to controls. Intracranial tumor growth was predominantly inhibited in the animals treated with p-MMP-2 alone or in combination with radiation compared to controls.
Conclusion/Significance
MMP-2 inhibition, mediated by p-MMP-2 and in combination with radiation, significantly reduced tumor cell migration, invasion, angiogenesis and tumor growth by modulating several important downstream signaling molecules and directing cells towards apoptosis. Taken together, our results demonstrate the efficacy of p-MMP-2 in inhibiting radiation-enhanced tumor invasion and progression and suggest that it may act as a potent adjuvant for radiotherapy in glioma patients.
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Introduction
Patients with glioblastoma multiforme (GBM), the most common type of human primary brain tumor and the most lethal of all human tumors, have a median survival of less than one year [1]–[3]. Despite advances in neurosurgery, radiation therapy, and chemotherapy, this prognosis has not changed significantly over the past two decades. Contributing to the deadly nature of the disease is the ability of glioblastoma cells to extensively invade normal brain tissue, thereby preventing a surgical cure and resistance of glioblastoma to existing therapeutic modalities, including radiotherapy [1]. Excessive proliferation, disseminated tumor growth, resistance to apoptotic stimuli, neovascularization, and suppression of anti-tumor immune surveillance are key biological features that contribute to the malignant phenotype of gliomas [4], [5]. Tumor cells obtain these invasive properties primarily because of their ability to secrete and activate proteolytic enzymes, such as serine, metallo, and cysteine proteases, which are capable of degrading extracellular matrix (ECM) components and breaking down other natural barriers to tumor invasion [6], [7].
Matrix metalloproteinases (MMPs) are zinc-dependent, proteolytic endopeptidases involved in cancer progression. Elevated levels of MMPs, in particular increased expression and activity of MMP-2 (72-kDa gelatinase A) and MMP-9 (92-kDa gelatinase B), have been correlated with an increased grade of glioma malignancy [8]–[10]. MMP-2 is highly expressed in gliomas as compared to normal brain tissue, and multiple roles have been indicated for this molecule in tumor progression. MMP-2 activates several key molecules leading to rapid cellular proliferation, increased motility, invasion, and angiogenesis of gliomas. Growth factor receptors, cell adhesion molecules, apoptotic ligands, angiogenic factors, chemokines, and cytokines are some of the diverse substrates targeted by MMPs [9], [11].
One of the major limitations in the treatment of glioma is the high prevalence of acquired resistance to radiation, which is likely associated with intrinsic cellular radioresistance, rapid cellular proliferation, and high invasiveness [12], [13]. Because radiation therapy continues to be the mainstay of cancer treatment, tumor resistance to ionizing radiation has been extensively studied. Of major interest is the role of MMPs in changing tumor cell properties and inducing an invasive phenotype after ionizing radiation [13]–[16]. Ionizing radiation induces an increase in MMP-2 levels in almost all human cancer types [17]–[23]. Radiation-enhanced expression as well as activation of the MMP-2 proteolytic system elevate or modify the bioavailability of several molecules that promote tumor progression. Enhanced MMP-2 secretion may increase tumor survival by decreasing apoptosis, stimulating proliferation, and increasing angiogenic and invasive potential. All these factors may contribute to the high vascularity as well as the high radioresistance of GBM [13]–[16].
There is strong biological background for the development of strategies targeting MMP-2. Nevertheless, several MMP inhibitors have proved to be disappointing in clinical trials due to either lack of benefits or major adverse effects [24]. Today, RNA interference-based, targeted silencing of gene expression is a strategy of potential interest for cancer therapy [25], [26]. Further, the specificity and potency of siRNA-MMP-2 in cell culture and in animal studies suggests that it has the potential to be a powerful therapeutic agent [27]–[29]. Viral vectors, particularly adenoviral ones, have been the primary gene transfer vehicle of choice. However, two concerns are the ability of adenoviral proteins to trigger an immune response and the limited length of time that the gene expression can be maintained in the target cells. Plasmid-based vectors have been considered to be more safe and efficient [30].
Since radiation is standard treatment for patients with GBM and because radiation-induced changes in invasive phenotype of glioma have been shown to be possibly due to changes in MMP activation, in the present study, we have tested the ability of a plasmid vector-mediated MMP-2 siRNA to modulate the ionizing radiation-induced invasive phenotype in human glioma cell lines, U-251 and U-87. We also investigated the anti-tumor effects of combining MMP-2 inhibition using the plasmid vector-mediated MMP-2 siRNA with radiation therapy in vivo. Our study shows that MMP-2 inhibition, mediated by plasmid DNA, in combination with radiotherapy reduces cell proliferation, tumor cell migration, invasiveness, and angiogenesis. We also show that inhibition of radiation-enhanced MMP-2 modulated important downstream signaling molecules, thereby directing the cells towards an apoptotic phenotype.
Results
p-MMP-2 transfection inhibits radiation-enhanced MMP-2 expression and cell viability
To examine the effect of radiation on MMP-2 expression, we determined MMP-2 activity in U-251 and U-87 cells irradiated with various X-ray doses. Radiation-induced MMP-2 activity increased up to 10 Gy for U-251 and up to 8 Gy for U-87 and decreased thereafter. The decreased levels of MMP-2 activity beyond 10 and 8 Gy for the two cell lines could be due to severe damage of DNA and other proteins. Maximal induction in pro-MMP-2 activity (72 kDa) was observed at 8 and 10 Gy when compared to the control, 0 Gy (Fig. 1A). Since MMPs play a role in the induction of malignant phenotype, we used a plasmid vector carrying siRNA against MMP-2 (p-MMP-2) to inhibit MMP-2 expression. We first determined the concentration of the plasmid DNA, which would give maximum inhibition of MMP-2 activity and levels when U-251 and U-87 cells were transfected. Figure 1B demonstrates that pro-MMP-2 activity was minimal when cells were transfected with 2 µg of the plasmid DNA. The figure shows a dose-dependent decrease in cellular MMP-2 levels with little or no MMP-2 detected when cells were transfected with 2 µg of the plasmid DNA. We therefore used this concentration (2 µg) of the plasmid DNA carrying MMP-2 siRNA or the scrambled sequence in combination with various doses of radiation for all experiments in the study. Western blots of cell lysates showed unchanged levels of MMP-9 when compared to the internal control GAPDH, thereby demonstrating the specificity of p-MMP-2. We next determined the effect of p-MMP-2 on radiation-induced MMP-2 expression. Figures 2A and B show pro-MMP-2 activity levels and protein levels, and mRNA levels of U-251 and U-87 transfected with p-MMP-2 or p-SV alone or in combination with various doses of radiation. Cells that were transfected with p-MMP-2 without radiation showed a significant decrease in activity/levels of MMP-2 when compared to the mock or p-SV controls or when compared to cells subjected to irradiation alone. The combination of p-MMP-2 and radiation caused a further decrease in MMP-2 levels. Maximal inhibition was observed with 6 Gy followed by 4 Gy. We further confirmed these results by immunofluorescence staining of the cells, which revealed similar results. There was a marked reduction in MMP-2 staining in all irradiated and concomitantly transfected cells, as well as in cells treated with p-MMP-2 alone (Fig. 2C). Cell viability/proliferation, as measured by MTT assay, was significantly reduced when U-251 and U-87 cells were concomitantly transfected with p-MMP-2 and irradiated or when cells were treated with p-MMP-2 alone when compared to the controls (Fig. 2D).
10.1371/journal.pone.0020614.g001Figure 1 Radiation enhances MMP-2 and p-MMP-2 inhibits MMP-2 activity and expression in glioma cell lines.
A, U-251 and U-87 cells were irradiated with 0–12 Gy X-ray, incubated for 24 h, and conditioned medium collected. MMP-2 activity was determined by gelatin zymography. The band intensities of MMP-2 activity were quantified by densitometry. Columns: mean of triplicate experiments; bars: SD; *p<0.01, **p<0.001, significant difference from non-irradiated (0 Gy) conditioned medium. B, U-251 and U-87 cells were transfected with mock (PBS), p-SV or p-MMP-2 (1, 2 or 3 µg). After 72 h of incubation, conditioned media was used to determine MMP-2 activity by gelatin zymography, and total cell lysates were used to determine MMP-2 and MMP-9 levels by Western blotting. The band intensities of MMP-2 activity as well as MMP-2 and MMP-9 protein levels were quantified by densitometry and normalized with the intensity of the mock bands. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a loading control. Columns: mean of triplicate experiments; bars: SD; *p<0.01, **p<0.001, significant difference from mock or p-SV.
10.1371/journal.pone.0020614.g002Figure 2 p-MMP-2 transfection inhibits radiation-enhanced MMP-2 activity and expression levels as well as cell viability.
A, U-251 and U-87 cells were transfected with mock (PBS), p-SV or p-MMP-2 (2 µg), and after 72 h of incubation, cells were irradiated with 0, 2, 4, 6 or 8 Gy and incubated for a further 24 h. Conditioned media was used to determine MMP-2 activity by gelatin zymography, and total cell lysates were used to determine MMP-2 levels by Western blotting. B, Total RNA was used to determine MMP-2 mRNA transcription levels by RT-PCR with gene-specific primers. GAPDH served as a loading control. C, U-251 and U-87 cells were transfected with mock, p-SV or p-MMP-2 and irradiated as described above. 24 h after radiation, the cells were fixed and processed to visualize MMP-2 expression. The cells were mounted using mounting media with DAPI to visualize the nucleus. D, U-251 and U-87 cells were transfected with mock, p-SV or p-MMP-2, and irradiated for 72 h after transfection. After a another 24 h of incubation, cell viability was analyzed by MTT assay (absorbance read at 550 nm). Columns: mean of triplicate experiments; bars: SD; *p<0.01, significant difference from mock, p-SV or irradiated controls.
p-MMP-2 inhibits radiation-enhanced glioma cell migration and invasion
Since MMP-2 has been implicated as an important factor in malignant glioma cell migration and invasion into the surrounding normal tissue, which is a major problem for any therapeutic modality, we studied the effect of p-MMP-2 on these processes. Since tumor spheroids mimic tumor growth, we allowed U-87 or U-251 cells to form spheroids, and then transfected and irradiated the cells. We observed a significant reduction in the migrating potency of cells transfected with p-MMP-2 and of cells that were concomitantly transfected and irradiated as compared to the controls. Cell migration was decreased five-fold and even more (particularly with 4 and 6 Gy) in the p-MMP-2 transfected and irradiated cells when compared to controls (Fig. 3). Matrigel invasion assay showed a significant inhibition of invasion by tumor cells that were transfected with p-MMP-2 without exposure to radiation and an even more significant dose-dependent inhibition when cells were concomitantly transfected and irradiated at various doses. Maximal inhibition was observed with 4 Gy followed by 6 Gy. Inhibition was significant when compared to mock or p-SV-transfected controls or to cells that were only subject to radiation. The normalization of the values as a percentage of the control (100%) cells treated with p-MMP-2 and concomitantly irradiated (6 Gy) showed a more than 90% inhibition (Fig. 4).
10.1371/journal.pone.0020614.g003Figure 3 p-MMP-2 transfection in combination with radiation inhibits glioma cell migration.
U-251 and U-87 cells were cultured for formation of spheroids as described in Materials and Methods. Spheroids were then transfected with mock, p-SV or p-MMP-2, and followed by irradiation as described earlier. At the end of the migration assay, spheroids were fixed and stained with Hema-3. Migration of cells from spheroids to monolayers was measured using a microscope calibrated with a stage and ocular micrometer. Columns: mean of triplicate experiments; bars: SD; *p<0.01, **p<0.001, significant difference from mock, p-SV or irradiated controls.
10.1371/journal.pone.0020614.g004Figure 4 p-MMP-2 transfection inhibits radiation-enhanced glioma cell invasion.
U-251 and U-87 cells were transfected with mock, p-SV or p-MMP-2, and irradiated as described earlier. Cells were trypsinized and counted, and 5×105 cells from each treatment condition were allowed to invade transwell inserts containing 12-µm-pore polycarbonate membranes pre-coated with Matrigel for 24 h at 37°C. Afterwards, cells were fixed and stained with Hema-3. Cells that had migrated to the lower side of the membrane were photographed under a light microscope at 20× magnification. Percentages of invading cells were quantified by counting five fields from each treatment condition. Columns: mean of triplicate experiments; bars: SD; *p<0.01, **p<0.001, significant difference from mock or irradiated controls.
p-MMP-2 inhibits tumor-induced and radiation-enhanced angiogenesis in vitro and decreases expression of angiogenesis-associated molecules
We next determined the ability of p-MMP-2 to inhibit angiogenesis. Glioblastoma is one of the most highly angiogenic tumors; MMP-2 is an important angiogenic molecule that promotes tumor vascularization, in turn rendering the tumor cells resistant to radiotherapy. Conditioned media from p-MMP-2-treated alone or in combination with radiation treated U-87 or U-251 cells failed to induce capillary-like formation when added to cultured human microvascular endothelial cells (HMECs) in contrast to conditioned media obtained from control and p-SV transfected cells, which was capable of triggering the angiogenic process. Mock and p-SV treated cells that were subject to radiation demonstrated enhanced angiogenesis, as is evident from the increase in percentage of branching when compared to their non-irradiated counterparts (Fig. 5A). Western blot analyses revealed enhanced levels of vascular endothelial growth factor (VEGF), a well-known inducer of angiogenesis, VEGF receptor-2 (VEGFR-2), and also a corresponding increase in phosphorylation of VEGFR-2 in lysates of irradiated glioma cells. p-MMP-2 treatment markedly reduced radiation-enhanced protein levels, which were almost completely inhibited when cells were simultaneously treated with the MMP-2 inhibitor and irradiated at 4 or 6 Gy (Fig. 5B). We also demonstrated the effect of radiation and the combined treatment on p38 and FAK, which are downstream molecules of VEGF signaling. Radiation-enhanced levels of phosphorylated p38 and phosphorylated FAK were largely inhibited when cells were simultaneously transfected with p-MMP-2. Levels of total p38 and FAK remained unchanged for the different treatment groups, when compared to the internal GAPDH control (Fig. 5B).
10.1371/journal.pone.0020614.g005Figure 5 p-MMP-2 inhibits radiation-enhanced tumor culture medium-induced microtubule network formation in endothelial cells and downregulates expression of angiogenesis-associated molecules.
A, Human microvascular endothelial cells (5×104) were seeded in 96-well plates and cultured with conditioned medium collected from U-251 and U-87 glioma cells transfected with mock, p-SV, and p-MMP-2, and irradiated as described earlier. 24 h after radiation treatment, the cells were washed, fixed and stained with Hema-3 and photographed. Percentages of branches were quantified by counting five fields in each condition. Columns: mean of triplicate experiments; bars: SD; *p<0.01, **p<0.001, significant difference from mock or irradiated controls. B, U-251 and U-87 transfection and radiation was carried out as described earlier. 24 h after radiation, whole cell lysates were prepared and analyzed by Western blotting for the angiogenic molecules VEGF, VEGFR-2 and p-VEGFR-2 as well as p-FAK, FAK, p-p38 and p38. GAPDH served as a loading control.
p-MMP-2 inhibits colony formation and induces apoptotic cell death in irradiated glioma cells
We performed the colony forming assay to determine the effect of p-MMP-2 and radiation on the survival of glioma cells. Treatment with p-MMP-2 followed by irradiation as well as treatment with p-MMP-2 alone significantly reduced clonogenic survival when compared to the irradiated cells (Fig. 6A). High levels of MMP-2 in gliomas and radiation-enhanced MMP-2 are known to render tumor cells less susceptible to apoptosis. TUNEL staining showed that radiation did not induce apoptosis in the glioma cells while a significant increase in TUNEL-positive cells was observed when irradiated cells were simultaneously transfected with p-MMP-2 as compared to the controls. Quantification revealed up to 80% induction of apoptosis in cells treated with the MMP-2 siRNA construct and irradiated at 6 Gy (Fig. 6B). Moreover, caspases 3, 8 and 9, which mediate the apoptotic mechanism, were cleaved and activated in irradiated cells transfected with p-MMP-2 as shown by comparison of Western blot analysis of cell lysates from the different treatment groups (Fig. 6C).
10.1371/journal.pone.0020614.g006Figure 6 p-MMP-2 inhibits colony formation and induces apoptosis and activation of pro-apoptotic molecules in glioma cells.
A, Clonogenic assay was performed as described in Materials and Methods. The cells were cultured, and colonies larger than 50 cells were counted. Columns: mean of triplicate experiments; bars: SD; *p<0.01, significant difference from radiation. B, U-251 and U-87 cells were transfected with mock, p-SV or p-MMP-2, and irradiated as described earlier. After 24 h of radiation, cells were evaluated with the TUNEL assay according to manufacturer's instructions and photographed under fluorescent microscopy. Apoptosis was measured by counting the percentage of cells that showed DNA staining in five different fields in each group. Columns: mean of triplicate experiments; bars: SD; *p<0.01, **p<0.001, significant difference from mock, p-SV or irradiated controls. C, Equal amounts of protein from whole cell lysates of control and treated cells were analyzed by Western blotting using caspase-specific primary antibodies. GAPDH served as a loading control.
p-MMP-2 combined with radiation inhibits tumor growth in vivo
For the intracerebral injections, we chose the glioblastoma cell line U-251, which forms highly invasive tumors in athymic mice in contrast with U-87 cells that form solid and well defined tumors. Ten days after tumors were induced, animals were treated on alternate days for a total of 4 doses with 60 µg per dose of p-SV or pMMP-2 or phosphate-buffered saline (mock). Mice were injected with stably transfected U-251 cells expressing a luciferase plasmid, which enabled us to monitor tumor growth by intraperitoneally injecting D-luciferin into the animals. Over a 6-week period, tumor growth was predominantly inhibited in the animals treated with p-MMP-2 alone or in combination with radiation when compared with the mock or p-SV controls as observed in the IVIS images and subsequently confirmed by hematoxylin and eosin (H&E) staining of the tumor sections (Figs. 7A–B). Control animals developed symptoms of weight loss and neurological degradation before they were euthanized, whereas animals treated with p-MMP-2 alone or concomitantly irradiated remained symptom-free. No significant differences were observed between animals treated with mock or p-SV in terms of tumor size or symptoms. Brain sections were analyzed for expression of MMP-2 by immunohistochemistry (Fig. 8A). Mice treated with p-MMP-2 alone or in combination with radiotherapy showed a decrease in MMP-2 expression. Conversely, in control and p-SV treated mice or even mice that were subject to only radiation in which MMP-2 expression was detected mostly at the invasive edge of the tumor and in cells surrounding the necrotic areas as well as reactive endothelial cells within and around the tumor area. Confirming the in vitro findings, VEGF and pFAK were also suppressed in the animals treated with MMP-2 siRNA alone or in combination with radiation (Fig. 8A). TUNEL staining of tissue sections showed that radiation did not induce apoptosis in the glioma cells while predominant TUNEL staining was observed in tissues of mice that were treated with p-MMP-2 alone and in those that were concomitantly irradiated (Fig. 8B). Further, we noticed that the invasiveness of the tumor cells into adjacent normal brain tissue of mice increased with radiation compared to mock and pSV controls, but decreased in mice that received p-MMP-2 alone or in combination of radiation (Fig. 8C). Further, tumor infiltrating cells were detected in non-tumor regions of brain sections from mice using Human nuclei (HuNu) antibody, a histological marker for identification of human cells (a specific human nuclear antigen). A considerable number of tumor infiltrating cells were observed in tumor sections from mice that received mock and pSV treatments. In addition, we observed a significant increase in the number of tumor infiltrating cells in tumor sections from mice that received radiation treatment. However, we observed a drastic reduction in radiation-induced tumor cell infiltration in tumor sections from mice that received pMMP-2 (Fig. 8D). The immunoreactivity of MMP-2 in tumor sections from mice that received both radiation and p-MMP-2 treatments also showed that p-MMP-2 inhibited radiation-induced MMP-2 expression. These results suggest that p-MMP-2 could serve as an adjuvant with low-dose radiation therapy against glioblastoma.
10.1371/journal.pone.0020614.g007Figure 7 p-MMP-2 combined with radiation inhibits tumor growth in vivo.
U-251 (1×106) cells were injected intracerebrally into athymic mice. After ten days, animals were separated into five groups and were treated on alternate days with intracerebral injections of p-SV or p-MMP-2 for a total of 4 doses (60 µg per dose) and 2 doses of radiation (4 Gy per dose) as described in Materials and Methods. Six weeks after the experiment was initiated, mice were euthanized with intracardiac perfusion of PBS, followed by formaldehyde. The brains were then removed. A, Six weeks after the experiment was initiated, an intraperitonal injection of 2.5 mg D-luciferin sodium salt diluted in 50 µL of PBS was given, and animals were photographed under the IVIS camera for fluorescent light emission. The brains were removed and fixed in 10% phosphate-buffered formaldehyde, and the fixed tissue samples were then processed into paraffin blocks. Brain sections (5 µM thick) were stained with hematoxylin and eosin (H&E), and photographed under a light microscope (4× and 40×). B, Every fifth or sixth brain section (5 µM thick) was stained with H&E solution, and the tumor masses (H&E-stained) were manually traced on the microscope attached computer screen. Areas were calculated using Image Pro Discovery Program software (Media Cybernetics, Inc., Silver Spring, MD). The total tumor volume was calculated as the summed area on all slices, multiplied by the slice separation. Columns: mean of area of tumor portion of all mice in the group (n = 8); bars: SD; *p<0.01, **p<0.001, significant difference from mock, p-SV or irradiated controls.
10.1371/journal.pone.0020614.g008Figure 8 p-MMP-2 combined with radiation enhances apoptosis in vivo.
A, Immunohistochemical analysis of brain sections using anti-MMP-2, anti-VEGF and anti-pFAK antibodies. Sections were photographed (60×). Also shown is the negative control where the primary antibody was replaced by non-specific IgG (insets). B, Tissue sections of mice were evaluated with the TUNEL assay according to manufacturer's instructions and photographed under fluorescent microscopy (60×). For the negative control, samples were incubated with label solution (without terminal transferase) instead of TUNEL reaction mixture (insets). C, siRNA against MMP-2 inhibits U251 tumor cell invasion in vivo. H&E staining was performed according to standard protocol, and representative pictures of tumor sections from mock, pSV, p-MMP-2-treated mice are shown (20× and 60×). D, Immunohistochemical analysis of brain sections using anti-human nuclei (HuNu) antibody, a histological marker for identification of human cells (a specific human nuclear antigen). Entire brain sections were photographed (4×; middle row); shown on the top row is a non-tumor region (40×; top row); and shown on the bottom row is tumor and non-tumor overlapping region (40×; bottom row). Also shown is the negative control where the primary antibody was replaced by non-specific IgG (inset).
Discussion
Findings of the present study demonstrate the anti-tumor efficacy of combining a plasmid vector-mediated MMP-2 inhibitor (p-MMP-2) with ionizing radiation to target radiation-induced invasiveness and angiogenesis in glioma cells. Our results show that p-MMP-2 significantly inhibited radiation-enhanced MMP-2 protein expression and activity and its associated tumorigenic properties, such as cell proliferation, tumor cell migration, invasion, and angiogenesis in two human glioma cell lines, U-251 and U-87. We also show that the decrease in MMP-2 expression/activity further modulated important downstream signaling molecules directing the cells towards an apoptotic phenotype when administered in combination with radiotherapy.
MMP-2 activation has been associated with invasive phenotype in glioma cells [31]–[33]. In our experiments, the MMP-2 inhibitor, p-MMP-2, significantly downregulated MMP-2 protein expression and activity and modulated multiple biological behaviors that determine the malignant progression of gliomas. Previous reports show that ionizing radiation increased MMP-2 activity and protein secretion along with invasiveness of glioma cells [15]. We observed that ionizing radiation induced a dose-dependent increase in MMP-2 activity in U-251 and U-87 cells. As demonstrated by Wick, et al., [34] we also observed that irradiation promoted the accumulation of MT1-MMP and loss of TIMP-2 protein, possibly shifting the balance of these molecules in favor of MMP-2. Treatment of cells with p-MMP-2 and in combination with irradiation seemed to show a slight, though not significant, decrease in levels of MT1-MMP and more or less unchanged levels of TIMP-2 when compared to irradiated cellular protein levels (data not shown). This was of interest as the activation and regulation of MMP-2 is closely associated with MT1-MMP and TIMP-2. Given the specificity of our p-MMP-2 siRNA and its ability to significantly downregulate radiation-enhanced invasiveness, angiogenesis, and cell survival, it is very evident that MMP-2 might be associated with the exceeding invasiveness after radiation. Several studies have shown that MMP-2 and MMP-9 promote cancer progression by increasing tumor cell growth, migration, invasion, metastasis, and angiogenesis [7], [31], [32], [35], [36]. Thus, following radiation, it is logical that the elevated levels of MMP-2 and a subsequent increase in activation of the MMP-2 proteolytic system would only lead to enhanced invasiveness. MMPs exert these effects by cleaving a diverse group of substrates, which include not only structural components of the extracellular matrix, but also growth factor binding proteins, growth factor precursors, receptor tyrosine kinases, cell adhesion molecules, and other proteinases [9]. There was a marked increase in VEGF and VEGFR-2 secretion and an increase in phosphorylated VEGFR-2 after irradiation of glioma cells, which was almost completely inhibited by p-MMP-2 treatment. Radiation therapy plays a role in upregulating the expression of molecules such as VEGF, VEGFR, and EGFR [37]–[39], all of which are considered key targets for novel anti-cancer therapies [40], [41]. Further, studies support a possible link between MMP-2 and the potent angiogenic factor, VEGF. MMPs may stimulate VEGF release from tumor cells either directly or by activating factors involved in VEGF release or by mobilizing VEGF from the extracellular compartments [42]–[44]. Thus, by decreasing the availability of VEGF, MMP-2 downregulation efficiently blocked radiation-enhanced angiogenesis in U-251 and U-87 cells in the present study. Several studies demonstrated a marked increase in VEGF secretion following irradiation of cancer cells [15]. Kaliski, et al. [45] showed that irradiated melanoma cells displayed enhanced invasive capacity with increased MMP-2 expression and subsequently induced VEGF protein secretion. Specific MMP-2 inhibition blocked VEGF upregulation and inhibited tumor growth and angiogenesis in vivo. Hovinga, et al. [46] showed that radiation-enhanced VEGF secretion increased angiogenesis and decreased apoptosis, both leading to GBM radioresistance. As a consequence of VEGF signaling, many proteins are activated by VEGFR-2 via an unknown mechanism; these proteins include Src, phosphoionisitide 3-kinase (PI3K), focal adhesion kinase (FAK), and p38 mitogen activated protein kinase (p38 MAPK). These downstream signal transduction molecules propagate a signal leading to several different endothelial cellular functions such as survival, permeability, migration, and proliferation [47]. In the present study, concomitant with the decrease in VEGF and VEGFR-2, p38 and FAK phosphorylation were also downregulated, with a corresponding inhibition of tumor cell migration and invasiveness. Radiation-enhanced VEGF has also been shown to induce anti-apoptotic pathways and thereby promote tumor growth after irradiation. VEGF is reported to reduce apoptosis after irradiation in human leukemia cells and in human and murine mammary adenocarcinoma cells [48]–[50]. The simultaneous administration of p-MMP-2 with radiation opposed the anti-apoptotic phenotype and led to cleavage/activation of the pro-apoptotic molecules caspase 3, caspase 8 and caspase 9. Studies have shown that virtually all glioma-derived cell lines exhibit resistance to radiation and do not undergo radiation-induced primary apoptosis [51], [52]. In the present study, U-251 and U-87 cells demonstrated very high levels of apoptosis as shown by TUNEL assay when irradiated cells were concomitantly administered p-MMP-2. These findings were further validated in vivo. In our in vivo studies, p-MMP-2 reduced tumor size by more than 80% compared to mock or pSV. Although the tumor size in mice that received radiation treatment alone did not increase significantly, the invasive nature of the tumor did increase drastically. Tumor cells that manage to escape the lethal effects of irradiation often adapt more aggressive properties, proliferate more rapidly and display enhanced migratory, invasive and angiogenic potential [53], [54]. Further, sub-lethal doses of radiation have a toxic effect on healthy tissues and may also result in a more aggressive and malignant tumor due to the development of resistant cancer cell sub-populations that have enhanced proliferative, invasive and angiogenic properties [54]. Treatment with both p-MMP-2 and radiation did reduce radiation-induced tumor cell infiltration and tumor size by ∼80% compared to mock and pSV controls (Figs. 7A–B).
Radiation-enhanced expression as well as activation of the MMP-2 proteolytic system elevate or modify the bioavailability of several molecules that promote tumor progression [17]–[23]. Further, enhanced MMP-2 may increase tumor survival by decreasing apoptosis, stimulating proliferation, and increasing angiogenic and invasive potential [13]–[16]. Since the downregulation of MMP-2 seems to cause the modulation of several other molecules and pathways, MMP-2 siRNA is effective in modulating radiation-enhanced molecules, invasiveness and altering signaling pathways, thereby driving the cells through apoptosis.
It is worth noting that in all of our experiments, glioma cells that were treated only with p-MMP-2 showed a significant downregulation of tumorigenic properties and were capable of acquiring an apoptotic phenotype. This proves the anti-tumor efficacy of p-MMP-2. Nevertheless, since radiotherapy is the standard treatment for glioma and malignant glioma is one of the most radioresistant tumor types, the administration of p-MMP-2 prior to radiotherapy could be a potent adjuvant therapeutic approach to improve the efficacy of radiotherapy for glioma. Adenoviral vectors have been widely used both in cells and in vivo for the delivery of MMP-2 siRNA [27]–[29]. Although they may be capable of efficiently delivering the gene to the target cells, adenovirus carries the risk of triggering an immune response, which limits the length of time that the gene expression can be maintained in the target cells. Plasmids, on the other hand, being non-infectious and non-immunogenic, could be much safer and provide stability of gene transfer [30]. The results of our study demonstrated the efficacy of p-MMP-2 in inhibiting radiation-enhanced tumor invasion and progression and suggest that it may act as a potent adjuvant for radiotherapy in malignant glioma.
Materials and Methods
Ethics Statement
The Institutional Animal Care and Use Committee (IACUC) of the University of Illinois College of Medicine at Peoria, Peoria, IL, USA, approved all surgical interventions and post-operative animal care. The consent was written and approved. The animal protocol number is 858, dated May 27, 2009, and renewed on April 27, 2010.
Cell cultures
Glioblastoma cell lines U-251 and U-87 obtained from American Type Culture Collection (ATCC, Manassas, VA) were grown in Dulbecco's modified Eagle's medium (DMEM). Cultures were supplemented with 1% glutamine, 100 µg/mL streptomycin, 100 U/mL penicillin and 10% fetal calf serum (FCS) and maintained in a humidified atmosphere containing 5% CO2 at 37°C.
Plasmid vector constructs, transient transfection, and radiation treatment
The plasmid constructs carrying siRNA against MMP-2 (p-MMP-2) and a scrambled MMP-2 sequence (p-SV) were designed and constructed as previously described [27]. pcDNA3.0 (Invitrogen) with a CMV promoter was used for the construction of vectors. The following siRNA sequences were cloned into the pcDNA3.0: aacggacaaagagttggcagtatcgatactgccaactctttgtccgtt for p-MMP-2 inverted repeat sequence and gcacggaggttgcaaagaataatcgattattctttgcaacctccgtgc for p-SV inverted repeat sequence. Annealed oligos were sequentially ligated to pcDNA3.0 at the Nhe I site. Briefly, 150,000 cells were seeded in 6-cm diameter dishes and grown in DMEM supplemented with 10% FCS. The cells were transfected with mock (PBS), p-SV, or p-MMP-2. Transfections were performed using FuGENE HD transfection reagent (Roche Applied Science, Indianapolis, IN) as per the manufacturer's instructions. After 72 h of transfection, cells were irradiated with a single dose of radiation (2, 4, 6, 8, 10 or 12 Gy). The RS 2000 Biological irradiator (Rad Source Technologies, Boca Raton, FL) X-ray unit operated at 150 kv/50 mA was used for radiation treatments. All experiments were performed 24 h after radiation.
Gelatin zymography
Cells were transfected with mock, p-SV or p-MMP-2 alone or in combination with various single doses of radiation (2, 4, 6, 8, 10 or 12 Gy) to which cells were exposed 72 h after transfection. Following radiation, cells were cultured for 24 h in serum-free DMEM/F-12 medium. The serum-free conditioned medium was collected and equal amounts of protein were used to determine MMP-2 activity. Gelatin zymography was performed as previously described [55].
Reverse transcription-PCR
Cells were transfected and irradiated as described above. Total RNA was extracted using TRIZOL reagent (Life Technologies, Rock-ville, MD) according to the manufacturer's protocol. RT-PCR was performed as described previously [27]. PCR products were resolved on 2% agarose gels and were visualized by ethidium bromide staining. To normalize for the amount of input RNA, RT-PCR was performed with primers for GAPDH. We used the following specific primers: MMP-2, forward 5′-GTGCTGAAGGACACACTAAAGAAGA-3′, and reverse 5′-TTGCCATCCTTCTCAAAGTTGTAGG-3′; GAPDH, forward 5′-TGAAGGTCGGAGTCAACGGATTTGGT-3′, and reverse 5′-CATGTGGGCCATGAGGTCCACCAC-3′. Images were generated by Alpha Innotech Image Acquisition and Analysis Software.
Western blot analysis
U-251 and U-87 cells were transfected and irradiated as described earlier. Equal amounts of total protein from cell lysates obtained by lysing cells in a suitable buffer [50 mM/L Tris-HCl (pH 7.4), 150 mM/L NaCl, 1% IGEPAL, 1 mM/L EDTA, 0.25% sodium deoxycholate, 1 mM/L sodium fluoride, 1 mM/L sodium orthovanadate, 0.5 mM/L PMSF, 10 µg/mL aprotinin, 10 µg/mL leupeptin] were separated by SDS–PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA). After blocking with 5% nonfat dry milk and 0.1% Tween-20 in PBS, membranes were incubated with 1∶1000 dilution of primary antibodies followed by incubation in HRP-conjugated secondary antibodies. Membranes were developed using the ECL system (Amersham Bioscience Corp., Piscataway, NJ). Primary antibodies used in this study were: MMP-2, MMP-9, VEGF, VEGFR-2, pVEGFR-2, pFAK, FAK, p-p38, total p38, caspase 3, caspase 8, caspase 9, GAPDH (used as a loading control), HRP conjugated secondary antibodies (Santa Cruz Biotechnology, SantaCruz, CA).
Cell proliferation assay
Cell growth was assessed by MTT assay. U-251 and U-87 cells (5×103 cells per well) were grown in 96-well plates, transfected and irradiated as described earlier. Proliferation rate was measured 24 h after treatment by adding 20 µL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution (R&D Systems, Minneapolis, MN) to each well and further incubating the plate for 1 h. Samples were read on a microplate reader using test wavelength of 550 nm and reference wavelength of 655 nm.
Immunofluorescence
U-251 and U-87 cells were cultured in 8-well chamber slides (Nalge Nunc International, Naperville, IL) (104 cells per well), transfected and irradiated as described earlier. After 24 h of treatment, cells were fixed in 3% formaldehyde and incubated with 0.2% Triton X-100 in PBS. Cells were then blocked with 3% bovine serum albumin (BSA) for 1 h and incubated with mouse anti-MMP-2 in 1% BSA in PBS (dilution 1∶500). Cells were washed with PBS and mouse fluorescein isothiocyanate (FITC)-conjugated secondary antibody was added for 1 h. Finally, slides were washed with PBS, mounted and examined under a fluorescent microscope connected to an Olympus camera.
Spheroid migration assay
Cells (4×104) were seeded in 96-well plates coated with 1% agarose in PBS and cultured on a shaker at 100 rpm for 48 h. After single spheroids formed, cells were transfected with mock, p-SV or p-MMP-2 alone or in combination with various doses of radiation as described earlier. Treated spheroids were placed in the center of each well of vitronectin-coated 96-well plates and were cultured at 37°C for 24 h in serum-free DMEM, after which they were fixed and stained with Hema-3. The migration of cells from the center of the spheroids to monolayers was measured using a microscope calibrated with a stage and ocular micrometer. We used Image Pro Discovery Program software to determine the index of cell migration (Media Cybernetics, Inc., Silver Spring, MD) as described previously [36].
Matrigel invasion assay
U-251 and U-87 cells were transfected and irradiated as described earlier. Transwell inserts (ThinCertTM; Greiner Bio-One, Monroe, NC) with 8 µm pores were coated with 200 µL growth factor reduced Matrigel (Collaborative Research, Inc., Boston MA) at a concentration of 0.7 mg/mL in DMEM serum-free medium for 3–4 h. 500 µL of cell suspension (5×105 cells) were added into wells in triplicate. Cells were allowed to invade through the Matrigel for 24 h. Cells in the upper chamber were removed by cotton swab. Cells adhered on the outer surface that had invaded though the Matrigel were fixed, stained with Hema-3, photographed and counted under a light microscope as previously described [36].
In vitro angiogenesis assay
Conditioned medium-induced microtubule network formation in vitro was determined. Cells were transfected and irradiated as described earlier. Conditioned medium from U-251 and U-87 control and treated cells was collected and added to HMEC (5×104 per well) seeded the previous day in 96-well plates. HMEC were incubated for 24 h and the formation of microtubule networks was examined after fixing and staining the cells with Hema-3. The angiogenic result was measured by counting the relative branch points in each field for the different treatment groups.
Clonogenic assay
U-251 and U-87 cells were transfected and irradiated as described earlier. Cells were trypsinized and 200 cells were seeded in 100-mm Petri dishes. On day 10 after irradiation, cells were fixed in methanol and stained with Giemsa. Colonies (>50 cells) were counted and survival fraction was calculated as number of colonies divided by the number of plated cells.
TUNEL assay and apoptosis
U-251 and U-87 cells were grown in 8-well chamber slides, transfected and irradiated as described earlier. Then, terminal deoxynucleotidyl transferase biotin-dUTP nick end-labeling (TUNEL) assay was performed using an apoptosis detection kit (Roche Applied Science, Indianapolis, IN) according to the manufacturer's instructions. Briefly, cells were fixed with 3% paraformaldehyde and incubated with 0.2% Tween-20, 0.2% BSA in PBS for 15 min at room temperature. Cells were washed with PBS and incubated with terminal deoxynucleotidyl transferase end-labeling cocktail for 60 min. The reaction was stopped with Tris-borate buffer, and cells were washed and incubated for 30 min in the dark with avidin-FITC solution diluted in the provided blocking buffer. Finally, cells were washed, mounted and photographed under a fluorescent microscope. Apoptosis was measured as the average percentage of positive nuclei per field for each treatment.
Animal studies
Experiments in nude mice were carried out according to the protocol approved by the IACUC of this institution. U-251 glioblastoma cells stably transfected with a luciferin-expressing plasmid were grown in serum-containing culture media. Cells were trypsinized and resuspended in PBS. 10 µL (1×106 cells) was injected intracerebrally into nude mice. To detect tumor growth, animals received an intraperitoneal injection of 2.5 mg of D-luciferin sodium salt (Gold Biotechnology, St Louis, MO) suspended in 50 µL of PBS, and tumor growth was monitored using IVIS-200 Xenogen imaging system (Xenogen Corporation, Alameda, CA). Tumors were allowed to grow for 10 days before animals were separated into five treatment groups of eight mice each. Two groups of animals were treated on alternate days with intracerebral injections of p-SV or p-MMP-2 for a total of 4 doses (60 µg per dose in 10 µL volume; flow rate ∼1 µL/10 sec). Between the first and the second injections and between the second and the third injections, the animals that were administered p-MMP-2 were also irradiated with a dose of 4 Gy each time. Another group of animals was subjected to only irradiation at 8 Gy. Only the tumor region was exposed to radiation while the rest of the mouse body was covered with lead sheet. Control animals were injected with PBS (mock) only. After 6 weeks and/or when the control animals started showing symptoms, animals were anesthetized and euthanized by intracardiac perfusion of PBS followed by formaldehyde. The brains were removed, and fixed in 10% phosphate-buffered formaldehyde and the fixed tissue samples were then processed into paraffin blocks. Every fifth or sixth section (5 µm thick) was stained with hematoxylin-eosin solution (H&E) and the tumor masses (H&E-stained) were manually traced on the microscope attached to a computer. Areas were calculated using Image Pro Discovery Program software (Media Cybernetics, Inc., Silver Spring, MD). The total tumor volume was calculated as the summed area on all slices, multiplied by the slice separation [56], and normalized to the volume of tumor in p-SV treated mice. For the immunohistochemical experiments, brain sections were deparaffinized in xylene and rehydrated through graded alcohol. Then, slides were incubated with 0.1% Triton X-100, blocked with 3% BSA in PBS, and incubated with anti-MMP-2, anti-VEGF, anti-pFAK (Santa Cruz Biotechnology, SantaCruz, CA) or anti-human nuclei (HuNu) antibody (Millipore, Temecula, CA) (1∶10 dilution). After a rinse in PBS, slides were incubated with HRP-conjugated secondary antibody for 1 h at a dilution of 1∶300, washed again with PBS and incubated with 0.05% 3,30-diaminobenzidine as chromogen. Finally, slides were counterstained with hematoxylin, mounted and observed under a light microscope. Tissue sections were also subject to TUNEL assay performed using an apoptosis detection kit (Roche Applied Science, Indianapolis, IN) according to the manufacturer's instructions.
Statistical analysis
Data are presented as the arithmetic mean ± standard deviation (SD) of at least three independent experiments, each performed at least in triplicate. Results were analyzed using a two-tailed Student's t-test to assess statistical significance. In the animal experiments, the mean differences in tumor volumes were compared among treatment groups using a one-way analysis of variance (ANOVA). Statistical differences are presented at probability levels of p<0.05, <0.01 and <0.001.
We thank Shellee Abraham for manuscript preparation and Diana Meister and Sushma Jasti for manuscript review.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This research was supported by a grant from the National Institute of Neurological Disorders and Stroke, NS064535 (to J.S.R.). The contents are solely the responsibility of the authors and do not necessarily represent the official views of National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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BMC GastroenterolBMC Gastroenterology1471-230XBioMed Central 1471-230X-11-602159994010.1186/1471-230X-11-60Research ArticleSlug down-regulation by RNA interference inhibits invasion growth in human esophageal squamous cell carcinoma Tang Peng [email protected] Zhentao [email protected] Kejun [email protected] Yu [email protected] Zhongliang [email protected] Shaoyan [email protected] Dong [email protected] Yanbing [email protected] Department of Esophegeal Oncology, Key Laboratory of Cancer Prevention and Therapy, Cancer Institute and Hospital of Tianjin Medical University, Tianjin 300060, R.P. China2 Department of surgery, the Affiliated Hospital of medical college, QingDao University, QingDao, Shan Dong Province, 266003. R.P. China3 Department of Laboratory, the Affiliated Hospital of medical college, QingDao University, QingDao, Shan Dong Province, 266003. R.P. China2011 20 5 2011 11 60 60 20 4 2010 20 5 2011 Copyright ©2011 Tang et al; licensee BioMed Central Ltd.2011Tang et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive carcinomas of the gastrointestinal tract. We assessed the relevance of Slug in measuring the invasive potential of ESCC cells in vitro and in vivo in immunodeficient mice.
Methods
We utilized RNA interference to knockdown Slug gene expression, and effects on survival and invasive carcinoma were evaluated using a Boyden chamber transwell assay in vitro. We evaluated the effect of Slug siRNA-transfection and Slug cDNA-transfection on E-cadherin and Bcl-2 expression in ESCC cells. A pseudometastatic model of ESCC in immunodeficient mice was used to assess the effects of Slug siRNA transfection on tumor metastasis development.
Results
The EC109 cell line was transfected with Slug-siRNA to knockdown Slug expression. The TE13 cell line was transfected with Slug-cDNA to increase Slug expression. EC109 and TE13 cell lines were tested for the expression of apoptosis-related genes bcl-2 and metastasis-related gene E-cadherin identified previously as Slug targets. Bcl-2 expression was increased and E-cadherin was decreased in Slug siRNA-transfected EC109 cells. Bcl-2 expression was increased and E-cadherin was decreased in Slug cDNA-transfected TE13 cells. Invasion of Slug siRNA-transfected EC109 cells was reduced and apoptosis was increased whereas invasion was greater in Slug cDNA-transfected cells. Animals injected with Slug siRNA-transfected EC109 cells exhihited fewer seeded nodes and demonstrated more apoptosis.
Conclusions
Slug down-regulation promotes cell apoptosis and decreases invasion capability in vitro and in vivo. Slug inhibition may represent a novel strategy for treatment of metastatic ESCC.
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Background
Despite improvements in detection, surgical resection, and (neo-) adjuvant therapy, the overall survival for esophageal squamous cell carcinoma (ESCC), one of the most aggressive carcinomas of the gastrointestinal tract, remains lower than that of other solid tumors due to distant metastasis [1]. Therefore, efforts are ongoing regarding exploration of novel targets and strategies for the management of ESCC, and gene targeting therapies in particular are promising.
Multiple studies focusing on the effects of various biological factors on the malignant potential of ESCC have been conducted [2-4]. One of those factors is E-cadherin as the loss of E-cadherin is an important step in the process of epithelial-to-mesenchymal transition (EMT) in cancer [5-8].. EMT is an embryonic process necessary for morphogenesis in multi-organ beings. Cells eventually lose E-cadherin and acquire a more mesenchymal, motile phenotype [3,4]. In ESCC, loss of E-cadherin expression is associated with tumor invasiveness, metastasis, and prognosis [2,9-11].
Slug is a member of the snail family of repressors, and is expressed in the neural crest and in mesodermal cells emigrating from the primitive streak in chick embryos [12]. Recently, it has been demonstrated that Slug is involved in the control of apoptosis and in the EMT that is linked to the acquisition of an invasive phenotype [13,14]. Uchikado et al. [11] recently found that E-cadherin and Slug expression were associated with ESCC properties, including depth of invasion, lymph node metastasis, stage, lymphatic invasion, and prognosis. Paras et al. [15] recently reported when the OE33 cell line was is transiently transfected with full length human Slug vector, the expression of E-cadherin was repressed and Slug was negatively correlated with E-cadherin expression. Bcl-2, an apoptosis-related genes, whose expression is Slug dependent in mice [16]. It has been suggested that Slug may be the upregulation gene of bcl-2. Therefore, we believe that blocking Slug expression could be applied to cancer targets.
In this study, we evaluated the effect of Slug blockade and Slug overexpression on survival and invasion in ESCC in vitro and vivo. In a pseudometastatic model of ESCC in mice we demonstrated the existance of an additive effect of Slug silencing in reducing metastatic burden. These data suggest that Slug is a relevant gene for regulation of the metastatic potential of ESCC cells and that Slug inhibition may be suitable as a treatment for metastatic ESCC.
Methods
Cell lines
EC109 were kindly provided by Dr. Li (The First Affiliated Hospital, Zhangzhou University, Henan Key Laboratory of Tumor Pathology, Zhangzhou, China). TE13 was obtained from the Japanese Cell Resource Center for Biomedical Research (Sdai, Japan);EC109 cell lines showed the highest level of Slug expression and TE13 cell lines showed less Slug expression [17].
Slug siRNA construction
Three different double strand siRNA oligonucleotides were successfully made. The sequence was submitted to a Blast search against the human genome sequence to ensure that only the Slug gene was targeted. The following sequences were as followed Sense:5-gatccgcgattgacaccagattcaagagatctgggtactgcattggtcttttttc-3;antisense:5-agctttccaaaaaagaccaatgcagtacccagatctcttgaatctgggtactgcattggtca-3;Sense:5-tgccctcctcacaatagtc tttcaagagaagactattgtgaggagggcttttttc-3;Antisense:5-gatcgaaaaaagccctcctcacaatagtcttctcttgaaagactattgtgaggagggca-3;Sense:5-tgcaaatgtacccaatgatattcaagagatatcattgggtacatttgcttttttc-3;Antisense:5-gatcgaaaaaagcaaatgtacccaatgatatctcttgaatatcattgggtacatttgca-3. A validated medium GC scramble (SCR) double strand siRNA oligonucleotide was used as control for transfection(data not shown). It does not match any mammalian sequences currently available on online databases. The double strand siRNA oligonucleotides were cloned into the pGCL-GFP vector. The pGCL-GFP vectors with double strand siRNA oligonucleotides inserts were used as entry clone vectors and transferred into the vector pDC316-EGFP-U6 [Invitrogen, Carlsbad, CA, USA] using the Gateway BamHI and HindIII enzyme mix according to the manufacturer's directions [Invitrogen] to generate pEGFP-siRNA-Slug (siRNA-Slug)or pEGFP-siRNA-mock(mock siRNA).
Slug cDNA construction
The full coding region of human Slug (GenBank, AF084243) was amplified by PCR using primers 5'-gctgtaggaaccgccgtgtc-3' and 5'-atttgtcatttggcttcggagtg-3' from cDNA of human EC109 cell line, employing human genomic DNA as the template. The DNA sequence was confirmed and he product was subcloned into the expression vector pGEM-T basic (Clontech, Palo Alto, CA), followed by digestion with BamHI to isolate the BamHI-BamHI fragment of the Slug DNA. The fragment was then subcloned into the vector pDC316-EGFP-U6 [Invitrogen, Carlsbad, CA, USA]and digested with BglII to construct the pSlug cDNA or pEGFP expression plasmid.
Transcient and stable transfection
The EC109 (TE13) cells were cultured in media for 7 days, plated in a six-well plate with 1 × 106 cells per well using 2 ml culture medium without antibiotics, and grown to 60-80% confluence for the time of transfection. The Lipofectamine 2000 reagent (Invitrogen; Carlsbad, CA) was used following the protocol set by the manufacturer to transfect the cells with siRNA(cDNA). The following groups were set: [1] RNAi (cDNA) group: the cells were transfected with vector siRNA-Slug(Slug cDNA; [2] Negative transfection group: the cells were transfected with vector siRNA-mock(pEGFP); [3] Control group: the cells were transfected with only liposome and no plasmid. The transfected cells were cultured for 24-72 h, then medium was removed. Stably expressed clones, used for in vivo study and invasion study, were selected by using medium containing G418(250-300 ug/ml) for 28 days. Cells were routinely maintained in selection media containing 200-250 ug/ml of G418-sulfate to avoid overgrowth of nontransfected cells.
Measurement of Apoptosis
The nucleosomal DNA degradation was quantified by Cell Death Detection ELISA kit using antihistone antibody (Roche, Mannheim, Germany) as described previously [18]. Briefly, 1 × 105 EC109 or TE13 cells were seeded in 5-cm culture dishes and allowed to adhere overnight. After treatment with siRNA-Slug (Slug cDNA) under the same schedule as described above for 72 h, following which both adherent and floating (apoptotic) populations were harvested. They were lysed in NP-40 lysis buffer and the nucleosomes in the supernatant were detected photometrically using an ELISA plate Reader (SpectraMax 190, Molecular Devices). The readings were expressed as degree of apoptosis considering the untreated control as 1.
To detect DNA fragmentation in cell monolayers after siRNA-Slug (Slug cDNA) treatment, TUNEL was performed by using the in situ Cell Death Detection kit (Roche, Inc.) that relies on fluorescent labeling of DNA strand breaks according to the manufacturer's instructions. Positive apoptotic nuclei were recorded by fluorescence microscopy.
In Vitro Cell Growth Assay
The in vitro growth effects of siRNA-Slug or Slug cDNA on EC109 or TE13 cells were assessed using MTT (Sigma Chemical Co.) Briefly, 1 × 104 cells were seeded in each well of 96-well microtiter plates and allowed to attach overnight. After treatment with siRNA-Slug (Slug cDNA) under the same schedule as described above for 24-72 h, 20 ml of 5 mg/ml MTT in PBS was added to each well, followed by incubation for 4 hours at 37°C. The formazan crystals were dissolved in DMSO. The optical density was determined with a microculture plate reader (Becton Dickinson Labware, Lincoln Park, NJ) at 540 nm. Absorbance values were normalized to the values obtained for the vehicle treated cells to determine the percentage of surviving cells. Each assay was performed in triplicate.
Clonogenicity Assay (Soft Agar Assay)
In vitro tumorigenecity and anchorage-independent growth were assayed using standard methods. Stably transfected cells were harvested and suspended in culture medium. One percent agarose was coated onto 35-mm plates, and further overlaid with cell suspension (5,000 cells in 0.5% agarose). Plates were incubated in humidified tissue culture incubator at 37°C, 5% CO2, for 2 weeks. Visible colonies were counted under a phase-contrast microscope.
Quantitative real-time PCR(Q-PCR)
Total RNA was isolated with TRIzol Reagent (Invitrogen) and first strand cDNA was synthesized from 1 ug total RNA using Oligo d(T) primer (Invitrogen) and ReveTra Ace (TOYOBO, Osaka, Japan). Q-PCR was done using 2 ul of first-strand cDNA to quantitatively determine the relative amounts of Slug cDNAs. Primers used were for Slug: F: 5-AGA TGC ATA TTC GGA CCC ACA-3 and R: 5-CCTCAT GTT TGT GCA GGA GAG-3. β-actin, 5-CAA CTG GGA CGA CAT GGA GA-3 and 5-CAG GCA GCT CGT AGC TCT TC-3 [Genebank accession BC_004251]. β-actin was used as internal control in all reactions. Real Time PCR reactions were carried out in triplicate in a volume of 25 μl containing 50 ng of cDNA template,1× SYBR Green Mix(Toyobo, Osaka, Japan), and 200 nM or 400 nM of forward and reverse primers. Samples were heated to 95°C for one and a half min, followed by 30 amplification cycles for 30 sec at 95°C and for 30 sec at 65°C for annealing and amplification. Product purity was controlled by melting point analysis. The amount of Slug mRNA was normalized to the endogenous reference β-actin and each experiment of ESCC cell line was used Gene Expression Macro Software (Version 1.1, BioRad).
Western blotting
Cellular proteins were extracted and separated on SDS-PAGE gels, and Western blotting analyses were carried out as described previously(19). Antibodies used were anti-Slug(G-18:1:200), anti-Bcl-2 (N-19: 1:250) and anti-E-cadherin(G-10: 1:200). All were from Santa Cruz Biotechnology; anti-β-actin (AC-15:1:500) from Sigma-Aldrich.
Subcutaneous xenografted tumor model
Tumor samples(metastasis node) fixed in 10% neutral buffered formalin were embedded in paraffin using automatic embedding equipment, after which 5-μm sections were prepared. Immunohistochemical analysis for Slug (dilution at 1:100; Santa Cruz Biotechnology)was done on paraffin-embedded sections of mice according to the manufacturer's instruction. After washing with phosphate-buffered saline, the sections were incubated with biotinylated secondary antibody. Positive staining of Slug was seen in the cytoplasm of tumor cells. The results of the immunohistochemical stainings were evaluated by the percentage of positively stained carcinoma cells. TUNEL staining for tumor tissue was based on the protocol of the Dead End Colorimetric TUNEL System (Promega). The tissue sections were viewed at ×100 magnification and images were captured with a digital camera. Percentage of apoptotic cells was defined as TUNEL-positive cells among 1000 tumor cells. Percentage of TUNEL-positive cells in each group were calculated from three tumor
specimens.
Tumor invasion in Matrigel-coated chambers
The BD BioCoat Matrigel™ invasion chambers with polyethylene terephthalate-filters coated with matrigel basement membrane matrix (6 wells, 8 μm pore size; BD Biosciences, Franklin Lake, NJ) were re-hydrated just before the assay using FBS-free DMEM according to the manufacturer's instructions. The chambers were assembled using freshly prepared matrigel-coated filters and DMEM containing 0.8 mL NIH-3T3 as a chemoattractant in the lower compartment. Stably siRNA Slug transfected or Slug cDNA-transfected cells were harvested by trypsinization, and suspended in DMEM containing 10% FCS. The cells (at a concentration of 1.25 × 105 cells/2 ml) were added to the invasion chamber containing a matrigel-coated filter. The assembled chambers were incubated for 24 h at 37°C. At the end of the incubation, nonmigrating cells, which remained on the upper surface of the filter, were completely removed by wiping with a cotton swab. The cells on the bottom surface of the filter were fixed with 100% ethanol for 30 sec and stained with toluidine blue for 10 min. Cells migrated in the lower chamber were counted. Experiments were repeated thrice.
In vivo analysis
Immunodeficient male mice, 4 to 6 weeks old, were purchased from Shang Hai Animal Center. All animals were maintained in a sterile environment and cared for within the laboratory animal regulations of the Ministry of Science and Technology of the People's Republic of China (http://www.most.gov.cn/kytj/kytjzcwj/200411/ t20041108). Autoclaved cages containing food and water were changed once a week. Mouse body weight was measured every 3 to 4 days. On the day of tumor cell inoculation, tumor cells at 60% to 80% confluence were trypsinized and resuspended in fetal bovine serum-free culture medium. Animals were injected in the abdominal cavity with 5 × 106 cells(siRNA Slug transfected cells, siRNA-mock transfected cells or control cells). The experiments were terminated 21 days. Animals were sacrificed by CO2 inhalation, autopsy was carried out for assessment of metastases macroscopically. The number of the seeded tumor in the abdominal cavity is used for assessment of metastases.
Statistical analysis
All statistical analyses were performed using the SPSS11.0 software. The results were presented as means ± SD of three replicate assays. Differences between different groups were assessed using ANOVA or Dunnett t-test. A P value of <0.05 was considered to indicate statistical significance.
Results
Slug modulates invasion in ESCC cells in vitro
Recent data indicate that Slug expression is relevant for melanoma metastasis [20]. We undertook experiments to assess whether Slug is involved in ESCC invasion and metastasis. We tested whether Slug knockdown affected the invasion capabilities of ESCC cells by using an in vitro invasion assay. In Slug-silenced EC109 cells, invasion was significantly reduced (Figure 1 A). Western blot analysis resulted in Slug protein at significantly lower levels in siRNA Slug-transfected EC109 cells (Figure 1 C, P < 0.05). The results indicate that Slug-silence affected the invasion capabilities of ESCC cells. We also tested the effects of Slug cDNA transfection on the invasion capability of ESCC cell line TE13. Expression of Slug was significantly increased in Slug cDNA-transfected cells(Figure 1 D, P < 0.05) and the invasion capability was significantly elevated(Figure 1 B, P < 0.05). Compared to untreated cells, no further decrease or increase in invasion capability was observed in mock transfected cells. Together, these data show that Slug modulates invasion of ESCC cells in vitro.
Figure 1 Slug regulates ESCC cells invasion in vitro. A,B, Stable Slug-transfected EC109 cells or Slug cDNA-transfected TE13 cells(1.25 × 105) were seeded into the upper chamber of the Transwell and the lower chamber was filled with 0.8 mL NIH-3T3 conditioned medium to induce chemotaxis. After 24 h of incubation at 37°C, the cells that invasion through the pores to the lower surface of the filter were counted under a microscope. Three invasion chambers were used per condition. The values obtained were calculated by averaging the total number of cells from three filters.C,D, Slug Protein expression was determined by Western blot in stable Slug-transfected EC109 cells or Slug cDNA-transfected TE13 cells.
Slug affects the in vitro growth of ESCC cell lines by affecting proliferation
This study analyzed the effect of Slug on the cell growth of EC109 and TE13 cell lines. EC109 cell lines were transfected with siRNA Slug, TE13 cell lines were transfected with pSlug cDNA, and their viability was followed for 72 hours using a MTT cell proliferation assay. Cell growth over the 72-hour period for the EC109 and TE13 cell lines transfected with siRNA Slug and Slug cDNA is shown in Figure 2A and 2B, respectively. Each value is measured in triplicate. Significant growth inhibition was found in siRNA Slug-transfected EC109 cell lines compated with mock siRNA control (P < 0.05)(Figure 2A). On the contrary, Slug cDNA promotes growth in TE13 cell lines compated with mock cDNA transfected TE13, although the difference was not significant (P = 0.085, Figure 2B). Figure 2A and 2B showed that the growth curves for Slug siRNA-transfected EC109 cells were significantly lower than those for control cells, and the growth curves for Slug cDNA-transfected TE-13 cells were significantly higher than those for control cells. Furthermore, colony formation assay in monolayer culture showed that the number of surviving colonies of Slug siRNA-transfected cells was markedly decreased compared to those of control cells, and the number of surviving colonies of Slug cDNA-transfected cells was increased compared to those of control cells, although the difference was not significant (P = 0.072, Figure 2C), suggesting that knockdown of Slug expression inhibits colony formation, and slug overexpression seems to enhance colony formation.
Figure 2 Slug affects proliferation in ESCC cells. A,B,MTT assay was done to investigate proliferation of siRNA-transfected EC109 cells and Slug cDNA-transfected TE13 cells. Every 24 h, the absorbencies of test well were read and semilogarithmic curves were drawn. Points, mean of three separate experiments; bars, SD. C,D, Down regulation of Slug inhibited colony forming ability, and upregulation of Slug promotes colonyforming ability. Histogram was the average number of colonies in each group.
Slug silencing induces apoptosis in EC109 cells
We evaluated the effects of Slug siRNA transfection on apoptosis induction in EC109 cells using an ELISA kit to quantitatively measure fragmented DNA. After the same treatment schedule described above, a significantly higher level of DNA fragmentation was detected after Slug siRNA transfection compared to those of control cells (Figure 3A, P < 0.05). We further evaluated the effects of Slug siRNA transfection on apoptosis induction using an TUNEL kit. After the same treatment schedule described above, a significantly higher level of cell apoptosis index was detected after Slug siRNA transfection compared to those of control cells (Figure 3.B, P < 0.05). Western blot shown a significantly higher level of Slug protein in Slug-transfected EC109 cells for 72 h compared with controls (Figure 3 C, *P < 0.05, *P < 0.01).
Figure 3 Effect of Slug silencing on EC109cell apoptosis. A, After the same treatment schedule as described above, DNA fragmentation was quantitatively measured by Cell Death Detection ELISA kit using antihistone antibody. Each column represents the mean value of three independent experiments with standard deviation. *Differs from control (P = 0.0043) by Student's t test. B, TUNEL analysis showed a higher percentage of positively stained cells in siRNA-transfected EC109(72 h) compared with control tumors, the difference was significant (P = 0.026). Bars, SEM. *, raw values multiplied by 100 for scale. Original magnification, ×200.C, Slug Protein expression was determined by Western blot in Slug-transfected EC109 cells for 72 h(*P < 0.05;**P < 0.01).
Slug modulates bcl-2 and E-cadherin in ESCC cell line
It has been demonstrated that Slug is involved in the control of apoptosis [14] and also involved in the EMT, linked to the acquisition of the invasive phenotype [19], we analyzed the expression of several Slug targets in stable siRNA Slug-transfected EC109 cells and Slug cDNA-transfected TE13 cells by western blotting and Q-PCR. In siRNA Slug-transfected EC109 cells, anti-apoptotic Bcl-2 was down-regulated (Figure 4 A, P < 0.05). By contrast, in Slug cDNA-transfected TE13 cells, Bcl-2 was up-regulated (Figure 4A, P < 0.05). E-cadherin, a well-known target of Slug previoussly discribed [20], was up-regulated in siRNA Slug-transfected cells(P < 0.05), but in Slug cDNA-transfected cells, E-cadherin was down-regulated (Figure 4 B, P < 0.05).
Figure 4 Slug modulates bcl-2 and E-cadherin expression in vitro. A, Q-PCR(top) and western blot (below) analysis for Bcl-2 in stable siRNA Slug-transfected EC109 cells and Slug cDNA-transfected TE13 cells. B, Q-PCR(top) and western blot (below) analysis for E-cadherin in Slug cDNA-transfected EC109 and Slug cDNA-transfected TE13 cells.. The experiment was repeated thrice with similar results.
Effect of Slug silencing in an in vivo pseudometastatic model
We first tested the effect of siRNA Slug-transfected cells in EC109 pseudometastatic models. The mice were injected with stable siRNA Slug-transfected (18 animals) or siRNA mock-transfected (18 animals) EC109 cells(5×106 )at the site of the abdominal cavity on day 0. After 21 days, the animals were sacrificed and autopsy was carried out to remove organs. Liver metastasis occurred in only four mice in parental groups, and three in mock-transfected mice, no liver metastasis occurred in siRNA Slug-transfected groups. There were many off-white nodus in peritoneum, retina, mesentery, intestinal and gastric wall(Figure 5 A-I, magnification ×200). The node that less than 2 mm is round, and irregular when the node is more than 3 mm. Autopsy showed a reduction of seeded tumor nodes in abdominal cavity organs in pEGFP-siRNA Slug-transfected groups. The number of seeded tumor was 91.16 + 8.7, 90.4 + 9.04 and 33.17 + 6.49 in control groups, siRNA mock groups and Slug siRNA groups respectively. Differences of seeded tumor reached statistical significance (Figure 5 J, P = 0.037).
Figure 5 Slug silencing inhibits seeded tumor in a pseudometastatic model of ESCC in vivo.. Seeded tumor in abdominal cavity organ(A-F)and Seeded tumor in liver (G-I) of each animal was evaluated for the extent of metastatic invasion. Seeded tumor in abdominal cavity was showed in parental (A)and siRNA mock-transfected mice(B), fewer seeded tumors in abdominal cavity were seen in siRNA Slug transfected mice(C). Seeded tumor in abdominal cavity organ was showed in parental (D)and mock transfected mice(E), fewer seeded tumors in abdominal cavity organ were seen in siRNA Slug transfected mice(F). Seeded tumor in liver was showed in control(G)and mock transfected mice(H), fewer seeded tumors in liver were seen in siRNA Slug transfected mice(I). Significant seeded tumor differences between the control and mock transfected groups are indicated(P < 0.05) (J),( magnification ×200).
Furthermore, we analyzed the expression of Slug in the tumor sections from control, mock and Slug siRNA transfected groups using immunohistochemistry (Figure 6 a and 6b). Positive staining of Slug was seen in the cytoplasm of tumor cells. We observed significant Slug expression in control and mock-transfected tumor sections. However, expression levels were drastically reduced in siRNA Slug-transfected sections of mice. TUNEL staining in the same samples was increased from 1.6% positive area in the mock siRNA transfected group to 14.2% positive areas in the siRNA Slug transfected groups(Figure 6 c and 6d).
Figure 6 Immunohistochemical and TUNEL staining in vivo xenograft model. A,Immunohistochemistry with monoclonal antibodies directed against human Slug was performed to compare the Slug expression profile of Slug- and vector-transfected
clones in the in vivo human xenograft model. Bars, 21 mm. (a) Slug is intensely expressed in siRNA mock-transfected group.(b)Slug is weakly expressed in siRNA Slug-transfected group, B, TUNEL staining in the mock siRNA(c) and the siRNA Slug transfected groups(d).
Discussion
Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive carcinomas of the gastrointestinal tract, and yet the molecular changes necessary for tumor cells to acquire metastatic competence are still not completely understood.
In addition to the important role during embryonic development, loss of cell adhesion and induction of EMT are required for tumor progression [22]. In fact, induction of EMT represents the first step in the metastatic cascade, allowing cells to delaminate from the primary tumor and to intravasate into lymphatic or blood vessels [3]. Snail, ZEB, and basic helix-loop-helix transcription factors activation represents one molecular switche whereby cells undergoing EMT lose intercellular connections and apico-basal polarity [20]. Recently it has been demonstrated that activation of microRNA-10b by Twist1, an EMT regulator, plays a role in breast cancer invasion and metastasis [23].
Slug is a member of the snail family of repressors, and is expressed in the neural crest and in mesodermal cells emigrating from the primitive streak in chick embryos. Recently, another critical role of Slug has been reported. Slug binds to E-box elements in the proximal E-cadherin promoter and represses transcription of the E-cadherin gene(24). Experimental data have led to the inclusion of Slug into the Snail family of transcription regulators involved in tumor progression and metastasis [11,25]. Previous data suggest that Slug expression significantly correlated with reduced E-cadherin expression in patients with esophageal squamous cell carcinoma, and patients with reduced E-cadherin expression or positive Slug expression have a poor clinical outcome [11].
In our study we found that the knockdown of Slug expression inhibited invasion capability in EA109 cell lines in vitro, and overexpression of Slug increased the invasion capability in TE-13 cell lines in vitro. It has been reported Slug is involved in cell invasion through direct E-cadherin regulation. We found Slug expression in EC109 and TE-13 cells to show a significant correlation with E-cadherin expression, which is in concordance with the proinvasive role of Slug in EC109 and TE-13 cells.
Zhang et al. [20] has previously found knockdown of Slug expression promotes apoptosis and inhibits cell proliferation in esophageal adenocarcinoma cell in vitro. In the present study, MTT and clonogenicity Assay revealed that overexpression of Slug significantly promotes cell proliferation of the TE-13 cell lines, and Slug inhibition significantly inhibits cell proliferation of the EC109 cell lines. ELISA and TUNEL staining revealed that apoptotic cell death was abundant in EC109 cells with slug inhibition, but almost completely absent in cells transfected with mock siRNA. These data demonstrate Slug silencing gene facilitates apoptosis.
Moreover, Slug is also involved in cell survival through the direct or indirect transcriptional regulation of anti-apoptotic genes [16]. In our studies, we detected a decrease of anti-apoptotic gene Bcl-2 expression in Slug siRNA-transfected EC109 cells, whereas Bcl-2 levels in Slug-cDNA-transfected TE-13 cells increased, findings that demonstrate the anti-apoptotic role of Slug in EC109 and TE13 cells. Although over expression of Slug inhibited apoptosis in TE-13 cells, no statistical significance was demonstrated in control TE-13 cells due to the low apoptosis rate (<5%) (data not shown).
In tumors, migrating cells that leave the primary mass must counteract anoikis [26], a form of apoptotic cell death triggered by the detachment of cells from the extracellular matrix, to arrive successfully at the final site of metastasis. Thus, the anti-apoptotic and proinvasive activities conferred by Slug to ESCC cells in vitro could act in concert to promote metastasis competence in vivo. To investigate whether the Slug knockdown can synergistically reduce the metastatic burden in ESCC, we used a EC109 pseudometastatic model in immunodeficient mice, Slug silencing did inhibit metastatic growth and the effects reached statistical significance. In the present study, we have investigated the effect of Slug on apoptosis and growth in EC109 cells in vivo. We found that knockdown of Slug expression promotes apoptosis in vivo.
Conclusions
Our data have shown for the first time the relevant role of Slug expression in apoptosis, invasion and metastasis of ESCC cells in vitro and in vivo. Although metastasis formation in ESCC as well as in other cancers is a highly complex and organized process that consists of multiple interrelated steps, Slug seems to play a central role, so that its inhibition significantly decreases, albeit does not eradicate, metastasis formation in vivo.
Competing interests
The authors promised there were not any possible conflicts of interest in this research.
Authors' contributions
TP and YZT participated in the design and coordination of the study and constructed the gene vector; It was TP and YZT to transfect the vector to the cells, carried out the proliferation assay and the apoptosis assay in vitro; ZSY carried out the Q-PCR assay;LXY and WY carried out the in vivo assay. Otherwise, it was TP and YZT to draft the manuscript and performed the statistical analysis. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-230X/11/60/prepub
Acknowledgements
We take this opportunity to specifically thank the reviewers and editors for their kind instructions that may be helpful for our further studies. We acknowledged Pro.Zhan L and Dr.Zhang K for their technical help.
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J Med LifeJ Med LifeJMedLifeJournal of Medicine and Life1844-122X1844-3117Carol Davila University Press Romania 21776295JMedLife-04-139ReviewManagement of small renal masses–update 2011
Surcel C Mirvald C Gingu C Stoica R Sinescu I ‘Fundeni’ Clinical Institute of Uronephrology and Renal Transplantation, BucharestRomania
Correspondence to:Cristian Surcel, MD, PhD, ‘Fundeni’ Clinic of Urology and Renal Transplantation, Bucharest, Romania
,e-mail
[email protected] 5 2011 25 5 2011 4 2 139 147 10 1 2011 04 5 2011 ©Carol Davila University Press
2011This is
an open-access article distributed under the terms of the Creative
Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work
is properly cited.Rationale: The management of renal parenchymal tumors has known many changes over time, a trend that continues today, as a result of technological advances, clinical research and improved diagnostic and therapeutic tools.
Unfortunately, conventional cancer treatments–chemotherapy and radiotherapy have proven ineffective and modern approaches such as immunotherapy, angiogenesis inhibitors, though they enjoyed an initial enthusiasm, subsequent studies have shown limited and controversial effects. Thus, surgery remains the gold standard therapy for this type of cancer.
The options for the treatment of RCC are numerous, with options that have advantages and disadvantages, with oncological results, in most cases, positive at five years and with different impact on cancer specific survival.
It is difficult to compare the results, as these are different techniques with various instruments and intraoperative steps, with more questionable inclusion criteria, selection biases and prosecution, with a tendency for preferential enrollment, different reasons to why randomized prospective studies have not been performed until today.
Objective: This article is a review of the diagnosis and methods of treatment of small renal masses 2011.
Conclusion: At the beginning of the new millennium, kidney cancer, with all the arsenal of techniques and methods of ablative surgery, remains a potentially fatal disease for a high percentage of patients, and the decision to choose a treatment or another should be taken with responsibility, depending on currently existing medical records, the degree of expertise and not based on subjective or other non–standard parameters.
renal cancercomputed tomographyradical nephrectomynephron sparing surgeryactive surveillance
==== Body
Introduction
The management of renal parenchymal tumors has known many changes over time, a trend that continues today as a result of technological advances, clinical research and improved diagnostic and therapeutic tools.
Unfortunately, conventional cancer treatments–chemotherapy and radiotherapy have proven ineffective and modern approaches such as immunotherapy, angiogenesis inhibitors, though they enjoyed an initial enthusiasm, subsequent studies have shown limited and controversial effects. Thus, surgery remains the gold standard therapy for this type of cancer.
Aggressive surgical approach to remove the whole tumor burden, here meaning both primary lesion and its extension–venous, lymphatic or metastatic–remains the only effective method that can ensure the cure, or, in some cases, the treatment of severe symptoms in order to increase the quality of life of these patients.
During the first half of the twentieth century, simple nephrectomy was the standard treatment of renal parenchymal tumors. The first radical nephrectomy with removal of the kidney, adrenal gland and fat tissue within the Gerota's fascia was reported by Mortensen in 1948 [1]. In 1960, Robson and colleagues added the retroperitoneal lymph node dissection to the radical nephrectomy, reporting high rates of survival at 5 years [2,3].
Although the incidence of kidney cancer has increased significantly worldwide in recent years, most tumors are detected in early stages, when the conservative treatment can be performed with curative aim. Extensive use of abdominal ultrasound plus evaluation of the detected lesions by computer tomography, represent the diagnostic imaging tools that had the greatest impact in the immediate change of the clinical evolution of renal tumors. Thus, the concept of incidentaloma arose, because kidney cancer is often detected incidentally in countries with a developed medical system.
In 1884, Wells performed the first partial nephrectomy for a kidney fibrolipoma and in 1887 Czerny did this type of intervention in a patient with solitary kidney (imperative indication). In 1950, Vermooten performed the first partial nephrectomy in a patient with normal controlateral kidney (elective indication). Subsequently, surgical technique has evolved, but it still remains a challenge, especially for central tumors or in patients with imperative indications. Although these operations have been globally popularized and recommendations have been established and published in medical practice guidelines, a study published in the U.S. paradoxically showed, that an extremely low number of partial nephrectomy are made even for small renal masses [4].
Percutaneus ablative techniques (cryotherapy and radiofrequency ablation) have been improved in the last decade, with deeper knowledge of cellular metabolism and implications of low temperatures, i.e. ultrasounds on tumor cells. However, long–term results are contradictory, with still unacceptable risks related to recurrence and metastatic progression of the disease.
Active surveillance, a relatively new concept in renal cancer, has an absolute indication in the presence of major comorbidities that prohibit surgical or percutaneous ablative techniques for patients with reduced life expectancy, with low performance status or patients who refuse surgery (relative indication).
Although the depth of tumor progression mechanisms has evolved and the urologist's armamentarium has been diversified, the question ‘why and how to treat all the kidney tumors?’ still stands.
Small renal tumors, defined as T1a in the international TNM staging, have a risk of death of 5% at 5 years without treatment and in T2 stage associates a risk of death of 27% over the same period of follow–up. On the other hand, approximately 30% of renal tumors, less than 2 cm are benign, so they are not life–threatening [5]. In a study published in the Journal of Urology in 2006 Remzi reported that 11% of the tumors less than 3 cm are actually pathological pT3a, 5% are G3 as histopathological grading and 2.5% are metastatic at diagnosis [6], in other words trying to differentiate the aggressive from, the mild forms still remain a challenge for the urologist today.
All these data confirm the lethal potential of small renal tumors, the possibility of local and systemic progression, with no mathematical correlation to the size.
What are small renal masses (SRM) and how do we diagnose them properly?
Imaging can provide objective answers to the clinician's questions such as whether it is a tumor or not and its degree of development, relationship with adjacent structures, presence or absence of metastatic sites. A special feature is the anatomical position of this type of tumors, most accurately described by the investigation that can access the retroperitoneum, such as computer tomography or magnetic resonance imaging.
Due to its accessibility and safety, renal ultrasonography became the first imaging method, essential in evaluating a patient with abdominal symptoms and the examination of a seemingly healthy individual who presents for a routine checkup or in postoperative protocols. The dependence on the expertise of physicians is the main disadvantage of ultrasound, plus the absence of direct information on renal function and excretory routes except for indirect signs such as uretero–and / or hydronephrosis.
Figure 1 Renal ultrasonography that detects a solid central mass in the left kidney (‘Fundeni’ Archives)
Intraoperative ultrasonography for detecting multicentric tumors or the parenchymal extension may be indicated in conservative renal surgery, but is not routinely applied.
Renal ultrasound is not considered an ideal imaging method for screening because the lesions smaller than 2 cm may remain undiagnosed, but it can be used on selected groups of risk patients and asymptomatic elderly population [7].
Doppler ultrasound can accurately certify the presence of tumor thrombus in the inferior vena cava and the cranial extension, including right heart cavities, in which the transoesophageal echocardiography is mandatory.
In fact, any renal mass with solid characters in the ultrasound exam has indication for contrast enhanced computer tomography examination, which is currently the election method for the detection, characterization and staging of renal masses. Spiral computer tomography with 5 mm sections allows the identification and correct description of renal lesions with a minimum size of 1.5 cm. We can use relatively thin slices of 2.5 mm in the kidney.
Not all kidney tumors are well defined during the corticomedular phase, so the images obtained during late phase (nephrographic or excretory phase) should be included to facilitate the detection of renal masses, particularly small ones [8,9].
In addition, acquisitions in excretory phase (normally obtained after a scan delay of 3 minutes) are helpful to describe the anatomical abnormalities or renal collecting system invasion [10].
We consider that any difference higher than 20 HU between SRM and the surrounding parenchyma with the presence of calcifications within a solid renal mass are imaging markers that suggest malignancy [11].
Figure 2 Mediorenal tumoral mass classified as T1, suggestive for RCC (‘Fundeni’ Archives)
Magnetic resonance imaging (MRI) is used complementary to CT imaging only in cases where we cannot perform computed tomography (chronic renal failure, contrast allergy, etc.).
Imaging SRM: is pathology correlated?
Oncocitomas could be confused with clear cell renal tumors in terms of imaging features and degree of contrast load [12].
In contrast, Cromophobe carcinomas, are much more varied in terms of levels and patterns of contrast load.
Figure 3 a) Right renal oncocitoma (with central scar)–nephrographic phase; b) Bilateral renal angyomiolipomas (‘Fundeni’ Archives).
Negative densities are considered the mark of angyomiolipomas. High, homogeneous attenuation on native scan and charge on contrast have been reported in cases of angyomiolipomas, which contain more muscles fibers and less fat, or in cases of sarcomatous degeneration [13].
We have the diagnosis, how do we treat?
The goal of treatment is to cure patients of cancer, preferably saving the kidney function and, if possible, with minimal perioperative morbidity.
The choice of treatment depends on many factors, which are listed below:
Tumor factors–stage, tumor size, location, Fuhrman's grading, histology, presence of tumor markers and receptors;
Factors related to the patient: performance status (ECOG or Karnofski), age, renal function (global and controlateral kidney), cardiac function, comorbidities, surgical history, etc;
Local facilities: open approach, laparoscopic, robotic and opportunities for cryoablation or radiofrequency, anesthetic and ICU support;
Active participation of the patient to treatment (regular follow–up, appropriate compliance).
Regardless of the treatment method we choose, the patient should be informed about the risks associated with each method: hemorrhagic risk, risk of positive margins, urinary fistula, the occurrence of postoperative acute kidney injury, intraoperative conversion from a laparoscopic/robotic approach to open and the need of conversion from partial to total nephrectomy.
Treatment of SRM
The options for the treatment of RCC are numerous, with options that have advantages and disadvantages, with oncological results, in most cases, positive at five years and with different impact on cancer specific survival.
Comparing the results is difficult, as these are different techniques and with various instruments and intraoperative steps, with more questionable inclusion criteria, selection biases and prosecution, with a tendency for preferential enrollment, different reasons to why randomized prospective studies have not been performed until today.
We have three clear answers so far:
Conservative surgery (NSS) has won the ‘battle’ with radical nephrectomy in the treatment of small renal tumors.
If we perform NSS in a patient with normal controlateral kidney, we assume the probability of 20% at three years to develop chronic kidney disease (glomerular filtration rate<60 ml / min) and 65% probability for radical nephrectomy. We also have the misfortune of 5% after NSS at 3 years of having a severely reduced glomerular filtration rate (below 45 ml/min) and 36% after a total nephrectomy. In a study published by Thompson et al. in the Journal of Urology in 2008, patients with ages less than 65 years old, who had radical nephrectomy for SRM, had a lower overall survival rate than patients who underwent NSS. Thus, mortality from cancer in certain situations differs from that determined by other comorbidities [15].
At a glomerular filtration rate between 60–45 ml/min, the relative risk of death from cancer is 1.2, the relative risk for cardiovascular events is 1.4 and for hospitalization is 1.1[16]. At a filtration rate between 45–30ml/min, the relative risk of death from cancer is 1.8, the relative risk of cardiovascular events is 2.0 and for hospitalization is 1.5 and at a filtration rate between 15–30 ml/min, the relative risk of death from cancer is 3.2, the relative risk of cardiovascular events is 2.8 and for hospitalization is 1.5.
Overall survival at 5 and 10 years is 85.5% for radical nephrectomy and 88.9% for NSS and 10 years 68.8% and 70.9%, respectively [16].Conclusions: The oncological results are similar for both treatment modalities for small renal tumors–classified T1a, but NSS reduces the risk of nontumoral death and maintains the renal function (80% for NSS vs. 35% for radical nephrectomy).
The role of adrenalectomy:Adrenalectomy is not routinely indicated except for the upper pole tumors or on suspicious adrenal lesions on CT or intraoperative.
According to EORTC, Locoregional lymph node dissection in renal cell cancer does not increase survival rate at 10 years [17].The report concluded that a correct lymph node dissection does not bring a higher morbidity risk for the patient and the risk of lymph node–positive in patients who did not have imaging or intraoperative suspicion is 3.3%, which, according to the authors of the study does not justify the procedure. In our experience, which includes approximately 3800 renal tumors in various stages, operated in the past 10 years, this is an inappropriate management. Routinely performing locoregional lymph node dissection induces additional expertise, which does not associate a higher morbidity, it is not time consuming and the oncological results, at least for the microscopic invasion, are excellent. This concept of radical surgery finds its counterpart absolutely in open surgery.
Radical surgery for SRM
Open radical nephrectomy: It has represented the gold standard procedure in the treatment of renal cell cancer over the years. However, in the last decade, due to good results reported by the NSS, its indication has become secondary. Global statistics for radical nephrectomy are presented as following [18]: mean hospital stay 6 days, 5% risk of readmission, mean intraoperative blood loss 325 ml, 16% risk of transfusion, 17% perioperative morbidity, mortality 0.3%. In addition, the risk of reintervention after a radical nephrectomy is of 0.6% and local recurrence rate reported is 0.3%. Regarding the oncological outcome, cancer specific survival at 5 years reaches rates of 94–98%. Radical nephrectomy is associated with a shorter hospitalization time, few type II and III complications on the Clavien scale, but it considerably increases the mortality from noncancerous specific events.
Laparoscopic radical nephrectomy faithfully reproduces the steps of open surgery and has established itself as the standard treatment for tumors up to 14 cm in diameter. The oncological results, functional and complications are comparable to those of open surgery, although there is no solid evidence to confirm this. Operative time is similar to that recorded for open nephrectomy, but has the advantage of a faster recovery. No recurrences were reported at the trocars sites and cancer specific survival rate at 5 years is of 94–97% [19].
NSS is the new gold standard for small renal tumors (SRM)
Regardless of the approach path and the available technology (open, laparoscopic, robotic), short–term oncological results are similar, but, unfortunately, the long term are contradictory or even unpublished. The need for long–term studies in a prospective randomized fashion is imperative in order to impose new standards for the treatment of RCC.
Open NSS has imperative, elective indications (normal controlateral kidney, accessible tumors under 4 cm) and relative indications (kidney disease associated tumors larger than 4 cm, etc.). Early complications reported, mostly due to a warm ischemia time (WIT) higher than 20 minutes, have a rate of 13.4% and the late complications are maintained at a high rate of 32%. Oncological results are translated into the local recurrence risk of 1.4% and 0.69% rate of systemic recurrence. Cancer specific survival for small renal tumors less than 4 cm is of 98% [20].
Figure 4 Open NSSߝintraoperative aspect (‘Fundeni’ Archives)
Laparoscopic NSS is a still technically inaccessible procedure, which requires special expertise. All the studies show a 12–32% rate of complications, increased bleeding risk associated even with small tumors, mean operative time of 3 hours, warm ischemia time in experienced hands of over 25 min [21, 22, 23, 24]
According to Gill's study, recently published with a remarkable honesty, the functional and oncological results obtained after 800 conservative operations laparoscopically performed, are improving, warm ischemia time (WIT) is reduced with increased risk of bleeding in early pedicle declamping, but, unfortunately, do not reach the results reported by open surgery. In addition, the learning curve even after this huge number of procedures is still on the ascendant trend [23].
In conclusion, laparoscopic NSS can be performed in centers of excellence, still technically difficult, and it is associated with a higher risk of complications. The need for new tools is imperative and well–managed long–term studies are required in order to establish this procedure as an indication in daily practice.
Comparisons between laparoscopic and open NSS are unbalanced; studies evaluating patients in open surgery are more difficult in terms of extension and tumor size, comorbidities, etc. Additionally, the risk of positive margin, intraoperative complications, urologic and non–urological surgery is higher after laparoscopy.
Data published in the literature report similar oncological results of both procedures: cancer specific survival rates are of 99% and of 99.2% for laparoscopy and open surgery, respectively [25].
Robotic NSS: Although promoted in recent years, conservative robotic surgery is still in evaluation, with scarce medium and long–term results. Certain procedures, such as hemostasis and intracorporeal sutures are easier to perform than laparoscopy, with lower WIT. Oncological results are contradictory and time tracking of patients is still insufficient to bring this technique into standard surgical practice.
Percutaneous ablative techniques in the treatment of small renal tumors (SRM), cryotherapy and radiofrequency ablation by HIFU.
The mechanism of action involves the use of agents, which determine the precipitatation of intracellular proteins, thereby causing cell apoptosis and necrosis. Ablation uses currents generated by radiofrequency (RFA) and is indicated in tumors with certain specific locations. It obtains diffuse necrosis, with secondary injury to the surrounding tissue. On the contrary, cryoablation limits its action as it is focused only on tumor tissue.
The need to introduce these procedures in the urological arsenal derives from the daily practice, as an alternative treatment for a difficult group of patients. Thus, those with low performance status, who present multiple comorbidities or those who refuse other more invasive procedures, have proved to be candidates for ablative techniques. In addition, tumors with a low malignant potential or those who have genetic syndromes such as Von Hippel–Lindau, can be candidates for one of these procedures.
HIFU can be performed through a percutaneous or laparoscopic approach, but the paucity of studies available in the literature, along with poor functional and oncological results, maintain this technique as an experimental procedure in the treatment of RCC [26].
Cryotherapy can be performed laparoscopically (60% of the cases), percutaneously (30%) or by an open approach. Regardless of the approach path, general anesthesia and longer operative time are required, together with longer hospitalization. Between percutaneous and laparoscopic approach, looking in particular at the differences regarding the costs, the percutaneous approach is cheaper, and the transfusion risk is higher for laparoscopic approach (11% vs. 28%) [27].
The temperature at which cell death occurs depends on the type of the cell. Thus, in the kidney it is of –19.4 C. In kidney cancer it is situated at a temperature below –10C, 96% of the cells survive, reducing the percentage to 15% at –20 degrees C [28].
In addition to lowering the temperature, the time also matters, and a longer period seems to provide a better oncological status. The size of necrosis area assessed at 2 weeks, shows similar values, regardless of the needles' size used in cryoablation [29].
Percutaneous radiofrequency ablation is performed in 90% of cases, so it requires sedation in most cases, which brings lower operative time and overall, lower costs.
Do these procedures imply no complications?
The answer is definitely not, some of them are severe and we question ourselves whether these are minimally invasive techniques. The following are reported in literature: kidney, liver or pancreas laceration, kidney hematoma, intestinal obstruction, pieloureteral junction syndrome, urinary fistula, conversion to open surgery, etc. Overall, the rate of major perioperative morbidity for both procedures is, regardless of the approach, between 0.8 and 4% (higher for percutaneous RFA). [30]
The Cryotherapy and radiofrequency ablation preserves pre–existing renal function. However, the studies analyzed, showed that 20% of the patients with chronic kidney failure who have a glomerular filtration rate below 60 ml / min, of which 15.5% had de novo CKD [31].
Long–term oncological results are scarce and contradictory; only one study has a follow–up period of 5 years on cryotherapy, the overall survival rate of 84% being reported, the cancer specific survival rate of 92% and 81% specific–disease free [32].
There are also two studies that reported data on cancer outcomes for patients treated with RFA, with a low number of individuals enrolled, with cancer specific survival rate of 94–100% and the risk of local and systemic recurrence of 5–10%, which is unacceptable from our point of view. The risk of local progression reported for cryotherapy is of 5.2% compared to 13% for radiofrequency ablation and the systemic progression of 1% and 2.5%. Regarding the risk of reablation, Lavinson et al. reported a rate between 1.3% to 8.5% for cryotherapy and RFA, respectively [33, 34].
Energy ablative techniques remain an alternative to radical/conservative surgery, but the results and criteria for evaluation of treatment response are still being under evaluation. The follow–up is done purely radiologically and, in many cases, they were positive after rebiopsy, with no corresponding imaging for active tumor. After such procedures, salvage surgery is ‘challenging’, whether we consider conservative or radical surgery.
Active surveillance, a relatively new concept in renal oncology, has an absolute indication in the presence of major comorbidities that prohibit any type of surgical approach or energy ablative techniques in patients with reduced life expectancy, with low performance status or patients who refuse surgery (relative indication).
The theory behind this procedure derives from the observation that 30% of the SRM do not grow in size, and from those that are still progressing, most of them slowly grow about 0.3 cm / year, few reaching rates higher than 1 cm/year. Also, there is no correlation between growth rate and prognosis of patients; in addition any degree of aggressiveness of the tumor cannot be forecasted based on this parameter [35].
However, there is no long–term data in order to establish the place of this method in the renal cancer therapeutic arsenal, but has a follow–up period higher than that of percutaneous ablative techniques, or even for laparoscopic NSS, and many clinicians consider active surveillance beneficial when compared to other procedures mentioned, at least from this point of view.
What do we know so far about the active surveillance of SRM?
In a study published in 2006 by Chawla, the authors report a systemic progression rate of 1% in the group studied [35]. A year later, Abou Youssif published an article, which reported that 5.7% of patients developed lung metastases during follow–up period [36], and Crispen reported in 2009 a 1.3% rate of systemic progression [37].
In conclusion, patients under active surveillance have an average risk of systemic progression of 1–2% and as a general observation, we can say that progressing tumors tend to grow faster. However, deaths during the studies were not caused by cancer, in other words, cancer specific survival was of 100%.
Unfortunately, several questions remain unanswered; one of the unknowns is when to decide to stop actively monitoring the patients and shifting them to surgical treatment. Thus, losing the window of opportunity for a radical curative therapy remains a constant concern. In addition, tumor aggressiveness, regardless of size, cannot be predicted earlier, and opponents of this method consider it hazardous to monitor this type of tumor taking into account its high risk of progression. Also, the proportion of benign lesions, which theoretically would be amenable for such therapy, is not known precisely.
When do we stop active surveillance?According to present recommendations for complex cases included in genetic syndromes (von Hippel–Lindau syndrome, etc.), active surveillance ceases when the tumor reaches 3 cm in diameter. Secondary renal tumors, with proven tumor progression in two successive evaluations, require at least caution, if not stopping of the active surveillance [37]. More subjective factors relating to the patients, their refusal to continue the monitoring and the decision to operate, must be taken into account in order to stop the surveillance and proceed to another therapeutic approach.
Renal biopsy has emerged as an effective diagnostic method and has lately improved due to the perfection of the technique itself, but also due to popularized conservative therapies. It can be performed under ultrasound guidance or under tomography, the first approach with a longer learning curve and can be technically difficult, especially in obese patients.
Depending on the manner of execution, such as needle type and the material collected, renal biopsies may be aspirative (FNA) or type core, both with high sensitivity (90%), except that the aspiration method has a lower sensitivity for Fuhrman grade and requires the interpretation from an experienced cytologist.
No major complications (transfusion, embolization, and surgical reinterventions) were reported, there are some are cited most frequently. According to an article published in 2009 by Lechevallier, kidney subcapsular hematoma is met in an alarming proportion of 44%, followed by arteriovenous fistula, pneumothorax, bowel perforation [38]. The risk of tumor seeding, uro–oncologists once feared has disappeared, being described in only six cases, the last one being reported in 1992.
Is renal biopsy useful?The method has a sensitivity of 70–100%, specificity of 100%, accuracy of 90%, performance parameters that have promoted this method in clinical practice. However, we believe that these indices are overrated and the abuse of such procedures is detrimental to the patient and to the oncological management of the tumor. In addition, the nephrology and transplantation daily practice and we agree that, in a significant proportion of cases, the pathologist interpretations are ambiguous. In large studies, this percentage increases to 25% of patients and therefore biopsy is nondiagnostic and induces major confusions with a delay of treatment with curative visa [39].
We believe that renal biopsy is useful whenever afterwards, the therapeutic attitude changes or in selected cases of secondary kidney tumors, if we suspect a metastasis from another cancer or lymphoma, before energy ablation therapy or prior to treatment with angiogenesis inhibitors. However, if the tumor is smaller and more centrally located, the more chances we have for a negative biopsy. In cases of large tumors, the harvesting of tissue from the necrotic or hemorrhagic areas poses a high risk for blending that needs to be taken into consideration.
Tumor markers have been studied extensively in the pathogenesis of RCC. The results are disappointing, the need for such biochemical parameters for the diagnosis and monitoring of patients is imperative. The following markers have been reported: carbonic anhydrase, Ki67, C–reactive protein, although initially they seemed promising, they have passed on the second place, never being evaluated prospectively.
Prognostic nomograms have been developed for the design development of kidney cancer patients. Most of them are based on tumor histology, but their clinical use is limited [40, 41, 42].
Table 1 NSS vs. cryotherapy vs. RFA vs. AS in the treatment of SRM
No patients Follow–up Local progression (relative risk) Systemic progression(relative risk)
NSS 5037 54 mths 1 1
Cryotherapy 496 18 mths 7,45 1,24
RFA 607 16,4 mths 18,23 3,21
Active surveillance 331 33,3 mths – 0,11
Conclusions
The risk of local recurrence is 7 and 18 times higher for cryotherapy and radiofrequency ablation versus conservative surgery or active surveillance; in addition, recurrences require radical surgery, which is difficult to perform after ablative techniques because of secondary fibrosis [43]. A high positive biopsy rate after RFA, which has no counterpart tumor imaging (MRI), has also been reported [44].
Partial nephrectomy, with all its technical forms (open/laparoscopic, enucleation/enucleoresection vs. wedge/partial nephrectomy) has become the new gold standard for the treatment of small renal tumors, although it remains underused. Approximately 27% of RCC T1 is treated by NSS in the U.S., the rest being solved by radical nephrectomy [45]. Partial nephrectomy brings a proven level of technical difficulty, especially for tumors on anterior valve, centered, large or upper renal pole, but overall perioperative morbidity related parameters remain within acceptable limits, which are improving with experience. However, in postoperative bleeding, the risk of urinary fistula are complications which the urologist must be familiar with, as coagulation and suturing techniques have improved, a significant percentage of patients, especially those with imperative indications, unfortunately have the risk to present such unwanted postoperative events.
Ablative techniques have not yet shown efficacy in patients without major contraindications for surgery and patients who choose such treatments must be informed of all treatment alternatives. Selecting an ablative technique is risky, both in short and long term, when it comes to cancer free survival rates. Pressure applied by patients who want the therapy with the lowest physical and psychological impact and by innovative companies should not be exercised in order to radically alter the therapeutic decision. The urologist must decide which approach is more familiar to him and which one has proven the best results for the patients, not letting the medical industry determine the method instead. A quick solution, with minimal impact on the health care system and patient's quality of life, does not always cure the patient or at least provide a good oncological prognosis.
At the beginning of the new millennium, kidney cancer with all the arsenal of techniques and methods of ablative surgery, remains a potentially fatal disease for a high percentage of patients, and, the decision to choose a treatment or another should be taken with responsibility, depending on currently existing medical records, the degree of expertise and not by subjective or other non–standard parameters.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 21738696PONE-D-11-0872610.1371/journal.pone.0021539Research ArticleBiologyMolecular Cell BiologyCellular TypesEpithelial CellsMedicineDermatologySkin NeoplasmsMalignant Skin NeoplasmsMelanomasOncologyBasic Cancer ResearchMetastasisGrape Seed Proanthocyanidins Inhibit Melanoma Cell Invasiveness by Reduction of PGE2 Synthesis and Reversal of Epithelial-to-Mesenchymal Transition Proanthocyanidins Inhibit Melanoma Cell MigrationVaid Mudit
1
Singh Tripti
1
Katiyar Santosh K.
1
2
3
*
1
Department of Dermatology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America
2
Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama, United States of America
3
Birmingham VA Medical Center, Birmingham, Alabama, United States of America
Zhang Lin EditorUniversity of Pennsylvania School of Medicine, United States of America* E-mail: [email protected] and designed the experiments: SKK MV TS. Performed the experiments: MV TS. Analyzed the data: MV TS SKK. Contributed reagents/materials/analysis tools: SKK. Wrote the paper: SKK.
2011 27 6 2011 6 6 e2153916 5 2011 1 6 2011 Vaid et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Melanoma is the leading cause of death from skin disease due, in large part, to its propensity to metastasize. We have examined the effect of grape seed proanthocyanidins (GSPs) on melanoma cancer cell migration and the molecular mechanisms underlying these effects using highly metastasis-specific human melanoma cell lines, A375 and Hs294t. Using in vitro cell invasion assays, we observed that treatment of A375 and Hs294t cells with GSPs resulted in a concentration-dependent inhibition of invasion or cell migration of these cells, which was associated with a reduction in the levels of cyclooxygenase (COX)-2 expression and prostaglandin (PG) E2 production. Treatment of cells with celecoxib, a COX-2 inhibitor, or transient transfection of melanoma cells with COX-2 small interfering RNA, also inhibited melanoma cell migration. Treatment of cells with 12-O-tetradecanoylphorbol-13-acetate, an inducer of COX-2, enhanced the phosphorylation of ERK1/2, a protein of mitogen-activated protein kinase family, and subsequently cell migration whereas both GSPs and celecoxib significantly inhibited 12-O-tetradecanoylphorbol-13-acetate -promoted cell migration as well as phosphorylation of ERK1/2. Treatment of cells with UO126, an inhibitor of MEK, also inhibited the migration of melanoma cells. Further, GSPs inhibited the activation of NF-κB/p65, an upstream regulator of COX-2, in melanoma cells, and treatment of cells with caffeic acid phenethyl ester, an inhibitor of NF-κB, also inhibited cell migration. Additionally, inhibition of melanoma cell migration by GSPs was associated with reversal of epithelial-mesenchymal transition process, which resulted in an increase in the levels of epithelial biomarkers (E-cadherin and cytokeratins) while loss of mesenchymal biomarkers (vimentin, fibronectin and N-cadherin) in melanoma cells. Together, these results indicate that GSPs have the ability to inhibit melanoma cell invasion/migration by targeting the endogenous expression of COX-2 and reversing the process of epithelial-to-mesenchymal transition.
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Introduction
Melanoma is the leading cause of death from skin disease due to its propensity to metastasis [1], [2], and is increasing rapidly in children [3]. Although, melanoma is less common than other types of skin cancers, it causes the majority (75%) of skin cancer-related deaths [1], [4]. The American Cancer Society estimated that in 2008, there were 8,420 melanoma-associated deaths in the U.S. and the number of new cases of invasive melanoma was estimated at 62,480 [1]. Solar ultraviolet (UV) radiation is a recognized risk factor for the development of skin cancers, including melanoma. Exposure of the skin to UV radiation induces an increase in the expression levels of cyclooxygenase -2 (COX-2), a rate-limiting enzyme that catalyzes the conversion of arachidonic acid to prostaglandins (PGs) [5], [6]. These inflammatory mediators have been identified as a risk factor for the development of skin cancers [5], [6], and thought to play a central role in orchestrating the multiple events involved in cancer invasion and metastasis [7], [8]. Since, melanoma is a highly malignant cancer with a potent capacity to metastasize distantly, an approach that decreases its metastatic or invasive ability may facilitate the development of an effective strategy for its treatment or prevention.
Dietary phytochemicals offer promising new options for the development of more effective strategies for the prevention of cancer cell invasion, migration, or metastasis, and thus can be utilized as complementary and alternative medicine. Grape seed proanthocyanidins (GSPs) are promising phytochemicals that have shown anti-carcinogenic effects in some murine models and exhibit no apparent toxicity in vivo
[9]–[11]. GSPs contain primarily proanthocyanidins (89%), which constitute dimers, trimers, tetramers, and oligomers of monomeric catechins and/or (-)-epicatechins, as described previously [10]. They are readily available as an extract of grape seeds and this extract, rather than the individual constituents, has been examined as an anti-carcinogenic agent against some forms of cancers [9]. It is believed that at least some of the constituents present in the extract may act synergistically and thus this product can be more effective than any single constituent. GSPs have been shown to inhibit UV radiation-induced skin cancer in mouse model [10] but its chemopreventive effect on the migration or invasive potential of melanoma cancer cells has not been explored.
In this study, we assessed the chemotherapeutic effects of GSPs on the migration potential of human melanoma cells, as the migration of cancer cells is a major event in the metastatic cascade. For this purpose, two highly metastasis-specific melanoma cancer cell lines were selected: one is A375 which is BRAF mutated and activating mutations of the protooncogene BRAF have been observed in approximately 50% of malignant melanomas. Second cell line is Hs294t, which is also highly metastatic but not BRAF mutated. In this study, we characterized the role of COX-2 and its metabolite PGE2 on the migration of human melanoma cancer cells and ascertained whether GSPs have any suppressive effects on the COX-2-mediated migration of these cells. Epithelial-to-mesenchymal transition (EMT), the process whereby epithelial cells transform into mesenchymal cells, has recently been shown to be relevant for cancer and cancer metastasis. During EMT, cancer cells lose expression of proteins that promote cell-cell contact such as E-cadherin and acquire mesenchymal markers such as vimentin, fibronectin and N-cadherin, which promote cell invasion and metastasis [12]. The EMT has also been associated with higher levels of inflammation or inflammatory mediators, and therefore we have also checked whether inhibition of COX-2 expression by GSPs in melanoma cells is associated with reversal of EMT and that leads to inhibitory effect on melanoma cell migration. Here, we present evidence that GSPs inhibit the invasiveness or migratory behavior of melanoma cancer cells through inhibition or reversal of EMT in melanoma cells and that GSPs do so through a process that involves the reduction in COX-2 expression and PGE2 production.
Materials and Methods
Source and composition of GSPs
GSPs were received from Kikkoman Corporation (Noda, Japan). Quality control of GSPs is maintained by the company on lot-to-lot basis. GSPs contain approximately 89% proanthocyanidins, with dimers (6.6%), trimers (5.0%), tetramers (2.9%) and oligomers (74.8%), as described earlier [10], [11], and are stable for at least two years when refrigerated at 4°C.
Cell lines and cell culture conditions
The human melanoma cells lines, A375 and Hs294, were purchased from the American Type Culture Collection (Manassas, VA). The cell lines were cultured as monolayers in RPMI 1640 culture medium supplemented with 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT), 100 µg/ml penicillin, and 100 µg/ml streptomycin and maintained in an incubator with 5% CO2 at 37°C. The GSPs were dissolved in a small amount of dimethylsulfoxide (DMSO), which was added to the complete cell culture medium [maximum concentration of DMSO, 0.1% (v/v) in media] prior to addition to sub-confluent cells (60–70% confluent). Cells treated with DMSO only served as a vehicle control. Normal human epidermal melanocytes (HEMa-LP, Cat. No. C-024-5C) were commercially obtained from Invitrogen (Carlsbad, CA), and were cultured in HMGS-2 medium supplemented with human melanocyte growth supplement provided by the supplier. To determine the effect of GSPs on 12-O-tetradecanoylphorbol-13-acetate (TPA)- or PGE2-mediated effects, GSPs were added in cell culture medium at least 30 minutes before the treatment of the cells with TPA, PGE2 or any other agent.
Antibodies, chemicals and reagents
Antibodies specific for COX-2 and an enzyme immunoassay kit for PGE2 analysis were obtained from Cayman Chemicals (Ann Arbor, MI). Celecoxib, PGE2 and 12-O-tetradecanoylphorbol-13-acetate (TPA) were purchased from Sigma Chemical Co. (St. Louis, MO). Boyden Chambers and polycarbonate membranes (8 µm pore size) for cell migration assays were obtained from Neuroprobe, Inc. (Gaithersburg, MD). The antibodies specific to N-cadherin, keratin-8, -18 and fibronectin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), while antibodies for vimentin, E-cadherin, NF-κB, IKKα and IκBα were purchased from Cell Signaling Technology (Beverly, MA) while desmoglein-2 was obtained from Abcam (Cambridge, MA). The secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 594 were purchased from Invitrogen (Carlsbad, CA).
Cell migration or invasion assay
The migration capacity of melanoma cancer cells was determined in vitro using Boyden Chambers (Gaithersburg, MD) in which the two chambers were separated with matrigel coated Millipore membranes (6.5 mm diameter filters, 8 µM pore size), as detailed previously [13]. Briefly, melanoma cells (1.5×104 cells/100 µL serum-reduced medium) were placed in the upper chamber of Boyden chambers, test agents were added alone, or in combination, to the upper (200 µL) chamber, and the lower chamber contained the medium alone (150 µL). Chambers were assembled and kept in an incubator for 24 h. After incubation, cells from the upper surface of Millipore membranes were removed with gentle swabbing and the migrant cells on the lower surface of membranes were fixed and stained with either hematoxylin or crystal violet. Membranes were then washed with distilled water and mounted onto glass slides. The membranes were examined microscopically and cellular migration was determined by counting the number of stained cells on membranes in at least 4–5 randomly selected fields using an Olympus BX41 microscope. Representative photomicrographs were obtained using a Qcolor5 digital camera system fitted to an Olympus BX41 microscope. Each cell migration experiment was repeated at least three times.
PGE2 immunoassay for quantitation of prostaglandin E2
The analysis of PGE2 in cell homogenates was performed using the Cayman PGE2 Enzyme Immunoassay Kit (Ann Arbor, MI) following the manufacturer's instructions. Briefly, at indicated time points, cells were harvested and homogenized in 100 mM phosphate buffer, pH 7.4 containing 1 mM ethylenediamine tetraacetic acid and 10 µM indomethacin using a homogenizer. Homogenates were centrifuged and the supernatants were collected and analyzed for PGE2 concentration according to the manufacturer's instructions.
COX-2-siRNA transfection of A375 and Hs294t cells
Human-specific COX-2 siRNA was transfected into A375 and Hs294t cells using the siRNA Transfection Reagent Kit (Santa Cruz Biotechnology, Inc.; Santa Cruz, CA) according to the manufacturer's protocol. Briefly, 2×105 cells/well were seeded in a 6-well plate and allowed to grow to 70% confluency. The COX-2 siRNA mix with transfection reagents was overlaid on the cells for approximately 6 h at 37°C and transferred into 2× growth medium for about 18–20 h. At 24 h post-transfection, fresh medium was added to the cells and the cells were incubated for an additional 48 h. Thereafter, cells were harvested and subjected to the cell migration assay. The knockdown of COX-2 expression in cells after transfection was confirmed using western blot analysis.
NF-κB/p65 activity assay
For quantitative analysis of NF-κB/p65 activity, the NF-κB TransAM Activity Assay Kit (Active Motif, Carlsbad, CA) was used following the manufacturer's protocol. For this purpose, the nuclear extracts of cells were prepared using the Nuclear Extraction Kit (Active Motif, Carlsbad, CA) following the manufacturer's instructions, and as performed previously [14]. Absorbance was recorded at 450 nm using absorbance at 650 nm as the reference. The results are expressed as the percentage of the optical density of the non-GSPs-treated control group.
Preparation of cell lysates and western blot analysis
Following treatment of melanoma cells for the indicated time periods with or without GSPs or any other agent, the cells were harvested, washed with cold PBS and lysed with ice-cold lysis buffer supplemented with protease inhibitors, as detailed previously [15]. Equal amounts of proteins were resolved on 10% Tris-Glycine gels and transferred onto a nitrocellulose membrane. After blocking the non-specific binding sites, the membrane was incubated with the primary antibody at 4°C overnight. The membrane was then incubated with the appropriate peroxidase-conjugated secondary antibody and the immunoreactive bands were visualized using the enhanced chemiluminescence reagents. To verify equal protein loading, the membrane was stripped and reprobed with anti-β actin antibody.
Immunofluorescent detection of EMT biomarkers in cells
In order to determine whether GSPs inhibit cell migration of melanoma cells is associated with reversal of epithelial-to-mesenchymal transition, the A375 melanoma cells were either treated with various concentrations of GSPs or celecoxib or TPA for 24 h. After 24 h, cells were harvested and cell lysates were prepared for western blotting for the analysis of epithelial and mesenchymal biomarkers. Cells were also used for cytostaining for the detection of EMT biomarkers such as vimentin, fibronectin and N-cadherin. Briefly, after harvesting the cells, cells were processed for cytospin (1×105 cells/slide). Cells were fixed with chilled methanol at −20°C for 10 minutes and non specific binding sites were blocked with 2% bovine serum albumin (Sigma, St. Louis, MO) in PBS for 30 min. Cells were then incubated with antibodies specific to EMT biomarkers for 2 h at room temperature. The cells were washed with PBS and antigen was detected by an Alexa Fluor-conjugated secondary antibody. Goat anti-rabbit IgG labeled with green-fluorescent Alexa Fluor 488 dye was used for detection of N-cadherin and vimentin, while donkey anti-mouse IgG labeled with red-fluorescent Alexa Fluor 594 was used for the detection of the expression of desmoglein 2. Cells were finally mounted with Vectashield mounting medium for fluorescence with DAPI (Vector Laboratories, Burlingame, CA) before they were observed under fluorescence microscope and photographed.
Statistical analysis
For migration assays, the control and GSPs-, TPA- or PGE2-treatment groups or combined-treatment groups separately were compared using one-way analysis of variance (ANOVA) followed by post hoc Dunn's test using GraphPad Prism version 4.00 for Windows, GraphPad Software, San Diego, California, USA, www.graphpad.com. All quantitative data for cell migration are shown as mean ± SD/microscopic field. In each case P<0.05 was considered statistically significant.
Results
Comparative invasiveness of human melanoma cells and normal human epidermal melanocytes
First, we examined the migration capacity of melanoma cells and compared this capacity with normal human epidermal melanocytes under identical conditions. For this purpose, cells were incubated in Boyden chambers for 24 h to assess their migration capability. As shown in Figure 1A, the cell migration capacity of melanoma cells was significantly higher (P<0.001) than normal human epidermal melanocytes. The migration of A375 cells, which are BRAF mutated, was greater (390±14 cells/microscopic field) than Hs294t cells (340±12 cells/microscopic field), which are metastasis-specific but not BRAF-mutated. Under identical conditions, migration of normal human epidermal melanocytes was lower (19±4 cells/microscopic field) than melanoma cells.
10.1371/journal.pone.0021539.g001Figure 1 Effect of GSPs on melanoma cell migration.
(A) Migration ability of human melanoma cells and comparison with normal human epidermal melanocytes (HEMa). Equal numbers of human melanoma cells (A375 and Hs294t) and HEMa were subjected to cell migration using standard Boyden chamber assay. Twenty four h later, migratory cells were detected on the membrane after staining with crystal violet. The migratory cells were counted and the results expressed as the mean number of migratory cells ± SD per microscopic field (n = 3). Significantly lower versus melanoma cells, *
P<0.001. (B) Treatment of human melanoma cancer cells (A375 and Hs294t) with GSPs for 24 h inhibit migration of cells in a concentration-dependent manner compared to non-GSPs-treated control cells. (C) The migratory cells were counted and the results expressed as the mean number of migratory cells ± SD/microscopic field. Significant inhibition versus non-GSPs-treated control, *
P<0.001.
GSPs inhibit human melanoma cancer cell migration
We determined whether treatment of A375 and Hs294t human melanoma cells with GSPs inhibited their invasiveness or migration using Boyden chamber cell migration assays. First, screening experiments were performed to determine the effects of lower concentrations of GSPs (µg/mL). The selection of the concentrations of GSPs was based on consideration of their relevance and achievability in vivo. As shown in Figure 1B, relative to untreated control cells, treatment of cells with GSPs at concentrations of 0, 10, 20 and 40 µg/mL reduced the invasive potential of A375 and Hs294t cells in a concentration-dependent manner. The density of the migrating cells on the membrane after staining with crystal violet is shown in Figure 1B, and the numbers of migrating cells/microscopic field are summarized in Figure 1C. The cell migration was inhibited by 22 to 64% (P<0.01−0.001) in A375 cells and by 29–69% (P<0.01−0.001) in Hs294t cells in a concentration-dependent manner after treatment with GSPs for 24 h. A similar but comparatively higher inhibitory effect on cell migration was observed at the 48 h time point (data not shown). To confirm that the inhibition of cancer cell migration by GSPs was a direct effect on migration ability, and that was not due to a reduction in cell viability, a trypan blue assay was performed using cells that were treated identically to those used in the migration assays. Treatment of A375 and Hs294t cells with various concentrations of GSPs (0, 10, 20 and 40 µg/mL) for 24 h had no significant effect on cell viability or cell death (data not shown).
The inhibitory effect of GSPs on invasiveness of melanoma cells is associated with the reduction of endogenous COX-2 expression and reduction of PGE2 synthesis
To determine whether the inhibitory effect of GSPs on the migration of the melanoma cells is associated with inhibition of endogenous COX-2 expression, we determined the levels of COX-2 in lysates of cells from the various treatment groups using western blot analysis. As shown in Figure 2A, treatment of A375 and Hs294t cells with GSPs reduced the levels of COX-2 expression in a concentration-dependent manner as compared to the expression in untreated controls. As the COX-2 metabolite, PGE2, has been implicated in COX-2-mediated effects including cancer cell metastasis; we determined the levels of PGE2 in the GSPs-treated cells. Our results revealed that treatment with GSPs for 24 h resulted in significant reduction in the production or synthesis of PGE2 in both A375 (19–76%, P<0.001) and Hs294t (18–71%, P<0.001) cells in a concentration-dependent manner (Figure 2B), suggesting that GSPs-induced reduction in PGE2 production is associated with an inhibitory effect of the GSPs on COX-2 expression and inhibition of cell migration in these cells.
10.1371/journal.pone.0021539.g002Figure 2 Effect of GSPs on COX-2 expression, PGE2 production and cell migration in melanoma cells.
(A) Effect of GSPs on the endogenous basal level of COX-2 in A375 and Hs294t cells. The levels of COX-2 were determined in cell lysates using western blot analysis. (B) Dose-dependent effect of GSPs on the levels of PGE2 in melanoma cells. The levels of PGE2 are expressed in terms of pg/mg protein± SD, n = 3 independent experiments. Significant inhibition of PGE2 level by GSPs versus non-GSPs-treated controls, *
P<0.001. (C) Down-regulation of endogenous COX-2 reduces melanoma cell migration. Treatment of A375 and Hs294t cells with celecoxib, a COX-2 inhibitor, inhibits cell migration in a dose-dependent manner. Significant difference versus control (non-celecoxib-treated) cells, ¶
P<0.05, *
P<0.001. (D) Transfection of cells, both A375 and Hs294t, with COX-2 siRNA significantly decreases cell migration. A375 and Hs294t cells were transfected with COX-2 siRNA to knockdown COX-2 expression. Significant reduction of cell migration versus control siRNA-treated cells: *
P<0.001. (E) Treatment of A375 and Hs294t cells with GSPs (20 and 40 µg/mL) inhibits PGE2-enhanced cell migration. The data on cell migration are summarized as a mean number of migratory cells ±SD/microscopic field. In each case, the migrating cells were counted at 4–5 different microscopic fields, and data are expressed as the mean number of migratory cells± SD/microscopic field, n = 3.
Selective COX-2 inhibitor inhibits melanoma cell migration
This experiment was performed to determine whether the inhibitory effect of GSPs on melanoma cell migration is mediated through its inhibitory effect on COX-2 expression. For this purpose, equal numbers of A375 and Hs294t cells were subjected to the cell migration assay after treatment with various concentrations of celecoxib (0, 5, 10, 20 µM), a well known inhibitor of COX-2, for 24 h. As shown in Figure 2C, treatment of the cells with celecoxib resulted in a dose-dependent reduction in the cell migration capacity of melanoma cells as compared with non-celecoxib-treated controls (P<0.05−0.001). These data suggested that the inhibition of constitutive levels of COX-2 expression is associated with the inhibition of melanoma cell migration.
siRNA knock-down of COX-2 leads to reduction of melanoma cell migration
We further verified the role of COX-2 in cell migration through siRNA knock-down of COX-2 in the melanoma cells and examined whether it would lead to the inhibition of the cell migration in these cells. The transfection of A375 and Hs294t cells with COX-2 siRNA resulted in significant reduction of cell migration in A375 (85%, P<0.001) and Hs294t (86%, P<0.001) cells after 24 h as compared to the migration of control siRNA-transfected A375 and Hs294t cells (Figure 2D).
GSPs inhibit PGE2-induced cell migration of melanoma cells
As the effects of COX-2 are mediated through its metabolites, such as PGE2, we examined whether GSPs inhibit PGE2-induced cell migration in human melanoma cells. For this purpose, A375 and Hs294t cells were treated with PGE2 (10 µM) with and without GSPs for 24 h and cell migration determined. We found that the treatment of melanoma cells with PGE2 resulted in a significant increase in cell migration (P<0.05) compared to the cells which were not treated with PGE2 (Figure 2E). Treatment of A375 and Hs294t cells with GSPs (20 or 40 µg/mL) resulted in a dose-dependent inhibition of PGE2 (10 µM)-induced cell migration (Figure 2E). As the inhibitory effect of GSPs on the migration of A375 and Hs294t cell lines was very similar, the subsequent studies were performed only with A375 cells.
TPA, an inducer of COX-2, enhances melanoma cell migration, and GSPs inhibit TPA-induced cell migration
Treatment of skin with TPA stimulates the levels of COX-2 expression [11], [16]; therefore, the melanoma cells were treated with TPA for COX-2 stimulation, and thereafter determined the effect of TPA on the migration of melanoma cells. As shown in Figure 3A, treatment of A375 cells with TPA for 24 h resulted in significantly enhanced cell migration (P<0.01) compared to non-TPA-treated cells. To determine whether GSPs inhibit TPA-induced cell migration in human melanoma cells, A375 cells were treated with TPA (40 ng/mL) with and without the treatment of GSPs for 24 h. We found that the treatment of A375 cells with GSPs resulted in a dose-dependent inhibition of TPA-induced cell migration. A summary of the cell migration data for the various treatment groups is provided in Figure 3A. Treatment of cells with GSPs at the doses of 20 µg/mL and 40 µg/mL inhibited TPA-induced cell migration by 50% (P<0.01) and >100% (P<0.001) respectively.
10.1371/journal.pone.0021539.g003Figure 3 Effect of GSPs and celecoxib on TPA-induced activation of ERK1/2 and melanoma cell migration.
(A) Effect of TPA or its combination with GSPs on melanoma cell migration. Treatment of A375 cells with TPA, a stimulator of COX-2, significantly enhances cell migration (†
P<0.001) compared with non-TPA-treated control cells. (B & C) Treatment of A375 cells with TPA results in activation of ERK1/2. Treatment of cells with GSPs (40 µg/mL) or celecoxib (40 µM) inhibits TPA-induced activation of ERK1/2 protein, and simultaneously inhibits TPA-enhanced migration of melanoma cells. The data on cell migration capacity are summarized in Panel C. Significant inhibition versus TPA-treatment alone, *
P<0.001. (D) Treatment of melanoma cells with MEK inhibitor (UO126, 80 µM) resulted in inhibition of the activation of ERK1/2 as well as inhibition of cell migration compared to non-MEK inhibitor-treated control cells. The data are expressed as the mean number of migratory cells± SD/microscopic field, n = 3. Significant difference versus controls *
P<0.001.
GSPs and celecoxib inhibit TPA-induced activation of ERK1/2 protein in melanoma cells
As activation of MAPK proteins has been implicated in the enhancement of COX-2 expression or an upstream regulator of COX-2, we examined the effect of TPA on activation of ERK1/2 in melanoma cells, and simultaneously checked the effect of GSPs or celecoxib on TPA-induced activation of ERK1/2. Western blot analysis revealed that treatment of A375 cells with TPA enhanced the activation of ERK1/2, however, treatment of cells with GSPs or celecoxib inhibited TPA-induced activation of ERK1/2, as shown in Figure 3B. We further checked the effect of GSPs and celecoxib on TPA-induced cell migration. Cell migration assay analysis revealed that both GSPs and celecoxib significantly inhibited TPA-induced cell migration of melanoma cells (Figure 3C). We further verified the role of activated ERK1/2 in melanoma cell migration by using the inhibitor of MEK (UO126). Cell migration assay revealed that treatment of A375 cells with UO126 significantly inhibited (P<0.001) melanoma cell migration (Figure 3D). A summary of data related with cell migration are also shown. Additionally, western blot analysis revealed that the level of activated ERK1/2 was also decreased after the treatment of cells with MEK inhibitor UO126, as shown in Figure 3D.
GSPs decrease the activation of NF-κB/p65 in melanoma cells: NF-κB is an important mediator of melanoma cell migration
COX-2 is a downstream target of NF-κB, therefore we assessed whether GSPs also affect the proteins of NF-κB family in melanoma cells. For this purpose, A375 cells were treated with various concentrations of GSPs (0, 10, 20 and 40 µg/mL) for 24 h, and thereafter cells were harvested and whole cell lysates and nuclear lysates were prepared. The results of western blot analysis revealed that treatment of cells with GSPs reduce the nuclear translocation of NF-κB/p65 in a dose-dependent manner (Figure 4A). The activity of NF-κB also was significantly reduced (25–70%, P<0.01 and P<0.001) after the treatment of cells with GSPs in a concentration-dependent manner (Figure 4B). The western blot analysis also revealed that treatment of GSPs resulted in the down-regulation of IKKα, an enzyme responsible for NF-κB activation, and degradation of IκBα (Figure 4A), which leads to the inactivation of NF-κB. To check whether NF-κB has a role in melanoma cell migration, A375 melanoma cells were treated with caffeic acid phenethyl ester (0, 5, 10 and 20 µg/mL), a potent inhibitor of NF-κB, and cell migration was determined. As shown in Figure 4C, treatment of cells with caffeic acid phenethyl ester resulted in a dose-dependent reduction of cell migration (24–78%) relative to untreated control cells, and it was similar to that observed on treatment of the cells with GSPs (Figure 1B).
10.1371/journal.pone.0021539.g004Figure 4 Effect of GSPs on NF-κB activation.
(A) Treatment of A375 cells with GSPs decreases the basal levels of NF-κB/p65 and IKKα while inhibiting the degradation of IκBα. After treatment of cells for 24 h with various concentrations of GSPs the cells were harvested and cytosolic and nuclear fractions were prepared and subjected to the analysis of NF-κB/p65, IKKα and IκBα using western blot analysis. Representative blot is shown from three independent experiments with identical results. (B) The activity of NF-κB/p65 in the nuclear fraction of cells after treatment with and without GSPs for 24 h was measured using NF-κB/p65-specific activity assay kit, n = 3. Activity of NF-κB/p65 is expressed in terms of percent of control (non-GSPs-treated) group. Significant decrease versus control: ¶
P<0.01, *
P<0.001. (C) Treatment of A375 cells with caffeic acid phenethyl ester (CAPE), an inhibitor of NF-κB, for 24 h inhibits cell migration in a concentration-dependent manner. Data on cell migration capacity are summarized as the mean number of migratory cells ± SD/microscopic field, n = 3. Significant inhibition versus non-CAPE-treated cells: *
P<0.001.
GSPs reverse epithelial-to-mesenchymal transition in melanoma cells
Activation of NF-κB has been implicated in inflammation-induced cancer development and progression, and has been identified as an important regulator of EMT in several cancer cell types [17]–[20]. As the inhibition of melanoma cell migration by GSPs is associated with the inactivation of NF-κB, we sought to determine whether GSPs also affect or reverse EMT in melanoma cells and that is responsible for their inhibitory effect on melanoma invasiveness. For this purpose, A375 cells were treated with GSPs for 24 h, and cell lysates were prepared for the western blot analyses of various epithelial and mesenchymal biomarkers. Our western blot analyses revealed that GSPs restored or increased the levels of the epithelial biomarkers, such as E-cadherin, keratin-18, keratin-8 and desmoglein 2 in melanoma cells compared to untreated controls. In contrast, the levels of mesenchymal biomarkers, such as N-cadherin, vimentin, fibronectin and SLUG, were reduced in melanoma cells after treatment with GSPs in a dose-dependent manner, as shown in Figure 5A. GSPs-induced changes or effects on these epithelial and mesenchymal biomarkers were also detected and analyzed using immunofluorescence staining (Figure 5B). Immunofluorescnce staining data revealed that treatment of A375 cells with GSPs for 24 h resulted in reduction of mesenchymal biomarkers, such as vimentin, fibronectin and N-cadherin which is evident by the intensity of staining of the cells. In contrast, GSPs enhanced the levels of epithelial biomarker, such as desmoglein 2, in melanoma cells which is evident by the strong intensity of fluorescence staining compared to untreated controls. Similar observations were also noted when cells were treated with celecoxib, a COX-2-specific inhibitor, in identical manner. Representative photomicrographs are shown from three independent experiments.
10.1371/journal.pone.0021539.g005Figure 5 Treatment of melanoma cells with GSPs results in reversal of epithelial to mesenchymal transition.
(A) Treatment of A375 cells with GSPs for 24 h enhances the levels of epithelial biomarkers in the cells, such as, the levels of E-cadherin, keratin-18, keratin-8 and desmoglein 2. Simultaneously the levels of mesenchymal biomarkers in melanoma cells, such as, vimentin, fibronectin, N-cadherin and SLUG were decreased dose-dependently. (B) Identification of the levels of epithelial and mesenchymal biomarkers in A375 cells after the treatment of cells with GSPs or celecoxib using immunocytostaining, as detailed in Materials and Methods. Treatment of A375 cells with GSPs (20 and 40 µg/mL) or celecoxib (20 µM) for 24 h resulted in reduced expression of vimentin, fibronectin and N-cadherine, while the level of desmoglein 2 was increased. Representative photomicrographs are representative of three independent experiments with similar results.
GSPs and celecoxib inhibit TPA-induced EMT biomarkers in melanoma cells
As TPA induces COX-2 expression as well as enhances cell migration in melanoma cells, we next examined whether TPA promotes EMT in melanoma cells and whether GSPs and celecoxib inhibit TPA-induced EMT in these cells. For this purpose, A375 melanoma cells were treated with either TPA or celecoxib alone or TPA with the treatment of GSPs for 24 h, cell lysates prepared and subjected to western blot analysis. As shown in Figure 6, TPA decreased the level of desmoglein 2 (an epithelial biomarker), while enhanced the levels of mesenchymal biomarkers (N-cadherin and vimentin) compared with untreated control cells. Celecoxib enhanced the level of desmoglein 2 while decreased the levels of N-cadherin and vimentin compared with untreated control melanoma cells. Further, as shown in Figure 6, GSPs increased or restore the level of desmoglein 2 in TPA-treated melanoma cells, while reduced TPA-induced levels of N-cadherin and vimentin on A375 cells. These data further support the evidence that GSPs function as a COX-2 inhibitor and have the ability to reverse EMT in melanoma cancer cells and thus lead to reduce the invasiveness of melanoma cells.
10.1371/journal.pone.0021539.g006Figure 6 The effect of GSPs, TPA and celecoxib on the EMT biomarkers in melanoma cells.
A375 cells were treated for 24 h and cell lysates were prepared for the analysis of N-cadherin, vimentin and desmoglein 2 using western blot analysis. Equal loading of proteins on the membranes were verified using β-actin antibody. Representative blots are shown from 3 independent experiments.
Discussion
Melanoma cells can metastasize rapidly and that is the leading cause of death. According to a World Health Organization report, 48,000 melanoma-related deaths occur worldwide per year [21]. Treatment is more difficult if it has spread beyond skin and lymph nodes [22]. Therefore, innovative strategies are required to be developed for the prevention of the invasive or the migratory potential of melanoma cells. Many human cancers express elevated levels of COX-2 and enhanced biosynthesis of PGs. COX-2 overexpression and abundant production of PGs, and particularly PGE2, have been linked with tumor progression, invasion and metastasis [23]. Because of its important role in tumor invasion and metastasis, COX-2 is always a promising target for cancer therapy [8], [24]; therefore, the search and development of potential COX-2 as well as PGE2 inhibitors for the prevention or treatment of melanoma may prove to be an important and effective strategy.
The significant findings in the present study are that the treatment of melanoma cells with GSPs inhibits cell migration in a dose-dependent manner, and that is associated with the inhibition of COX-2 expression and PGE2 production. The melanoma cells overexpress COX-2, and the inhibition of COX-2 by GSPs contributes to the inhibition of cell migration of these cells. This concept is supported by the evidence that treatment of the melanoma cells with celecoxib, a potent COX-2 inhibitor, resulted in a reduction in cell migration. Similar effects were also noted when the melanoma cancer cells, A375 and Hs294t, were transfected with COX-2 siRNA. It has been shown that TPA promotes COX-2 expression, and we found that treatment of melanoma cells with TPA enhances cell migration, and that this TPA-induced cell migration was blocked by the treatment of cells with GSPs. These observations support the evidence that inhibition of melanoma cell migration by GSPs requires the inhibition of COX-2 expression. It has been reported that COX-2 inhibitors can inhibit cell migration; however, they may also induce some form of toxicity. This possibility is not found in GSPs as these are dietary components and toxicity has not been observed in animal models [10], [11].
It is well known that PGE2 exerts its biologic functions by stimulating epithelial cell growth, invasion potential and cellular survival signals [25], [26]. Singh et al. [27] have shown that PGE2 treatment enhanced melanoma cell migration and that berberine, a phytochemical, inhibits PGE2-induced migration of melanoma cells. Punathil and Katiyar [28] have examined the effect of GSPs on non-small cell lung cancer cell migration, and found that GSPs inhibit the migration of these cells by targeting nitric oxide, guanylate cyclase and ERK1/2 pathways. As COX-2 is a downstream target of NF-κB pathway, we further checked the effect of GSPs on the basal levels of NF-κB in melanoma cells, and found that treatment of melanoma cells with GSPs results in inactivation of NF-κB pathway in a dose-dependent manner. GSPs down-regulate the levels of IKKα which is responsible for NF-κB activation. Treatment of melanoma cells with caffeic acid phenethyl ester, an inhibitor of NF-κB, resulted in an inhibitory effect on melanoma cell migration. These observations support the concept that the inhibitory effect of GSPs on melanoma cell migration is mediated through the downregulation of COX-2 and PGE2, which are the downstream targets of NF-κB. Our study also demonstrates the requirement of activated ERK1/2 in melanoma cancer cell migration. Our results show that inhibition of melanoma cell migration by GSPs is associated with the inhibition of ERK1/2 phosphorylation. The inhibition of MEK with UO126, a MEK inhibitor, blocked the migration capacity of melanoma cells which is similar to the action of GSPs. Treatment of A375 cells with TPA increased ERK1/2 phosphorylation and subsequently enhanced cell migration, while treatment of cells with celecoxib decreased ERK1/2 phosphorylation and subsequently decreased cell migration. These observations suggest a possible involvement of MAPK pathway (which is an upstream regulator of NF-κB) in inhibition of melanoma cell migration by GSPs.
The transcription factor NF-κB regulates a wide spectrum of biological processes, including inflammation, cell proliferation and apoptosis. Additional roles of NF-κB in cancer biology, such as in tissue invasion, cell migration and metastasis, have been investigated recently. Importantly, NF-κB is involved in inflammation-induced cancer development, and has been identified as an important regulator of EMT in several cancer cell types [17]–[20]. EMT has been observed to play a major role in invasion and metastasis of epithelial tumors. EMT can render tumor cells migratory and invasive through the involvement of all stages, invasion, intravasation and extravasation [12]. During the process of EMT, cells can change from an epithelial to a mesenchymal state. They lose their characteristic epithelial traits and instead gain properties of mesenchymal cells. This process is primarily coordinated by the disappearance or loss of epithelial biomarkers such as E-cadherin and certain cytokeratins with the concomitant appearance or gain of mesenchymal markers such as vimentin, fibronection and N-cadherin, etc. In the present study, GSPs treatment of melanoma cells showed the suppression of mesenchymal biomarkers, such as vimentin, fibronectin and N-cadherin while restored the levels of epithelial biomarkers such as, E-cadherin, desmoglein 2, keratin-8 and -18, etc, in melanoma cells which suggest that GSPs have the ability to reverse the EMT process in melanoma cells and this may also be one of the possible mechanisms through which GSPs reduce the invasiveness of melanoma cells and that lead to inhibition of melanoma cell migration in our system.
In summary, the results from this study have identified for the first time that GSPs inhibit the invasiveness of melanoma cells or inhibit the ability of melanoma cell migration and that involves: (i) the inhibitory effect of GSPs on endogenous COX-2 overexpression and successive down-regulation of PGE2 synthesis, (ii) the inhibitory effect of GSPs on the activation of NF-κB and the proteins of MAPK family, which are the upstream regulators of COX-2 and PGE2, and (iii) the reversal of EMT process. More detailed studies are needed to develop GSPs as a pharmacologically safe agent either alone or in combination with other anti-metastatic drugs for the treatment of metastatic melanoma in humans.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by funds from the Veterans Administration Merit Review Award (SKK). There is no grant number. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Int J Mol SciijmsInternational Journal of Molecular Sciences1422-0067Molecular Diversity Preservation International (MDPI) 10.3390/ijms12042325ijms-12-02325ArticleDextran Sulfate Sodium Inhibits Alanine Synthesis in Caco-2 Cells Ye Zhong 1Mishchuk Darya O. 2Stephens Natasha S. 2Slupsky Carolyn M. 12*1 Department of Nutrition, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA; E-Mail: [email protected] Department of Food Science and Technology, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA; E-Mails: [email protected] (D.O.M.); [email protected] (N.S.S.)* Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +1-530-752-6804; Fax: +1-530-752-8966.4 4 2011 2011 12 4 2325 2335 5 2 2011 12 3 2011 28 3 2011 © 2011 by the authors; licensee MDPI, Basel, Switzerland.2011This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).To understand and characterize the pathogenic mechanisms of inflammatory bowel disease, dextran sulfate sodium (DSS) has been used to induce acute and chronic colitis in animal models by causing intestinal epithelium damage. The mechanism of action of DSS in producing this outcome is not well understood. In an effort to understand how DSS might impact epithelial cell metabolism, we studied the intestinal epithelial cell line Caco-2 incubated with 1% DSS over 56 hours using 1H NMR spectroscopy. We observed no difference in cell viability as compared to control cultures, and an approximately 1.5-fold increase in IL-6 production upon incubation with 1% DSS. The effect on Caco-2 cell metabolism as measured through changes in the concentration of metabolites in the cell supernatant included a three-fold decrease in the concentration of alanine. Given that the concentrations of other amino acids in the cell culture supernatant were not different between treated and control cultures over 56 hours suggest that DSS inhibits alanine synthesis, specifically alanine aminotransferase, without affecting other key metabolic pathways. The importance of alanine aminotransferase in inflammatory bowel disease is discussed.
metabolomicsmetabonomicsIBDDSSNMRCaco-2alanine transaminaseCrohn’s diseaseALATALT
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1. Introduction
Crohn’s disease (CD) and ulcerative colitis (UC), both inflammatory bowel diseases (IBD), involve chronic inflammation of the gastrointestinal tract. The etiology and mechanisms of IBD remain unclear, but it is generally agreed to be a complex interplay between the immune system, genetics, and environmental factors. To aid in the understanding of the pathogenesis of the disease, dextran sulfate sodium (DSS) has been used to induce colitis in experimental animal models [1,2]. DSS is a water-soluble polymer of glucose containing up to 20% sulfur with molecular weights ranging from 5,000 to 1.4 million Da. DSS is poorly absorbed after oral administration of enteric coated tablets, and no evidence of systemic absorption has been observed in humans [3]. Depending on the concentration, molecular weight, sulfation, and length of exposure, oral administration of DSS to rodents has been shown to induce acute or chronic colitis that resembles UC [1,4,5]. When supplied with DSS in their drinking water, mice develop colonic mucosal inflammation with ulcerations, body weight loss, and bloody diarrhea that resolves after DSS removal [4]. Chronic inflammation may be induced by administration of a further three to five cycles of DSS [6,7].
Intestinal epithelium damage is a key feature of DSS-induced colitis, characterized by multi-focal areas of mucosal erosion, epithelial cell injury, and significant mucosal infiltration of neutrophils [8]. A recent study involving a mouse model of DSS-induced colitis showed that epithelial apoptosis increased approximately five-fold, mitotic cells decreased by approximately half, and cells with cell cycle arrest at G0 increased two-fold in DSS treated mice as compared to control mice [9].
The effects of DSS have also been studied in cell-culture models. For instance, it has been shown that DSS alters Caco-2 tight junctions, cell cycle metabolism, as well as cytokine release at concentrations ranging from 0.3% to 5% w/v [7,10]. DSS at higher molecular weights and higher concentrations tended to have a greater effect on cell viability [10]. However, it is unclear what metabolic changes happen to colon epithelial cells in the absence of bacteria. In the present study, we apply 1H NMR-based metabolomics to study how DSS affects the extracellular metabolites of Caco-2 cells in an effort to understand the mechanism of action of DSS on epithelial cells.
2. Results and Discussion
2.1. Cell Viability of Caco-2 Cells Treated with DSS Is Similar to Control
To determine whether treated cells were viable after incubation with DSS, a trypan blue dye exclusion assay was performed (Figure 1). At specific time points from 2 to 56 h, numbers of viable cells exposed to 1% DSS were compared to those of controls. No difference in cell viability was found over 56 h. In addition, microscopy did not reveal any significant morphological changes between treated and untreated Caco-2 cells.
2.2. Interleukin-6 Level Increases with DSS Incubation
To determine the effect of 1% DSS on expression of interleukin-6 (IL-6), IL-6 assays were performed and compared between control and DSS-treated Caco-2 cells (Figure 2). At all timepoints, the concentration of IL-6 in the cell supernatant was determined to be higher for the DSS-treated cells.
2.3. DSS Induces Changes in 1H NMR Spectra of Supernatant Derived from Culture of Caco-2 Cells
To determine the effect of 1% DSS on metabolism of Caco-2 cells, 1H NMR spectroscopy was performed and compared between control and DSS-treated Caco-2 cell supernatants. Representative spectra from control and 1% DSS-treated cells at 56 h are shown in Figure 3. The concentration of alanine was higher in the control culture in comparison with the DSS-treated culture. Interestingly, no changes in lactate were observed upon incubation with 1% DSS, however, glucose concentrations appeared to be slightly higher in DSS-treated cells, but due to variability between samples, the difference was not significant (Figure 4). Ethanol was a contaminant in all samples, and its concentration was determined to not be significantly different between treated and untreated cells.
Comparison of metabolite concentrations in the cell culture media between the control and DSS-treated Caco-2 cells revealed statistically significant higher concentrations of alanine in the control culture supernatant (Figure 4), with a concentration approximately three times greater than the concentration of alanine in the supernatant of DSS-treated cells. In the media alone, the concentration of alanine is approximately 100 μM. However, in both control and DSS-treated cells the concentration of alanine increases over time to nearly 2 mM for the control, and 600 μM for the DSS-treated cells suggesting that alanine is exported from the cell. Interestingly, the concentration of lactate in the cell culture supernatant was similar between the control and DSS-treated cells. Comparison of metabolites imported into the Caco-2 cells (including glucose, glutamine, and pyruvate) revealed no significant differences between control and DSS-treated cells (Figure 4). Glutamate concentrations were not significantly different between treated and control cells, and α-ketoglutarate was undetectable in the cell culture supernatant.
2.4. Discussion
DSS is often used in animal studies to induce colitis [1,2]. However, the metabolic effects of DSS on intestinal epithelial cells have not been characterized to date. In this study, we applied 1H NMR spectroscopy to study the effect of DSS on a cell-culture mimic of the human small intestine, Caco-2. Utilizing 1% DSS, we determined that cell viability was unaffected over 56 h, and that a 1.5-fold increase in IL-6 production by Caco-2 cells occurred upon incubation of Caco-2 cells with 1% DSS. This is in agreement to Araki et al. [10]. Furthermore, a significant decrease in alanine production was observed when Caco-2 cells were incubated with DSS, but no significant differences were observed in the concentrations of lactate or pyruvate. These observations suggest one of two mechanisms: either the blocking of the alanine transporter, or the inhibition of the enzyme alanine aminotransferase (ALAT) either through blocking of transcription or blocking of the enzyme itself.
Alanine transport in Caco-2 cells occurs via system B, which is a sodium dependent chloride-independent transporter that also transports glutamine [11]. If DSS were blocking this transporter, changes in the transport of glutamine would be expected (Figure 5). However, no significant change in the concentration of glutamine was observed in the cell supernatant of DSS-treated versus control cells. Thus the lower concentration of alanine is unlikely due to the blockage of the alanine transporter. Although system B is a sodium-dependant transporter, it has been previously determined that sodium concentration does not affect maximal alanine influx [12,13]. Even so, the addition of 1% DSS to the cell culture only changed the conductivity by a small amount (16.7 mS/cm versus 15.4 mS/cm in the control media).
Alanine aminotranferase (ALAT) is an enzyme that catalyzes the transfer of the α-amino group from glutamate to pyruvate forming alanine and α-ketoglutarate. Inhibition of this enzyme would impact synthesis of alanine, but not necessarily change the concentration of other metabolites or affect other cellular pathways as pyruvate has many fates in the cell including the formation of lactate, other amino acids, and can enter the TCA cycle. α-Ketoglutarate can also enter the TCA cycle. If the reason for decreased alanine in the cell culture media is due to the inhibition of ALAT, it is likely that it occurs either through direct inhibition of the enzyme or through inhibition of transcription. In either case, the enzyme still functions as the concentration of alanine in the medium of DSS-treated cells does increase from the baseline level in the media over 56 hours.
The fact that ALAT is somehow affected by DSS is an interesting finding. In a study involving 123 IBD patients, it was determined that 49/50 CD patients had subnormal serum ALAT levels, whereas only 1/67 patients with UC had subnormal ALAT on one or more occasions [14]. Interestingly, however, in a study of 544 patients, it was determined that ALAT was increased in concentration in the serum, with no specific association to IBD activity [15]. In another study, total enteral nutrition of pediatric patients was shown to be associated with a transient hypertransaminasemia and no other evidence of liver disease [16]. In a study involving IL-10 gene deficient mice, it was determined that in both wild-type and gene-deficient mice, treatment of mice with dinitrobenzene sulfonic acid (DNBS) resulted in a substantial increase in serum ALAT [17]. However serum ALAT did not appear significantly different from control in 5% DSS-treated mice [18]. Of importance, it was determined that the concentration of alanine in the colonic mucosa of patients with both UC and CD was decreased as compared to normals [19]. Moreover, alanine was shown to be significantly higher in fecal samples from CD patients, but not UC patients, as compared to control [20].
Taken together, regulation of ALAT activity, either through direct inhibition of the enzyme or inhibition of transcription, has an association with inflammatory bowel disease, and in particular Crohn’s disease. Whether it is directly related to the pathogenesis of CD, or is a consequence of the disease itself remains to be elucidated. The fact that in human patients serum ALAT deviations from normal may be transient in nature, and in most cases do not appear to be associated with liver disease suggests that there may be a dietary or bacterial flora connection. Although ALAT activity has often been thought of as an indicator of hepatic function, the increase in serum values of ALAT may be related to changes in the intestinal tissue itself, with changes in intestinal metabolism potentially signaling changes in hepatic ALAT expression. Indeed it has been shown that patients with CD have increased insulin secretion [21], and that higher ALAT levels are associated with impaired glucose tolerance [22]. Of significance, it has been shown that p300 and c-Myb regulate ALAT gene transcription, and that insulin levels affect expression of these factors [23] thereby affecting ALAT gene expression. We are currently investigating whether DSS directly inhibits ALAT or inhibits transcription, and whether serum levels of ALAT can affect the action of DSS.
3. Experimental Section
3.1. Caco-2 Preparation and Reagents
The human Caco-2 cell line has been widely used as an in vitro model of the intestinal epithelium [24]. In this study, Caco-2 cells were obtained from American Tissue Culture Collection (ATCC, Manassas, VA, USA) at passage 18 and experiments were performed with cells from passages 25–30. Caco-2 cells were cultured in Dulbecco’s modified Eagle’s minimum essential medium (DMEM, HyClone, Logan, UT, USA) supplemented with 25 mM glucose, 10% fetal bovine serum (FBS), 1% nonessential amino acids, 4 mM L-glutamine and 1% penicillin-streptomycin solution at 37 °C with 5% CO2. Dextran sulfate sodium (DSS, MW 36,000–50,000, MP Biomedicals LLC, Solon, OH, USA) was dissolved in culture media and filter-sterilized using a 0.2 μm filter. To test the effect of DSS on Caco-2 cells, cells were seeded onto 24-well plates (Costar, Corning, NY, USA) at a density of 1 × 104 viable cells/cm2. After 90–100% confluency, the Caco-2 cell monolayers were allowed to differentiate for an additional 14 days. Fully differentiated cell monolayers were incubated with or without 1% DSS in cell culture media for 2 to 56 h. The Caco-2 cells at different time points after DSS addition were observed under an Olympus IX71 inverted microscope equipped with a digital camera using MetaMorph software. Images of Caco-2 cells were taken at 40X magnification. 1.0 mL aliquots of supernatant samples were collected at different time points, centrifuged at 14,000 rpm for 20 min to remove cellular debris, and stored at −80 °C until further analysis.
3.2. Caco-2 Cells Viability Test
Caco-2 cells were incubated with DSS in 24 well plates as described above. At each time point, cells were collected from the wells using 0.5 mL of 0.25% trypsin with 0.2 g/L EDTA (HyClone, Logan, UT, USA) and re-suspended in 1 mL of serum-free medium. The viability of control cells and cells incubated with DSS were determined using a Bright Line hemacytometer (Hausser Scientific, Horsham, PA, USA) and the trypan blue dye exclusion test. Results of viability are expressed as the percentage of the values obtained for control cells. All experiments were performed four times.
3.3. IL-6 Assay
Caco-2 cells were incubated in 24-well plates (Costar) and cell culture supernatants were collected as described above. IL-6 assays were performed using human IL-6 ELISA Ready-Set-Go kit (eBioscience, Inc., San Diego, CA, USA) according to manufacturer instructions.
3.4. NMR Sample Preparation, Spectroscopy and Analysis
Sample preparation: Samples were prepared by thawing the frozen supernatant and filtering though a 3000 MW cutoff filter (Pall Life Sciences, Ann Arbor, MI, USA). 585 μL of filtered sample was mixed with 65 μL of Internal Standard (IS) (5mM DSS-d6 (3-(trimethylsilyl)-1-propanesulfonic acid-d6) with 0.2% NaN3, in 99.8% D2O and the pH was adjusted to 6.8 ± 0.1. A 600 μL aliquot of each sample was transferred to a 5-mm NMR tube and stored at 4 °C until NMR data acquisition.
NMR spectroscopy: All one-dimensional NMR spectra of the samples were acquired using the first increment of the standard NOESY pulse sequence on a Bruker AVANCE 600 MHz NMR spectrometer equipped with a SampleJet. All spectra were recorded at 25 °C with a 12 ppm sweepwidth, 2.5 s recycle delay, 100-ms τmix, an acquisition time of 2.5 s, 8 dummy scans, and 32 transients. 1H saturation of the water resonance was applied during the recycle delay and the 100 ms τmix. All spectra were zero-filled to 128k data points and multiplied by an exponential weighting function corresponding to a line-broadening of 0.5 Hz.
Spectral analysis: Analysis of the NMR data was accomplished through targeted profiling using the Chenomx NMRSuite v6.1 (Chenomx Inc., Edmonton, Canada) [25]. A total of 39 metabolites were identified and quantified representing 99% of the spectral area.
3.5. Statistical Analysis
All data, including the concentrations derived from the 1H NMR spectra, IL-6 ELISA results, and viability of Caco-2 cells, are presented as mean ± SD. The difference in levels of variable between treatment and control were evaluated for individual values using the Student’s t-test. P-values of <0.05 were considered to be statistically significant.
4. Conclusions
The goal of this study was to understand the effect of DSS on Caco-2 cell metabolism. Although cell viability was similar, and IL-6 production was increased approximately 1.5 times, the only major metabolite difference observed when Caco-2 cells were incubated with 1% DSS was a decrease in alanine concentration in the cell culture medium as compared with controls. Since the concentration of glutamine and other amino acids were unaffected, we ruled out the possibility that DSS inhibited alanine transport across the membrane. These results suggest that either transcription of alanine aminotransferase is inhibited, or the enzyme itself is inhibited. This study emphasizes that alanine aminotransferase has a direct relationship with inflammatory bowel disease, and in particular CD, and may provide a more thorough understanding of the pathogenesis of CD in addition to the metabolic mechanisms for DSS-induced colitis in animal models. Work is currently under way to determine how alanine aminotransferase is inhibited by DSS.
This work was supported by UC Davis NMR award program. The UC Davis NMR facility is supported by the National Institutes of Health Grant RR11973. NSS is supported from a grant by the Broad Foundation.
Figure 1. Incubation of Caco-2 with dextran sulfate sodium (DSS) does not affect viability or morphological characteristics of Caco-2. (A) Numbers of viable cells were determined using the trypan blue dye exclusion method. Percent viability was expressed as the percentage of growth compared to total cells at each time point; (B) Images of Caco-2 cells using microscopy at 40× were acquired using an Olympus digital camera. Each time point for both treated and untreated groups represents the mean of four determinations.
Figure 2. Secretion of IL-6 in cell culture medium by control (□) and 1% DSS-treated (
) Caco-2 cells. Supernatants were collected for each of control and DSS-treated cells at 2, 6, 8, 10, 24, 32, and 56 hours, and IL-6 levels were measured, and averaged. Results therefore represent the mean of 28 determinations ± SD, p = 0.00003.
Figure 3. Representative 600 MHz 1H NMR spectra obtained from cell supernatant extracts from control and DSS-treated Caco-2 cells. IS (internal standard) represents sodium 2, 2-dimethyl-2-silapentane-5-sulfonate used as chemical shift reference. Ethanol is a contaminant.
Figure 4. Comparison of the concentration of metabolites in control (□), and 1% DSS-treated (
) Caco-2 cell culture supernatants. All metabolites in cell culture supernatants were collected for each of control and DSS-treated cells at 10, 24, 32, and 56 hours, and metabolites were measured, and averaged. Results therefore represent the mean of 16 determinations ± SD, and * p < 0.00001.
Figure 5. Schematic of alanine metabolism in Caco-2 cells. Here it is shown that Caco-2 cells absorb glucose, glutamine, and pyruvate from the medium and produce lactate and alanine that are released. Over time, glucose, glutamine, and pyruvate concentrations decrease in the medium while lactate and alanine concentrations increase.
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Proteome SciProteome Science1477-5956BioMed Central 1477-5956-9-272163577110.1186/1477-5956-9-27ReviewClinical and Technical Phosphoproteomic Research López Elena [email protected]ópez Isabel [email protected] Antonio [email protected]í Julia [email protected] Inflammatory core, Centro de Investigación i+12 del Hospital Universitario 12 de Octubre, Avda de Córdoba s/n 28041, Madrid, Spain2 Hematology Department, Hospital Universitario 12 Octubre, Avda de Córdoba s/n 28041, Madrid, Spain3 Immunology Department, Hospital Universitario La Paz, P° de la Castellana 261-28046, Madrid, Spain4 Immunology Department, Hospital Carlos III, Sinesio Delgado 28029, Madrid, Spain2011 2 6 2011 9 27 27 31 1 2011 2 6 2011 Copyright ©2011 López et al; licensee BioMed Central Ltd.2011López et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.An encouraging approach for the diagnosis and effective therapy of immunological pathologies, which would include cancer, is the identification of proteins and phosphorylated proteins. Disease proteomics, in particular, is a potentially useful method for this purpose. A key role is played by protein phosphorylation in the regulation of normal immunology disorders and targets for several new cancer drugs and drug candidates are cancer cells and protein kinases. Protein phosphorylation is a highly dynamic process. The functioning of new drugs is of major importance as is the selection of those patients who would respond best to a specific treatment regime. In all major aspects of cellular life signalling networks are key elements which play a major role in inter- and intracellular communications. They are involved in diverse processes such as cell-cycle progression, cellular metabolism, cell-cell communication and appropriate response to the cellular environment. A whole range of networks that are involved in the regulation of cell development, differentiation, proliferation, apoptosis, and immunologic responses is contained in the latter. It is so necessary to understand and monitor kinase signalling pathways in order to understand many immunology pathologies. Enrichment of phosphorylated proteins or peptides from tissue or bodily fluid samples is required. The application of technologies such as immunoproteomic techniques, phosphoenrichments and mass spectrometry (MS) is crucial for the identification and quantification of protein phosphorylation sites in order to advance in clinical research. Pharmacodynamic readouts of disease states and cellular drug responses in tumour samples will be provided as the field develops. We aim to detail the current and most useful techniques with research examples to isolate and carry out clinical phosphoproteomic studies which may be helpful for immunology and cancer research. Different phosphopeptide enrichment and quantitative techniques need to be combined to achieve good phosphopeptide recovery and good up- and-down phospho-regulation protein studies.
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Background
Importance of the field
The personalized management of diseases has and is being extended and this implies the prescription of specific therapeutics best suited for the individual patient and his/her type of illness. With the combination of different proteomic strategies this can be improved, and this would imply the coupling of proteomic and clinical research.
Areas covered in this review
Clinical and Proteomic research can be carried out in a complementary manner in order to advance and innovate therapies and diagnostics. We also point out the importance of immunology diseases including cancer, especially those which are directly connected to phosphorylated protein kinases and the way in which to isolate and methodologically analyse phosphoproteins-phosphopeptides, with their advantages and disadvantages, when using proteomic tools.
What the reader will gain
An overview of various types of different proteomic strategy-combinations personalized for specific diseases. The principles of phosphoproteomic techniques with examples are also presented in a simple manner. In addition, important mass spectrometry clues will be detailed in order to identify and correctly assign a phosphate group in a phosphorylated protein.
Take home message
A high number of proteomic-combination-approaches are available for clinical research. It is always necessary to test different proteomic tools in order to raise a greater level of efficiency for your clinical proteomic study, especially those related to phosphorylated proteins which are poorly expressed as some kinases. Nowadays it is essential that clinicians and proteomic experts work together in order to improve the therapies and drug candidates development.
We aim to detail the current and most useful techniques with research examples to isolate and carry out clinical phosphoproteomic studies which may be helpful for immunology and cancer research. Different phosphopeptide enrichment and quantitative techniques need to be combined to achieve good phosphopeptide recovery and good up- and-down phospho-regulation protein studies.
(A) Phosphorylation's role in immune disorders and cancer
Phosphorylation and de-phosphorylation at serine, threonine and tyrosine residues are one of the most common mechanisms of activation and/or inactivation signalling pathways. A variety of cellular processes including cellular growth, proliferation, cell cycle control, cytoskeletal mobility and receptor regulation [1] are controlled by this type of modification. Phosphorylation leads to allosteric modifications that may result in conformational changes sufficient to cause activation or inactivation of various proteins and associated altered functioning. It is our hypothesis that identification of phosphoproteins associated with the various stages of different immunological disorders, including cancer, may provide information on the development of the pathology. In addition the mechanism of tumorigenesis gives us insight into the development of diagnostic and therapeutic procedures.
The mitogen activated protein kinase (MAPK) pathways are known to be deregulated in many human malignancies [2-5]. With relation to malignancy, the best studies MAPKs are the extracellular signal regulated protein kinases (ERK). ERKs phosphorylate cytoplasmic targets migrate to the nucleus where they can activate transcription factors involved in cellular proliferation. Eractic signalling in the MAPK/ERK pathways has been described in prostate, breast and colon cancers in vitro as well as in vivo models [6-9]. In cervical cancer, furthermore, a study has described decreased activation of ERK1/2 in invasive cervical carcinoma [10]. Annexin A1, which is a calcium dependent phospholipid binding protein that has been linked to membrane trafficking through exocytosis and endocytosis [11], is a second relevant example. Other studies have evaluated the role of annexin A1 in the modulation of the MAPK/ERK [12]. In fact, many members of the Annexin family are known to undergo alternative splicing yielding a number of isoforms. The resulting variant forms may have different functions and binding capacity in comparison to the native forms [13]. The DNA-Protein Kinase catalytic subunit (DNA-PKcs) - another relevant example - a macromolecule found to be involved in the repair of double stranded DNA fractures through activation of p53, found to be expressed in cancer specimens in its tyrosine phosphorylated and cleaved form [14]. In contrast, in normal specimens DNA-PKcs existed in its whole, full length in non-phosphorylated form. This study was aimed at identifying differential expression and modification of proteins that could suggest erratic pathways which could serve as novel targets for developing new therapies in the treatment of cervical cancer and help in monitoring disease recurrence or progression. The general principles of signalling pathways are illustrated (Figure 1) and also, an example of the structure of a relevant phosphorylated protein kinase (Figure 2).
Figure 1 Signalling pathways: general principles. Followed by communication of the signal to different cellular compartments are signal processing and amplification by plasma membrane proximal events. The activation of multiple signal cascades by receptors, different protein post-translational modifications (PTMs), crosstalk between signalling pathways and feedback loops to ensure optimal signalling output are involved in this process. The binding of receptor Tyr kinases (RTKs) to their cognate ligands at the cell surface results in receptor dimerization and autophosphorylation. Phosphorylated Tyr residues subsequently serve as docking sites to recruit signalling mediators, such as growth factor receptor-bound protein 2 (GRB2). Multiple signalling cascades such as the phosphoinositide-3 kinase (PI3K)-AKT, Ras-Raf- extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK), and signal transducer and activator of transcription (STAT) pathways are activated by the assembly of these signalling complexes. Casitas B-lineage lymphoma (CBL)-mediated ubiquitylation of RTKs controls their endocytosis and the duration of receptor signalling. In addition, binding of tumour necrosis factor-α (TNFα) to its receptor, TNFR1, induces trimerization of the receptor and recruitment of the adaptor protein TNFR1-associated death domain (TRADD) This functions as a hub to assemble a multiprotein signalling complex containing TNFR-associated factor 2 (TRAF2), receptor interacting Ser/Thr protein kinase 1 (RIPK1) and nuclear factor-κB (NF-κB) essential modulator (NEMO). The result is the activation of different signalling networks, such as the ERK MAPK, p38 MAPK and NF-κB pathways. Proteins in the MAPK signalling pathways are activated by both RTKs and TNFα, which allows cells to integrate multiple signals. [Dotted lines indicate indirect activation of signalling pathways or translocation of proteins into the nucleus. IκB, inhibitor of NF-κB; IKK, inhibitor of NF-κB kinase; JNK1, Jun N-terminal kinase 1; MEK, MAPK ERK kinase; mTOR, mammalian target of rapamycin; p70S6K, p70 ribosomal S6 kinase-α; RSK, ribosomal protein S6 kinase-α].
Figure 2 Example of a phosphorylated protein kinase. The location of phosphorylated Ser-279 in the protein structure of human MAP kinase p38beta (p38B) is shown in this figure. A model for phosphorylated serine was located in the structural position of residue Ser-279 in the 3D crystallographic coordinates of p38B (Protein Data Bank code: 3GC8). Position of the ATP binding site is indicated. Plot was generated using PyMOL (DeLano Scientific, San Carlos, CA). The p38 pathway is one of the mitogen-activated protein kinase (MAPK) signalling cascades along with the extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways. Similar to other MAPK pathways, the p38 signalling cascade involves sequential activation of MAPK kinases (MAP3Ks) and MAPK kinases (MKKs) including MKK3, MKK4, and MKK6, which directly activate p38 through phosphorylation in a cell-type- and stimulus-dependent manner. Once activated, p38 MAPKs phosphorylate serine/threonine residues on their substrates, such as transcription factors, cell cycle regulators as well as protein kinases. By the p38 signalling pathway cells can interpret a wide range of external signals, such as inflammation, hyperosmorality, UV radiation and oxidative stress and they respond appropriately by generating an excessive abundance of different biological effects.
On the other hand, the CDC25 family of proteins consists of dual specificity phosphatases which regulate cell cycle transitions, and they are key targets for the checkpoint machinery to maintain genome stability during DNA damage. Three isoforms of CDC25 have been identified in mammalian cells: CDC25A, CDC25B, and CDC25C. CDC25A and CDC25B over-expression has been reported in many types of human cancers, but these are insufficient to cause cancer, and the mechanism responsible for CDC25 over-expression is unclear [15,16]. The study of dose-response effects of the anti-cancer drug rapamycin on the phosphoproteomics level has identified hundreds of novel rapamycin-targeted cellular proteins and their phosphorylation sites. This information has enabled us to identify CDC25B as the key enzyme in mediating rapamycin induced oncogenic AKT activation. It is important to point out that we can demonstrate that phosphoproteomic profiling of a certain therapeutic agent does not only identify potential drug target(s) to improve the efficiency of that therapeutic approach in disease treatment, but it can also provide cellular information about possible beneficial and adverse side effects of a specific disease therapy when treating patients [17].
In addition, primary immunodeficiencies (PID) are "nature's experiments" which have allowed, not only the elucidation of many signalling pathways, but also their function an clinical relevance. Bruton's tyrosine kinase, is an interesting example: (Btk; member of the Tec family of kinases) [18,19], important in B-lymphocyte development, differentiation, and signalling. Btk is predominantly expressed in B lymphocytes and monocytes but not in plasma cells [20,21]. Btk expression in the B-cell lineage is also developmentally regulated, with bone marrow derived hematopoietic stem cells, common lymphoid progenitor cells, developing B and myeloid lineages showing the highest levels, whereas the remaining mature cells prior to activation have reduced cellular Btk. What remains to be established is the physiological significance of Btk expression in other cell types as B lymphocytes are the only cells known to be affected in X-linked agammaglobulinemia (XLA). Mutations in the Btk gene lead to XLA in humans and X-linked immunodeficiency (Xid) in mice. Activation of Btk triggers a cascade of signalling-events that culminates in the generation of calcium mobilization and fluxes, cytoskeletal rearrangements, and transcriptional regulation involving nuclear factor-κB (NF-κB) and nuclear factor of activated T cells (NFAT). In B cells, NF-κB was shown to bind to the Btk promoter and induce transcription, whereas the B-cell receptor dependent NF-κB signalling pathway requires functional Btk. In addition, Btk activation is strictly regulated by a plethora of other signalling proteins including protein kinase C (PKC), Sab/SH3BP5, and caveolin-1. Additionally, the prolyl isomerase Pin1 negatively regulates Btk by decreasing tyrosine phosphorylation and uniform state levels of Btk [22]. It is of great interest that PKC and Pin1, both of which are negative regulators of Btk, bind to the pleckstrin homology domain of Btk. For this purpose, novel mutations in the pleckstrin homology are under research, in order to design selective and novel drugs [23]. Common variable immunodeficiency (CVID) is a PID disease. CVID is the result of intrinsic deficits affecting immunologic functions. Moreover lymphomas and neoplams are found to be related to CVID. CVID is heterogeneous, can be present early or late in life, and it is associated with specific comorbidities [24,25]. Efforts to subcategorize CVID to predict outcomes and comorbid-condition, both clinically and based on immunologic phenotypes, are ongoing [26]. B cell-activating factor of the TNF family receptor [27], transmembrane activator, calcium modulator, cyclophilin ligand interactor (TACI) [28-30], and certain HLA haplotypes [31,32] have been identified as potential gene candidates for susceptibility to CVID. Inducible costimulator [33,34], CD81 [35], CD19 [36,37] and CD20 [38] harbour disease-causing mutations that presently explain only a small percentage of cases [39]. Recently, a genome-wide association work [40] has identified diverse causes of common variable immunodeficiency providing new mechanistic insights into immunopathogenesis based on these unique genetic variations. A highly significant number of subjects with duplications in ORC4L, a gene previously associated with B-cell lymphoproliferative disorders was observed. All these new insights could be susceptible to phosphoproteomic analysis in order to clarify the clues of the different pathologies [41].
(B) Analytical techniques used in phosphoproteomics
B.1. Preparation of samples
The key to any successful analysis is good sample preparation; phosphorylated proteins are quite stable, chemically, but there are highly susceptible to enzymatic modification. We emphasize the importance of phosphorylation of protein kinases due to the fact that they modulate many immunology diseases and they are usually poorly expressed. Moreover, the human genome contains around 500 kinases and over 100 phosphatases [42], so that when tissues or cells are lysed and extracted, it is highly probable that further enzymatic reactions will occur. Samples have to be prepared (a) quickly, (b) generally be snap-frozen and (c) treated with phosphatase inhibitors to avoid modification of phosphopeptides during sample work-up [43,44]. Phosphopeptides generally constitute a small portion of the peptides in a given protein sample, making them difficult to detect by MS; their enrichment helps to overcome this problem. It is important (d) to avoid salts and detergents, which can decrease the recovery of phosphopeptides and/or interfere with subsequent analysis [45].
B.2. Enrichment of phosphoprotein and phosphopeptide
The aim in many focuses, including the study of immune disorders, is to generate a global view of serine, threonine and tyrosine phosphorylation within the sample, concentrating specifically on the selected subset of phosphopeptides. Since the detection of phosphopeptides by MS is often hindered by suppression effects, many different strategies have been established: for the removal of unphosphorylated peptides: (I) immunoprecipitation by antibodies, (II) pre-fraction systems such as ionic chromatographic exchange (SCX/SAX), calcium phosphate precipitation and hydrophilic interaction chromatography (HILIC) (III) metal affinity chromatography i.e. IMAC, TiO2, ZrO2, and (IV) reverse phase chromatography (RP). (V) Immunoprecipitation of phosphotyrosine coupled or not to polyacrylamide gels, is still much more frequent [43] than immunoprecipitation using phospho- serine or threonine antibodies. This is because affinity chromatography such as IMAC or titanium dioxide has higher a capacity for phosphoserine and phosphothreonine peptide binding.
• Antibody purification and Polyacrylamide gels
Affinity purification, a method for purifying proteins, can be used together with SDS-PAGE or alone. Antibodies raised against a protein can be used to immunoprecipitate the protein and search for phosphorylation sites. Immunoprecipitation permits the isolation of a protein under a variety of biological conditions to assess changes in phosphorylation on that protein. In the same way, antibodies raised against a specific phosphosite on a protein can be used for immunoprecipitation. Assessment of other phosphosites on a protein is possible when one phosphosite is known (the epitope of the antibody) under this scenario. However, care must be taken when a protein is phosphorylated at multiple serines as certain phosphorylation events could be mutually exclusive and be obliterated during subsequent analysis. Phosphospecific antibodies can be used to determine the proteins that bind to a phosphoprotein (protein-phosphoprotein interactions) using phosphosite-specific immunoprecipitation followed by analysis of the binding partners. Furthermore, antibodies specific for phosphotyrosines, not affected by the surrounding amino acids, have been successfully used to immunoprecipitate the "phosphotyrosineome" of cells. Since phosphoserine and phosphothreonine are much more abundant in cells and these antibodies seem to have less specificity, phosphoproteome-wide experiments are much more complicated [46].
Moreover, phospho-specific antibodies against a consensus sequence-motif for a specific kinase-motif (for example SXR, where × is any amino acid) can also be used to immunopurify all proteins that contain this motif. This form of phosphorylation has been enriched by the use of antiphosphotyrosine antibodies. It is an interesting strategy as phosphotyrosine is far less common than phosphoserine or threonine; the antibodies generally have a higher specificity and tyrosine kinases play a prominent role in human cancer. The isolated proteins are enzymatically cleaved with trypsin and analysed by MS or the phosphopeptides can be further enriched for analysis by MS. Tyrosine phosphorylated proteins are enriched by these methods to levels sufficient for detection and sequencing by MS [47-52]. Antiphosphoserine and antiphosphothreonine antibodies have been also generated [43] but have not been widely used due to their low specificity.
We would like to mention the scientific study of Kemna and co-workers (2007) [53], who used immunocapture, and tandem MS to identify and characterize hepcidin in serum and urine. In addition to diagnostic application, they investigated analytical reproducibility and biological and preanalytical variation for both serum and urine sample fluids. Samples were obtained from healthy controls and patients with documented iron-deficiency anaemia, inflammation-induced anaemia, thalassemia major, and hereditary hemochromatosis. This important proteomic technique showed that hepcidin-20, -22, and -25 isoforms are present in urine. Hepcidin-25 in serum had the same amino acid sequence as hepcidin-25 in urine, whereas hepcidin-22 was not detected in serum. In this work, Kemna and co-workers (2007) also observed that urine hepcidin is more affected by multiple freeze-thaw cycles and storage conditions, but less influenced by diurnal variation, than serum hepcidin.
Barbey and co-workers (2009) [54] also produced another interesting scientific work where they described the results of a proteomic analysis based on SDS-PAGE, immunoblot and mass spectrometry, aimed at the identification of secreted proteins that are differently expressed at 30°C versus 37°C and at mid-exponential versus early-stationary growth phase and antigenic proteins from Rhodococcus equi ATCC 33701. A total of 48 proteins were identified irrespective of growth conditions. Cholesterol oxidase ChoE appears to be the major secretory protein. Four proteins, in addition, revealed high homologies with the mycolyl transferases of the Ag85 complex from Mycobacterium tuberculosis. 24 proteins are transported by a signal peptide-dependent pathway according to the prediction of the sequence analysis. Moreover, five antigenic proteins of R. equi were identified by immunoblot, including a novel, strongly immunoreactive protein of unknown function. In conclusion, the elucidation of the secretome of R. equi identified several proteins with different biological functions and a new candidate developing vaccines against R. equi infection in horses.
Radio-labelling polyacrylamide gels (P32 & 2DE) and 2D phosphopeptide mapping P32 labelling has long been used, on the other hand, for the analysis of immuno-precipitated and gel-separated signalling complexes and for quantify cation of differentially phosphorylated proteins by two-dimensional gel electrophoresis (2DE) of total cell lysates. The latter technique uses differential labelling of cells with P32 and P33 in a control and experimental group respectively. The samples are combined, and then separated by 2DE before the gels are exposed twice to radio-sensitive film. Comparison of these two exposures will reveal spots that are specifically phosphorylated under the experimental conditions tested [55,56]. It measures the incorporation of the label but not the phosphorylation level, although interesting studies can be carried out to study different isoforms of the same protein [57]. Two-dimensional (2D) phosphopeptide mapping by electrophoresis is another useful technique combined with thin-layer chromatography of peptides derived by proteolysis of a phosphoprotein. The number of spots detected indicates the number of sites of phosphorylation, but it is not easy to determine the position of the phosphorylation sites. Nevertheless, analysis of temporal and positional changes in a protein phosphorylation pattern under different physiological conditions [58] is permitted by this technique. Immunoprecipitation with specific antibodies against phosphopeptides can be used to immunoprecipitate phosphoproteins from the cell lysates [59].
• Immobilised metal ion affinity chromatography (IMAC)
IMAC [60] is an enrichment technique that makes use of metal ions to capture and enrich negatively charged phosphopeptides prior to mass spectrometric analysis [61-66]. Simple and complex samples containing phosphopeptides and non-phosphorylated peptides are dissolved in an acidic solution to stimulate the electrostatic interactions between the negatively charged peptides, mainly phosphopeptides, and the metal ions [64]. The phosphopeptides are eluted from the stationary phase using alkaline buffers. It is also possible to bind peptides containing the acidic amino acid residues glutamic acid and aspartic acid to the metal ions. Ficarro and co-workers [67] bypassed this problem with IMAC (Fe3+) by converting acidic amino acid residues to methyl esters. They were able to purify and sequence hundreds of phosphopeptides from yeast, although there was a strong tendency towards phosphoproteins highly expressed within the cell.
Collins and co-workers (2008) [68] analyzed the mouse forebrain cytosolic phosphoproteome using sequential (protein and peptide) IMAC purifications, enzymatic dephosphorylation, and targeted tandem mass spectrometry analysis strategies (MS clues will be detailed later) which we consider a relevant biological study. To summarize, Collins et al., (2008) [68] with the use of complementary phosphoenrichment and LCMS/MS strategies, 512 phosphorylation sites on 540 nonredundant phosphopeptides from 162 cytosolic phosphoproteins were characterized. Analysis of protein domains and amino acid sequence composition of this data set of cytosolic phosphoproteins revealed that it is significantly enriched in intrinsic sequence disorder, which enrichment is associated with both cellular location and phosphorylation status. The majority of phosphorylation sites found by MS were located outside structural protein domains (97%) They were mostly located in regions of intrinsic sequence disorder (86%). 368 phosphorylation sites were located in long regions of disorder (over 40 amino acids long), and 94% of proteins contained at least one such long region of disorder. In addition, it was found that 58 phosphorylation sites in this data set occur in 14-3-3 binding consensus motifs; linear motifs that are associated with unstructured regions in proteins. These results demonstrate that in this data set protein phosphorylation is distinctively depleted in protein domains and distinctively enriched in disordered protein sequences and that enrichment of intrinsic sequence disorder may be a common feature of phosphoproteomes. This goes to support the hypothesis that disordered regions in proteins allow kinases, phosphatases, and phosphorylation-dependent binding proteins to gain access to target sequences to regulate local protein conformation and activity.
• Titanium dioxide metal-based chromatography (TiO2)
TiO2 is also capable of binding negatively charged phosphate groups from aqueous solutions [65,69,70]. TiO2, like IMAC, experiences the problem of binding acidic non-phosphorylated peptides (negatively charged peptides). Heck and co-workers [65] observed a number of non-phosphorylated peptides in their analysis and recommended esterification of the acidic residues prior to the MS analysis. Larsen et al. [45,71,41] used 2,5-dihydroxybenzoic acid (DHB) with TiO2 and achieved higher specificity and yield compared to IMAC (Fe3+) for the selective enrichment of phosphorylated peptides from model proteins. It was also demonstrated that by the use of glycolic acid in the loading buffer, more phosphopeptides are bound to the metal ions and more phosphopeptides can be eluted by using ammonium hydroxide as the eluent. TiO2 binds multi-phosphorylated peptides in a strong way, thus their elution is difficult. However, this is a very effective method for the isolation of singly phosphorylated peptides [72].
The research work of Craft and co-workers (2007) [73] is an interesting example of the application of TiO2 technique coupled to other proteomic tools. Amphiphysin I (amphI) is dephosphorylated by calcineurin during nerve terminal depolarization and synaptic vesicle endocytosis (SVE). Some amphI phosphorylation sites (phosphosites) have been identified with in vitro studies or phosphoproteomics screens. A multifaceted strategy including 32P tracking to identify all in vivo amphI phosphosites and determine their relative abundance and potential relevance to SVE was used. AmphI was extracted from 32P-labeled synaptosomes; phosphopeptides were isolated from proteolytic digests using TiO2 chromatography, and mass spectrometry revealed 13 sites: serines 250, 252, 262, 268, 272, 276, 285, 293, 496, 514, 539, and 626 and Thr-310. These were distributed into two clusters around the proline-rich domain and the C-terminal Src homology 3 domain. Hierarchical phosphorylation of Ser- 262 preceded phosphorylation of Ser-268, -272, -276, and -285. Off-line HPLC (High-performance liquid chromatography or high-pressure liquid chromatography separation and two-dimensional tryptic mapping of 32P-labeled amphI revealed that Thr-310, Ser-293, Ser-285, Ser-272, Ser-276, and Ser-268 contained the highest 32P incorporation and were the most stimulus-sensitive. Individually Thr-310 and Ser-293 were the most abundant phosphosites, incorporating 16 and 23% of the 32P. The multiple phosphopeptides containing Ser-268, Ser-276, Ser-272, and Ser-285 had 27% of the 32P. Evidence for a role for at least one proline-directed protein kinase and one non-proline-directed kinase was obtained. Four phosphosites predicted for non-prolinedirected kinases, Ser-626, -250, -252, and -539, contained low amounts of 32P and were not depolarization-responsive. At least one alternatively spliced amphI isoform was identified in synaptosomes as being constitutively phosphorylated because it did not incorporate 32P during the 1-h labeling period. Multiple phosphosites from amphIco- migrating synaptosomal proteins were also identified, including SGIP (Src homology 3 domain growth factor receptor-bound 2 (Grb2)-like (endophilin)-interacting protein 1), AAK1, eps15R, MAP6, α/β-adducin, and HCN1. Their results revealed two sets of amphI phosphosites that are either dynamically turning over or constitutively phosphorylated in nerve terminals and they improve the understanding of the role of individual amphI sites or phosphosite clusters in synaptic SVE.
• IMAC (SIMAC) Sequential elution
Sequential elution from IMAC is useful for purifying, detecting and characterising phosphorylated peptides from complex biological samples [72]. It makes use of the observation that mono-phosphorylated peptides tend to elute from IMAC (Fe3+) under acidic conditions whereas multi-phosphorylated peptides elute at high basic pH. TiO2 is used to capture and purify the unbound mono-phosphorylated peptides in the combined IMAC flowthrough and washings. SIMAC has been used successfully in the study of human stem cells (~300 μg) with more than 300 phosphopeptides, including the identification of mono and multiply phosphorylated peptides [74].
This technology was developed by Tine E. Thingholm and co-workers (2007) [74]. They reported a simple and rapid strategy, SIMAC (sequential elution from IMAC), using stem cells as a sample to be studied, for sequential separation of monophosphorylated peptides and multiply phosphorylated peptides from highly complex biological samples. This research study, allowed individual analysis of different pools of phosphorylated peptides using mass spectrometric parameters differentially optimized due to their unique properties. They compared the phosphoproteome identified from 120 μg of human mesenchymal stem cells using SIMAC and an optimized titanium dioxide chromatographic method. More than double the total number of identified phosphorylation sites was obtained with SIMAC, primarily from a 3-fold increase in recovery of multiply phosphorylated peptides.
• Zirconium dioxide (ZrO2)
The utility of ZrO2 for phosphopeptide isolation prior to mass spectrometric analysis has been demonstrated [75]. When compared with TiO2 using is α and ß casein as protein models, ZrO2 was capable of isolating singly phosphorylated peptides more selectively than TiO2. An interesting research study was carried out by Houjiang Zhou et al (2007) [76] where the high specificity of this approach was also demonstrated by the isolation of phosphopeptides from the digests of model phosphoproteins. The strong affinity of ZrO2 nanoparticles to phosphopeptides enables the specific enrichment of phosphopeptides from a complex peptide mixture in which the abundance of phosphopeptides is two orders of magnitude lower than that of nonphosphopeptides. ZrO2 nanoparticles were further applied to selectively isolate phosphopeptides from the tryptic digestion of mouse liver lysate for phosphoproteome analysis by nanoliter LC MS/MS (nano-LC-MS/MS) and MS/MS/MS. Manual validation, using a series of rigid criteria, identified a total of 248 defining phosphorylation sites and 140 phosphorylated peptides. Therefore, ZrO2 has been successfully used in the large-scale characterisation of phosphoproteins from mouse liver samples (~1 mg) [76]. A total of 248 phosphorylation sites and 140 phosphorylated peptides were identified in this study.
• Calcium phosphate precipitation
This is a strategy providing a useful pre-fractionation step to simplify and enrich phosphopeptides from complex samples. Zhang and co workers [77] have demonstrated that phosphopeptide precipitation by calcium phosphate combined with a two step IMAC (Fe3+) procedure resulted in the observation of an increased number of phosphopeptides. This method consists of precipitating phosphopeptides by adding 0.5 M NaHPO4 and 2 M NH3OH to the peptide-mixture followed by 2 M CaCl2. The sample is vortexed and centrifuged, and, subsequently, the supernatant is removed before washing the pellet with 80 mM CaCl2. The washed pellet is dissolved in 5% of formic acid and the resulting peptide mixture is desalted through reversed phase chromatography before isolating the phosphopeptides by IMAC (Fe3+).
Zhang and co workers [77] point out that even with very complex biological samples such as the total enzymatic digest of rice embryo proteins, high enrichment of the phosphopeptides can be achieved with minimal contamination with non-phosphopeptides. In addition, it could be possible to reduce the complexity of the samples by successive IMAC enrichments using a limited amount of IMAC material at each step. This technique demonstrates that serial phosphopeptide enrichment initiated by a precipitation step improves the selectivity of phosphopeptide enrichment and allows identification of more phosphopeptides. In addition, Zhang and co workers say that further analyses to examine the rice phosphoproteome in detail are now underway. Moreover, it can be applied for clinical phosphoproteomics clinical research.
• Strong cation and anion exchange (SCX and SAX)
The principle of SCX/SAX phosphopeptide enrichment is based on the negative charged phosphate group (PO4-) of the phosphopeptides. In cation exchange chromatography, a positively charged analyte is attracted to a negatively charged solid-support; whilst in anion exchange chromatography negatively charged molecules are attracted to a positively charged solid-support. SAX has previously been successfully combined with IMAC [66] and has resulted in greater recovery and identification by MS of mono-phosphorylated peptides originating from membrane proteins. In a similar way, SCX has been combined with IMAC (Fe3+) and MS analysis, allowing the identification of thousands of phosphorylated residues from complex biological samples [78]. Moreover, Gruhler and co-workers [78] demonstrated that use of the SCX/IMAC combination is consistent with their previous study where strong anion exchange chromatography/IMAC was used. Thus, either strong anion exchange chromatography (SAX) or SCX can be used to reduce the sample complexity prior to IMAC enrichment of phosphopeptides in large scale phosphoproteomics.
As practical issues, Nuhse et al., 2003 [66], investigated and presented a scheme for two-dimensional peptide separation using SAX chromatography prior to IMAC (Fe3+) in order to decrease the complexity of IMAC-purified phosphopeptides, obtaining a wide coverage of monophosphorylated peptides. Nuhse and co-workers did, in fact, obtain a high yield in identifying phosphopeptides from membrane proteins. SCX has also been successfully used coupled to IMAC (Fe3+) and MS analysis allowing the identification of thousands of phosphorylated residues from biological complex samples [78,79]. Gruhler and co-workers showed that performing SCX at low pH (2.7-3.0), phosphorylated peptides are separated from nonphosphorylated species according to the charge difference associated with the negatively charged phosphate group. Therefore, net charged peptides (+1) were collected in the first fractions of the SCX prefraction step containing mainly single phosphorylated peptides. These first fractions were then loaded onto IMAC (Fe3+) micro tips in order to recover a large number of phosphopeptides from biologically complex samples.
• Hydrophilic interaction chromatography (HILIC)
Hydrophilic interaction chromatography (HILIC) is a less commonly used method for peptide fractionation despite the fact that it is often used to fractionate small metabolites. HILIC is commonly described as partition chromatography or liquid/liquid extraction system between the mobile and stationary phase. A water-poor layer of mobile phase versus a water-rich layer on the surface of the polar stationary phase is formed. Thus, a distribution of the analytes between these two layers will occur. In addition, HILIC includes weak electrostatic mechanisms as well as hydrogen donor interactions between neutral polar molecules under high organic elution conditions. This distinguishes HILIC from ion exchange chromatography - the main principle for HILIC separation is based on the compound's polarity and degree of salvation. More polar compounds will have stronger interaction with the stationary aqueous layer than less polar compounds - resulting in a stronger retention. In addition, HILIC shows a very good separation and peak shape for critical compounds like adenosine and its phosphate derivatives.
It is of interest to note that Alburquerque and co-workers (2008) [80] carried out a study related to the separation of unphosphorylated peptides using SCX, HILIC, and RP-HPLC, indicating that a better orthogonal separation could occur between HILIC and RP-HPLC for unphosphorylated peptides. The observed orthogonal separation between HILIC and RP-HPLC is probably a reflection of their different mechanisms of separation. Although RP-HPLC depends on interaction with the hydrophobic amino acid side chains, HILIC depends on interaction with those hydrophilic and possibly charged amino acid residues via hydrogen bonding and ionic interactions. Moreover, because phosphopeptides are generally hydrophilic and charged, one would expect that phosphopeptides should interact more strongly with HILIC than do unphosphorylated peptides. Thus, it should be possible to separate phosphopeptides using HILIC.
Dean E. McNulty and Roland S. Annan (2008) [81] reported the use of hydrophilic interaction chromatography (HILIC) as part of a multidimensional chromatography strategy for proteomics. Analysis of tryptic digests from HeLa cells yielded numbers of protein identifications comparable to those obtained using strong cation exchange. They also demonstrate that HILIC represents a significant advance in phosphoproteomics analysis. In fact, they exploited the strong hydrophilicity of the phosphate group to selectively enrich and fractionate phosphopeptides based on their increased retention under HILIC conditions. In addition, in this study IMAC enrichment of phosphopeptides from HILIC fractions showed more than 99% selectivity. This was achieved without the use of derivatization or chemical modifiers. In a 300 μg equivalent of HeLa cell lysate over 1000 unique phosphorylation sites were identified. More than 700 novel sites were added to the HeLa phosphoproteome.
• Reverse phase chromatography
All the phosphorylated proteins and phosphopeptides isolations can be coupled to reverse phase chromatography. Subsequently, most phosphoprotein-phosphopeptide analyses are performed nowadays by MS. As the MS technique is sensitive to contaminants such as salts, it is necessary to clean the samples prior to analysis, generally by reversed phase chromatography combining POROs R3 with C18 Disks and also graphite powder [82-84]. Poros R3, C18 Disks and graphite powder are materials containing long hydrocarbon chains, proven to be effective for the desalting and cleaning of very hydrophilic peptides, including phosphopeptides [71,85]. In 1999, Gobom and co-workers [82] introduced a micro column purification method in which a chromatographic resin was packed in the tip of a small constricted GELoader tip, creating a micro-column. With GELoader tips packed with R3, C18 or graphite material, contaminants like salts can be separated from the phosphopeptides using a chromatographic approach. In fact, using RP chromatography, molecules such as proteins, peptides and nucleic acids are separated according to their hydrophobicity. In addition to the removal of salts, these techniques also facilitate a concentration of the sample by the use of a low elution volume. This is an additional improvement for the sensitivity and quality of the subsequent mass spectrometric analysis. RP chromatography is usually coupled to all the phosphoproteins and phosphopeptides enrichment-methods previously described.
• Current methodologies for the detection of phosphorylated proteins - Advantages and limitations
There are several analytical techniques for the analysis of phosphorylation, i.e., Edman sequencing and 32P-phosphopeptide mapping for localization of phosphorylation sites; however, these methods do not allow high-throughput analysis or imply very high- labour operations [86], whereas with the use of Mass Spectrometry (MS) high-throughput analysis of phosphorylated protein residues can be developed [67,78]. On the other hand, phosphospecific antibodies are routinely used to immunoprecipitate and therefore enrich in phosphorylated proteins from complex mixtures [87], but, currently, no commercial antibodies are available which are suitable for enriching all proteins that are phosphorylated, and thus, these proteins must be purified or enriched from complex mixtures using alternative methods [88]. By carrying out in-gel or in-solution trypsin digestion of protein complex mixtures, the resulted phosphopeptides and non-phosphopeptides can be loaded into different metal ion chromatographies (i.e. Immobilized Metal ion Affinity Chromatography IMAC (Fe3+), and Titanium Dioxide TiO2 [71] in order to enrich in phosphopeptides. The enriched solution can also be submitted into different reverse-phase chromatographies (i.e. Graphite powder [89], POROS R3 [88] in order to clean and desalt those phosphopeptides previously eluted. In addition these kinds of chromatographies will reduce the suppression of phosphorylated peptides in the mass spectra.
Using IMAC (Fe3+) and also (TiO2) [71] and (ZrO2) [1], the negatively charged phosphopeptides are purified by their affinity to positively charged metal ions, but some of these methods experience the problem of binding acidic, non-phosphorylated peptides. Ficarro and co-workers [67] bypased this problem on IMAC (Fe3+) by converting acidic peptides to methyl esters but increased the spectra complexity and requiring lyophilization of the sample, which causes adsorptive losses of phosphopeptides in particular [90]. Ficarro et al., were able to sequence hundreds of phosphopeptides from yeast, including Slt2p kinase, but the level of phosphorylated residues identified from kinases were low compared to those from phosphoproteins highly expressed within the cell. Recently, TiO2 chromatography using 2,5-dihydroxybenzoic acid (DHB) was introduced as a promising strategy by Larsen et al., [71]. TiO2/DHB resulted in a higher specificity and yield as compared to IMAC (Fe3+) for the selective enrichment of phosphorylated peptides from model proteins (i.e. lactoglobulin bovine, casein bovine). Moreover, SIMAC has been developed in order to get a higher efficiency than IMAC and TiO2 for the isolation of as many phosphopeptides as possible [74].
The fact that mainly phosphopeptides from highly expressed proteins within cells can be purified, while those from phosphorylated proteins with low level expression (i. e. kinases) do not bind so well to those resins, constitutes another important limitation concerning phosphoenrichment methods This is due to the low proportion of this kind of protein, or, on the other hand, their available amount binds to metal ions although it is not sufficient to be detected by MS. The combination of SCX with IMAC (Fe3+) has been proven on yeast, resulting in a huge number of phosphorylated residues identified (over 700 including Fus3p kinase) [78]. Although more than 100 signalling proteins and functional phosphorylation sites, including receptors, kinases and transcription factors, were identified, it was clear that only a fraction of the phosphoproteome was revealed [78]. In addition, recent combinations of HILIC with IMAC (Fe3+) have been proven in clinical studies (HeLa samples), with the result of the identification of a large number of phosphorylated residues (1000) [81].
Improvement in methodologies to enrich for phosphorylated residues from kinases is clearly necessary. However, this is not straightforward for several reasons: (a) The low abundance of those signalling molecules within cells, (b) The stress/stimulation time-duration, as only a small fraction of phosphorylated kinases are available at any given time as a result of a stimulus. The time adaptation over signalling pathways is also a relevant and fast factor for kinases phosphorylation [91].
• Summary - phosphoprotein and phosphopeptide enrichments based on electrostatic interactions
The most common techniques for enrichment for individual and/or global phosphorylation are IMAC and Titanium Dioxide (TiO2) [45], which are based on the high affinity of positively charged metal ions. However, conversion of carboxylate groups to esters effectively eliminates nonspecific retention of non-phosphorylated peptides, although this constitutes a drawback due to increased complexity in the subsequent MS analysis.
During the last five years, titanium dioxide (TiO2) has emerged as the most common of the metal oxide affinity chromatography (MOAC) based phosphopeptide enrichment methods. This technique offers increased capacity compared to IMAC resins in order to bind and elute mono-phosphorylated peptides. TiO2 exploits the same principle as IMAC, and is similarly prone to nonspecific retention of acidic nonphosphorylated peptides. However, when loading peptides in 2, 5-dihydroxybenzoic acid (DHB) [71], glycolic and phthalic acids, nonspecific binding to TiO2 is reduced, thereby improving phosphopeptide enrichment without a chemical modification of the sample. In addition, TiO2 is often considered to be interchangeable with IMAC. It works on similar levels of sample amounts (e.g., micrograms of protein) for the identification of phospho-sites by MS analysis. Recently, SIMAC [72,74] appeared as a phosphopeptide enrichment tool which exploits the properties of IMAC coupled to TiO2, making it possible to carry out more refined studies. Another phosphopeptide enrichment prior to mass spectrometric analysis is ZrO2 [75] and its principle is based on metal affinity chromatography like IMAC and TiO2. ZrO2 permits the isolation of single phosphorylated peptides in a more selective manner than TiO2. It has, in fact, been successfully used in the large-scale characterization of phosphoproteins [66,78-80]. Furthermore, strategies which consist of fractionating and subsequently enriching phosphopeptides on a proteome wide scale are based on strong cation/anion exchange (SCX and SAX) chromatography and HILIC interaction chromatography. Calcium phosphate precipitation is also a useful pre-fractionation step to simplify and enrich phosphopeptides from complex samples which can be coupled to IMAC [77].
B.3. Phosphopeptides isolated by Proteomic techniques - MS analysis
Phosphorylation on serine and threonine residues are labile and conventional fragmentation CID (Collision Induced Dissociation) typically results in the partial neutral loss of phosphoric acid (H3PO4, 98/z) in MS2 mode, due to the gas phase β-elimination of the phosphor-ester bond. Therefore, dehydroalanine and dehydroaminobutyric acid are generated. When peptide ions are fragmented by CID, series of y- and b- ions are formed [92,93]. By correlating mass difference between peaks in the y-ion series or between peaks in the b- ion series with amino acid residue masses the peptide sequence is obtained. The CID fragmentation occurs on the peptide backbone, and only limited sequence information is obtained. This event can also compromise the identification of phosphorylation sites. In relation to phosphotyrosine residues, partial neutral loss is also observed (HPO3, 80/z) in MS2 mode, but the phosphate group on tyrosine residues is more stable than on serine and threonine residues. In addition, the phospho-finger-print characteristic of phosphotyrosine, is the phosphotyrosine immonium ion (~216 Da), this being a positive indicator for the presence of a peptide phosphorylated on tyrosine [94,95]. The ion originating from neutral loss of phosphoric acid (H3PO4) can be selected for further fragmentation by MS3 mode. After neutral loss fragmentation, the selected ion is automatically selected for further fragmentation. This makes it possible to add extra energy for the fragmentation of peptide backbone. However, the MS3 mode requires that the phosphorylation on serine and threonine residues are labile and conventional fragmentation CID (Collision Induced Dissociation) typically results in the partial neutral loss of phosphoric acid (H3PO4, 98/z) in MS2 mode, due to the gas phase β-elimination of the phosphor-ester bond. Therefore, dehydroalanine and dehydroaminobutyric acid are generated. When peptide ions are fragmented by CID, series of y- and b- ions are formed [92,93]. The peptide sequence is obtained by correlating mass difference between peaks in the y-ion series or between peaks in the b-ion series with amino acid residue masses. The CID fragmentation occurs on the peptide backbone, and only limited sequence information is obtained. This event can also compromise the identification of phosphorylation sites. In relation to phosphotyrosine residues, partial neutral loss is also observed (HPO3, 80/z) in MS2 mode, but the phosphate group on tyrosine residues is more stable than on serine and threonine residues. In addition, the phospho-finger-print characteristic of phosphotyrosine, is the phosphotyrosine immonium ion (~216 Da), which is a positive indicator for the presence of a peptide phosphorylated on tyrosine [94,95]. The ion originating from neutral loss of phosphoric acid (H3PO4) can be selected for further fragmentation by MS3 mode. The selected ion, after neutral loss fragmentation, is automatically selected for further fragmentation. This makes it possible to add extra energy for the fragmentation of peptide backbone. However, the MS3 mode requires that the selected ion is abundant in order to observe the fragmented ions. A pseudo-MS3 development is MultiStage Activation (MSA) [96], which was implemented on quadrupole-IT and linear IT-orbitrap. In MSA, the fragmentation of the precursor ion occurs simultaneously with the fragmentation of the ion originating from the neutral loss. The MS2 and MS3 mass-data are then combined in a hybrid spectrum, resulting in improved sequence information and also in an improvement of reliance for the phosphorylation site assignment. Alternative fragmentations to CID are ECD (electron capture Dissociation) and ETD (Electron transfer dissociation). By ECD, radical peptide ions are obtained when multiplycharged peptide ions are rationed with low-energy thermalelectrons. In addition, this fragmentation occurs in the peptide between the backbone amide and the alpha carbon, generating c- and z-ions [97]. An advantage of ECD is that it only occurs on the peptide backbone, and labile phosphate groups remain intact on the resulting c- and z- fragment ions, thus enabling the identification of the specific phosphorylation sites. Therefore, it is extremely useful for the analysis of multiply-phosphorylated peptides. A disadvantage of ECD is its selectivity for disulfide bonds, due to the high radical affinity of the bond [98,99]. The main drawback of ECD is that it is solely used in the Fourier transform-Ion Cyclotron Resonance (FT-ICR) instruments due to the requirement of a static magnetic field for the thermal electrons, meaning high costs and high specialization. c- and z- ions are also generated by ETD. This fragmentation was actually developed in order to carry out ECD-like dissociation experiments, in a Quadrupole Linear Ion Trap [96,100]. ETD is a chemical process in which reaction with fluoranthene radical anions disrupts the peptide backbone at regular intervals. ETD preserves the intact information about labile modifications, which are not observed directly when using CID. For instance, phosphate groups are good leaving groups, which mean that they are easily lost in the excitation process. However, by using ETD one can directly observe fragments that contain the intact phosphopeptides. The drawback of ETD is that it is less sensitive compared to CID, because of lower ionization efficiency. As a result, we recommend using CID to start with, and would recommend switching to ETD in case you are not able to determine the phosphorylation site.
(C) Quantitative proteomic methodologies used in clinical research; examples of relevant phosphorylated proteins studied
For phosphopeptides proteins containing amino acids with one or more of the stable isotopes of 2H, 13C, 15N or 18O can be used as internal standards by addition, at an early stage of the analysis, of a complex protein sample. There are two approaches for introducing a stable isotope into proteins or peptides: metabolic labelling using whole cells grown in culture (e.g. SILAC) or chemical labelling (e.g. iTRAQ, ICAT). Since protein phosphorylation is very dynamic and constantly changing throughout the life of a cell, measuring the changes in phosphorylation is critical for understanding the biology of a phosphorylation event, We restrict the discussion here to four MS based quantitation strategies which have direct utility towards measuring changes in protein phosphorylation extensively: SILAC, iTRAQ, AQUA and MRM. Other chemical labelling techniques which rely on stable isotope incorporation using e.g. 18O labelled water during trypsin digestions and stable isotope incorporation ICAT can also be considered to contain relevant information, but will not be described here. In addition, we will also include the explanation and examples of 2-D Fluorescence Difference Gel Electrophoresis (2D_DIGE) quantification methodology, which nowadays also provides interesting research studies.
C.1. Stable Isotope Labelling with Amino acid in cell Culture (SILAC)
Stable isotope labelling by amino acids in cell culture (SILAC) is a quantitative method based on in vivo labelling of proteins in cell cultures with amino acids that contain stable isotopes (non radioactive, e.g. 2H, 13C and 15N) [57,101]. In its simplest form, two separated cell cultures are grown in a pair-wise fashion; for example, culture A might be yeast cells grown under "normal" conditions (light conditions) while culture B might be yeast cells grown in the presence of a stress condition. The growth conditions of the cells are identical (except for the presence of the stress stimuli), but the growth media of culture B has an essential amino acid (one not synthesized by the cell) replaced with an isotopically "heavy" form of that amino acid (e.g. 13C6-arginine). A number of cell lines have been used for SILAC experiments, and the growth and morphology of the cells have not been affected by the isotopically labelled amino acid [78,101,102].
After approximately five rounds of doubling, cellular proteins are essentially 100% labelled with the selected amino acid. After culturing, the light and heavy cell populations are combined (1:1) into one pool and the proteins are isolated. The protein pool is then digested with a protease, typically trypsin, to form a peptide pool that is analyzed by MS. Each peptide analyzed will be present in two forms: the light and the heavy form. They are distinguishable based on the mass difference due to the heavy isotope incorporation in the selected amino acid. The SILAC method is compatible with the above mentioned enrichment of phosphoproteins/phosphopeptides including the immunoprecipitation of a target protein [103]. One of the first research studies which carryied out this technology was provided by Gruhler and co-workers (2005) [78]. In this study, more than 700 phosphopeptides from Sacharomyces cerevisiae were identified, 139 were differentially regulated at least 2-fold in response to mating pheromone. Components belonging to the mitogen-activated protein kinase signalling pathway and to downstream processes including transcriptional regulation, the establishment of polarized growth, and the regulation of the cell cycle were among these regulated proteins.
C.2. Isobaric Tag for Relative and Absolute (iTRAQ)
The second method for the global quantification of proteins and protein modifications is an in vitro chemical labelling procedure called iTRAQ. The iTRAQ reagent consists of two to eight isobaric tags that can be used to label two to eight separate protein samples. The iTRAQ tags contain three regions: a peptide reactive region, a reporter region, and a balance region [104]. The peptide reactive region of the tag consists of an NHS ester and is designed to react with the N-termini and lysines of peptides after protease digestions. In the case of 4-plex iTRAQ, the four reporter groups appear in the tandem mass spectrum at m/z 114, 115, 116, and 117. The attached balance groups are designed to make the total mass of the balance and reporter group 145 Da for each tag, which results in balance groups of 31 Da, 30 Da, 29 Da, and 28 Da, respectively. Protein samples for quantification are separately isolated and digested proteolytically, and each sample is chemically labelled with one of the iTRAQ reagents. After labelling, the samples are combined and subsequently analyzed by MS. Identical peptides from each sample will have identical masses as the iTRAQ reagents are isobaric The iTRAQ reagent labels phosphopeptides to the same degree as nonphosphorylated peptides and it does not affect the stability of phosphopeptides. Enrichment strategies, such as IMAC [44,105] or immunoprecipitation with anti-phosphotyrosine antibodies [44], have been used to remove non-phosphorylated peptides to focus the analysis on site-specific phosphorylation. Since iTRAQ is an in vitro labelling procedure it can also be applied to clinical samples such as tumour tissues and fluids (e.g. serum, urine, blood). iTRAQ has been described as a very powerful method for the quantification of phosphorylation on a proteomic scale. As a relevant example we mention that Boja and co-workers (2009) [106] successfully monitored phosphorylation sites of mitochondrial proteins including adenine nucleotide translocase, malate dehydrogenase and mitochondrial creatine kinase, etc. Among them, four proteins exhibited phosphorylation changes with these physiological stimuli: (a) BCKDH-E1α subunit increased phosphorylation at Ser337 with DCA and de-energization; (b) apoptosis-inducing factor phosphorylation was elevated at Ser345 with calcium; (c) ATP synthase F1 complex α subunit and (d) mitofilin dephosphorylated at Ser65 and Ser264 upon de-energization. This screening validated the iTRAQ/HCD technology as a method for functional quantitation of mitochondrial protein phosphorylation as well as providing insights into the regulation of mitochondria via phosphorylation.
C.3. Absolute Quantitation (AQUA)
The AQUA strategy provides an absolute quantification of a protein of interest [107]. In the AQUA method, a peptide from the protein of interest is constructed synthetically containing stable isotopes, and the isotopically labelled synthetic peptide is called AQUA peptide. The synthetic peptides can be synthesized with modifications such as phosphorylation to allow for the direct, quantitative analysis of post-translationally modified proteins. The stable isotopes are incorporated into the AQUA peptide by using isotopically "heavy" amino acids during the synthesis process of the interesting peptide (native peptide). In this way, the synthetic peptide has a mass increase of e.g. 10 Daltons, due to the incorporation of a 13C6 and 15N4-arginine into the synthetic peptide, compared to the native peptide. Although the mass difference between the native and the synthetic peptide allows the mass spectrometer to differentiate between the two forms, both forms have the same chemical properties, resulting in the same chromatographic retention, ionization efficiency, and fragmentation distribution. In AQUA experiments, a known amount of the isotopically labelled peptide is added to a protein mixture, which is proteolytically digested, and later analyzed by MS. Since the native peptide and its synthetic counterpart have the same chemical properties, the MS signal from the quantified synthetic peptide can be compared to the signal of the native peptide. The absolute quantification of the peptide to be determined [108] is thus finally permitted. Multiple AQUA peptides can be used to quantify multiple proteins in a single experiment.
Ziwei Yu and co-workers (2007) [109] using AQUA as a novel system of in situ quantitative protein expression analysis, studied the protein expression levels of phosphorylated Akt (p-Akt). Activation of Akt in tumours is mediated via several mechanisms, including activation of cell membrane receptor tyrosine kinases such as EGFR and loss of phosphatase PTEN with dephosphorylation of phosphoinositol triphosphate. Ziwei and co-workers (2007) discovered that Akt activation in oropharyngeal squamous cell carcinoma (OSCC) is associated with adverse patient outcome, indicating that Akt is a promising molecular target in oropharyngeal squamous cell carcinoma.
C.4. Multiple Reaction Monitoring (MRM)
MRM is a very sensitive method for detecting phosphorylated peptides on a hybrid triple quadrupole linear ion trap mass spectrometer (qTRAP). This method requires that the sequence of the protein be known in order to calculate precursor and fragment ion values, which can be used to trigger dependent ion scans in a qTRAP instrument [110,111]. This technique can also be used to perform a precursor ion and neutral loss scan, to identify unknown phosphopeptides from a complex mixture, and is a powerful method for the identification and quantification of post-translational modifications in proteins. MRM has recently been used by White and co-workers [112,113] to identify and quantify tyrosine phosphorylated kinases for hundreds of nodes within a signalling network and across multiple experimental conditions. Moreover, White and co-workers [112,113] applied iTRAQ combined with MRM for phospho quantitative analysis of signalling networks, identifying and quantifying 222 tyrosine phosphorylated peptides, obtaining an extremely high percentage of signalling nodes covered. They defined the mechanisms by which EGFRvIII protein alters cell physiology, as it is one of the most commonly mutated proteins in GBM and has been linked to radiation and chemotherapeutic resistance. They performed a phosphoproteomic analysis of EGFRvIII signalling networks in GBM cells. The results of this study provided important insights into the biology of this mutated receptor, including oncogene dose effects and differential utilization of signalling pathways. In addition, clustering of the phosphoproteomic data set revealed a previously undescribed crosstalk between EGFRvIII and the c-Met receptor. Treatment of the cells with a combination using both EGFR and c-Met kinase inhibitors dramatically decreased cell viability in vitro.
C.5. 2-D Fluorescence Difference Gel Electrophoresis (DIGE)
In DiGE, proteins extracted from a control extract are labelled with one CyDye (Cy3 or Cy5 conjugated), and proteins isolated from a test extract labelled with the other colour of CyDye fluorophore, which are size and charge matched. These labelled protein extracts are mixed and co-resolved (often with the addition of an internal standard, which can be labelled with Cy2) on large-format two-dimensional gels for analysis of expression changes in the resulting pattern of spots ('spot maps') [114]. In comparison with two-dimensional gel electrophoresis, DiGE offers the advantage that multiple samples could be compared on a single gel ('multiplexing'), and made it possible to stain control and test samples with different fluorescent dyes prior to electrophoresis. This advance alleviated issues of gel-to-gel comparison and decreased the number of gels required. The capability to include an internal standard, composed of an equal fraction of all the samples in an experiment, also improved intergel matching and facilitated normalization of matched spots in replicate samples on multiple gels. The use of CyDyes to label proteins, in place of non-fluorescent post-stains, can give a large enhancement of sensitivity for protein detection [115] and constitutes the crucial advantage of the DiGE approach for biomaterial applications. This enables analysis of even very scarce protein samples, including small areas of laser-microdissected tissue [116,117]. Two-dimensional difference gel electrophoresis (2D-DIGE) with novel ultra high sensitive fluorescent dyes (CyDye DIGE Fluor saturation dye) enables the efficient protein expression profiling of laser-microdissected tissue samples. The combined use of laser microdissection allows accurate proteomic profiling of specific cells including tumour tissues [118].
As an example, differential protein analysis was performed using 2-dimensional differential in-gel electrophoresis (2D-DIGE) by Yefei Rong and co-workers (2010) [119]. They found that 16 protein spots were differently expressed between the two mixtures (a comparison was made with serum samples from five individuals with pancreatic cancer and five individuals without cancer). Yefei Rong and co-workers [119] demonstrated that eight proteins from these fluids were up-regulated and 8 were down-regulated in cancer. Mass spectrometry and database searching allowed the identification of the proteins corresponding to the gel spots. Up-regulation of mannose-binding lectin 2 and myosin light chain kinase 2, which had not previously been implicated in pancreatic cancer, were observed. In an independent series of serum samples from 16 patients with pancreatic cancer and 16 non-cancerbearing controls, increased levels of mannose-binding lectin 2 and myosin light chain kinase 2 were confirmed by western blot.
Moreover, Nagano (2010) [120] has recently developed the technology named "antibody proteomics technology". This technology can screen for biomarker proteins by isolating antibodies against each candidate in a rapid and comprehensive manner. He applied "antibody proteomics technology" to breast cancer-related biomarker discovery and evaluated the utility of this novel technology. Cell extracts derived from breast tumour cells (SKBR3) and normal cells were analyzed by two-dimensional differential gel electrophoresis (2D-DIGE) in order to identify proteins over-expressed in the tumour cells. Candidate proteins were extracted from the gel pieces, immobilized onto a nitrocellulose membrane using a dot blot apparatus and then used as target antigens in scFv-phage enrichment and selection. scFvs binding to 21 different over-expressed proteins in tumor cells were successfully isolated within several weeks following this in vitro phage selection procedure. The expression profiles of the identified proteins were then determined by tissue microarray analysis using the scFv-phages. Consequently, three breast tumour-specific proteins were identified. His data demonstrated the utility of an antibody proteomics system for discovering and validating tumour-related proteins in pharmaceutical proteomics. Currently, he and other related groups are analyzing the functions of these proteins in order to be able to confirm and use them as diagnostic markers or therapeutic targets.
(D) Phosphorylated proteins related to different diseases and the benefits of proteomic strategies in such clinical studies
In a recent study Steen et al. examined the role of phosphorylation in the dynamics of the anaphase promoting complex (APC) [121]. Some drugs that bind to microtubules and block mitosis are ineffective in cancer treatment; others show inexplicable focal efficacy. For example, the vinca alkaloids are useful for treating lymphoma, neuroblastoma and nephroblastomas, whereas taxol is useful for advanced breast cancer and ovarian cancer. It is not known why these drugs are not all equally effective, nor why they have different therapeutic value against different cancers. The authors observed distinct phosphorylation states of the APC in response to different antimitotic drugs and suggest they may explain some of these differences. They also propose it is possible that cells from different tissues, or cells harbouring different mutations, or cells under different physiological stresses, such as hypoxia, may differ in their response to spindle poisons and would thus reflect those differences in different sites of phosphorylation. Differences in spindle checkpoint phosphorylation may reveal new features of the mitotic state. The categorisation of drugs, the discrimination of the response of tumours to drugs and the identification of new means of checkpoint control may be facilitated by the ability to characterise drug candidates based on the spectrum of APC phosphorylations The authors further suggest that the results of the study indicate that the term mitotic arrest is a misnomer: arrest is a dynamic state in which some cells enter apoptosis and other cells revert to interphase. The ability to observe biochemical events during arrest could be very important for understanding antiproliferative treatments. The exploration of the dynamics of phosphorylation, however, makes great demands on the accuracy of quantitation. Most mass spectrometric based quantitative approaches, including stable isotope labelling with amino acids in cell culture (SILAC) and isobaric tag for relative and absolute quantitation (iTRAQ), give relative data, meaning that one state of phosphorylation is determined relative to another phosphorylation state [122-124]; these data can help to establish the kinetics of a pathway. The method used in this work offers a significant advance over earlier techniques. It allowed the measurement of specific quantitative changes in APC phosphorylation in cells arrested in nocodazole for varying periods. If these dynamics can be correlated with the process by which the arrested state is resolved, we may be provided with new tools to understand the mitotic process and to find more effective drug targets in cancer.
The long-held belief in the cancer research community that a precise molecular understanding of cancer can result in cancer therapy is validated by the development of drugs for specific biological pathways with increased specificity and reduced toxicity. The development of Herceptin, a monoclonal antibody against the HER2 receptor for breast cancer therapy is one of the most successful recent examples of cancer-specific drugs. HER2 is an important target in cancer because its overexpression increases tumour cell proliferation, vessel formation and invasiveness, and predicts poor prognosis. Wolf-Yadlin and other scientists [111,112], [121-124] have used phosphoproteomics and MS to investigate the role of phosphorylation in the effects of HER2 overexpression on EGF- and HRG-mediated signalling of erbB receptors. Identification was possible of specific combinations of phosphorylation sites that correlate with cell proliferation and migration and that potentially represent targets for therapeutic intervention. Unfortunately, owing to sensitivity limitations, only 68 out of 322 phosphorylation sites could be analysed kinetically, so the study does not provide a comprehensive analysis of the multitude of effects produced by HER2 overexpression. It does, however, mark an important breakthrough in the characterisation of the erbB receptor signalling network in tumours and illustrates the importance of understanding protein phosphorylation.
A central role is played by mitochondria in energy metabolism and cellular survival, and consequently mitochondrial dysfunction is associated with a number of human pathologies. Moreover, mitochondrial dysfunction is linked to insulin resistance in humans with obesity and type 2 diabetes. Recently, Zhao and co-workers (2011) [125], studied the phosphoproteome of the mitochondria isolated from human skeletal muscle. Zhao and co-workers revealed extensive phosphorylation of inner membrane protein complexes and enzymes combining titanium dioxide (TiO2) protocols with reverse phase chromatography coupled to MS analysis. 155 distinct phosphorylation sites in 77 mitochondrial phosphoproteins, including 116 phosphoserine, 23 phosphothreonine, and 16 phosphotyrosine residues were identified. Phosphorylation sites in mitochondrial proteins involved in amino acid degradation, importers and transporters, calcium homeostasis, and apoptosis were also assigned. Furthermore, many of these mitochondrial phosphoproteins are substrates for protein kinase A, protein kinase C, casein kinase II and DNA-dependent protein kinase. The high number of phosphotyrosine residues suggests that tyrosine phosphorylation has an important role in mitochondrial signalling. Many of the mitochondrial phosphoproteins are involved oxidative phosphorylation, tricarboxylic acid cycle, and lipid metabolism, i.e. processes proposed to be involved in insulin resistance. It is well known that mitochondria dysfunction is centrally involved in a number of human pathologies, such as type 2 diabetes, parkinson's disease and cancer [126]. In this study, the most prevalent form of cellular protein post-translational modifications (PTMs), reversible phosphorylation [127-134][135-139], emerges as a central mechanism in the regulation of mitochondrial functions [130,131]. The steadily increasing numbers of reported mitochondrial kinases, phosphatases and phosphoproteins also imply the important role of protein phosphorylation in different mitochondrial processes [132-134]. The prototypical proteomics pipe-line useful for clinical research is illustrated (Figure 3).
Figure 3 A prototypical proteomics pipe-line useful for clinical research. Depending on the application, different samples processed and fed into the proteomics pipeline yield different results. The pipeline's several steps are listed in the different panels: (1) proteolytic digest, (2) the separation and ionization of peptides, (3) their analysis by MS, (4) fragmentation of selected peptides and analysis of the resulting MS/MS spectra and, (5) (6) data-computer analysis, which includes identification and quantification of proteins from several detected peptides.
(E) Observations and Future Needs
Cancer and immune disorders are still among the leading causes of death worldwide. Therefore, there is still a need for the identification of useful biomarkers and the improvement of the understanding of the development of these diseases. The immune system is readily affected by the existence of cancer in the body, even at a preclinical stage, and these studies should be expanded and extended in the future to answer the numerous questions concerning (a) the roles of immune cells in cancer surveillance (b) the characteristics of inflammation in association with cancer development, (c) the effects of environment/lifestyle factors on the immune system, and (d) the interaction between aging diseases. The importance of protein kinase-regulated signal transduction pathways in immunology disorders and cancer has led to the development of drugs that inhibit protein kinases at the apex or intermediary levels of these pathways. Protein phosphorylation assignment studies of these signalling pathways will provide important insights into the operation and connectivity of these pathways that will facilitate the identification of the best targets for cancer therapies and immunology diseases (e.g. the identification of a phosphate group on a specific serine, threonine or tyrosine by phosphoenrichments combined with MS). Phosphoproteomic analysis of individual tumours, blood, sera, tissues will also help match targeted therapeutic drugs to the appropriate patients. It is now generally accepted that no single proteomic method is comprehensive, but combinations of different enrichment methods produce distinct overlapping phosphopeptide datasets to enhance the overall results in phosphoproteome analysis. During the last decade, phosphopeptide sequencing by mass spectrometry has seen tremendous advances. For example, MS/MS product ion scanning, multistage activation and precursor ion scanning are effective methods for identifying serine (Ser), threonine (Thr) and tyrosine (Tyr) phosphorylated peptides.
The current phosphoproteomic goals imply the identification of phosphoproteins, mapping of phosphorylation sites, quantitation of phosphorylation under different conditions, and the determination of the stoichiometry of the phosphorylation. In addition, knowing when a protein is phosphorylated, which kinase/s is-are involved, and how each phosphorylation fits into the signalling network, are also important challenges for researchers in order to understand the significance of different biological events. The new MS technologies are fundamental for cataloguing all this information, and it is heading towards the collection of accurate data on phosphopeptides on a global scale. In addition, the possible difficulties to get sufficient amount of specific phosphorylated proteins of specific low abundant protein-kinases in vivo which might limit the usability of the phosphoproteome analysis, must be pointed out.
Finally, it is important to state that to develop clinical proteomic applications using the identified proteins and phosphoproteins, collaboration between research scientists, clinicians and diagnostic companies, and proteomic experts is essential, particularly in the early phases of the biomarker development projects. The proteomics modalities currently available have the potential to lead to the development of clinical applications, and channelling the wealth of the information produced towards concrete and specific clinical purposes is urgent.
Abbreviations
Note: These abbreviations are useful proteomic abbreviations; some of them are mentioned and described in this Review, and they are also described in the References of this article.
AQUA: Absolute Quantitation; CID: Collision-Induced Dissociation; Da: Dalton (molecular mass); DIGE 2-D: Fluorescence Difference Gel Electrophoresis; ECD: Electron Capture Dissociation; ESI: Electron Spray Ionization; ETD: Electron Transfer Dissociation; FT-ICR: Fourier transform-Ion Cyclotron Resonance; HILIC: Hydrophilic interaction chromatography; HPLC: High-performance liquid chromatography or high-pressure liquid chromatography; H3PO4: Phosphoric acid; ICR: Ion Cyclotron Resonance; IMAC: Immobilized Metal Affinity Capture; IT: Ion Trap; iTRAQ: Isobaric Tag for Relative and Absolute Quantitation; kDa: kilodalton (molecular mass); LC: Liquid Chromatography; MALDI: Matrix-Assisted Laser Desorption/Ionization; MOAC: Metal Oxide Affinity Chromatography; Mr: Relative molecular mass (dimensionless); MRM: Multiple reaction monitoring; MS: Mass Spectrometry; MSA: MultiStage Activation; MS/MS: tandem mass spectrometry; m/z: Mass to charge ratio; PID: Primary Immunodeficiencies; PTM: Post-Translational Modification; SILAC: Stable Isotope Labelling with Amino acid in cell Culture; SIMAC: Sequential Elution from IMAC; TiO2: Titanium dioxide; TOF: Time Of Flight; ZrO2: Zirconium dioxide
competing interests
The authors declare that they have no competing interests.
Authors' contributions
EL carried out the proteomics, phosphoproteomics and mass spectrometry studies for this review. IL, AF, JS carried out the clinical studies for this review. EL, IL, AF and JS carried out these complementary studies in order to develop clinical phosphoproteomics research and publish this article. All authors read and approved the final manuscript.
Acknowledgements
EL PhD was a recipient of a Post-doctoral fellowship of Ministerio de Ciencia e Innovación de España. IL PhD was a recipient of a FLL (Fundación Leucemia y Linfoma) grant. AF and JL are M.D. PhD with permanent positions from Spanish National Hospitals La Paz and Carlos III respectively.
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Evid Based Complement Alternat MedEvid Based Complement Alternat MedECAMEvidence-based Complementary and Alternative Medicine : eCAM1741-427X1741-4288Hindawi Publishing Corporation 1924429610.1093/ecam/nen090nen090Original ArticleEvidences of Protective Potentials of Microdoses of Ultra-High Diluted Arsenic Trioxide in Mice Receiving Repeated Injections of Arsenic Trioxide Banerjee Pathikrit
1
Bhattacharyya Soumya Sundar
1
Pathak Surajit
1
Boujedaini Naoual
2
Belon Philippe
2
Khuda-Bukhsh Anisur Rahman
1
*1Department of Zoology, University of Kalyani, Kalyani-741235, India2Boiron Lab, 69110 Sainte-Foy-Les-Lyon, France*Anisur Rahman Khuda-Bukhsh: [email protected] 14 2 2011 2011 3917524 1 2008 19 12 2008 Copyright © 2011 Pathikrit Banerjee et al.2011This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.The present study was undertaken to examine if microdoses of ultra-high diluted arsenic trioxide (a potentized homeopathic remedy, Arsenicum Album 200C, diluted 10−400 times) have hepatoprotective potentials in mice subjected to repeated injections of arsenic trioxide. Arsenic intoxicated mice were divided into: (i) those receiving Arsenicum Album-200C daily, (ii) those receiving the same dose of diluted succussed alcohol (Alc 200C) and (iii) another group receiving neither drug nor succussed alcohol. Two other control groups were also maintained: one fed normal diet only and the other receiving normal diet and Alc-200C. Toxicity biomarkers like aspartate and alanine aminotransferases, glutathione reductase, catalase, succinate dehydrogenase, superoxide dismutase and reduced glutathione contents were periodically assayed keeping the observer “blinded”. Additionally, electron microscopic studies and gelatin zymography for matrix metalloproteinases of liver tissues were made at day 90 and 120. Blood glucose, hemoglobin, estradiol and testosterone contents were also studied. Compared to controls, Arsenicum Album-200C fed mice showed positive modulations of all parameters studied, thereby providing evidence of protective potentials of the homeopathic drug against chronic arsenic poisoning.
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1. Introduction
Microdoses of highly diluted arsenic trioxide (As2O3 10−400) was chosen to validate its ameliorative potential against repeated arsenic intoxication employing acceptable toxicity biomarkers including specific hepato-toxicity denoting protocols. Some of these biomarkers were chosen because they reflected degree of hepatotoxicity, directly or indirectly being influenced by the extent of toxicity caused by arsenic poisoning. Further, tissue damage and necrosis, often inflicted as a result of arsenic poisoning, largely contribute to improper liver function, that in turn, may cause hepatotoxicity. In view of this, liver function tests, which include the assay of enzymes like AST and ALT, were performed. These tests can throw light on the extent of liver malfunctioning and tissue damage in arsenic intoxicated mice. Therefore, positive modulation of these parameters, if any, achieved by the administration of the remedy, can be significant and supportive of its hepato-protective potential.
Inorganic arsenic (iAs) acts as a tumor-promoting agent by inducing a rapid burst of reactive oxygen species (ROS) in mammalian cells, resulting in oxidative stress [1, 2] and carcinogenesis in man [3]. Cooperative defense systems that protect the body from free radical damage include the anti-oxidant enzymes [(superoxide dismutase (SOD), catalase (CAT) and indirectly glutathione reductase (GRD) and also GSH)] and nutrients. The study of these anti-oxidant enzymes was therefore considered very pertinent. Succinate dehydrogenase, an enzyme of the inner mitochondrial membrane is concerned with energy generation and respiration; hence is essential for cell survival. This parameter was therefore also included in this study. The hematological parameters like blood glucose, hemoglobin and hormonal studies were performed since they depict the physiological status of the body. Additionally, scanning and transmission electron microscopic analyses and gelatin zymography for analyzing expression pattern of matrix metalloproteinases (MMPs) were performed at day 90 and 120.
2. Materials
2.1. Animals
Thirty-six healthy adult Swiss albino mice (Mus musculus) of both sexes (30–40 g) served as materials for each series, six sacrificed at each fixation interval. All animal experiments were performed based on animal ethics guidelines of Institutional Animal Ethics Committee, University of Kalyani, under the supervision of Animal Welfare Committee.
2.2. Experimental Design
Mice were randomized and sub-divided into the following sub-sets.
2.3. Arsenic Intoxicated and Drug Fed Series
In all groups, injection (intraperitoneal) with 0.016% As2O3 solution (aqueous) at the rate of 1 mL/100 g body weight was performed at an interval of 7 days (Gr-1) and sacrificed at 7, 15, 30, 60, 90 and 120 days, respectively, for all groups.
A sub-group was fed Ars Alb-200C (Gr-2) once daily till sacrificed, which comprised the intoxicated drug fed series. Another sub-group (Gr-3) was fed Alc 200C. Potentized Ars Alb-200C was procured from Boiron Laboratory, Lyon, France, as also the placebo (Alc-200 C), prepared with 70% ethanol.
2.4. Control Series
A group of healthy mice was maintained on normal low protein diet (normal control—Gr -4). Another group of healthy mice were fed diluted succussed alcohol (Alc-200 C) at the same rate as that of As203, because the “vehicle" of the homeopathic remedy was ethyl alcohol (alcohol control Gr-5).
Another group of mice was injected 0.016% As203 every 7th day and fed Alc-200 C (intoxicated control) till they were sacrificed, to learn about modulated effect, if any, induced by ethyl alcohol on arsenic intoxicated mice.
3. Methods
3.1. Preparation of Tissue Homogenates
Liver and spleen tissues were homogenized in phosphate buffer saline (pH 7.4) and centrifuged at 5000 g for 60 min. The supernatant was used for assay of the marker enzymes (glutathione reductase, superoxide dismutase, catalase and succinate dehydrogenase), reduced glutathione, aspartate aminotransferase, alanine aminotransferase and protein estimation.
3.2. Biochemical Analysis
The tissue homogenate was used for the assessment of AST and ALT [4], reduced glutathione content [5], catalase [6], SOD [7], SDH [8], GRD [9, 10] and protein [11].
3.3. Pathological Parameters
Blood glucose content was assayed by the GOD-POD End point Colorimetric assay kit supplied by Span Diagnostics Limited, Baroda (Code-B0112), India.
Hemoglobin content was determined by Sahli's method with the help of a hemometer (Marienfield, Germany).
Serum estradiol and testosterone content was assayed by using the appropriate diagnostic kit (EQUPAR srl, Saronno, Italy) with the aid of an ELISA Reader (ELDEX 3.8, USA) at 450 nm.
3.4. Electron Microscopic Studies
The detailed procedure of fixation and section cutting for scanning and transmission electron microscopy (SEM and TEM, respectively) have been described elsewhere [12].
3.5. Level of MMP Expression
For the study of MMP activity, hepatic tissue was homogenized with phosphate buffer saline (pH 7.4), followed by centrifugation in a cooling centrifuge (REMI, C 24, India). The supernatant was collected and subjected to gelatin zymography [12, 13].
3.6. Statistical Analysis and Scoring of Data
Statistical significance of the difference between experimental groups was calculated using Student's paired t-test [14] for two means. A P < .05 was considered significant. One way ANOVA has also been performed. The observers were “blinded" if the sample belonged to “experimental" or “control" group at the time of scoring the data.
4. Results
4.1. Biochemical Parameters
Arsenic intoxication caused a significant rise in AST (P < .05) (Table 1), ALT (P < .05) (Table 2), AcP (Table 3) and AlkP (Table 4) activity levels in liver and spleen tissues when compared to the control group. The same was true for LPO activity (Table 5). Administration of Ars Alb-200C brought about considerable recuperation in the activities of AST (.05 < P < .001), AcP, AlkP and ALT (.01 < P < .001) and also of LPO. There was a palpable reduction in GSH contents of both the tissues in arsenic intoxicated mice (Table 6). These values were significantly increased (.05 < P < .001) in drug fed mice. Catalase activity (Table 7) significantly declined (.05 < P < .001) after arsenic trioxide treatment in both the liver and spleen tissues. However, there was a considerable increase of values in the drug fed mice (P < .001) as compared to that of control. Similarly, both the tissues revealed a substantial depletion (.01 < P < .001) in the activities of SDH enzyme (Table 7) after repeated arsenic trioxide intoxication. But Ars Alb-200C supplementation produced considerable repletion and the values were comparable to that of controls (both Gr-1 and Gr-3; P < .001).
The SOD levels (Table 8) showed a significant decline in its activity in both liver and spleen tissues after arsenic trioxide treatment. In Ars Alb-200C fed mice, there was considerable revival (P < .05) and the values were comparable to that of controls. Arsenic intoxication resulted in significant decrease (.05 < P < .001) of total GRD content in both liver and spleen tissues when compared with normal group. Administration of Ars Alb-200C resulted in significant increase (.05 < P < .001) in total GRD content (Table 8) in both liver and spleen tissues as compared to arsenic intoxicated group.
4.2. Pathological Changes
4.2.1. Blood Hemoglobin
The hemoglobin content was reduced in As2O3 + Alc200 C fed mice as compared to that of normal controls. In Ars Alb-200C fed mice (Table 9), an increase in hemoglobin level was obtained and the values were comparable to that of controls.
4.2.2. Blood Glucose Content
In As2O3 and As2O3 + Alc 200C fed mice, an increase in blood sugar content was observed at successive fixation intervals, the increase being significant (P < .001) at day 90 only. The increase in blood sugar content (Table 9) was so high that it exceeded the normal level. In Ars Alb-200C fed group, there was also an increase (P < .001) in blood sugar content except at day 60, where a slight decrease in the said level was observed. Here, the increase was within the permissible limits and not as high as that of As2O3 and As2O3 + alcohol fed group.
4.2.3. Serum Estradiol and Testosterone Concentration
Compared to intoxicated controls (Gr-1), a decrease in serum estradiol and testosterone levels (Table 10) was obtained in As2O3 and As2O3 + Alc200 C fed mice at day 90 and 120. Administration of Ars Alb-200C resulted in an increase in serum estradiol and testosterone levels in comparison to arsenic trioxide treated group.
4.3. Transmission Electron Microscopic Study
Contrary to the intact nuclear membrane observed in liver cells of normal (Figure 1(a)) mice and arsenic intoxicated (Figures 1(b)–1(d)) mice administered Ars Alb-200C (Figures 1(e)-1(f)), the nuclear membrane in liver cells of As2O3 intoxicated mice seemed to be broken. Destructive changes were prominent in the cristae of arsenic intoxicated mice. Furthermore, number of vacuoles was noticed in arsenic intoxicated mice. Cisternae of the Golgi bodies of arsenic intoxicated mice were absent. In arsenic intoxicated mice fed Ars Alb-200C, these features were less conspicuous.
4.4. Scanning Electron Microscopic Study
The hepatocytes in normal controls are shown in Figures 2(a) and 2(b). Damaged hepatocytic cells were found in the arsenic intoxicated series (Figure 2(c)). Relatively little damaged hepatocytes were found in the intoxicated group that was fed Ars Alb-200C (Figure 2(d)).
4.5. Metalloproteinase Activity
At 90- and 120-day fixation intervals, in As2O3 and As2O3 + Alc 200C fed mice, only one band was expressed near 77 kDa (Figure 3) which presumably belonged to MMP family. However, in some mice, this band was near 97 kDa, presumably belonging to a different MMP member. In the drug fed series, the expression of the single band appeared to be somewhat less.
4.6. Statistical Analysis
Many of the differences in data revealed by Student's t-test have been found to be statistically significant at different levels, as shown in Tables 1–10. Similarly, results of one-way ANOVA also revealed significant differences among the different parameters studied (Tables 11–13).
5. Discussion
Arsenic compound is a protoplasmic poison that can bind to human sulphydryl-containing proteins with high affinity. Arsenic trioxide (As2O3), extracted from arsenic compound, is a powerful ancient medication for a variety of ailments with the principle of “using a toxic against another toxic" in traditional Chinese medicine. Strikingly, As2O3 treatment in a regime of 10 mg/day of intravenous infusion for 28–60 days is effective in patients with acute promyelocytic leukemia (APL) without viable toxicity in refractory to the all-trans retinoic acid (ATRA) and the conventional chemotherapy by inducing apoptosis of APL cells [15]. Notably, As2O3 exerts a broader anti-inflammatory activity by inhibition of nuclear factor, NF-κB activation through induction of inhibitor of κ-B expression in the airways [16]. Low dosage of As2O3 may have a potential benefit in treating patients with asthma, especially in those with steroid-dependent and resistant asthma. Overall, existing data suggest a beneficial effect of As2O3 for the treatment of cancer and even inflammatory diseases. However, that even a non-material dose derived from dilution and succussions of As2O3 could show some beneficial effects in the present study is of considerable significance and of some practical application as well. To ascertain the exact amount of arsenic pushed inside the body and simulate a condition of chronic arsenic intoxication, mice were subjected to repeated injections of arsenic trioxide that inflicted hepatic damage and oxidative stress that was observed from a substantial increase in the activities of the hepatic enzymes, namely ALT and AST. An increase in the activity of these marker enzymes portrays possibility of tissue necrosis and loss of functional integrity of hepatocyte membrane. Reduction in the levels of ALT and AST towards the respective normal values by administration of Ars Alb-200C is an indication of the protective ability of the potentized homeopathic drug as well as its ability to repair hepatic tissue damage inflicted by As2O3. During hepatic injury, superoxide radicals are known to be generated at the site of damage, which in turn modulate activities of SOD and CAT, resulting in the loss of activity and accumulation of superoxide radical, which are responsible for damage of liver tissue. Decreased CAT activity is linked to the exhaustion of the enzyme as a result of oxidative stress caused by As2O3. SOD and CAT activities were brought down to near normal after treatment with Ars Alb-200C. This gives a positive indication regarding the protective efficacy of the potentized homeopathic drug.
Reduced glutathione (GSH) constitutes the first line of defense against free radicals. It plays an important role in the regulation of cell proliferation and cellular defense [17]. Exposure of cells to arsenic trioxide leads to the depletion of GSH, which may also signify damage to the hepatic cells. Positive alterations in endogenous GSH have been encountered in the present investigation after administration of the homeopathic remedy, which is in line with the activities of other biomarkers. The availability of sufficient amount of GSH increased the detoxification of active metabolites of As2O3. Succinate dehydrogenase is a membrane-bound dehydrogenase linked to the respiratory chain and is a member of the Krebs' cycle [18]. Positive modulations encountered in the activity of this enzyme and those of SOD and CAT in the drug fed mice lends support to the protective ability of the remedy in cellular and sub-cellular environments. GSSG is reduced to GSH by glutathione reductase, which is NADPH-dependent. It plays a significant role in maintaining adequate amounts of GSH. Accordingly, the reduction of GRD results in decreasing GSH [18]. In As2O3 treated mice, the activity of GRD is markedly decreased. An increase in GRD activity highlights the hepato-protective ability of the remedy.
Elevated blood glucose levels in As2O3 and As2O3 + alcohol fed mice was also considerably reduced in the drug fed series. The level of blood hemoglobin was markedly decreased in arsenic intoxicated group, but in the drug fed mice the level slightly increased. Similarly, serum estradiol and testosterone levels decreased markedly in arsenic trioxide injected mice. Testosterone, an anabolic steroid, is responsible for the development and maintenance of male secondary sex characters as well as growth promoting effects. Decreased testosterone levels in males may indicate hypothalamic or pituitary disorders or damage to the testis. Alternatively, decrease in the level of serum testosterone could also be implicated to a direct relationship with the increase in toxicity due to arsenic intoxication. A positive correlation can be drawn between increased SHA and an increase in testosterone level. Positive changes in the levels of these hormones were encountered in the drug fed mice. Eagon et al. [19] showed an increased level of estradiol in females of a rat model of hyperplasia.
An increase in the number of mitochondria, distorted nuclei and large black lipid droplets was observed in TEM study of liver tissues of arsenic treated mice. Similarly, in SEM study, damaged hepatocytes and hepatic chords were observed in arsenic intoxicated group. In the drug fed series, however, these features were less marked, which would also support the positive effects of the drug in ameliorating arsenic induced effect at the ultrastructural level. MMPs hydrolyze components of the extracellular matrices; these proteases play a central role in many biological processes. MMPs facilitate invasion and metastization of carcinoma cells [20]. MMPs are a family of Zn2+- and Ca2+-dependent endopeptidases secreted by both normal and transformed cells, and capable of degrading collagenous and non-collagenous components of extracellular matrix [21, 22]. Results of the present study revealed a high level of expression of metalloproteinases. This is in conformity with high levels of expression of metalloproteinases reported in pathological conditions such as wound healing, angiogenesis, tumor invasion, metastasis [23–26], arthritis, emphysema and apoptosis [27, 28]. In the drug fed mice, no overexpression of metalloproteinases was noticed. Therefore, the expression of MMPs or rather the lack of expression provides substantial evidence in favor of their anti-tumorigenic effects at the gene expression level.
How the ultra low doses of the potentized remedy could bring about multiple changes in both enzymatic and pathological parameters as well as many biomarkers is rather unclear at the present state of our knowledge. Incidentally, Khuda-Bukhsh [29–32] proposed a working hypothesis that one mechanism through which the potentized homeopathic drugs act could be through regulation of expression of certain relevant genes. This explanation seems plausible because all the biomarkers tested are regulated by specific genetic regulatory mechanisms, and without involvement of these regulatory mechanisms, such positive results could not have been achieved.
6. Conclusion
The results of the present study suggests that Arsenicum Album 200C can be used as an interim relief measure, especially in high-risk arsenic contaminated areas, at least till arsenic-free drinking water or better strategies to manage this dreadful problem are available. However, this would need further clinical trial, preferably by others, to validate the ameliorative potentials for its homeopathic use in large scale in the arsenic contaminated areas.
Acknowledgments
A research grant from Boiron Lab, Lyon, France to
Prof. A. R. Khuda Bukhsh is gratefully acknowledged.
The authors are thankful to Dr T.C. Nag, AIIMS,
New Delhi for providing necessary infrastructural support
and expertise for electron microscopic studies.
Figure 1 Representative photomicrographs under TEM of liver sections of normal (a), arsenic intoxicated (b–d) and arsenic intoxicated drug fed mice (e–f). BD, black droplet; ER, endoplasmic reticulum; NM, nuclear membrane; NU, nucleus; WD, white droplet.
Figure 2 Representative photomicrographs under scanning electron microscopy of liver sections of normal (a–b), arsenic intoxicated (c) and arsenic intoxicated drug fed (d) mice. (Magni- 1KX); H, hepatocyte; MD, mechanical disorder.
Figure 3 Gelatin zymogram of liver samples showing the expression of MMP in experimental mice at 120 days l. Lane 1, normal; lane 2, As2O3; lane 3: As2O3 + Alc-200; lane 4, As2O3 + Ars Alb-200; M, molecular weight marker.
Table 1 Mean activities of Aspartate amino transferase (AST) (nM/mg protein/min) in liver and spleen of different series of mice at different fixation intervals.
Fixation intervals in days 30 Days 60 Days 90 Days 120 Days
Series Activity ± SE Activity ± SE Activity ± SE Activity ± SE
Liver
Negative control 0.014 ± 0.001 0.020 ± 0.000 0.011 ± 0.001 0.012 ± 0.001
Alcohol control 0.015 ± 0.002 0.022 ± 0.004 0.013 ± 0.000 0.013 ± 0.002
As2O3
0.03 ± 0.001 0.027 ± 0.001 0.035 ± 0.001 0.038 ± 0.002
As2O3 + Alcohol 0.033 ± 0.001 0.031 ± 0.003 0.040 ± 0.003 0.041 ± 0.000
As2O3 + Ars Alb-200C 0.026 ± 0.001** 0.032 ± 0.002 0.024 ± 0.003** 0.016 ± 0.004***
Spleen
Negative control 0.011 ± 0.000 0.008 ± 0.001 0.016 ± 0.003 0.011 ± 0.000
Alcohol control 0.012 ± 0.001 0.005 ± 0.002 0.026 ± 0.001 0.012 ± 0.002
As2O3
0.025 ± 0.000 0.022 ± 0.001 0.031 ± 0.001 0.043 ± 0.000
As2O3 + Alcohol 0.027 ± 0.001 0.026 ± 0.001 0.033 ± 0.001 0.045 ± 0.001
As2O3 + Ars Alb-200C 0.02 ± 0.000*** 0.012 ± 0.000*** 0.022 ± 0.001*** 0.018 ± 0.000***
SE, Standard error; **P < .01, ***P < .001, determined versus arsenic intoxicated Alcohol control.
Table 2 Mean activities of Alanine amino transferase (ALT) (nM/mg protein/min) in liver and spleen of different series of mice at different fixation intervals.
Fixation intervals in days 30 Days 60 Days 90 Days 120 Days
Series Activity ± SE Activity ± SE Activity ± SE Activity ± SE
Liver
Negative control 0.006 ± 0.001 0.008 ± 0.000 0.005 ± 0.004 0.005 ± 0.002
Alcohol control 0.008 ± 0.002 0.005 ± 0.001 0.006 ± 0.001 0.006 ± 0.003
As2O3
0.012 ± 0.000 0.015 ± 0.000 0.019 ± 0.001 0.020 ± 0.000
As2O3 + Alcohol 0.016 ± 0.002 0.019 ± 0.001 0.021 ± 0.000 0.025 ± 0.005
As2O3 + Ars Alb-200C 0.014 ± 0.001 0.012 ± 0.000*** 0.009 ± 0.001*** 0.005 ± 0.000***
Spleen
Negative control 0.003 ± 0.000 0.005 ± 0.001 0.002 ± 0.001 0.003 ± 0.000
Alcohol control 0.007 ± 0.001 0.005 ± 0.002 0.002 ± 0.000 0.006 ± 0.001
As2O3
0.011 ± 0.001 0.015 ± 0.001 0.020 ± 0.001 0.024 ± 0.000
As2O3 + Alcohol 0.020 ± 0.002 0.020 ± 0.000 0.021 ± 0.003 0.028 ± 0.001
As2O3 + Ars Alb-200C 0.012 ± 0.002* 0.007 ± 0.001*** 0.005 ± 0.002** 0.004 ± 0.000***
SE, Standard error; *P < .05, **P < .01, ***P < .001, determined versus arsenic intoxicated Alcohol control.
Table 3 Mean activities of Acid phosphatase (AcP) (nM/mg protein/min) in liver and spleen of different series of mice at different fixation intervals.
Fixation intervals in days 30 Days 60 Days 90 Days 120 Days
Series Activity ± SE Activity ± SE Activity ± SE Activity ± SE
Liver
Negative control 0.041 ± 0.003 0.047 ± 0.001 0.010 ± 0.000 0.022 ± 0.011
Alcohol control 0.034 ± 0.005 0.046 ± 0.002 0.031 ± 0.003 0.035 ± 0.000
As2O3
0.076 ± 0.001 0086 ± 0.003 0.054 ± 0.003 0.066 ± 0.000
As2O3 + Alcohol 0.085 ± 0.001 0.090 ± 0.001 0.057 ± 0.003 0.079 ± 0.000
As2O3 + Ars Alb-200C 0.083 ± 0.002 0.069 ± 0.001*** 0.046 ± 0.001** 0.044 ± 0.001***
Spleen
Negative control 0.03 ± 0.001 0.021 ± 0.003 0.035 ± 0.000 0.03 ± 0.001
Alcohol control 0.036 ± 0.002 0.22 ± 0.005 0.031 ± 0.008 0.038 ± 0.002
As2O3
0.074 ± 0.001 0.077 ± 0.003 0.085 ± 0.001 0.092 ± 0.003
As2O3 + Alcohol 0.076 ± 0.002 0.086 ± 0.004 0.089 ± 0.005 0.098 ± 0.001
As2O3 + Ars Alb-200C 0.068 ± 0.001** 0.057 ± 0.002*** 0.054 ± 0.005*** 0.051 ± 0.003**
SE, Standard error; **P < .01, ***P < .001, determined versus intoxicated Alcohol control.
Table 4 Mean activities of Alkaline phosphatase (AlkP) (nM/100 mg protein/min) in liver and spleen of different series of mice at different fixation intervals.
Fixation intervals in days 30 Days 60 Days 90 Days 120 Days
Series Activity ± SE Activity ± SE Activity ± SE Activity ± SE
Liver
Negative control 0.018 ± 0.001 0.012 ± 0.001 0.018 ± 0.001 0.018 ± 0.002
Alcohol control 0.037 ± 0.001 0.013 ± 0.002 0.024 ± 0.002 0.024 ± 0.003
As2O3
0.031 ± 0.002 0.035 ± 0.000 0.036 ± 0.002 0.042 ± 0.001
As2O3 + Alcohol 0.035 ± 0.003 0.039 ± 0.006 0.041 ± 0.001 0.046 ± 0.000
As2O3 + Ars Alb-200C 0.033 ± 0.001 0.025 ± 0.002 0.023 ± 0.000*** 0.026 ± 0.001***
Spleen
Negative control 0.025 ± 0.002 0.022 ± 0.003 0.012 ± 0.001 0.014 ± 0.001
Alcohol control 0.031 ± 0.001 0.026 ± 0.002 0.021 ± 0.001 0.021 ± 0.000
As2O3
0.030 ± 0.001 0.034 ± 0.004 0.043 ± 0.002 0.043 ± 0.002
As2O3 + Alcohol 0.037 ± 0.002 0.038 ± 0.002 0.045 ± 0.001 0.039 ± 0.003
As2O3 + Ars Alb-200C 0.034 ± 0.006 0.031 ± 0.002* 0.037 ± 0.001** 0.028 ± 0.001**
SE, Standard error; *P < .05, **P < .01, ***P < .001, determined versus intoxicated Alcohol control.
Table 5 Mean lipid peroxidation (LPO) (nm/MDA/mg wet tissue) in liver and spleen of different series of mice at different fixation intervals.
Fixation intervals in days 30 Days 60 Days 90 Days 120 Days
Series Activity ± SE Activity ± SE Activity ± SE Activity ± SE
Liver
Negative control 0.089 ± 0.020 0.05 ± 0.002 0.04 ± 0.001 0.044 ± 0.000
Alcohol control 0.094 ± 0.003 0.056 ± 0.005 0.094 ± 0.002 0.080 ± 0.009
As2O3
0.262 ± 0.013 0.277 ± 0.031 0.292 ± 0.025 0.372 ± 0.012
As2O3 + Alcohol 0.281 ± 0.010 0.246 ± 0.028 0.298 ± 0.003 0.416 ± 0.009
As2O3 + Ars Alb-200C 0.229 ± 0.010* 0.235 ± 0.028 0.139 ± 0.002*** 0.093 ± 0.005***
Spleen
Negative control 0.17 ± 0.002 0.105 ± 0.02 0.105 ± 0.002 0.030 ± 0.011
Alcohol control 0.185 ± 0.003 0.087 ± 0.004 0.118 ± 0.003 0.022 ± 0.005
As2O3
0.242 ± 0.041 0.272 ± 0.028 0.282 ± 0.002 0.296 ± 0.015
As2O3 + Alcohol 0.262 ± 0.040 0.278 ± 0.006 0.296 ± 0.023 0.396 ± 0.010
As2O3 + Ars Alb-200C 0.232 ± 0.034 0.239 ± 0.014* 0.165 ± 0.004** 0.109 ± 0.006***
SE, Standard error; *P < .05, **P < .01, ***P < .001.
Table 6 Mean reduced glutathione (GSH) (nm/mg tissue) in liver and spleen of different series of mice at different fixation intervals.
Fixation intervals in days 30 Days 60 Days 90 Days 120 Days
Series Activity ± SE Activity ± SE Activity ± SE Activity ± SE
Liver
Negative control 0.006 ± 0.001 0.006 ± 0.002 0.007 ± 0.001 0.009 ± 0.001
Alcohol control 0.003 ± 0.001 0.003 ± 0.001 0.007 ± 0.002 0.007 ± 0.001
As2O3
0.004 ± 0.000 0.002 ± 0.001 0.001 ± 0.000 0.001 ± 0.000
As2O3 + Alcohol 0.005 ± 0.000 0.003 ± 0.001 0.002 ± 0.000 0.001 ± 0.000
As2O3 + Ars Alb-200C 0.005 ± 0.001 0.006 ± 0.000* 0.006 ± 0.000** 0.008 ± 0.000***
Spleen
Negative control 0.005 ± 0.001 0.007 ± 0.001 0.009 ± 0.001 0.009 ± 0.002
Alcohol control 0.006 ± 0.002 0.005 ± 0.001 0.008 ± 0.001 0.008 ± 0.000
As2O3
0.004 ± 0.000 0.003 ± 0.000 0.002 ± 0.001 0.001 ± 0.000
As2O3 + Alcohol 0.004 ± 0.000 0.004 ± 0.000 0.002 ± 0.000 0.002 ± 0.000
As2O3 + Ars Alb-200C 0.004 ± 0.000 0.006 ± 0.001 0.007 ± 0.001** 0.008 ± 0.001***
SE, Standard error; *P < .05, **P < .01, ***P < .001, determined versus intoxicated Alcohol control.
Table 7 Catalase and Succinate dehydrogenase activities in liver and spleen of different series of mice at 90 and 120 days fixation interval.
Table-1 Succinate dehydrogenase (μmol/mg protein) Catalase (unit enzyme/mg protein)
Series 90 days 120 days 90 days 120 days
Liver Spleen Liver Spleen Liver Spleen Liver Spleen
Negative control 530.00 ± 1.58 487.00 ± 0.32 550.00 ± 1.26 500.00 ± 0.63 8.80 ± 0.003 5.50 ± 0.00 8.70 ± .001 8.20 ± 0.002
Alcohol control 541.00 ± 1.26 488.00 ± 0.63 557.00 ± 0.63 498.00 ± 0.95 8.60 ± 0.001 5.30 ± 0.01 8.50 ± 0.001 8.40 ± 0.001
As2O3
283.00 ± 1.90 272.00 ± 1.59 262.00 ± 0.63 261.00 ± 1.26 4.36 ± 0.005 2.30 ± 0.02 2.95 ± 0.003 1.86 ± 0.002
As2O3 + Alcohol 270.00 ± 0.63 256.00 ± 0.95 268.00 ± 0.95 230.00 ± 1.30 3.11 ± 0.002 1.90 ± 0.01 2.50 ± 0.01 1.40 ± 0.001
As2O3 + Ars Alb-200C 495.00 ± 0.013*** 428.00 ± 0.01*** 512.00 ± 0.33*** 453.00 ± 1.38*** 6.1 ± 0.001*** 5.29 ± 0.003*** 6.69 ± 0.002*** 6.21 ± 0.002***
SE, Standard error; ***P < .001, determined versus intoxicated Alcohol control.
Table 8 Glutathione reductase (μmol/mg protein/min) and Superoxide dismutase (unit enzyme/mg protein) in liver and spleen of different series of mice at 90 and 120 days fixation interval.
Table-1 Glutathione reductase (μmol/mg protein/min) Superoxide dismutase (unit enzyme/mg protein)
Series 90 days 120 days 90 days 120 days
Liver Spleen Liver Spleen Liver Spleen Liver Spleen
Control 24 ± 0.010 20 ± 0.201 23.63 ± 0.010 20.85 ± 0.010 0.052 ± 0.031 0.050 ± 0.010 0.049 ± 0.010 0.051 ± 0.007
Alcohol control 21 ± 0.020 22 ± 0.110 22 ± 0.010 22 ± 1.30 0.056 ± 0.011 0.051 ± 0.012 0.051 ± 0.007 0.048 ± 0.010
As2O3
9.2 ± 0.122 10 ± 0.211 8 ± 0.158 8.9 ± 0.066 0.011 ± 0.006 0.015 ± 0.002 0.010 ± 0.001 0.012 ± 0.131
As2O3 + Alcohol 10 ± 0.447 12 ± 0.103 9 ± 0.224 11 ± 1.33 0.014 ± 0.010 0.013 ± 0.007 0.009 ± 0.003 0.010 ± 0.004
As2O3 + Ars Alb-200C 19 ± 0.316*** 16 ± 0.010*** 22 ± 0.158*** 18.6 ± 1** 0.037 ± 0.012 0.031 ± 0.010 0.061 ± 0.008*** 0.052 ± 0.007**
SE, Standard error; **P < .01, ***P < .001, determined versus intoxicated Alcohol control.
Table 9 Mean hemoglobin and Mean Blood sugar (mg/dl) content in different series of mice at different fixation intervals.
Fixation intervals in days 30 Days 60 Days 90 Days 120 Days
Series Activity ± SE Activity ± SE Activity ± SE Activity ± SE
Mean hemoglobin
Negative control 14.1 ± 0.006 13.8 ± 0.007 16.6 ± 0.410 17.1 ± 0.170
Alcohol control 14.0 ± 0.009 14.6 ± 0.019 15.9 ± 0.039 17.0 ± 0.190
As2O3
12.8 ± 0.008 11.9 ± 0.004 11.2 ± 0.710 10.17 ± 0.190
As2O3 + Alcohol 11.6 ± 0.012 11.1 ± 0.004 10.7 ± 0.470 10.14 ± 1.490
As2O3 + Ars Alb-200C 14.0 ± 0.007*** 12.7 ± 0.014*** 15.17 ± 0.750*** 16 ± 0.710**
Mean Blood sugar
Negative control 84.731 ± 0.082 82.156 ± 0.054 85.76 ± 0.970 82.240 ± 0.840
Alcohol control 86.426 ± 0.056 84.286 ± 0.076 90.84 ± 1.760 80.740 ± 1.460
As2O3
111.743 ± 0.121 109.410 ± 0.238 162.45 ± 2.470 175.080 ± 2.890
As2O3 + Alcohol 117.412 ± 0.185 121.146 ± 0.089 180.48 ± 1.850 182.790 ± 1.050
As2O3 + Ars Alb-200C 104.131 ± 0.120*** 97.420 ± 0.423*** 99.78 ± 0.240*** 92.460 ± 1.080***
SE, Standard error; **P < .01, ***P < .001, determined versus intoxicated Alcohol control.
Table 10 Mean Serum testosterone (ng/mL) and Mean Serum Estradiol (pg/ml) levels in different series of mice at different fixation intervals.
Fixation intervals in days 90 Days 120 Days
Series Activity ± SE Activity ± SE
Mean Serum testosterone
Negative control 5.10 ± 0.160 5.40 ± 0.030
Alcohol control 5.60 ± 0.130 5.50 ± 0.040
As2O3
0.98 ± 0.010 1.21 ± 0.020
As2O3 + Alcohol 2.54 ± 0.020 2.68 ± 0.010
As2O3 + Ars Alb-200C 1.87 ± 0.020*** 1.43 ± 0.015***
Mean Serum Estradiol
Negative control 42.00 ± 1.900 46.00 ± 1.260
Alcohol control 48.00 ± 0.840 51.00 ± 1.580
As2O3
33.00 ± 0.630 32.00 ± 0.710
As2O3 + Alcohol 31.00 ± 1.260 28.00 ± 1.210
As2O3 + Ars Alb-200C 39.00 ± 1.260** 48.0 ± 0.840***
SE, Standard error; **P < .01, ***P < .001, determined versus intoxicated Alcohol control.
Table 11 One way ANOVA for different hepatic enzymes.
Source of AST ALT LPO GSH Catalase SDH SOD
variation df F Sig. df F Sig. df F Sig. df F Sig. df F Sig. df F Sig. df F Sig.
Between groups 23 14.72 0.000* 23 35.93 0.000* 23 990.89 0.000* 23 2.51 0.001* 23 54.52 0.000* 23 172.80 0.000* 23 2.62 0.000*
Within groups 72
72
72
72
72
72
72
*P < .05.
Table 12 One way ANOVA of different spleen enzymes.
Source of variation AST activity ALT activity GSH activity CAT activity SDH activity SOD activity
df F Sig. df F Sig. df F Sig. df F Sig. df F Sig. df F Sig.
Between groups 23 39.131 0.000* 23 30.120 0.000* 23 3.160 0.000* 23 100.361 0.000* 23 91.231 0.000* 23 3.002 0.000*
Within groups 72
72
72
72
72
72
*P < .05.
Table 13 One way ANOVA of blood parameters.
Source of Variation Blood Hb Blood Sugar Serum Testosterone Serum Estadiol
df F Sig. df F Sig. df F Sig. df F Sig.
Between groups 23 63.268 0.000* 23 750.120 0.000* 23 28.160 0.000* 23 20.361 0.000*
Within groups 72
72
72
72
*P < .05.
==== Refs
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Evid Based Complement Alternat MedEvid Based Complement Alternat MedECAMEvidence-based Complementary and Alternative Medicine : eCAM1741-427X1741-4288Hindawi Publishing Corporation 1961720310.1093/ecam/nep084nep084Original ArticleModulation of Signal Proteins: A Plausible Mechanism to Explain How a Potentized Drug Secale Cor 30C Diluted beyond Avogadro's Limit Combats Skin Papilloma in Mice Khuda-Bukhsh Anisur Rahman
1
*Bhattacharyya Soumya Sundar
1
Paul Saili
1
Dutta Suman
1
Boujedaini Naoual
2
Belon Philippe
2
1Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani 741235, West Bengal, India2Boiron Laboratory, Lyon, France*Anisur Rahman Khuda-Bukhsh: [email protected] 18 6 2011 2011 28632024 2 2009 28 5 2009 Copyright © 2011 Anisur Rahman Khuda-Bukhsh et al.2011This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.In homeopathy, ability of ultra-high diluted drugs at or above potency 12C (diluted beyond Avogadro's limit) in ameliorating/curing various diseases is often questioned, particularly because the mechanism of action is not precisely known. We tested the hypothesis if suitable modulations of signal proteins could be one of the possible pathways of action of a highly diluted homeopathic drug, Secale cornutum 30C (diluted 1060 times; Sec cor 30). It could successfully combat DMBA + croton oil-induced skin papilloma in mice as evidenced by histological, cytogenetical, immunofluorescence, ELISA and immunoblot findings. Critical analysis of several signal proteins like AhR, PCNA, Akt, Bcl-2, Bcl-xL, NF-κB and IL-6 and of pro-apoptotic proteins like cytochrome c, Bax, Bad, Apaf, caspase-3 and -9 revealed that Sec cor 30 suitably modulated their expression levels along with amelioration of skin papilloma. FACS data also suggested an increase of cell population at S and G2 phases and decrease in sub-G1 and G1 phages in carcinogen-treated drug-unfed mice, but these were found to be near normal in the Sec cor 30-fed mice. There was reduction in genotoxic and DNA damages in bone marrow cells of Sec Cor 30-fed mice, as revealed from cytogenetic and Comet assays. Changes in histological features of skin papilloma were noted. Immunofluorescence studies of AhR and PCNA also suggested reduced expression of these proteins in Sec cor 30-fed mice, thereby showing its anti-cancer potentials against skin papilloma. Furthermore, this study also supports the hypothesis that potentized homeopathic drugs act at gene regulatory level.
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1. Introduction
Convincing positive effects of homeopathic drugs in prevention and treatment of various cancers like prostate [1], liver [2–5] and Ehrlich ascites carcinoma and Dalton's lymphoma [6, 7] have been documented earlier in laboratory animals. However, the mechanisms of chemoprevention or intervention by homeopathic remedies have been debated over and again and some critics even suggested that the actions of these drugs are no better than the “placebo” effects [8, 9].
In recent past, clinical trials and laboratory-based experiments have provided evidences that these drugs interfere with cancerous growth through genetical and physiological pathways [10–12].
However, despite scientific evidences and arguments for and against the efficacy of potentized homeopathic drugs diluted beyond Avogadro's limit (potency 12 and above), the issue has not yet been fully resolved, particularly because such ultra-high diluted drugs cannot theoretically have even a single molecule of the original drug substance. But more recent works [6, 7, 13] pointing to the ability of highly diluted (dynamized) drugs showing positive effects on the face of their physical non-existence have reopened the search for other possible molecular pathways of activities. Sunila et al. [6, 7] addressed this issue in in vitro experimental model (cancer cell line) and suggested that potentized homeopathic drugs diluted beyond Avogadro's limit can modulate certain signal proteins.
Analysis of expression of signal proteins, one of the state-of-the art techniques to understand signaling pathways [14], has been employed to understand the mechanism of chemoprevention or intervention by anti-cancer drugs employing HeLa cancer cell line [15, 16]. However, the exact signaling pathway through which they possibly act still remains rather obscure.
With this view in mind, the present work was aimed at testing the hypothesis if (i) Secale cornutum 30C (Sec cor-30, diluted 1060 times) could show amelioration of skin cancer and (ii) to examine if some relevant signal proteins were suitably modulated during this process. This drug was chosen because Sec cor-30 is claimed in homeopathic literature [17] to have great action against any stubborn case of skin lesions, particularly of hemorrhagic and wrinkled ulceration of skin. A DMBA + croton oil-induced mouse skin cancer model was chosen for this study, primarily because the cytokine-mediated cell signaling pathway is well defined in this model. DMBA is known to induce skin cancer through ligand-activation of the Aryl hydrocarbon receptor (AhR) [18–20] and by up-regulation of proliferating cell nuclear antigen (PCNA) [21, 22]. Thus, the study of expression of other downstream proteins like IL-6, NF-κB, Bcl-2, Bcl-xL, Bad, Bax, p53, caspase-3 and -9 could throw significant light on the pathway of carcinogenesis by DMBA + croton oil application. Furthermore, this could highlight how the homeopathic drug could, if it did, combat the carcinogenesis process.
2. Methods
Inbred Swiss albino mice (Mus musculus) of both genders reared in the animal house under standard hygienic conditions and food were used. The experiments were performed with clearance from the Ethical Committee, University of Kalyani and as per the stipulated guidelines. Healthy mice 8–10 weeks old, weighing between 22 and 26 g were selected randomly for use in the experiment. The selected mice were allocated to four groups, each comprising six mice (in two replicates of three mice each kept in separate cages).
Group 1 (Negative control)
mice fed with normal diet and water ad libitum without any exposure to DMBA + croton oil.
Group 2 (Treated)
100 μg of DMBA was applied once a week and 1% croton oil was applied twice a week to skin on dorsal side of the mouse for 24 weeks. Instead of the generally adopted practice of treating mice with DMBA for once or twice a week for 4 weeks (for initiation) [23], followed by regular application of croton oil (for promotion of papilloma) [24] as a complete carcinogenesis protocol, the mice in the present investigation were repeatedly treated with DMBA, once every week for 24 weeks, because: (i) that would further ensure development of papilloma (with possibility of many of them turning malignant) and (ii) we were interested in conducting studies on modulation of signal protein expression (particularly AhR), if any, as a result of DMBA and croton oil treatment, and any further modulation caused by the homeopathic drug [25]. DMBA is known to activate AhR by acting as a ligand.
Group 3 (positive control)
100 μg of DMBA was applied once a week and 1% of croton oil was applied twice a week to skin on dorsal side of the mouse for 24 weeks and fed a daily dose (two times) of Alcohol-30C, (Alc-30; placebo) through gavage with the aid of a fine tube.
Group 4 (drug-fed)
this group was similarly treated as in Group 2 but, in addition, they were treated with Sec cor-30. Each mouse was fed a daily dose (two times) of 0.06 ml of stock solution of Sec cor 30 through gavage with the aid of a fine tube.
2.1. Preparation of the Potentized Sec cor 30
The potentized homeopathic drug, Sec cor 30 was procured from HAPCO (BB Ganguly Street, Kolkata). The placebo, Alc-30, was prepared from the same stock of alcohol used for preparation of Sec cor 30, as per the standard procedure of homeopathic principle of “succussions and dilution.” One milliliter of each of Sec 30 and Alc-30 was diluted separately with 20 ml of double distilled water to make the stock solution of Sec 30 and Alc-30, respectively.
2.2. Incidence of Skin Tumors
The numbers of tumor formation in four groups were recorded weekly.
2.3. Histology and Immunofluorescence
Back skin samples were dissected from the lesion and/or tumor regions of the mid-dorsal back skin of both the drug-fed and positive controls (and also of normal back skin of negative control) and fixed in normal buffer formalin, followed by dehydration treatment, as per the standard practice [21]. After storing at room temperature for less than a week in 70% alcohol, the tissues were washed two times each for 30 min in xylene, and then transferred to the embedding bath. The paraffin blocks were cut into ribbon like pieces and placed on to slides after stretching into warm water. The paraffin was removed by xylene (two washes each for 10 min), then the xylene was removed by alcohols. Processed tissues were stained in haematoxylin for 2 min, followed by washing in distilled and tap water, respectively. Then tissues were brought into alcohol medium (70%, 90% two washes each for 2 min), followed by staining with eosin (30 s) and dehydrated into 90% and absolute alcohol (two washes each for 2 min). Finally, xylene treatment (two washes each for 2 min) was carried out, followed by mounting into DPX. For immunofluorescence study, the technique of Arabzadeh et al. [26] was adopted with a little modification. Briefly, cut tissue sections were de-paraffinized and incubated separately for 12 h with mouse monoclonal AhR and PCNA antibodies, respectively, procured from Abcam International, USA. Then after blocking with 3% BSA, the tissue sections were incubated for 2 h with fluorescence isothiocyanite (FITC) conjugated secondary antibody purchased from Sigma, St. Louis, MO, USA. The fluorescence intensity of FITC was measured under ZEISS fluorescence microscope, Germany, Carl Zeiss MicroImaging, Inc., Thornwood, New York.
2.4. Signal Proteins
These were either detected by ELISA method (caspase-3) or by western blots (the remaining ones).
2.4.1. Western Blot
It was performed as per protocol described previously [27]. Briefly, mouse skin tissues were homogenized in lysis buffer containing 50 mM Tris-HCl, pH 8.0, 1% Nonidet P-40, 125 mM NaCl, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 μg ml−1 aprotinin, 1 mM Na3 VO4 and 10 mM sodium pyrophosphate [14]. Equal amount of protein as determined by Lowry assay [28] were diluted with 5× sample loading buffer, boiled and loaded onto 12% polyacrylamide gels. After electrophoresis, the protein bands were transferred to a nitrocellulose membrane, and membranes were blocked in 5% milk in 1 × TBS, 0.05% Tween, then the transferred bands of AhR, PCNA, p53, Bcl-2, Bcl-xL, Bad, Bax, NF-κB (p65 sub-unit of nuclear extract), cytochrome c, IL-6 and caspase-9 were bound to their respective monoclonal antibodies purchased from Abcam, Kendall Square, Cambridge, USA and Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA. Antibodies for caspase-9, Akt and cytochrome c were purchased from BD Bioscience, San Jose, CA USA. The bound proteins were treated with the appropriate ALP-conjugated secondary antibodies (Sigma Chemicals, St. Louis, MO, USA). The same membranes were also immunoblotted against β-actin (house-keeping protein) for data normalization. For quantitative analysis of each band, density was determined using Gel Doc System; Ultra Lum, East Uwchlan Ave, Exton, PA, USA; Nonlinear Dynamic Ltd, Newcastle, UK.
2.4.2. ELISA
The activity of caspase-3 was measured using the colorimetric substrate method from skin tissue employing an ELISA reader (Thermo Scientific, Vantaa, Finland), following Das et al. [24]. Cytosolic extracts were prepared by homogenization of dorsal shaved area of skin tissues in extraction buffer containing 25 mM HEPES (pH 7.5), 5 mM MgCl2, 1 mM EDTA, 0.1% (w/v) CHAPS and 10 μg aprotinin. Subsequently, the homogenates were centrifuged at 13 000 × g for 15 min at 4°C. The supernatant fraction was used to determine caspase-3 activity. In brief, caspase-3 colorimetric substrates (Ac-DEVD-pNA), procured from Sigma, were incubated with cell lysates for 1 h at 37°C, then the amount of chromophore, p-nitroaniline (p-NA), released by caspase-3 activity was quantified by measuring the optical density at 405 nm. Caspase-3 activity was expressed as μM p-NA released per hour per milligram cellular protein.
2.5. Cytogenetic Assay
The standard cytogenetic protocols like assays of chromosome aberrations (CA), micronuclei (MN), mitotic index (MI) and sperm head anomaly (SHA) have been adopted in the present study [2, 3].
2.5.1. CA
Slides were prepared by the conventional flame-drying technique followed by Giemsa staining for scoring bone marrow CA. A total of 300 bone marrow cells were observed.
2.5.2. Micronucleus Preparation
One part of the suspension of bone marrow cells in 1% sodium citrate was smeared on clean grease-free slides, briefly fixed in methanol and subsequently stained with May-Grunwald followed by Giemsa. Approximately 3000 bone marrow cells, comprising both polychromatic erythrocytes and normochromatic erythrocytes were scored.
2.5.3. MI
It was determined from the same slide that was scanned for MN, and a total of 5000 cells were examined from each series. The non-dividing and dividing cells were recorded and their ratios ascertained.
2.5.4. SHA
The technique of Wyrobek [29] was adopted. 5000 sperms were examined in each series.
2.5.5. Estimation of DNA Damage
Comet assay was performed with single-cell gel electrophoresis, as per protocol described elsewhere [30]. Briefly, peripheral blood nuclear cells (PBMC) (1 × 104) were suspended in 0.6% low-melting agarose and layered over a frosted microscopic slide previously coated with a layer of 0.75% normal-melting agarose to ensure firm gripping. The slides were then kept at 4°C for solidification. Subsequently, slides were immersed in a lysis buffer of pH 10 and kept overnight for lysis of cell and nuclear membranes. The following day slides were transferred into a horizontal electrophoresis chamber containing electrophoresis buffer (300 mM NaOH, 1 mM Na2EDTA; pH 13.0) and presoaked for 20 min in order to unwind DNA. Electrophoresis was then carried out for 20 min (300 mA, 20 V). Slides were then washed thoroughly with neutralizing buffer (Tris 0.4 M, pH 7.5), stained with ethidium bromide (final concentration of 40 μg ml−1) and examined under a Lyca fluorescence microscope. The percentage of DNA breakage was determined by measuring the comet tail length using the software Motic Image China.
2.5.6. Cell-Cycle Analysis
Cellular DNA was stained with propidium iodide and quantified by flow cytometry according to Nicoletti's procedure [31]. Briefly, cells were fixed with 70% (v/v) cold aqueous ethanol (−20°C) and stored at 4°C for at least 24 h. The cells were washed in phosphate buffer saline, after cell centrifugation, cell pellets were stained with propidium iodide staining solution containing 10 μg ml−1 propidium iodide, 5 K units of RNase, and 0.1% (v/v) Triton X-100. The cell suspension was incubated in the dark at room temperature for 30 min. DNA content was determined by using a FACS Calibur flow cytometer (Becton Dickinson, Mountain View, CA). A total of 40 000 events were acquired, and MOD-FIT LT software (Becton Dickinson) was used.
2.6. Statistical Analysis
The significances of differences between data of carcinogen plus 2% alcohol administered mice and carcinogen administered Sec cor 30-fed mice were analyzed by the Student's t-test. For all quantitative analyses, data were expressed as mean values obtained from triplicate experiments (on blot and immunofluorescence studies) showing standard errors and levels of significance. Additionally, the data were also analyzed using one-way ANOVA (Turkey Method; SPSS version 11).
3. Results
3.1. External Morphology
The changes in external morphology of skin in mice subjected to: (i) no treatment (Figure 1(a); negative control: Group 1), (ii) carcinogen treatment (Figure 1(b); Group 2), (iii) carcinogen plus alcohol treatment (Figure 1(c); Group 3) and (iv) carcinogen plus Sec cor 30 treatment (Figure 1(d); Group 4) have been provided. While Groups 2 and 3 mice showed distinct growth of papilloma, both the Sec cor 30-fed groups showed much less growth of papilloma as well as loss of hair than their counterparts who did not receive Sec cor 30 treatment. The incidence of papilloma growth in different weeks in control and treated mice has been provided in Figure 2. Papillomas observed in DMBA + croton oil-treated group were significantly bigger in size (70% papillomas of ≥2.5 mM in diameter) as that of drug-fed series (8%–10%).
3.2. Histology
As compared to the skin section with normal histological features (Figure 3(a)), irregular distribution of different cell types (e.g., squamous epithelial cells) and finger-like projections (papilloma) indicative of cancerous growth were found in the skin sections of DMBA administered mice (Figure 3(b)). Tumors of animal belonging to DMBA + croton oil-treated group were composed of focal proliferation of squamous cells, presence of some necrotic cells, keratinization and epithelial pearls, indicative of the malignant nature of tumors that is characterized by well-differentiated squamous cell carcinoma. On the other hand, in the Sec cor 30-fed mice, these features were visible to a much lesser extent (Figure 3(c)).
3.3. Immunofluorescence Studies
The AhR-specific immunochemical staining reveals lack of expression in normal skin (Figure 3(d)), while in DMBA-treated mice, AhR receptor was seen to take up higher level of green fluorescence in DMBA-treated mice without Sec cor 30 feeding (Figure 3(e)), indicating localization of AhR protein in carcinogen-treated mice, while in Sec cor 30-fed mice, the intensity of fluorescence was much less (Figure 3(f)). Immunofluorescence analysis for PCNA was also used to assess the proliferation activity in normal (Figure 3(g)), carcinogen-treated (Figure 3(h)) and in Sec cor 30-fed groups (Figure 3(i)) during tumor promotion. PCNA is a proliferating cell nuclear antigen associated with S phase of DNA replication. It was observed through immunofluorescence localization that PCNA expression was higher in DMBA administrated group, a characteristic intense staining, as compared to control, was observed (Figure 3(h)) and, also, there was a significant modulation of this expression in Sec cor 30-fed mice. The data on proportion of fluorescence intensity represented in Figure 3(h) prepared from three replicates would also validate the above observation.
3.4. Immunoblots
The expression of different signal proteins in normal healthy mice (Group 1) can be observed in the lane 1 of Figure 4. Of these, the expression level of β-actin, AhR, Akt, IL-6, NF-κB, PCNA, Bcl-2 and Bcl-xL was of low level, while it was moderate in Bad, Bax, cytochrome c, Apaf, p53 and caspase-9. In the DMBA + croton oil-induced mice, the expression levels were increased in AhR, Akt, IL-6, NF-κB, PCNA, Bcl-2 and Bcl-xL, while in the carcinogen administered Sec cor 30-fed mice (Group 4), there was suppression of the enhanced expression of these signal proteins. Correspondingly, there was decrease in expression of proteins like Bad, Bax, Apaf, cytochrome c, caspase-9 and p53 in the DMBA + croton oil-induced mice, which were increased in the Sec cor 30-fed mice. Quantification of proteins was done by densitometry using image analyzer, shown in Figure 5.
3.5. Caspase-3 Activity through ELISA
The caspase-3 activity was higher in Sec cor 30-fed mice as compared to the DMBA-administered mice (Figure 6).
3.6. Cytogenetical Studies
The frequency distributions of CA, MN have been provided in Table 1. While the percentage of CA, MN, MI and SHA (Figure 7) were significantly elevated in the DMBA-induced mice as compared to normal untreated control, the values were significantly decreased by the feeding of Sec cor 30. The same was supported by the data of Comet assay (Figure 7) that indicated a much more amount of broken DNA in mice treated with DMBA + croton oil, but much less in the Sec cor 30-fed mice.
3.7. Cell-Cycle Analysis by FACS
The FACS analysis revealed that in the Sec cor 30-fed mice, the cell cycle was arrested more in the sub-G1 stage (32%) than that of the DMBA treated mice (23%). Furthermore, DMBA showed higher percentage of S phase (25%) in contrast to the Sec cor 30 that showed 19% of cells in S phase (Table 2).
4. Discussion
As far as authors are aware, this is the first demonstration where a potentized homeopathic drug diluted beyond Avogadro's limit, Secale cor 30, successfully reduced the incidence of DMBA-induced skin papilloma in mice. The histological features of skin of the drug-fed group showed evidence of amelioration rendered by the administration of the homeopathic drug. Similar findings of amelioration were also reported in case of other types of cancer like liver in mice through the administration of some crude plant extracts used as homeopathic mother tincture [2, 3] or in skin cancer by an anti-mitotic agent like 4-demethyl epipodophyllotoxin [32].
DMBA is a pro-carcinogen that requires metabolic conversion to its ultimate carcinogenic metabolite 3,4-dihydrodiol-1,2-epoxide [33]. These metabolites damage DNA presumably by formation of adducts, which could be evidenced by the increase in comet tail lengths and elevated frequencies of chromosomal aberrations in the carcinogen-administered mice. The DNA damage, CA and, consequently, disorder in metabolic functioning, contributed to the initiation of the tumorigenic process, through generation of ROS. The generation of ROS contributed further to the up-regulation of several downstream signaling proteins like NF-κB, Akt and PCNA. Up-regulation of NF-κB would induce the anti-apoptotic Bcl-2 and Bcl-xL genes through up-regulated Akt and thereby prevent the cells to undertake the apoptotic pathway via down-regulation of pro-apoptotic genes Bad, Bax and Apaf, caspase-3 and -9.
The AhR is known to be involved in the induction of several enzymes that participate in xenobiotic metabolism. Induction of enzymes involved in xenobiotic metabolism occurs through binding of the ligand-bound AhR to xenobiotic response elements in the promoters of gene for these enzymes [34, 35]. The AhR protein contains several domains critical for function and act as a variety of transcription factors. Two PAS domains are also present in AhR, PAS-A and PAS-B [36]. The ligand-binding site of AhR is contained within the PAS-B domain and contains several conserved residues critical for ligand binding [37]. There is also a Q-rich domain located in the C terminal region of the protein and is involved in the co-activator recruitment and trans-activation [38].
Toxicity results from two different ways of AhR signaling. The first is a function of the adaptive response in which the induction of metabolizing enzymes result in the production of toxic metabolites, as for example the PAH, benz[a]pyrine, a hydrocarbon for AhR induces its own metabolism and bioactivation to toxic metabolites via the induction of CYP1A1 and CYP1B1 in several tissues [18]. The second approach to the toxicity is the result of aberrant changes in global gene transcription beyond those observed in the “AhR gene battery.”
Our present findings agree well to the reported fact that the DMBA-induced cell signaling mechanism is mediated by up-regulation of AhR. Our studies on several anti-oxidant markers like catalase, superoxide dismutase, lipid peroxidation and reduced glutathione (results unpublished) also suggested that there was the generation of reactive oxygen species (ROS) on application of DMBA. We found up-regulation in expression of some genes like IL-6, NF-κB, Bcl-2 and Bcl-xL that may be responsible for leading to proliferation of cells, which in turn is reflected on an increase in PCNA in the drug-untreated mice. The increase in PCNA was accompanied by down-regulation of p53, Bax and caspase-3 genes, for which epidermal skin cells (keratinocytes) started uncontrolled divisions to subsequently form papilloma. The immunochemical localization also suggested that there was excess amount of the enzymes involving the products of unregulated genes.
Interestingly, in the drug-fed mice, very clear indication of modulation in the expression of the genes was noticed. The AhR expression was significantly lowered. Significant changes were also noted in the expression of IL-6, NF-κB, Bcl-2, Bcl-xL and PCNA, which were reversed. Secale cor 30 administration caused up-regulation in the expression of p53, Bax, Apaf, caspase-3 and -9 and down-regulation of PCNA. This was a good indicator of the lower state of proliferation of the cells. This was further confirmed by our analysis of the FACS data, which showed more number of cells in the sub-G1 and G1 stages of the cycle. This tempted us to believe that the potentized homeopathic drug Sec cor-30 was possibly tagged in an unknown manner to some regulatory proteins. This binding with an unknown protein could give it the ability to bind on the AhR, thereby competitively reducing space for DMBA-binding that was responsible for lowering activity of AhR in the drug fed mice. Alternatively, it prevented the conversion of DMBA into the lesser amount of carcinogenic metabolites. Thus, the homeopathic drug apparently fulfilled the function of `ligand' by its ability to modulate the AhR activity. This was further confirmed by results of our immunofluorscence assay. Incidentally, nanoparticles associated with molecules of drug substance have been shown to affect the physicochemical property of potentized homeopathic drugs [15]. Nanoparticles of glass vial and other containers are also known to form complexes with various proteins and in some cases, in the process change their physicochemical properties [39]. Therefore, it is possible that the potentized homeopathic drug Secale cor 30, theoretically devoid of the presence of even a single molecule of the original drug substance, was still capable of apparently initiating binding to AhR, presumably through some activated proteins, because it is a pre-condition for Ahr to be ligand-activated before it could stimulate downstream proteins. However, the other possibility that cannot be excluded is the ability of Sec cor 30 to have regulatory influences on the other gene products directly or through indirect means. But one thing is clear from this study that potentized homeopathic drug does elicit regulatory influences on expression of certain relevant genes that can interfere with the carcinogenic effect of DMBA + croton oil in ameliorating/protecting skin cells from being transformed into skin papilloma (as evidenced from the difference in expression of several signal proteins), although one may still argue that it is difficult to attribute the biochemical differences between tumor and normal cells to a causal mechanism or secondary consequences for cell transformation. Therefore, further in-depth study, preferably with global micro-array methodology, may transpire that expression of many other relevant downstream protein genes can also be modulated. Incidentally, like what has been found in the present study in regard to specific signal proteins, modulation of some 147 downstream genes by a homeopathic drug has been reported by analysis of microarray [40], giving much credentials to the postulation of Khuda-Bukhsh [10–12, 41].
Funding
Grant from Boiron Laboratory, Lyon, France (to A.R.K.-B.).
Acknowledgments
The authors are thankful to Dr P.K. Das, Former Director of Vector Control Research, Government of India, Pondicherry, for his helpful criticism of the work.
Figure 1 External morphology of skin of mice: (a) normal; (b) DMBA + croton oil; (c) DMBA + croton oil + alcohol; (d) DMBA + croton oil + Sec cor 30.
Figure 2 Number of tumors/mouse plotted as a function of weeks on test. Each value represents the mean number of tumor per mouse. **P < .01, ***P < .001.
Figure 3 Histology of skin stained with Haematoxylin/Eosin: (a) normal, (b) DMBA + croton oil + alcohol, (c) DMBA + croton oil + Sec cor 30-fed; immunofluorescence expression of AhR protein: (d) normal, (e) DMBA + croton oil + alcohol, (f) DMBA + croton oil + Sec cor 30-fed; and PCNA protein localization: (g) normal, (h) DMBA + croton oil + alcohol, (i) DMBA + croton oil + Sec cor 30-fed; (j and k) represent proportion of average fluorescence intensity of AhR and PCNA protein in different groups. **P < .01.
Figure 4 Immunoblots of β-actin, AhR, Akt, IL-6, NF-κB, PCNA, Bcl-2, Bcl-xL, Bad, Bax, Apaf and p53, caspase-3 and cytochrome c. Ln1 represents normal; Ln2 represent DMBA; Ln3 represents DMBA + croton oil + alcohol; Ln4 represents DMBA + croton oil + Sec cor 30.
Figure 5 Densitometric data expressed as mean ± SE of the three independent experiments; *P < .05, **P < .01, ***P < .001. of β-actin, AhR, Akt, IL-6, NF-κB, PCNA, Bcl-2, Bcl-xL, Bad, Bax, Apaf, p53, cytochrome c and caspase-9. Each experiment is representative of three different sets of experiments. Ln1 represents normal; Ln2 represents DMBA + croton oil; Ln3 represents DMBA + croton oil + alcohol; Ln4 represents DMBA + croton oil + Sec cor 30.
Figure 6 Showing ELISA assay of caspase-3. The rate of Ac-DEVD-pNA cleavage was measured at 405 nm. Value represents mean ± SE (n = 3); ***P < .001., represent highly significant differences from alcohol control.
Figure 7 Showing percentage of CA, MN, MI, SHA and DNA damage in different groups.*P < .05, **P < .01.
Table 1 Percentage of CA, MN in normal, DMBA + croton oil, DMBA + croton oil + alcohol and DMBA + croton oil + Sec cor 30-fed series.
Series Major CA (%) Other CA (%) Total CA (%) MN (%)
Normal 2.45 2.15 4.60 ± 0.40 0.225 ± 0.08
DMBA + croton oil 12.1 9.7 21.8 ± 0.54 1.13 ± 0.09
DMBA + croton oil + alcohol 14.0 10.3 24.3 ± 0.67 1.22 ± 0.05
DMBA + croton oil + Sec cor 30 10.0 6.67 16.67 ± 1.02** 0.68 ± 0.06**
MCP-1, monocyte chemoattractant protein-1; VCAM-1, vascular cell adhesion molecule-1; TGF-β
1, transforming growth factor-β
1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Table 2 Flow cytometric analysis of normal, DMBA + croton oil, DMBA + croton oil + alcohol and DMBA + croton oil + drug-treated cells showing data of percentage of cells in different phases of cell cycle.
Series Cell-cycle analysis by FACS
SubG1 (%) G1 (%) S (%) G2/M (%)
Normal 32.07 ± 0.375 32.73 ± 0.565 17.44 ± 1.24 2.14 ± 0.020
DMBA + croton oil 23.39 ± 0.005 23.56 ± 0.560 25.73 ± 0.610 6.625 ± 0.375
DMBA + croton oil + alcohol 22.15 ± 0.105 28.41 ± 0.410 28.74 ± 0.640 4.76 ± 0.040
DMBA + croton oil + Sec cor 30 32.68 ± 0.310* 31.88 ± 0.995* 19.44 ± 1.16** 2.18 ± 0.28***
Values are expressed as mean ± SE. Data are the means of the results from two independent experiments. Comparisons are made between carcinogen treated and drug fed mice. *P < .05, **P < .01; ***P < .001.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 21779402PONE-D-11-0318910.1371/journal.pone.0022244Research ArticleBiologyMolecular Cell BiologyNeuroscienceMedicineDrugs and DevicesDrug Research and DevelopmentNeuropharmacologyNeurologyNeuro-OphthalmologyNeuropharmacologyOphthalmologyRetinal DisordersInhibition of Reactive Gliosis Prevents Neovascular Growth in the Mouse Model of Oxygen-Induced Retinopathy Targeting Reactive Gliosis by YC-1DeNiro Michael
1
2
*
Al-Mohanna Falah H.
2
Al-Mohanna Futwan A.
3
1
Research Department, King Khaled Eye Specialist Hospital, Riyadh, Saudi Arabia
2
Department of Comparative Medicine, King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia
3
Department of Biological and Medical Research, King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia
Sharif Naj EditorAlcon Research, Ltd., United States of America* E-mail: [email protected] and designed the experiments: MD FHA-M FAA-M. Performed the experiments: MD FHA-M FAA-M. Analyzed the data: MD FHA-M FAA-M. Contributed reagents/materials/analysis tools: MD FHA-M FAA-M. Wrote the paper: MD FHA-M FAA-M.
2011 14 7 2011 6 7 e2224410 2 2011 20 6 2011 DeNiro et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Retinal neovascularization (NV) is a major cause of blindness in ischemic retinopathies. Previous investigations have indicated that ischemia upregulates GFAP and PDGF-B expression. GFAP overexpression is a hallmark of reactive gliosis (RG), which is the major pathophysiological feature of retinal damage. In addition, PDGF-B has been implicated in proliferative retinopathies. It was the aim of this study to gain insights on the possible pharmacological interventions to modulate PDGF-B and GFAP expression, and its influence on RG and NV. We used an array of assays to evaluate the effects of YC-1, a small molecule inhibitor of HIF-1 and a novel NO-independent activator of soluble guanylyl cyclase (sGC), on RG and NV, in vivo and in vitro. When compared to the DMSO-treated retinas, dual-intravitreal injections of YC-1, in vivo: (1) suppressed the development and elongation of neovascular sprouts in the retinas of the oxygen-induced retinopathy (OIR) mouse model; and (2) reduced ischemia-induced overexpression of GFAP and PDGF-B at the message (by 64.14±0.5% and 70.27±0.04%) and the protein levels (by 65.52±0.02% and 57.59±0.01%), respectively. In addition, at 100 µM, YC-1 treatment downregulated the hypoxia-induced overexpression of GFAP and PDGF-B at the message level in rMC-1 cells (by 71.42±0.02% and 75±0.03%), and R28 cells (by 58.62±0.02% and 50.00±0.02%), respectively; whereas, the protein levels of GFAP and PDGF-B were reduced (by 78.57±0.02% and 77.55±0.01%) in rMC-1cells, and (by 81.44±0.02% and 79.16±0.01%) in R28 cells, respectively. We demonstrate that YC-1 reversed RG during ischemic retinopathy via impairing the expression of GFAP and PDGF-B in glial cells. This is the first investigation that delves into the reversal of RG during ischemic retinal vasculopathies. In addition, the study reveals that YC-1 may exert promising therapeutic effects in the treatment of retinal and neuronal pathologies.
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Introduction
Diabetic retinopathy [DR] is a leading cause of visual disturbance in adults and is the leading cause of blindness in Americans between the ages of 20 and 74 years [1]. DR has been regarded as a retinal microvascular disease, which develops in two stages: an early, non-proliferative stage, and a later, proliferative stage. In the early non-proliferative stage, retinal vascular permeability can increase even before the appearance of clinical retinopathy [2]. Currently, this stage is diagnosed by dilation of retinal veins, retinal microaneurysmas, intraretinal microvascular abnormalities, areas of capillary nonperfusion, retinal hemorrhages, cotton wool spots, edema, and exudates. All of these signs indicate regional failure of the retinal microvascular circulation, which presumably results in ischemia. In contrast, proliferative DR's [PDR] diagnosis is based on the ischemia-induced formation of new blood vessels [BVs] on the surface of the retina. New vessels can extend into the vitreous cavity of the eye and can hemorrhage into the vitreous, resulting in visual loss [3]. They also can cause tractional retinal detachments from the accompanying contractile fibrous tissue. Furthermore, during this stage, over-proliferation of capillary endothelial cells [ECs] results in retinal neovascularization [NV], abnormal formation of new vessels in the retina and the vitreous, leading to PDR [4]. As a consequence retinal edema may ensue [5]. Retinal edema involves the breakdown of the blood-retinal barrier, with leakage of plasma from small BVs.
Platelet-derived growth factors (PDGFs) are growth-regulatory molecules that stimulate chemotaxis, proliferation, and increased metabolism of primarily connective tissue cells [6]. In addition, PDGF is a potent mitogen for mesenchymal cells and glial cells, which may act as a neuronal regulatory agent. Neuronal release of PDGF could contribute to nerve regeneration and to glial proliferation that leads to gliosis and scarring [6]. Furthermore, glial cells could utilize the elevated levels of PDGF to proliferate in RG, and under these conditions PDGF may also augment the immune responses [7]. It is also possible that PDGF increases the survival, and promotes the neurite outgrowth from grafted dopaminergic neurons [7]. PDGF-B is a vasoactive factor that possesses both chemotactic and mitogenic properties to vascular ECs in vitro
[67] and may also have angiogenic effects in vivo
[8]. Previous data have indicated that the distributions of PDGF B-chain-related immunoreactivity [PBRI] in the cerebral wound lesion were closely related to the NV and astrogliosis [9]. The same study has demonstrated that cerebral wound induced the expression of PDGF B-chain in neurons and macrophages in the lesion area. This increased expression of PDGF B-chain at a stab wound was proven to be related strongly to RG [9]. Moreover, increased expression of PDGF-B in the retina causes plays an important role in the pathogenesis of PDR and retinal detachment [10]. PDGF-B promotes the recruitment, proliferation and survival of pericytes; recruits glial cells [8] and retinal pigment epithelial [RPE] cells [11] that instigates scarring, which is ultimately the major cause of permanent loss of vision. Previous data have revealed that PDGF-B may play a crucial role in RG following injury [12], as well as it may lead to astrocyte and glial cell chemotaxis and proliferation [13].
The retina contains two types of macroglial cells. The most abundant are the Müller cells, which project from the retinal ganglion cell layer [GCL] to the photoreceptors. While the astrocytes, which originate in the optic nerve and migrate into the retina during development [14], reside as a single layer adjacent to the inner limiting membrane [ILM]. Glial cells provide structural and metabolic support for retinal neurons and BVs. These cells become reactive in certain injury states [15]. Several studies suggested that glial reactivity and altered glial metabolism are early pathological events in the retina during diabetes [16]. The most constant manifestation of reactivity is the increase in immunoreactivity for the intermediate filament protein [glial fibrillary acidic protein] [GFAP] [17]. GFAP is mainly expressed in astrocytes for which it constitutes a selective marker. Previous reports have demonstrated that upregulation of astrocytic intermediate filaments is a crucial step and a hallmark of RG [18]. RG is one of the pathophysiological features of retinal damage. The vertebrate retina contains a specialized type of glia, the Müller glia. Like other glial cells of the CNS, Müller cells undergo RG following acute retinal injury or chronic neuronal stress [19]. The overexpression of GFAP is the most sensitive non-specific response to retinal disease and injury, and it may be considered as the universal hallmark of retinal stress; such as retinal injury and Müller cell activation [20]. GFAP, which is located primarily in Müller cells, has specific immunoreactivities that occur in all retinal eccentricities. Moreover, virtually every pathologic alteration in the retina is accompanied by RG, that is, by distinct changes of the Müller cells properties [20]. Under these pathological conditions, Müller cells exhibit three crucial nonspecific gliotic responses, which are considered as the “hallmarks of glial cell activation”, these are: [i] cell proliferation [21]; [ii] changes in cell shape [hypertrophy] due to alterations in intermediate filament [22]; and [iii] the upregulation of the intermediate filament system composed of GFAP, vimentin, nestin and synemin [23], [24]. There are other gliotic characteristics such; targeted cellular migration [25], changes in ion transport properties [26], and secretion of signaling molecules such as VEGF [27]. Successful inhibition of GFAP using antisense oligonucleotides has also been reported by several groups [28], [29], [30]. Ostensibly, gliosis is important for the protection and repair of retinal neurons, yet some pathologies such as DR may be exacerbated by RG properties [26], [27].
YC-1; [3-[5′-hydroxymethyl-2′furyl]-1-benzyl indazole], is a small molecule that inhibits cGMP breakdown. YC-1 is an agent that has been considered as a novel type nitric oxide (NO)-independent activators of soluble guanylate cyclase (sGC) [31], [32], [33], [34]. YC-1 is not an NO donor, however, it causes activation of sGC especially in the presence of NO [35], [36], while binding to sGC at a different site from the heme [37]. We have previously shown that YC-1 suppressed retinal new vessel growth and formation in human retinal microvascular ECs, and retinal explants [38]. Furthermore, we have demonstrated that YC-1 downregulates HIF-1α, HIF-2α, VEGF, EPO, ET-1, and MMP-9 protein expression in the human retinal microvascular ECs [38], [39], [40]. The primary objectives of this investigation were to determine; [i] whether ischemic exposure in the OIR mouse model induces RG; [ii] whether YC-1 could target and hence reverse RG, and [iii] whether YC-1's effect is mediated through the inhibition of the length and numbers of neovascular sprouts.
Materials and Methods
Ethics Statement
All experiments were conducted in compliance with the laws and the regulations of the Kingdom of Saudi Arabia. In addition, all animal protocols were approved by the Institutional Review Board and conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research statement of the Association for Research in Vision and Ophthalmology.” This research study was approved by the HEC/IRB Committee (Human Ethics Committee/Institutional Review Board Committee) at the King Khaled Eye Specialist Hospital (KKESH). The permit number/approval ID is “RP 0630-P”.
Materials
YC-1 was purchased from A.G. Scientific [San Diego, CA] and dissolved in sterile dimethyl sulfoxide [DMSO]. Fluorescein isothiocyanate [FITC]-dextran 2,000,000 was purchased from Sigma-Aldrich [St. Louis, MO]. Rabbit Anti-PDGF-B polyclonal antibody was obtained from Abbiotec [San Diego, CA]. The GFAP labeling was carried out by primary rabbit-anti rat GFAP polyclonal antibody [Sigma, catalog number G9269] during immunohistochemistry; or a monoclonal GFAP antibody produced in mouse [Sigma, catalog number G3893] for Western Blot analysis. Polyclonal rabbit anti-β-actin antibody was purchased from MBL Intl [Woburn, MA].
Tissue Culture
A transformed Müller cell line [rMC-1] was kindly sent to us by Dr. V.J. Sarthy. Müller cell cultures were grown in DMEM supplemented with 15% FBS, as well as with a fungicide mixture and 0.5% gentamicin in a humidified atmosphere of 5% CO2/95% air. Medium was changed every 2–3 days, and cells were grown to confluence in a 150-mm dish. Cells were split into 60-mm dishes and were used in the experiments when confluent.
R28 cells are immortalized rat retinal neurosensory/neuoroglial progenitor cells, by transfection with Adenovirus 12S E1A into the neonatal retinal tissue. The cells were a kind gift from Dr. Gail M. Seigel [SUNY, Buffalo, NY]. R28 cells express genes characteristic of neurons, as well as functional neuronal properties. R28 cells were cultured in DMEM/F12 medium in a 1∶1 mixture, supplemented with 5% FBS, 1.5 mM L-glutamine, 7.5 mM sodium pyruvate, 0.1 mM nonessential amino acids, 1× MEM, 0.37% sodium bicarbonate and 10 µg/ml gentamicin. Cells were incubated at 37°C in the presence of 5% CO2.
Human retinal microvascular endothelial cells [hRMVEC] were cultured in 150 µl of CS-C medium supplemented with 10% FBS and in the presence of 1–100 µM YC-1 or DMSO [0.2%] and incubated at 37°C.
In Vitro Hypoxia
Cells were placed in airtight chambers [BioSpherix, Redfield, NY] and the O2 tension was maintained at 1.2% by using Pro-Ox Model 110 O2 regulator [BioSpherix, Redfield, NY]. The chamber was purged with a gas mixture of 5.32% CO2, and 93.48% N2.
Cell Proliferation in a Co-culture System Model
To test the influence of Müller cells [rMC-1] on the hRMVECs proliferation, rMC-1 [2×105 cells cm−2] were plated in a transwell insert [Millipore, Billerica, MA] with 0.4 mm pores and allowed to adhere overnight in 150 µL of CS-C complete medium [Cell Systems, Kirkland, WA], supplemented with 10% FBS and incubated at 37°C under normoxic [5% CO2/95% air]. The rMC-1 cells were then incubated under normoxic [5% CO2/95% air], or hypoxic conditions. To establish hypoxic conditions, cells were placed in airtight chambers [BioSpherix, Redfield, NY] that were flushed with a gas mixture of 5% CO2 and 95% N2. Oxygen concentrations within these chambers were maintained at 1.2% using Pro-Ox Model 110 O2 regulators. After treating the r-MC-1 cells with YC-1 [100 µM] for 24 hours, the insert was then placed into 24 well-plates, in which hRMVECs were plated at 5×103 cells cm−2 and allowed to adhere overnight. hRMVECs proliferation was evaluated using 3, [4,4-dimethylthiazol-2-yl]-2,5- diphenyl tetrazolium bromide [MTT] colorimetric assay, at 24, 48, 72, and 96 hours after coculture. During the last 4 hours of each day, 100 ml of 5 mg/ml MTT [Millipore, Billerica, MA] was added in each well. Formed Formazan crystals were dissolved in 600 ml DMSO and optical density was recorded at 492 nm. Experiments were performed on at least three independent occasions. Data were presented as a percentage of negative control proliferation with **P<0.01 being significant.
Cell Migration in a Co-culture System Model
Migration assay of hRMVECs was carried out using the transwell insert with 8 mm pore size. The inserts were coated with Extracellular matrix [ECM] [Millipore, Billerica, MA] and air-dry up. Chemotaxis was induced by the control r-MC1cells or the r-MC1 cells that were treated with YC-1 [100 µM], which were situated in the lower compartment. hRMVECs suspension [final concentration, 5×104 cells/well] was added to the upper compartment. After incubated for 24 hours, the filters were washed and then fixed and stained with crystal violet [0.5% crystal violet and 20% methanol] for 30 min at room temperature. The filters were washed with distilled water, and the cells on the upper surface of the inserts were wiped with a cotton swab. The number of cells per field that migrated to the lower surface of the filters was determined microscopically. Five randomly chosen fields were counted per filter. Data were presented as a number of migrated cells [**P<0.01].
Quantitative RT-PCR by Molecular Beacon Assays
The message levels for PDGF-B and GFAP were quantified by Real time RT-PCR. Gene-specific molecular beacons and primers were designed to encompass the genes of interest, with beacon's annealing site to overlap with the exon-exon junctions for additional specificity [Beacon Designer 6.0, Premier Biosoft International, Palo Alto, CA, USA]. Threshold cycle [Ct] values for the different samples were utilized for the calculation of gene expression fold change using the formula 2 to the minus power of delta delta ct. Fold changes in the PDGF-B and GFAP gene relative to the β-actin endogenous control gene were determined by the following equation: fold change = 2−Δ [ΔC
T
], where change in threshold cycle [ΔC
T] = C
T [gene of interest]−C
T [β-actin] and Δ [ΔC
T] = ΔC
T [treated]−ΔC
T [untreated].
Western Blot
Müller cells [rMC-1] and R28 cells were seeded overnight in 6-well plates [3×105 cells/well]. Cells were treated with either YC-1 [25–100 µM] or DMSO [0.2% v/v] for 72 hours under normoxic or hypoxic environments. Reactions were terminated by addition of lysis buffer [Cell Signaling, Beverly, MA]. Protein content of the cell lysates was determined according to the Bradford method [Bio-Rad, Hercules, CA]. Aliquots [40 µg] of whole-cell lysates were separated on 7.5% SDS-PAGE, and electro-transferred onto polyvinylidene membranes [Amersham Pharmacia Biotech, Little Chalfont]. After blocking with 5% nonfat dry milk in TBS-T, the blots were incubated overnight with anti-[GFAP, PEDG-B, and β-actin (internal control)] antibodies. Then blots were washed 3×10 min washes in PBS/tween and subsequently incubated with peroxidase-conjugated anti-mouse IgG secondary antibody at 1∶3000. The signals were obtained by enhanced chemiluminescence [Amersham Biosciences], and visualized by exposure to X-ray film. Upon completion of chemiluminescence, equal lane loading was checked by Ponceau S Solution [Sigma, St. Louis, MO]. X-ray films were scanned with a computer-assisted densitometer [model G-710; Bio-Rad] to quantify band optical density [Quantity One software; Bio-Rad].
GFAP Immunofluorescence
Müller cells [rMC-1] and R28 cells [2×104 cells per well] were grown on 8-well chamber slides and cultured in 300 µl of their growing media, which contained YC-1 [25–100 µM] or DMSO [0.2% v/v] and incubated under normoxia or hypoxia for 48 hours at 37°C. YC-1 or DMSO was added 5 minutes prior to the hypoxic incubation. Immunofluorescence of rMC-1 and R28 cells was conducted by blocking slides in 0.1% blocking solution (Invitrogen/Molecular Probes TSA Kit #2: T-20912) followed by incubation for two hours with the primary rabbit-anti rat GFAP polyclonal antibody [Sigma, catalog number G9269], diluted in 0.1% blocking solution in a humid chamber for 2 hours at 4°C. Negative controls included omission of the primary antibody or its substitution with phosphate-buffered saline (PBS). This was followed by incubating the slides with the horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (H+L) secondary antibody (#A-11008) in conjunction with a dye-labeled tyramide [AlexaFluor 488] (Invitrogen/Molecular Probes Carlsbad, California] [TSA Kit #2] for 1 hour in a dark humid chamber at room temperature. These processes results in localized deposition of the activated tyramide derivative (Stage 1). Hence, further dye deposition, and therefore higher levels of signal amplification, can be generated by detecting dye deposited in “Stage 1” with a horseradish peroxidase (HRP)-labeled anti-dye antibody in conjunction with a dye-labeled tyramide “Stage 2”. Immunofluorescence was visualized by using a Zeiss microscope [Zeiss LSM 510 META inverted confocal microscope equipped with argon and helium/neon lasers using excitation wavelengths 488, on Axiovert 200 M, Thornwood, NY]. Digitized images were acquired utilizing excitation wave length 488 nm. Intensity values of immunofluorescence staining of GFAP in rMC-1 and R28 cells was analyzed and quantified using Metamorph™ imaging analysis software version 6.0 [Universal Imaging, Sunnyvale, CA]. The staining intensity in our series ranged from a weak blush to moderate or strong. The amount of cells staining with the antibody was further categorized as focal [<10%], patchy [10%–50%], and diffuse/multifocal [>50%]. For semiquantitative analysis, focal and/or weak staining was considered equivocal staining, and patchy or diffuse/multifocal staining was subcategorized as either moderate or strong.
Animals and Experimental Design
C57BL/6J mice, from Jackson Laboratory [Bar harbor, ME] were used in these experiments. All animal protocols were approved by the Institutional Review Board and conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research statement of the Association for Research in Vision and Ophthalmology. Mice were divided into four separate groups; [1] Non-treated mice grown under ambient conditions [negative control]; [2] non-treated hyperoxia-exposed mice [positive control]; [3] DMSO- treated hyperoxia-exposed mice [sham-treated]; and [4] YC-1-treated hyperoxia-exposed mice [drug-treated].
Mouse Model of Oxygen Induced Retinopathy
Retinal NV was induced in newborn mice as described previously [41]. Briefly, P7 mice were exposed with their nursing mother, for 5 days [between P7 and P12] to hyperoxic conditions, by incubating them in an airtight chamber [PROOX 110 chamber O2 controller; Biospherix Ltd., Redfield, NY] ventilated by a mixture of O2 and air to a final oxygen fraction of 75±2%. These incubation conditions induced vaso-obliteration and subsequent cessation of vascular development in the capillary beds of the central retina [41]. At P12, the mice were allowed to recover in normal room air conditions and maintained for another 5 days [till P17], the day in which peak disease occurs. A condition of relative hypoxia resulted between P12 and P17, and extensive retinal NV developed in 100% of the mice. Age-matched animals with the hyperoxia-exposed groups were maintained identically, except they were exposed to room air [21% O2, 79% N2] for the duration of the experiment. Animals were examined and sacrificed on the same days.
Intravitreal Drug Injections
A group of hyperoxia-exposed animals [n = 15] were injected intravitreally [into both eyes] at P12 and P15 with 3 µl of YC-1 [100 µM] [drug-treated group]. Another group of hyperoxia-exposed mice [n = 15] were injected intravitreally [into both eyes] at P12 and P15 with 3 µl of DMSO [0.2% (v/v)]. Non-treated mice grown under ambient conditions, non-treated hyperoxia-exposed mice, DMSO- treated hyperoxia-exposed mice and YC-1-treated hyperoxia-exposed mice, were all examined at different critical time points for qualitative assessment of the retinal vasculature by fluorescein angiography.
Retinal Fluorescein Angiography and Visualization of Retinal Vascularization
Deep anesthesia was induced by intraperitoneal injection of ketamine [1%], xylazine [0.1%], and sodium chloride [0.9%] in a concentration of 0.1 mL/10 g mouse body weight. Sternotomy was performed, the mice were perfused through the left ventricle with 600 µL of high-molecular-mass [2×106 Da] FITC-dextran in PBS [50 mg/ml], which was allowed to circulate for 2 minutes before the animals were euthanatized and the eyes enucleated and the flat mounts were prepared. Subsequent to retinal extraction, all retinas were fixed in 4% paraformaldehyde for 24 hrs at 4°C. A dissecting microscope was used to dissect the cornea with a circumferential limbal incision, followed by removal of the lens and vitreous. Microscissors were used to make four radial incisions of the retinal eyecup in order to prepare retinal flat mounts on glass slides. Flat mounts were immersed in Aquamount mounting medium [Polysciences, Warrington, PA], coverslips were placed over the retina, and the edges of the coverslips were sealed.
Immunohistochemistry
Mouse retinas were dissected and prepared for immunohistochemical analysis, fixed in 4% paraformaldehyde in 0.1 M PBS for 15 min at room temperature and embedded in paraffin, sectioned [5 µm]. Tissue sections were deparaffinized, hydrated, and later exposed to heat-induced antigen retrieval using a microwave oven [three 5-minute cycles in citrate buffer, pH 6.0], endogenous peroxidase was abolished with methanol, and hydrogen peroxide and nonspecific background staining was blocked by incubating the tissue sections for 5 minutes in normal swine serum. Subsequently, all slides were washed three times in PBS, and incubated for 1 hour with primary anti–[PDGF-B, GFAP, and β-actin] antibodies. The sections were washed with TBST and incubated with EnVision Polymer HRP secondary antibody [DAKO, Carpinteria, CA] for 30 minutes. All slides were stained with DAB solution and counterstained with hematoxylin. Slides were cover slipped [Permount; Fisher Scientific, Fairlawn, NJ] and examined by light microscopy. Negative controls included omission of the primary antibody or its substitution with phosphate-buffered saline (PBS). Sections were photographed under a microscope [Zeiss Axiovert 135, Thornwood, NY], and images were acquired a digital camera [AxioCam, NY]. All retinas were examined at ×60 objective. The staining intensity in our series ranged from a weak blush to moderate or strong. The amount of cells staining with the antibody was further categorized as focal [<10%], patchy [10%–50%], and diffuse/multifocal [>50%]. For meaningful semiquantitative analysis, focal and/or weak staining was considered equivocal staining, and patchy or diffuse/multifocal staining was either subcategorized as either moderate or strong staining. All Immunohistochemical analyses were measured by Metamorph digital image software [Molecular Devices, Sunnyvale, CA].
Quantitative Image Analysis of Immunohistochemical Staining
GFAP and PDGF-B positive immunostaining in retinal tissue sections (immunohistochemistry), as well as GFAP positive immunolabeling in rMC-1 and R28 cells (immunocytochemistry), were all visualized and captured using AxioCam digital microscope camera. The filter was set with excitation wavelengths 488. MetaMorph image analysis software (version 7.1, Universal Imaging, Downingtown, PA) was used for image processing and quantitative analysis of GFAP and PDGF-B positive immunostaining. MetaMorph tools were used to set the threshold and regions of interest (ROIs). All images were captured at identical time and exposure settings, and they were all processed to the same scale. Images were first segmented on the basis of pixel intensity, which was done on a plane-by-plane basis for an image stack. Briefly, each retinal section was scanned into Metamorph, and five (5) fields/slide were chosen from each section for analysis. One hundred and fifty (150) cells from each field were selected. The saved file was used to calibrate each image for specific pixel size. With the help of a free drawing tool, GFAP and PDGF-B-stained areas were chosen and measured in total-pixels area. A threshold encompassing an intensity range of 100–250 gray-scale values was applied to the ROIs in the least brightly stained condition first. The data were also read and investigated by Matlab v6.5 script file software, which counted the total number of pixels that were above threshold value. This number was divided by the total number of pixels in each image to yield percent fluorescent pixels. To correct for background fluorescence, the threshold was adjusted for each experimental series, with concomitantly processed negative controls used as the guide for setting background fluorescence. The background fluorescence intensities per pixel were subtracted from the experimental data by using a one-step erosion procedure, and then all remaining objects were counted. The same threshold was subsequently applied to all images. GFAP and PDGF-B was considered to be positive only when it exceeded the established threshold. Percent GFAP and PDGF-B expression above threshold in the total area selected was then calculated. The total GFAP and PDGF-B fluorescence intensity per cell was calculated, and the average fluorescence intensity per pixel was determined by dividing the total intensity by the area of the cell measured in pixels. This was followed by measuring the average fluorescence intensity in each field. Data from multiple fields as indicated over several experiments were used to obtain the final results. The number of immunopositive-stained cells per image was then expressed per um2, and the average number per section was determined among five separate fields.
Statistical Analysis
Analysis was performed utilizing ANOVA for multiple variables and with t–Tests for comparison of 2 groups with normal distribution. For the analysis of Real Time RT-PCR data; immunohistochemistry data; Western Blot data, analysis was performed with ANOVA for multiple variables and with t–Tests. Data are expressed as mean ± SEM from at least 3 independent experiments. Significance was defined as *P<0.05; **P<0.01; ***P<0.001.
Results
Analyses of the Retinal Vasculature and the Progression of Retinal NV
At P2 only budding superficial vessels were observed occupying a single plane around the optic disc [Fig. 1; P2]. In P4 wild type mice, the retinal vasculature was organized uniformly and was evenly spaced over the superficial retina and vessels grew into the deeper layers of the retina [Fig. 1; P4]. The vascular density and the number of branch-surrounded spaces increased at P4. In addition, the margin of the developing BVs on the surface of the retina is located halfway between the optic nerve and the peripheral edge of the retina. This is followed by development of a capillary network at P7. During P7, superficial vessels cover about 80% of the retina [Fig. 1; P7]. At this stage sprouts from superficial vessels begin to grow into the retina to form the intermediate and deep capillary beds. In addition, the hyaloidal vessels start to regress. Over the course of the next week, the primary superficial network extended toward the periphery, reaching the far periphery at P12. Angiogram of P12 FITC-dextran-perfused retinal flat mounts of the OIR model preparations displays the effects of 5 days of hyperoxic-exposure [Fig. 1; P12]. On return to normoxia at P12, a relative state of ischemia in the poorly vascularized retina is associated with the excessive re-growth of superficial vessels, leading to abnormal sprouting at the interface between retina and vitreous. Retinas at P12 exhibit typical signs of central non-perfusion of the retina and a drastic regression in the vascular network, leaving only the major vessels and practically no capillary network. The peripheral retina still showed evidence of a vascular network, but, in general, the deep vascular plexuses had completely failed to form. By P15 the retinal ischemia initiates an aggressive neovascular response at the interface of the perfused retinal periphery and the ischemic central capillary beds [Fig. 1; P15]. On P17, the vascular network of the O2-injured retinas was significantly altered as demonstrated by an increase in retinal NV. These retinas displayed the features indicative for a strong ongoing vasoproliferative response: central capillary-free regions, vessel tortuosity and BV tufts [Fig. 1; P17].
10.1371/journal.pone.0022244.g001Figure 1 Analyses of Retinal Vascular Development in the Normal Retinal Vasculature and the Neovascular Retina.
Mice were perfused with fluorescein-labeled dextran. Normal mice were analyzed P2 [A], P4 [B], and P7 [C]. Exposure of mice to 75% O2 between P7–P12 led to rapidly progressive vaso-obliteration of the retinal vasculature over. Retinal NV was analyzed in the OIR mice on P12 [D], P15 (OIR) [E], and P17 (OIR) [F]. [n = 15 per group]. bar: 300 µm.
YC-1 Attenuates Neovascular Sprouting in the OIR Mouse Model
Data from [Figs. 2A–F] demonstrated that retinal NV in the O2-injured and the DMSO-treated retinas was associated with the presence of neovascular sprouts and the formation of cordlike or tube-like structures. However, YC-1 treatment has disturbed and suppressed the development and elongation of neovascular sprouts [Figs. 2C–F]. YC-1 significantly and dose-dependently reduced the total length [Fig. 3A] and the number [Fig. 3B] of neovascular sprouts, as compared to DMSO-treated retinas.
10.1371/journal.pone.0022244.g002Figure 2 The Influence of YC-1 on Neovascular Sprouts Number and Length.
Images exhibit a higher magnification of growing tips of BVs that reveal multiple neovascular sprouts growing at multiple angles including both along and above the plane of the section. Image along the leading edge of vascularization from; [A] Non-treated ischemic retinas; [B] DMSO-treated retinas; [C, D, E, and F] represent retinas from animals that were injected with YC-1 [25–100 µl]. neovascular sprouts lengths and numbers were not influenced by DMSO treatment. Whereas treatment with YC-1 significantly and dose-dependently inhibited the number and the length of neovascular sprouts. [n = 5 per group].
10.1371/journal.pone.0022244.g003Figure 3 YC-1 Reverses Retinal Reactive Gliosis and Retinal Neovascularization, in vivo and in vitro.
Neovascular sprouts from DMSO-treated retinas had approximately similar lengths as the non-treated ischemic retinas [A]. YC-1 treated animals exhibited a significant and a dose-dependent decrease in neovascular sprouts lengths as compared to DMSO-treated retinas. [ANOVA, ***P<0.001; **P<0.01 between YC-1 and DMSO] [A]. DMSO has no influence on the number of neovascular sprouts [B]. Whereas YC-1 exhibited a significant and a dose-dependent decrease in neovascular sprouts number [B]. [ANOVA, ***P<0.001; **P<0.01 between YC-1 and DMSO]. There were 24 neovascular sprouts/retina that were averaged. [n = 5 per group]. By using primer sets [C]; Real Time RT-PCR analyses, in vivo [D and E] indicate that the levels of GFAP and PDGF-B mRNA were increased in the non-treated ischemic retinas, while non-treated normoxic retinas exhibited extremely low levels. Treatment of ischemic retinas with dual injections of YC-1 resulted in a significant knockdown of PDGF-B [**P<0.01] and GFAP [**P<0.01] gene expression when compared with DMSO-treated controls. ANOVA; Mean ± SEM of mRNA level normalized to β-actin were calculated, [***P<0.001 and **P<0.01, as compared to DMSO-treated retinas]. Data are representative of 3 independent experiments. Real Time RT-PCR analysis, in vitro [F] indicate that post-culturing R28 and rMC-1 cells under normoxic and hypoxic conditions; the mRNA levels of GFAP and PDGF-B were upregulated in all non-treated hypoxic cells, while normoxic cells exhibited remarkable low mRNA levels. Treatment of hypoxic R28 and rMC-1 cells with various concentrations of YC-1 resulted in a significant inhibition of GFAP and PDGF-B, mRNA expression as compared to DMSO-treated controls. ANOVA was used for statistical analyses. Mean ± SEM of mRNA level normalized to β-actin were calculated, [***P<0.001 and **P<0.01 as compared to DMSO-treated hypoxic control. Data are representative of 3 independent experiments].
YC-1 Reverses Retinal Reactive Gliosis and Retinal Neovascularization, in vivo and in vitro
In order to investigate the mechanisms via which YC-1 reverses retinal RG and NV in the OIR mouse model, we measured the transcription of key genes that are associated with both; RG and NV, as markers of a putative recovery. By using the primers, which were summarized in [Fig. 3C]; Real Time RT-PCR data have indicated that there was a significant enhancement of GFAP gene expression levels in the non-treated ischemic retinas as compared with the retinas from animals that were placed under ambient conditions. The effects of DMSO-treatment on the gene expression profiles paralleled those seen in the non-treated ischemic retinas. YC-1 treatment significantly attenuated [**P<0.01] the message levels of GFAP expression [Fig. 3D]. YC-1 treatment [100 µM] reduced ischemia-induced overexpression of GFAP at the message [by 64.14±0.5%], as compared to DMSO-treated controls. Furthermore, there was a significant alteration in the mRNA levels of PDGF-B, between YC-1-treated groups and non-treated and DMSO-treated controls. There was a significant upregulation in the mRNA levels of PDGF-B in the ischemic retina as compared to normoxic retinas [Fig. 3E]. Treatment of the animals with YC-1 [100 µM] significantly [**P<0.01] attenuated the ischemia-induced overexpression of PDGF-B at the message level of [by 70.27±0.04%], as compared to DMSO-treated retinas.
Our in vitro studies have revealed that post hypoxic exposure, the level of GFAP mRNA expression was significantly increased over the normoxic control, which displayed a basal expression levels [Fig. 3F]. The effects of DMSO-treatment on the gene expression patterns in both cell types paralleled those seen in the non-treated ischemic retinas. Treatment of r-MC1 and R28 cells with 25–100 µM YC-1 resulted in significant dose-dependent attenuations in the message levels of GFAP expression, when compared with DMSO-treated hypoxic cells. At 100 µM, YC-1 treatment downregulated the hypoxia-induced overexpression of GFAP at the message level in rMC-1 cells [by 71.42±0.02%], and R28 cells [by 58.62±0.02%], respectively. Data were normalized to β-actin mRNA levels. In addition, Real Time RT-PCR data have demonstrated that after 48 hours of hypoxic exposure, the levels of PDGF-B mRNA expression were significantly increased over the normoxic control, which displayed low gene expression levels [Fig. 3F]. Treatment of both cell lines YC-1 [25–100 µM] resulted in significant dose-dependent attenuations in the message levels of PDGF-B expressions, when compared with DMSO-treated hypoxic cells. Treatment of r-MC1 and R28 cells with YC-1 at 100 µM downregulated the hypoxia-induced overexpression of PDGF-B at the message level in rMC-1 cells [by 75±0.03%], and R28 cells [by 50.00±0.02%], respectively.
Immunohistochemical Expression and Localization of PDGF-B, and GFAP, in vivo
In the Non-Treated Normoxic Retinas [Fig. 4], PDGF-B expression was focal and sparse, and it was predominantly localized in the ILM, NFL, GCL, and INL. Furthermore, the Non-Treated Ischemic Retinas exhibited a pattern of PDGF-B overexpression and immunohistolocalization, which was particularly prominent in the ganglion cells [GCL], as well as a significant upregulation in the positive immunoreactivities of PDGF-B, which was present in the ILM, and within the NFL, and the inner segments of the photoreceptor cells [INL]. The immunoreactivity was diffuse with strong PDGF-B overexpression that was primarily augmented in the NFL, GCL, and INL, as compared to Nontreated Normoxic Retinas and the YC-1-Treated Retinas. In the DMSO-Treated Retinas, the immunohistolocalization pattern of PDGF-B was identical to the pattern that was shown in the ischemic controls. In the YC-1-treated retinas; PDGF-B immunoreactivity was significantly inhibited [by 57.59±0.01%], as compared with DMSO-treated retinas, and the immunostaining was primarily focally localized in the ILM, GCL, and INL [Fig. 4].
10.1371/journal.pone.0022244.g004Figure 4 Immunohistochemical Analysis of PDGF-B, in vivo.
Photomicrographs of retinas from various OIR groups that were immunostained for PDGF-B. The expression of PDGF-B was upregulated in the non-treated ischemic and DMSO-treated groups, compared with non-treated normoxic group. While all protein immunoreactivities were downregulated in the YC-1-treated group, compared with DMSO-treated groups. Data are representative of 3 independent experiments. Scale bar: 140 µm.
Our immunohistochemistry data have demonstrated that the Nontreated Normoxic Retinas exhibited staining signals for GFAP immunoreactivity, which tended to be sparse and focal and primarily localized in the ILM and GCL [Fig. 5]. However, the Nontreated O2-Injured and the DMSO-Treated O2-Injured Retinas exhibited strong staining of GFAP expression, primarily in the ILM, nerve fiber layer [NFL], GCL, and inner nuclear layer [INL] of the ischemic retinas. In addition, there was a strong staining for GFAP immunoreacreactivity in Müller cell processes throughout the retina. In contrast, YC-1-Treated retinas displayed a significant downregulation in GFAP immunoexpression as compared to DMSO-Treated Retinas [Fig. 5]. in addition, GFAP expression was weak “focal”, sparse, sporadic and primarily localized in the ILM, NFL, and the GCL regions of these retinas. Our data has indicated that YC-1 treatment [100 µM] has reduced ischemia-induced overexpression of GFAP protein levels [by 65.52±0.02%], as compared to DMSO-Treated Controls [Fig. 5].
10.1371/journal.pone.0022244.g005Figure 5 Immunohistochemical Analysis of GFAP, in vivo.
Photomicrographs of retinas from OIR groups that were immunostained for GFAP. GFAP was overexpressed in the non-treated ischemic and DMSO-treated groups, compared with non-treated normoxic group. Whereas all GFAP immunoreactivities were downregulated in the YC-1-treated group, compared with DMSO-treated groups. Data are representative of 3 independent experiments. Scale bar: 140 µm.
Our data analysis of PDGF-B and GFAP expression in the retinal tissues indicated that YC-1 significantly reduced the number of PDGF-B (+) cells and GFAP (+) cells as compared to DMSO-treated retinas [Fig. 6A], which indicated that YC-1 exhibited anti-reactive gliosis and anti-angiogenic properties, in vivo.
10.1371/journal.pone.0022244.g006Figure 6 YC-1 Exhibits Anti-Reactive Gliosis and Anti-Angiogenic Properties, in vivo and in vitro.
Assessments of retinal immunohistochemical analysis exhibit the values that were obtained from at least 5 retinal fields were used to calculate the average pixel intensity value/retina [A]. Bar graphs exhibit the area of staining of GFAP and PDGF-B in all four groups. Values [mean ± SEM], from 3 separate experiments from at least 10 images from 4 different eyes/group. [***P<0.001 and **P<0.01 as compared to DMSO-treated retinas]. [B] Coculture models. [i] Cell proliferation assay model. [ii] Cell migration assay model; were both utilized to investigate the effects of YC-1 on the proliferation [C] and migration [D] of ECs in a coculture system. hRMVECs growth curves from four groups were depicted. Coculture group had a higher proliferation and migration rate of hRMVECs cells than that of hRMVECs solo [**P<0.01]. Hypoxia significantly increased hRMVECs proliferation and migration rate in the coculture system [**P<0.01, rMC-1/hypoxia vs. rMC-1/normoxia]. After rMC-1 cells were treated with YC-1 [100 µM], the proliferation and the migration rate of hRMVECs were significantly inhibited compared with the rMC-1/hypoxia group. [**P<0.01, YC-1-treated rMC-1/hypoxia vs. nontreated rMC-1/hypoxia]. Data are representative of 3 independent experiments.
Inhibition of ECs Proliferation and Migration in the Coculture System via the Anti-angiogenic Effects of YC-1 on Müller Cells
Our proliferation assay in a coculture system model has demonstrated that rMC-1cells-hRMVECs coculture significantly increased hRMVECs proliferation compared to solo hRMVECs culture [Fig. 6B and 6C] under normoxia and hypoxia. Data have indicated that coculture under hypoxic conditions had a synergistic effect. Although there was insignificant difference in the hRMVECs proliferation while being cocultured with nontreated rMC-1 cells under normoxic or hypoxic conditions; the proliferation of hRMVECs was significantly suppressed when rMC-1 cells were treated with YC-1, under normoxic and hypoxic conditions. Furthermore, our migration assay in a coculture system model has shown that hRMVECs were found to extend through 8.0 mm Transwell pores in a transmigration assay with Müller cells grown in the well [Fig. 6C and 6D]. Coculture of hRMVECs with Müller cells under hypoxia resulted in a significant increase in hRMVECs migratory activity over the levels of [rMC-1 cells/normoxia group]. Whereas the rMC-1 cells-induced hRMVECs migration was significantly attenuated by YC-1 treatment under normoxic [rMC-1 cells/hRMVECs (normoxia group)] and hypoxic conditions [rMC-1 cells/hRMVECs (hypoxia group)] [Fig. 6C and 6D].
YC-1 Restrains Hypoxia-Induced Upregulation of GFAP and PDGF-B Protein Levels, in Glial Cells
In order to investigate the mechanisms via which YC-1 reverses retinal RG and NV in our cell culture models, we quantified the expression of GFAP and PDGF-B, respectively, as key protein molecules that are associated with RG and NV. Our Western immunoblot data have indicated that rMC-1 and R28 cells cultured under normoxia exhibited suppressed protein levels of GFAP protein, whereas GFAP was overexpressed 72 hours post-hypoxic exposure [Fig. 7A]. In the DMSO-treated hypoxic cells, GFAP protein expression remained relatively stable, when compared with non-treated hypoxic cells. This increase in GFAP expression was measured by densitometry [Fig. 7B]. In both cell types and under hypoxic conditions; YC-1 treatment dose-dependently inhibited the hypoxia-induced GFAP protein levels in by [78.57±0.02%] in rMC-1cells, and by [81.44±0.02%] in R28 cells, respectively. Furthermore, Western immunoblot analysis have indicated that rMC-1 and R28 cells cultured under normoxia exhibited low immunoreactivity signals of PDGF-B protein expression, while this signal was overexpressed after 72 hours of hypoxic exposure [Fig. 7A]. There was a significant increase in PDGF-B protein expression, as measured by densitometry [Fig. 7B], compared to normoxia. In both cell types and under hypoxia, YC-1 inhibited the hypoxia-induced upregulation of PDGF-B protein levels in a dose-dependent manner, compared with DMSO-treated hypoxic cells. At 100 µM, YC-1 treatment inhibited the hypoxia-induced protein overexpression of PDGF-B [by 77.55±0.01%] in rMC-1cells, and [by 79.16±0.01%] in R28 cells, respectively.
10.1371/journal.pone.0022244.g007Figure 7 YC-1 Restrains Hypoxia-Induced Upregulation of GFAP and PDGF-B Protein Levels, in Glial Cells.
Western Blot analysis indicated that protein expression levels were elevated markedly in the non-treated hypoxic cells [A]. In YC-1-treated hypoxic cells, GFAP and PDGF-B protein expression were significantly decreased in a dose-dependent fashion, compared with DMSO-treated hypoxic cells. Statistical significance was determined by ANOVA [**P<0.01]. Data are representative of 3 independent experiments. The densitometric analysis of Western Blot data [B] represents the intensities of GFAP and PDGF-B protein expressions in rMC-1 and R28 cells relative to those of β-actin expression, whereas the relative ratio of hypoxia control was defined as 100. Values, shown as the mean ± SEM, from 3 separate experiments with a total sample size of 6. [**P<0.01 as compared to DMSO-treated hypoxic control].
Our immunofluorescence staining of GFAP demonstrated that non-treated rMC-1 and R28 cells cultured under hypoxia displayed enriched GFAP protein fluorescence immunoreactivity, with strong cytoplasmic staining of both cell types [Fig. 8; positive control]. Hypoxic cells exhibited significant (**P<0.01) increase in GFAP protein levels, as compared with normoxic cells, which exhibited limited areas of very weak “blush” GFAP staining [Fig. 8; negative control]. Furthermore, there was a strong positive GFAP staining signal deposited over the cytoplasms of the DMSO-treated cells cultured for 48 hours under hypoxia [Fig. 8; DMSO]. This intense positive staining was diminished with YC-1 treatment. No GFAP staining was observed in experiments in which the primary antibody was omitted and/or substituted with PBS (data not shown). rMC-1 cells treated with 25 µM YC-1 displayed the presence of cytoplasmic localization but then with weaker equivocal “weak” staining intensity. Whereas, a stronger diffuse cytoplasmic GFAP staining was observed in R28 cells. Treatment of rMC-1 and R28 cells with 25 µM YC-1 under hypoxia for 48 hours displayed a significant inhibitory effects (42.10% and 48.78% inhibition, respectively), compared to DMSO-treated hypoxic controls. At 50 µM YC-1, GFAP cytoplasmic staining levels was drastically reduced in both cell types, as compared with non-treated controls cultured under hypoxia. At [50 and 75 µM], YC-1 had significant inhibitory effects on GFAP protein expression in both cell types, as compared to hypoxic controls. There was a strong reduction of GFAP cytoplasmic staining levels in both cell types, as compared with non-treated controls cultured under hypoxia. Treatment of rMC-1 and R28 cell lines with 50 µM YC-1 for 48 hours under hypoxia, caused a significant inhibition of 44.73% (**P<0.01) and 50% (*P<0.05) respectively, as compared with non-treated hypoxic controls. Whereas treatment of cells with 75 µM YC-1 for 48 hours under hypoxic conditions had significant (**P<0.01) down-regulatory effects on GFAP protein expression in both cell types, whereas GFAP inhibition in rMC-1 and R28 cells were 52.63% and 56.09%, respectively, as compared with non-treated hypoxic controls. At 100 µM YC-1, there were few stained regions that were still detected in the cytoplasm of YC-1-treated cells. At 100 µM, YC-1 displayed significant (**P<0.01) 78.94% and 70.73% folds of inhibition in rMC-1 and R28 cells, respectively, as compared with non-treated hypoxic controls. Our data indicated that YC-1 significantly reduced GFAP protein levels in R28 and rMC-1 cells under hypoxia and in a dose-dependent fashion [**P<0.01] [Fig. 8 and bottom graphs in Fig. 8].
10.1371/journal.pone.0022244.g008Figure 8 Immunofluorescence Analysis of GFAP Expression, in vitro.
Photomicrographs show the rMC-1 and R28 cells, which were immunostained with anti-GFAP antibody. Intense staining was considered a positive signal. Yellow arrows indicate GFAP immunostaining. Under hypoxia, non-treated hypoxic cells exhibited extremely high GFAP immunoreactivity. Treatment of cells with YC-1 [25–100 µM] under hypoxia for 48 hours resulted in a dose-dependent inhibition of GFAP expression. Images are representatives of 3 independent experiments. Scale bar: 40 µm. Graphs in the bottom represent the assessments of the cellular immunocytochemical analysis of GFAP. Graphs showing the intensity of GFAP staining in rMC-1 and R28 cells after treatment with YC-1 relative to that measured in DMSO-treated hypoxic control. The areas of staining to GFAP/µm2 in all four groups were measured. Values, shown as the mean ± SEM, from 3 separate experiments.
Discussion
Reactive gliosis [RG] is one of the pathophysiological features of retinal damage. RG includes morphological, biochemical, and physiological changes of Müller cells; these alterations vary with type and severity of damage. The molecular mechanism(s) leading to RG is not fully elucidated. In this study we have investigated the effects of YC-1, a small molecule inhibitor of hypoxia induced factor -1 (HIF-1), on experimentally induced retinopathy using the OIR mouse model. YC-1 is a novel type nitric oxide (NO)-independent activators of soluble guanylate cyclase (sGC) [31], [32], [33], [34]. YC-1 is not an NO donor, however, it causes activation of sGC especially in the presence of NO [35], [36], while binding to sGC at a different site from the heme [37]. Data has previously demonstrated that sustained hyperoxia effectively preserved astrocytes in the central retina in spite of extensive damage to the capillary networks and prevented the reactive expression of GFAP in the Müller glia [42]. In addition, the OIR mouse model has been extensively used because it constitutes several clinical manifestations that are analogous to the retinopathy of maturity [ROP], and mimics the important aspects of PDR; the most common ischemic retinopathy in patients [43]. Since (i) YC-1 induces physiological RV in the OIR model [39]; (ii) a significant segment of this process seems to occur in the INL, OPL, ONL, and OLM retinal cell layers; and (iii) the fact that Müller cells processes span the entire retina and extend from the INL to the OLM, the possibility existed that YC-1 maybe acting directly on retinal glial cells. We found that GFAP [the hallmark of RG] was expressed at low levels under normoxic conditions, in vitro and in vivo, which is in concordance with what's been previously reported [44]. However, upon exposure to hypoxic/ischemic conditions, GFAP levels were increased both at the message and the protein levels. These results are in agreement to what has been previously reported by other studies, which indicated that hypoxic subculture of Müller cells causes a dramatic upregulation of GFAP in vitro
[45]. Retinal ischemia/hypoxia have been shown to cause an increase in gene and protein expression of GFAP in Müller cells within 1–3 h and that expression extended the inner processes of Müller cells [up to the INL] [46], [48]. It has been noted that astrocytes are the only cells in the retina that express GFAP, but after various insults such as retinal detachment, ischemia, light damage, or genetic photoreceptor degeneration, Müller glia become GFAP-positive [49], [50], whereas Normoxic Retinas expressed low levels of GFAP expression that was confined to the ILM and GCL regions of the retina, with no Müller cell processes staining. In the Nontreated Ischemic Retinas and DMSO-Treated Ischemic Retinas; GFAP was significantly upregulated in the ILM, GCL, IPL, and INL with clear staining at Müller cell processes, and was overly expressed due to the damage to the retina by ischemia after vascular pruning, which indicates glial reactivity.
YC-1 displays antiangiogenic activities in vitro and in vivo
[38], [39], [40]. YC-1 possesses novel pleiotropic effects pertaining the making, toning, maintaining the structural and functional integrities of BVs. Our results show that YC-1 repressed GFAP expression at the message and the protein levels, in the ischemic retinas and in glial cells, suggesting a reversal of RG. To our knowledge no other report to date has demonstrated the ability of small anti-angiogenic molecules to target and reverse RG.
Previous studies have demonstrated that levels of PDGF-B were increased after ischemic injury [51]. Since YC-1 inhibits platelet aggregation, it is plausible that some of its anti-angiogenic effects in retinal vasculopathies are mediated through PDGF-B. Our data demonstrate that under hypoxic conditions PDGF-B is expressed at significantly higher levels compared to normoxic retinas. YC-1-treated retinas exhibited a significant inhibition of PDGF-B mRNA and protein levels as compared to DMSO-treated retinas. It is noteworthy that other groups have attributed the retinal production of PDGF-B to the ganglion cells [52]. Our data indicated Nontreated Ischemic Retinas and DMSO-Treated Ischemic Retinas have both exhibited an overexpression of PDGF-B, which was predominantly localized within the cell bodies of the retinal neurons; and it was mostly localized to the Müller cell end-feet at the ILM with diffuse/multifocal overexpression at the NFL, GCL, and INL. Our data corroborated previous reports [47], which indicated that PDGF-B immunoreactivity was observed in Müller cell end-feet of the neural retina. The source of this PDGF-B was next investigated in a series of in vitro experiments in which glial cells were subjected to hypoxic conditions for 72 hours. Such treatment caused a profound increase in PDGF-B at the message and the protein levels. Taken together, these findings suggest the contribution of Müller cells to retinal PDGF-B and implicate an autocrine mechanism of PDGF-B on Müller cells. It is noteworthy that our results are in concordance with previous studies [8], which demonstrated that glial cells may utilize the elevated levels of PDGF-B to proliferate, and consequently causing RG. In this manuscript, we demonstrate that hypoxia has triggered RG within 72 hours of exposure in vitro [rMC-1 and R28 cells], whereas the ischemic retina has fully developed RG at P17 in vivo [OIR mouse model]. These findings are in concordance with previous studies [20].
Hypoxia inducible factor-1 [HIF-1] is a master regulator that controls the transcriptional activation of VEGF signaling and other hypoxia-inducible genes [53]. Like VEGF; PDGF-B is a hypoxia-regulated gene [54]. Our previous studies have revealed that YC-1 inhibits HIF-1 and other proangiogenic factors in the ischemic retinas of the OIR mouse model [38], [39], [40]. It is therefore possible that YC-1's effect on RG may be mediated through the inhibition of HIF-1-signaling, since PDGF-B protein molecule is downstream of HIF-1. Throughout our current and previous investigations, we have demonstrated that targeting hypoxia and HIF-1 signaling maybe considered as a therapeutic modality to target several downstream angiogenic molecules, such as PDGF-B, which play crucial roles in ischemic retinopathies. Moreover, sprouting angiogenesis involves collective migration processes [55]. Tip cells are distinguished by their strong expression of PDGF-B mRNA and VEGFR2 mRNA and protein, implying that tip cells may have a distinct gene expression profile [56] and regulate the coordinated processes of neovascular sprouting. These findings would be consistent with our current data, which revealed that the decrease in the length and number of neovascular sprouts in YC-1-treated retinas, were both highly associated with the downregulatory effects of YC-1 on PDGF-B expression. Taken together, this study has demonstrated that YC-1 could be exploited as valuable therapeutic modality in the treatment of RG in the ischemic retina. Antagonists of PDGFs may help to reduce scarring, but may also synergize with VEGF antagonists to reduce NV possibly through pericytes, which provide survival signals for ECs of new vessels [57]. Kinase inhibitors that block both VEGF and PDGF receptors are some of the most efficacious drugs for the treatment of ocular NV in animal models [58], [59]. Our current study has revealed that YC-1 inhibited GFAP and PDGF-B, while our previous studies have indicated that YC-1 inhibited VEGF [38], [39], [40]; such data corroborate other studies, which demonstrated that combining antagonism of PDGFs with blockade of VEGFs may be a useful strategy for treatment of ocular NV [59]. Successful inhibition of GFAP using antisense oligonucleotides has also been reported by other investigators [28], [30].
Our previous investigations have demonstrated the presence of spatial overexpression of VEGF, ET-1, iNOS, and MMP-9 in the neovascular retina of the OIR mouse model [39]. Whereas in our current study, we demonstrate a concomitant expression of GFAP and PDGF-B, which occur during ischemic retinal NV. It was reported that neoangiogenesis was associated with reactive astrogliosis and was correlated to increased reactive astrocytosis and associated VEGF expression [60]. In addition, multiple lines of evidence have demonstrated a pivotal role of the endothelin system in stimulating RG. The production of ET-1 is stimulated at injured brain sites [61], and cerebral ischemia specifically triggers astrocytes to release it [62]. Additionally, ET-1 is highly expressed in reactive astrocytes from patients who have suffered from various neurological disorders, including cerebral infarcts, and Alzheimer's disease [63]. Moreover, it was suggested that ET-R-associated pathways might represent important targets to control RG [64]. In injured nerve tissues, astrocytes become the reactive phenotype, which causes the overexpression of MMP-9 [65]. In addition, the same study has suggested that ETs are one of factors, which stimulate astrocytic MMP-9 productions. Moreover, previous studies have suggested that iNOS expression may serve as an index of RG [66], [3]. Taken together, our findings and those of others suggest a crosstalk between RG and NV. Our study demonstrates that the neuroprotective mode of YC-1's action, which blunts the ischemic insult to the retina may have similar role in protecting ischemia-induced neural damage.
We are tremendously grateful to Adara DeNiro for her valuable technical assistance in the quantification of the immunohistochemical and immunocytochemical staining. We owe special thanks to Jörgen Larsson, MD [King Khaled Eye Specialist Hospital] for his positive and careful review of the manuscript. We would like to thank Gail Seigel, PhD [Ross Eye Institute] for providing us with R28 cells and VJ Sarthy, PhD [Northwestern University] for providing us with rMC-1 cells.
Competing Interests: The authors have declared that no competing interests exist.
Funding: The study was funded by King Khaled Eye Specialist Hospital and King Faisal Specialist Hospital & Research Centre. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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ISRN Obstet GynecolISRN Obstet GynecolOBGYNISRN Obstetrics and Gynecology2090-44362090-4444International Scholarly Research Network 2180494510.5402/2011/983542Clinical StudyA Survey of Home Delivery and Newborn Care Practices among Women in a Suburban Area of Western Nigeria Adelaja Lamina Mustafa
1, 2
*1Department of Obstetrics and Gynaecology, Olabisi Onabanjo University Teaching Hospital, P.M.B. 2001, Sagamu 12001NG, Ogun State, Nigeria2Department of Obstetrics and Gynaecology, Gizan General Hospital, Gizan, Jasan Region, Saudi Arabia*Lamina Mustafa Adelaja: [email protected] Editor: H. C. Wallenburg
2011 7 6 2011 2011 9835421 3 2011 7 4 2011 Copyright © 2011 Lamina Mustafa Adelaja.2011This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Context. Information about reasons for delivering at home and newborn care practices in suburban areas of Western Nigeria is lacking, and such information will be useful for policy makers. Objectives. To describe the home delivery and newborn care practices and to assess the reasons for delivering at home. Study Design, Setting, and Subjects. A cross-sectional survey was carried out in the immunization clinics of Sagamu local government, Western part of Nigeria during January and February 2008. Two trained health workers administered a semistructured questionnaire to the mothers who had delivered at home. Main Outcome Measures. Planned or unplanned home delivery, reasons for delivering at home, the details of events that took place at home from the onset of labour pains till delivery and after birth till initiation of breast-feeding, attendance at delivery, cleanliness and hygiene practices during delivery, thermal control, and infant feeding. Results. A total of 300 mothers were interviewed. Planned home deliveries were 200 (66.7%) and 100 (33.3%) were unplanned. Only 13.4% of deliveries had a skilled birth attendant present, and 47 (15.7%) mothers gave birth alone. Only 51 (16.2%) women had used a clean home delivery surface. Majority (98.2%) of the newborns were given a bath soon after birth. Initiation rates of breast-feeding were 65.3% within one hour and 95.7% within 24 hours. Conclusion. High-risk home delivery and newborn care practices are common in semiurban population also. Community-based interventions are required to improve the number of families coming to health facilities and engaging a skilled attendant and hygiene during delivery.
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1. Background
Of the approximately four million global neonatal deaths that occur annually, 98% occur in developing countries, where most newborns die at home while they are cared for by mothers, relatives, and traditional birth attendants [1]. During the past two decades, infant mortality rate has declined very slowly. This is as a result of a very slowly declining neonatal mortality rate. There has been relatively little change in neonatal mortality despite proven cost-effective solutions to reduce neonatal mortality, such as promoting tetanus toxoid immunisation, skilled attendance during delivery, immediate and exclusive breastfeeding, and clean cord care [2, 3]. In Nigeria, approximately 67% of births occur at home [4]. In 2005, the infant mortality rate in Nigeria was 64 per 1000 live births, and the neonatal mortality rate was 39 per 1000 per live births [5]. In rural areas of Nigeria, the proportion of institutional deliveries is as low as four percent [4, 6]. Even in urban areas like Lagos, a significant proportion of women (19%) still deliver at home [7]. This is in spite of a relatively easy access to institutional maternity services in urban areas. Previous studies about home deliveries in urban and periurban areas of Abeokuta have reported poor maternal education, multiparity, and low socioeconomic status as the predictors of home deliveries [6]. A study carried out in Sagamu reported “cost” and “convenience” as the reasons for delivering at home [8].
The World Health Organization (WHO) guidelines for essential newborn care include the following: hygiene during delivery, keeping the newborn warm, early initiation of breastfeeding, care of the eyes, care during illness, immunization, and care of low-birth-weight newborns [9]. Therefore it is necessary for the mother and her family to understand these aspects of childbirth and newborn care and be prepared to react to the potential danger signs. A study from rural areas of Oyo State, Nigeria reported that a very large proportion (>90%) took place at home. The study also reported that only six percent of home deliveries were attended by skilled government health workers and newborn care practices were unhygienic and of high risk [10]. Such high-risk practices have also been reported from remote Osun State, Nigeria [4]. Newborn care practices may change over time and may be different in urban areas. Studies from low socioeconomic settlements of Karachi, Pakistan [11], Bangladesh [12] and Delhi, India [13] have reported that traditional newborn care practices were of high risk and emphasized the need for community-based interventions to promote proper newborn care practices in urban areas.
Implementation of an effective program for promotion of childbirth and newborn care practices requires understanding of the community and household traditional newborn care practices. Such information will enable the development of programs to promote culturally sensitive and acceptable change in practices. Information about the reasons for delivering at home is also necessary for health care planners to design appropriate maternity services. Information about reasons for delivering at home, home delivery, and newborn care practices in suburban areas of Nigeria is lacking. Therefore, this study was undertaken in a suburban population of Western Nigeria with the following objectives:
to describe the home delivery and newborn care practices,
to assess the reasons for delivering at home.
2. Methods
2.1. Study Setting
Nigeria has a population of about 135 million people, 70% of whom live in rural areas [5]. With an estimated per capital income of $1000 per year, Nigeria is a “poor developing country” in Sub-Saharan region of Africa because of uneven distribution of wealth—few are extremely stinking rich while the majority are languishing in abject poverty. Life expectancy at birth of 47.5 years is low, and it is still lower than neighboring West African countries. Infant mortality rate is among the highest in the region. Due to high maternal mortality rate in women and high mortality from road traffic accident in men, life expectancy for women is slightly higher than that of men (49 versus 47). Gender disparities are also common in terms of literacy. The literacy rate of Nigerian women as a percentage of men is 77% [5]. Curative and preventive health care is organised primarily by the Ministry of Health through hospitals located at federal, state, and local government levels, and primary health centres, health posts, and subhealth posts located at the community level. Private hospitals and clinics exist mostly in urban areas. Missionary and nonprofit hospitals operate in a few areas [14]. Nigeria spends about five percent of its gross domestic product on health of which only one fifth comes from the public sector, and the remainder is paid for by the individual households [5].
Ogun State is one of the 6 states in the western region of Nigeria. The state has a land area of 2017 square kilometers and a population of 380,527. Ogun State has 20 local government areas of which Sagamu, with a population of 156,312, is one. Sagamu local government is administratively divided into 18 wards. In each of these wards immunisation clinics are conducted once a month in the child health clinics of public and private health institutions. The child health clinics are managed by the national, state, and local governments, and private hospitals, each providing manpower, vaccines, medicines, and technical input, respectively. Since primary immunisation in Nigeria is completed at one year of age, the majority of the children attending these clinics were infants. A few children who missed Measles vaccine between 9 and 12 months are older than a year when they attend the immunisation clinic.
2.2. Study Design and Participants
The institutional ethics committee of Olabisi Onabanjo University Teaching Hospital, approved this study. A cross-sectional study was carried out in the immunisation clinics of Sagamu local government. We included the mothers of all the infants who were brought for immunisation during the months of June and July 2008. The framework used for the design and presentation of our study was based on a similar study carried out in rural area of Nepal [14]. A semistructured questionnaire was developed for the purpose of this study and was pretested among 25 mothers during the month of June 2008. After pretesting, the questionnaire was modified according to local traditions and cultural sensitivity. The questionnaire sought information about sociodemographic characteristics of the family, planned or unplanned home delivery, reasons for delivering at home, and the details of events that took place at home from the onset of labour pains till delivery and after birth till initiation of breastfeeding. The details included attendance at delivery, cleanliness and hygiene practices during delivery, thermal control, and infant feeding. The information about the reasons for delivering at home was sought by both open- and closed-ended questions. Those women who reported that they had decided to deliver in a hospital but could not reach the hospital after the onset of labour pains due to various reasons were categorized as unplanned home deliveries. Those women who reported that they had decided to deliver at home were categorized as planned home deliveries.
Two health workers were trained to administer the questionnaire during a one-day training conducted by the investigator at OOUTH. The health workers were stationed at the registration counter and enquired from the mothers about place of delivery. The mothers of the infants who reported that they delivered at home were invited to participate. Verbal consent was sought and the respondents were assured that the interviewers were not a part of the health service team and services would not be denied if they declined to participate in the interview. After obtaining verbal consent, the health worker carried out the interview and recorded the necessary information on a semistructured questionnaire. The chief investigator and a staff nurse who trained the interviewers supervised all the interviews. The data were coded and analyzed using the SPSS 15.0 for Windows Evaluation Version package (Statistical Package for Social Sciences). Frequencies and percentages of different variables were calculated.
3. Results
Three hundred and seven out of 1420 infants were brought to the mmunization clinics during the study period. Three hundred and seven were born at home. Seven infants were brought by a family member other than the mother or a relative, and they could not provide reliable information. Hence they were excluded from the analysis. Two hundred (66.7%) of these 300 home deliveries were planned whereas 100 (33.3%) were unplanned.
3.1. Sociodemographic Profile of the Respondents
The median age of the infants was 4 months (interquartile range: 4 months). One hundred and forty seven (49%) infants were males and 153 (51%) were females. The median age of the mothers was 26 years (interquartile range: 8 years). More than half of the mothers were Christians (171, 57%), followed by Muslims (99, 33%) and traditional religion worshipers (30, 10%). Forty-three (14.3%) respondents were illiterate, and the majority of the mothers (202, 67.3%) had education of high school and above. The mean monthly family income was 18,360 naira (approximately 122.4 USD).
3.2. Antenatal Care and Past Obstetric Performance of the Respondents
Out of the 300 mothers interviewed, 76 (25.3%) had not gone for antenatal visit and about 91 (30.3%) mothers had at least four antenatal visits as recommended by National Safe Motherhood Program of Nigeria. The majority of women received antenatal care from the publicly funded Sagamu local government health centers. Fifty (16.7%) mothers did not receive tetanus toxoid vaccine during their previous pregnancies; >48 hours and 210 (70%) received at least two doses of tetanus toxoid as recommended by National Safe Motherhood Program. Of the multiparous women, 181 (83.8%) had delivered at home at least once before. Only 100 (46.3%) mothers had at least one institutional delivery in the past. Nine mothers reported of having had a stillbirth (3.0%), 18 a neonatal death (6.0%), and eight a postnatal death (2.7%) after their previous home deliveries.
3.3. Birth Place and Attendance at Delivery
The majority (277, 92.3%) of the deliveries took place either in a separate room or inside the house, and the remaining 23 deliveries (7.7%) took place outside the house, either at the backyard or other places. One hundred (33.3%) deliveries were attended by neighbors, 70 (23.3%) were attended by traditional birth attendants and 47 (15.7%) were auxiliary nurse midwife or health assistant, and 47 (15.7%) were attended by auxiliary nurse midwife or health assistant, and 40 (13.3%) were attended by family members (Table 1).
3.4. Cleanliness and Hygiene Practices during Delivery
One hundred and twenty (40%) mothers recalled that the birth attendants had washed their hands, and 152 (50.7%) recalled that they did not do so. Twenty eight (9.3%) mothers could not remember at all. Clean home delivery surfaces are being encouraged in Nigeria. Emphasis is laid on a clean sheet, a clean razor blade, a clean surface for cutting cord, soap, and a cord tie. Fifty one (17%) mothers responded that a clean surface was used, and 165 had not used clean surface during their last delivery. The umbilical cord was cut after the expulsion of placenta in 203 (67.7%) deliveries. The umbilical cord was cut with a new, boiled blade or sterile scissors in 279 (93.0) deliveries, and in 12 (4.0%) deliveries a sickle/household knife or an old unboiled blade was used. The stump of umbilical was left undressed in 50 (16.7%) deliveries. But oil was applied in 14 (4.7%) deliveries. In 211 (70.3%) deliveries, methylated spirit was used to dress the umbilical stump. Applications like antiseptics were also reported by the mothers. The newborn was often wrapped in an old washed cloth (208, 69.3%) (Table 2).
3.5. Maintenance of Warm Chain for the Newborn
Since more than 90% of the deliveries took place in a room or inside the house, information about heating of the birthplace was also asked. In 247 (82.3%) deliveries the birthplace was heated only after birth. There was no heating of birthplace before or throughout birth. The heating of birthplace was not statistically significant between planned and unplanned home deliveries. The time taken to wrap the baby was usually prolonged. Only 150 (50%) newborns were wrapped within 10 minutes, and 287 (95.7%) newborns were wrapped within 30 minutes after birth. By one hour all the newborns were wrapped. Two hundred and seventy seven (98.2%) out of 300 newborns were bathed after birth. Almost all of these newborns were bathed within six hours after birth. More than a third of them were bathed within ten minutes, 95.8% within half an hour, and 98.2% within one hour (Table 3). The massage of the newborn with oil was a common practice, and 184 (61.3%) newborns received an oil massage any time after birth.
3.6. Newborn Feeding
All the newborns were breast-fed. Plain water, sugar, or animal milk was sometimes given to the newborns (9/300, 3.0%) before the initiation of breastfeeding. Overall, 291 (97%) mothers had given colostrums or breast milk to their babies as the first feed. Only one (0.3%) mother had given breast milk from other lactating mother when there was delay in initiation of breastfeeding. Six (2.0%) out of 300 mothers had discarded colostrums before initiating breastfeeding. The rates of initiation of breastfeeding were 65.3% within one hour and 95.7% within 24 hours (Table 4).
3.7. Reasons for Delivering at Home
In our study, 200 (66.7%) out of 300 home deliveries were planned, and, in 153 (76.5%) of these planned home deliveries, the reasons cited by the mothers were “I prefer home delivery,” “home delivery is easy and convenient,” and “all my previous deliveries were at home.” In our study, 100 (33.3%) home deliveries were unplanned. The common reasons cited for unplanned home deliveries were “precipitate labor” (57.0%), “lack of transportation” (21.0%), and “lack of escort during labor” (5.0%). “Worries about cost of care in the hospital” and “financial problems at home” (13.7%), “distance of the hospital” (2.7%), family members preference for home delivery (1.7%), and “before the expected date” (0.7%) were also mentioned as the reasons for delivering at home (Table 5).
4. Discussion
This study shows that home deliveries are not only common in rural areas but also in semi-urban areas where maternity services are relatively easily accessible. An earlier study carried out in Ogun State reported that 90.2% of deliveries took place outside health facility [6]. The proportion of home deliveries in the present study are similar to that reported from earlier studies in Sagamu and its surrounding areas. These studies reported that the proportion of home deliveries increased the farther one gets from urban areas [4, 6].
Interestingly, some findings of the present study are similar to the previous study from rural Nigeria [4]. It was surprising that skilled attendance health workers or auxiliary nurse midwife, use of clean surfaces, and hygiene practices during delivery were low in semi-urban areas also. Practices like cutting the cord with new or boiled blade, application of methylated to the umbilical stump, bathing the baby soon after birth, and delay in wrapping the baby after birth were common in semi-urban areas. Early initiation of breastfeeding and use of prelacteal feeds are also practices prevalent in semi-urban areas.
4.1. Attendance during Delivery
Most deliveries took place either in a separate room or some place inside the house which is similar to the report from a Nepal study [14]. Studies from areas had reported prominent role played by the mothers and mother-in-law during delivery and care of new born [4, 7]. But in our study, mothers and mother-in-law were present in only a small proportion (10%) of home deliveries. One-third of the deliveries were attended by neighbors. Such a difference may be due to demographic structure of the semi-urban population in which many families may be economic migrants and nuclear families. This is similar to the findings reported by a study in Nepal [14]. Earlier studies have confirmed the extremely low presence of skilled government health staff or auxiliary nurse midwife during delivery in rural areas of Nigeria [4, 6]. Registered nurse midwives and community extension health workers who are identified as key birth attendants by the policy makers were not present at delivery during our study either. This study highlights that skilled attendance at home deliveries is very low in semi-urban areas also. Previous studies found that about 15% of the mothers had delivered alone at home [4, 14]. This may emphasize the low status of women in the society and the gender inequalities in health. For many of these semi-urban families, pregnancy and delivery may not be taken seriously. It will take huge efforts to change the tradition of home deliveries and lack of skilled attendance during delivery in home setting. There is an ongoing debate about reinforcing home-based delivery strategies by training the traditional birth attendants who are closer and more acceptable to women who prefer home delivery in developing countries [12]. Such a study might cost huge amount of money. Therefore, there is a need for research comparing the feasibility, cost-effectiveness, cost-efficiency, acceptability, and equity implications of skilled home-based and facility-based obstetric care [12].
4.2. Hygiene and Thermal Control
Studies from Nigeria and India had reported that infection accounts for up to 40% of neonatal deaths [15, 16]. Hence, WHO emphasizes on five cleans during the delivery. The “five cleans” are a clean place, a clean surface, clean hands, clean cord and dressing, and a clean tie. In this study, forty percent of the attendants had washed their hands before delivery, a proportion which is less satisfactory. Clean surfaces were used only in 45% of the deliveries, which is higher than that reported in a study in rural Nepal [14]. Despite perceived usefulness and awareness, the use of clean surface was low. The reason for this was not clear. In this study, despite the low usage of clean surface, new razor blade/sterilized scissors were used to cut the cord in a majority (93%) of deliveries. This practice is encouraging as compared to practice in rural areas where sickle or kitchen knife was used in nearly one-third of deliveries and old/unbolted blade in 23% [17]. This practice was complemented by leaving umbilical stump undressed which is similar to the practice in rural areas [18]. The practice of applying substances like oil or animal dung is a more important risk factor than the means of cutting the cord as reported in earlier studies [18–20]. The common substances applied to the cord were “spiritual” oil and disinfectants. This practice was similar to the reports from earlier studies from urban settlements of Karachi Pakistan, semi-urban Nigeria, and rural Nepal [11, 17, 18]. The WHO has focused on thermal control of newborn care [21]. Previous studies from Nigeria, Nepal, India, and Bangladesh have reported on health beliefs about pregnancy and childbirth. The common view is that pregnancy is a “hot” state while postpartum is a “cold” one [15, 22–24]. Neonatal hypothermia has been described earlier in Nigeria and Nepal [7, 25]. In this study, it was found that in 83.2% of the instances birthplace was heated only after the delivery. Heating of the birthplace before birth is not practiced in Nigeria because it is a tropical country where warm weather is experienced in most of the months of the year. However, it is a fairly common practice to ensure warmth is provided for the baby and hence the reason for having the birthplace heated in over four-fifth of the deliveries. The practice of waiting for the placenta to deliver before cutting the umbilical cord was observed in 67.7% of the deliveries, a rate similar to that reported in a similar study in Nepal [14]. This practice delays immediate wrapping of the baby. This was further compounded by bathing the baby soon after birth which seems to be a universal practice. In this study, 98.2% of the newborns were given a bath within one hour after delivery. Similar practices were also reported in urban Nepal [11, 14, 17].
4.3. Infant Feeding
In this study, the only traditional newborn care practice which seems healthy and encouraging is breastfeeding. As reported in earlier studies, rates of initiation and exclusive breastfeeding are high [7, 17, 26–28]. However, practices like prelacteal feeding and discarding colostrum which still persist in urban areas are a cause for concern. Qualitative studies suggest that the traditional practice is to give a taste of non-breast-milk food and usually only once [7, 22]. In our study, 3% of the newborns received a prelacteal feed though low but significant, this is lower than that reported in Nepal (17,26). In this study, only two percent of the mothers discarded colostrum which is similar to 3% in rural area in Nigeria but lower than that reported in rural areas in Nepal [10, 17]. Use of formula feeds was minimal and feeding with bottles and nipple almost nonexistent in Nigeria. A recent study from urban area in Nigeria reported that grand mothers held colostrum in high regard, did not use prelacteal feeds, and also supported early initiation of breastfeeding [10]. These findings have positive implications on child nutrition.
4.4. Reasons for Delivering at Home
A study carried out in Sagamu and its surrounding areas has reported socioeconomic status and multiparity as strong predictors of the place of delivery. In this study, the reasons for planned home deliveries were related to “preference” for home delivery, perception of home deliveries as “easy” and “convenient,” and experience of previous home deliveries. For unplanned home deliveries, the reasons cited were “precipitate labor,” “lack of transport,” and “lack of escort” during labor. Similar findings were reported from earlier studies in Sagamu and urban Nepal [8, 14]. In semi-urban areas, there is a mix of traditional families and recent economic immigrant families. In rural areas, women have a strong cultural preference for home deliveries because institutional deliveries are inaccessible. This could be the reason for women indicating “preference” as the reason for delivering at home. The decision making process in the family about the place of delivery is also an important aspect of the reasons for home deliveries. The details of this aspect could not be explored in this study. Therefore, a plan to undertake an in-depth qualitative study to explore the reasons for delivering at home is on the pipeline. The findings of this study suggest that easy access to maternity services may not be enough to ensure the use of such services. Lack of utilization may be influenced by income, education, and cultural beliefs. In Sagamu, institutional delivery facilities are available at the University Teaching Hospital, maternity centers, and many private hospitals. Since the services are “cash and carry,” financial constraints may be the main reason for not using these facilities. A large section of this semi-urban population may be recent economic migrants from rural areas. This may be the reason for semi-urban-rural similarities observed in home delivery and newborn care practices in this study. In this study, two-thirds (33.3%) of the home deliveries were unplanned. These women would have sought institutional delivery if an ambulance service or local facility for delivery was made available. In this respect it will be worth investing on satellite maternity services run by midwives. Mothers might prefer to utilize such local and user-friendly services than a tertiary care hospital. In addition, mothers need to have information about how to access a trained community health worker or a midwife during delivery. In this study, a quarter of the mothers had not gone for antenatal checkups and only 16.3% of them received two doses of tetanus toxoid during their previous pregnancy. There may be cultural constraints for use of maternity services, for example, decision of the husband or mother-in-law which often overrides that of the mother. The reasons for low uptake of maternity services in the urban/semi-urban population may be due to socioeconomic and cultural factors [29–31]. Therefore, interventions should address not only the medical problems but also need to deal with wider social problems. Intervention should also be targeted at improving the status of women in the society including increasing female literacy and economic empowerment to tackle the maternal health problems [32]. Recent studies carried out in Ilesa, Nigeria and Kathmandu, and Nepal reported that attitudes of pregnant women, husbands, and service providers were favorable towards encouraging greater male participation in maternal health services [29, 33]. Further qualitative studies are needed about quality of available maternal services and cultural beliefs about pregnancy and childbirth.
The present study may have both selection and information biases. Since this survey was carried out in immunization clinics, selection bias cannot be ruled out. Those mothers who delivered at home and did not attend immunization clinics could have been missed. However, the sample of mothers interviewed may be representative of the semi-urban population since the immunization coverage is more than 90% in semi-urban areas [5]. The high infant mortality rate in the study area means that twelve percent of the children who were born at home may have not reached their first birthday. Therefore, twelve percent of the mothers did not attend the immunization clinics. Only those deliveries that took place within one year of the study were included in order to avoid recall bias over a longer period of time. However, some amount of recall and report bias cannot be completely ruled out. Hence, the interpretation and generalisability of this study is limited.
In this study, all the women who delivered at home agreed to participate in the study. The interviewers identified themselves as independent researchers rather than as part of health service team present in the immunization clinics. The respondents were assured of being provided health services irrespective of their decision to participate in the interview, and verbal consent was sought. All the women agreed to participate in the study after receiving such assurance. The possibility that the women considered interviewers as a part of health service cannot still be rued out. The mean monthly family income of the respondents was approximately US$122. But the annual per capital income of Nigeria is US$640 [5]. The reported monthly family income of this semi-urban population is higher than national average. The families in this semi-urban population may be more affluent than an average Nigerian the the family since Nigeria is an agrarian economy. In Nigeria, the majority of the population is mainly dependent on subsistence farming with constant migration to other economically perceived better countries.
Despite the above-mentioned limitations, this study has obtained important information about home delivery and newborn care practices and reasons for delivering at home. This information has many policy implications about the ongoing safe motherhood and child survival programmes in Nigeria. There is a need to focus more on the skilled attendance hygiene during delivery and the use of clean surfaces for delivery in semi-urban/population. Some high-risk newborn care practices like delayed wrapping, immediate bathing, oil application to the cord, prelacteal feeding, and discarding colostrum need more attention. This information will assist in planning public health interventions aimed at changing the behavior. Expanding skilled attendance during delivery is an important issue since these semi-urban women “prefer” home deliveries and home deliveries are perceived as “easy” and “convenient.”
5. Conclusions
There is a need for community-based interventions to improve the uptake of publicly funded maternity services. Health promotion interventions are required to improve the number of families engaging a skilled attendant and hygiene during delivery. High-risk traditional newborn care practices need to be addressed by culturally acceptable community-based health programmes to improve newborn care practices.
Conflict of Interests
The author declares that he does not have any conflict of interests.
Author's Contribution
The author was the primary researcher who conceived the study, responsible for the design and protocol of the study; design and development of the questionnaire; data collection and analysis; training and supervision of health workers during data collection; paper drafting and preparation for publication.
Acknowledgments
The author is grateful to the two health workers who assisted in data collection during the study. He is thankful to the staff of the immunization clinics used to conduct the interview. He also thanks all the mothers who participated in the study.
Appendix
Questionnaire on a Survey of Home Delivery and Newborn Care Practices among women in South Western Nigeria:
No.
Age:
Marital status:
Occupation:
Parity:
Religion:
Level of education:
(1) Where exactly at home did you deliver?
- in a room
- inside the house
- outside the house
- backyard of the house
- others (state)
(2) Who assisted or attended to you during delivery?
- no attendant
- neighbour
- mother
- mother-in-law
- other family member
- auxiliary nurse midwife
- health assistant
- traditional birth attendant
- others (state)
(3) What instrument was used to cut the cord?
- new or boiled blade
- old unboiled blade
- household knife
- unknown
- others (state)
(4) How was the umbilical stump treated/dressed?
(5) What kind of cloth was used to wrap the baby?
- old unwashed cloth
- old washed cloth
- others
- new unwashed cloth
- new washed cloth
- unknown
(6) Was the birthplace heated?
- none
- before birth
- after birth
- -throughout birth
(7) How soon after delivery was the baby wrapped?
- ≤5 minutes
- ≤10 minutes
- ≤30 minutes
- ≤60 minutes
- >60 minutes
- unknown
(8) What was the baby fed on first?
- breast milk/colostrums
- breast milk from other woman
- formula feed
- cow's milk
- glucose water
- plain water
- honey
- others (state)
(9) How soon after birth was the baby breastfed?
- immediately after birth
- ≤15 minutes
- ≤30 minutes
- ≤60 minutes
- ≤24 minutes
- ≤48 minutes
- >48 minutes
(10) Did you plan to deliver at home?
- Yes
- No
(11) Why did you deliver at home?
- preference for home delivery
- home delivery is easy and convenient
- all my previous deliveries were at home
- worries about cost in the hospital
- health worker lives close to house
- onset of labour before the expected date
- lack of transport during labour
- -others (state)
- hospital is too far
- financial problems at home
- family members prefer home delivery
- fear of hospital
- precipitate labour
- lack of escort during labour
Table 1 Place of delivery and attendance during delivery.
Place of delivery Number of births (N = 300) Percentage
In a room 181 60.3
Inside the house 96 32.0
Outside the house 12 4.0
Backyard of the house 8 2.7
Others 3 1.0
Attendant during delivery*
Neighbor 100 33.3
No attendant 47 15.7
Mother 15 5.0
Mother-in-law 15 5.0
Other family members 10 3.3
Auxiliary nurse midwife 32 10.7
Health assistant 8 2.7
Traditional birth attendant 70 23.3
Others 3 1.0
*More than one attendant might have been present.
Table 2 Cleanliness and hygiene practices during delivery.
Instrument used for cutting umbilical cord Number of births (N = 300) Percentage
New or boiled blade 209 69.7
Sterilized scissors 70 23.3
Household knife 8 2.7
Old unboiled blade 4 1.3
Unknown 6 2.5
Dressing applied to umbilical stump
Nothing 50 16.7
Methylated spirit 211 70.3
Oil 14 4.7
Antiseptic 4 1.3
Unknown 21 7.0
Cloth used for wrapping the baby
Old washed cloth 208 69.3
Old unwashed cloth 23 7.7
New unwashed cloth 26 8.7
New washed cloth 41 13.7
Unknown 2 0.6
Table 3 Practices related to maintenance of the warm chain for the newborn.
Heating of the birthplace Number of births (N = 300) Percentage
None 53 17.7
Before birth 0 0.0
After birth 247 82.3
Throughout birth 0 0.0
Time to wrapping the baby
≤5 minutes 43 14.3
≤10 minutes 150 50.0
≤20 minutes 287 95.7
≤30 minutes 297 99.0
≤60 minutes 300 100.0
Time to bathing
≤5 minutes 43 15.3
≤10 minutes 200 70.9
≤30 minutes 270 95.8
≤60 minutes 277 98.2
≥60 minutes 300 100.0
Table 4 Type and timing of first feed.
Newborn's first feed Number of newborns Percentage
Breast milk/colostrum 290 96.7
Breast milk from other woman 1 0.3
Glucose water 5 1.7
Plain water 2 0.7
Formula feed 2 0.7
Time to breast-feed
Immediately after birth 15 5.0
≤15 minutes 45 15.0
≤30 minutes 90 30.0
≤60 minutes 196 65.3
≤24 hours 287 95.7
≤48 hours 293 97.7
≥48 hours 300 100.0
Table 5 Reasons for choice of planned and unplanned home deliveries.
Reason given Planned Unplanned Total (%)
Preference for home delivery 77 — 77 (25.7)
Home delivery is easy and convenient 66 — 66 (22.0)
All my previous deliveries were at home 10 — 10 (3.3)
Hospital is too far 5 3 8 (2.7)
Worries about cost in the hospital 24 7 31 (10.3)
Financial problems at home 7 3 10 (3.3)
Family members prefer home delivery 4 1 5 (1.7)
Fear of hospital 2 — 2 (0.7)
Health worker lives close to house 5 — 5 (1.7)
Precipitate labor* — 57 57 (19)
Lack of transport during labor — 21 21 (7.0)
Lack of escort during labor — 5 5 (1.7)
Onset of labor before the expected date — 2 2 (0.7)
Other reasons — 1 1 (0.3)
Total
200
100
300
*Precipitate labor: labor results in rapid expulsion of fetus.
==== Refs
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 21829495PONE-D-11-0653810.1371/journal.pone.0022730Research ArticleBiologyGenomicsGenome Expression AnalysisMicrobiologyVirologyImmunodeficiency VirusesHost-Pathogen InteractionPathogenesisSystems BiologyComparative Expression Profile of miRNA and mRNA in Primary Peripheral Blood Mononuclear Cells Infected with Human Immunodeficiency Virus (HIV-1) miRNA and mRNA Regulation during HIV-1 InfectionGupta Ankit
1
Nagilla Pruthvi
1
Le Hai-Son
2
Bunney Coulton
1
Zych Courtney
1
Thalamuthu Anbupalam
3
Bar-Joseph Ziv
2
Mathavan Sinnakaruppan
3
Ayyavoo Velpandi
1
*
1
Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America
2
Department of Machine Learning, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States of America
3
Human Genetics, Genome Institute of Singapore, Singapore
Mammano Fabrizio EditorINSERM, France* E-mail: [email protected] and designed the experiments: AG VA. Performed the experiments: AG PN CB CZ. Analyzed the data: AG PN H-SL CB AT ZB-J SM VA. Wrote the paper: AG CZ ZB-J VA.
2011 28 7 2011 6 7 e2273011 4 2011 29 6 2011 Gupta et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Host cells respond to exogenous infectious agents such as viruses, including HIV-1. Studies have evaluated the changes associated with virus infection at the transcriptional and translational levels of the cellular genes involved in specific pathways. While this approach is useful, in our view it provides only a partial view of genome-wide changes. Recently, technological advances in the expression profiling at the microRNA (miRNA) and mRNA levels have made it possible to evaluate the changes in the components of multiple pathways. To understand the role of miRNA and its interplay with host cellular gene expression (mRNA) during HIV-1 infection, we performed a comparative global miRNA and mRNA microarray using human PBMCs infected with HIV-1. The PBMCs were derived from multiple donors and were infected with virus generated from the molecular clone pNL4-3. The results showed that HIV-1 infection led to altered regulation of 21 miRNAs and 444 mRNA more than 2-fold, with a statistical significance of p<0.05. Furthermore, the differentially regulated miRNA and mRNA were shown to be associated with host cellular pathways involved in cell cycle/proliferation, apoptosis, T-cell signaling, and immune activation. We also observed a number of inverse correlations of miRNA and mRNA expression in infected PBMCs, further confirming the interrelationship between miRNA and mRNA regulation during HIV-1 infection. These results for the first time provide evidence that the miRNA profile could be an early indicator of host cellular dysfunction induced by HIV-1.
==== Body
Introduction
There is remarkable variation in the onset of disease in HIV-1 infected individuals. The replication, spread, and immune evasion of the virus and the progression of disease depend on host cellular transcription and gene regulation in virus-specific target cells and immune cells [1], [2], [3]. Both viral and host cellular factors have been shown to contribute to infection, virus replication, and disease progression. Viral factors include free-extracellular (cell- and virus-free), virion-associated, and infected cell-associated viral antigens, as well as infectious and noninfectious virus particles. HIV-1 virus with defective expression of viral proteins such as Nef, Vpr, Gag, and Pol are shown to induce differential gene expression [4], [5], [6]. Host factors include host genetics (determined by HLA alleles), polymorphisms in HIV receptors and coreceptors, and genes involved in innate and adaptive immune responses [7], [8], [9], [10]. Following the landmark discovery of the CCR5-Δ32 mutation that protects against HIV infection [11], [12], [13], many other genetic variants have been shown to affect HIV infection and AIDS pathogenesis [7], [14], [15]. It is likely that, in addition to incomplete immunological control, host genetic variation and differences in gene expression in the infected host cells may also contribute to the differential disease pattern [16], [17], [18]. Along these lines, attempts were made to identify host cellular proteins associated with HIV-1 infection [19], [20], [21], [22]. There is limited information, however, regarding the regulation of host cellular genes at the transcriptional, post-transcriptional, and translational levels.
Several studies have shown that HIV-1 infection differentially regulates host cellular genes and pathways, suggesting that differential gene expression in infected individuals either accelerates disease progression or enhances resistance to the development of disease [23], [24], [25]. Genome-wide association studies have not fully defined the resistance in exposed uninfected sex workers or elite controllers, indicating a role for other host cellular factors [26]. Studies delineating the cellular factors in individuals who remain uninfected despite repeated exposures to the virus have found that they over-express anti-viral innate factors, such as RANTES, SLPI, and other chemokines [27]. It is important to note that the differential expression of host cellular genes is not universal, resulting in disease progression at variable rates in infected individuals [28], [29], [30].
Gene expression in general is regulated at transcriptional, post-transcriptional, and translational levels. Recent discoveries have emphasized a central role for the new class of small non-coding RNA in gene expression controlling growth, development, and immune response in vivo
[31], [32], [33]. These non-coding RNAs, which include microRNA (miRNA), partition function for interacting RNA (piRNA), and small interfering RNA (siRNA), are emerging as a major component of the cellular regulatory pathways that underlie the development and physiology of complex organisms [34], [35], [36]. Regulation of gene expression by miRNA occurs primarily at the post-transcriptional level [37], [38]. Recent studies have shown that miRNA have a unique expression profile in cells of the innate and adaptive immune systems, CNS, and cancers [39], [40], [41], [42]. Based on these observations, we suggest that pathogens including viruses could potentially modulate host cellular transcription at multiple levels by targeting various factors including miRNAs.
Studies previously have evaluated the expression of either miRNA or mRNA in cells isolated from HIV-1 infected subjects [43], [44], [45], [46]. Their results are likely to be influenced by variabilities in host genetics and viral heterogeneity in addition to other factors including viral burden. In an effort to understand host cellular gene regulation during HIV-1 infection, we performed a comparative global miRNA and mRNA microarray profiling in PBMCs derived from multiple donors upon infection with HIV-1. Our results indicate that HIV-1 infection differentially regulated several miRNAs that could potentially regulate host cellular pathways such as cell cycle, apoptosis, T cell signaling and cytokine/chemokine responses. Taken together, these results for the first time provide evidence that the miRNA profile could be an early indicator of HIV-1 induced host cellular dysfunction.
Materials and Methods
Generation of infectious HIV-1
The infectious HIV-1 particles were generated by using the proviral DNA construct pNL4-3 obtained from the National Institutes of Health AIDS Research and Reference Reagent Program (NIH AIDS RRRP). Two million HEK293T cells (a kind gift from Dr. Michelle Calos, Stanford University) were transfected with 5 µg of proviral DNA using Polyjet (SignaGen) as suggested by the manufacturer. Virus titer was measured by p24 antigen ELISA, and infectivity was assessed by determining multiplicity of infection (MOI) using the HIV-1 reporter cell line TZM-bl (NIH AIDS RRRP) as described earlier [47].
Isolation and infection of PBMCs with HIV-1 virus
We purchased normal donor blood from the American Red Cross Blood Bank in Pittsburgh using appropriate IRB approval forms from the University of Pittsburgh. PBMCs were isolated by Ficoll-Hypaque gradient centrifugation. Freshly isolated normal donor PBMCs (5×106/mL) were stimulated with 5 µg/ml PHA-P (Sigma, St. Louis, MO) for three days. Cells were washed, divided into two parts and cultured in RPMI medium (GIBCO, CA) containing 10% FBS (Hyclone, Logan, UT), 1% L-glutamine (Cambrex, MD), 1% penicillin-streptomycin (GIBCO, CA), and IL-2 (200 U/mL, Chiron, Emoryville, CA). One half of the cells were subsequently infected with 0.1 MOI of virus particles using standard protocols as described [48] and the remaining half of the culture is maintained under similar conditions and used as control. Seven days post infection (pi), culture supernatants were assessed by p24 ELISA for infection and virus replication [47]. Cells were collected and frozen for miRNA and mRNA isolation. We performed a total of 16 independent experiments using donor PBMCs (n = 16).
Total and miRNA isolation
PBMCs (both infected and control) were collected, washed with PBS, and lysed for RNA isolation. Total RNA was isolated using the TRIzol method (InVitrogen) as suggested by the manufacturer. Next, to enhance the sensitivity and detection, we enriched the small RNA using a microRNA isolation kit (SABiosciences) with two separating columns as per the manufacturer's instructions. This allowed us to isolate both miRNA and mRNA, which were used in the miRNA array and gene expression arrays, respectively. RNA quality was determined by chip-based capillary electrophoresis using Agilent Bioanalyzer 2100 (Agilent, CA), according to the manufacturer's instructions.
MiRNA profiling using RT2 MicroRNA PCR Array
For miRNA profiling studies, we used the SABiosciences RT2 MicroRNA PCR Array system, an optimized real-time PCR assay, which allows the simultaneous detection of 704 miRNAs, representing most functional miRNAs, as well as appropriate housekeeping assays and RNA quality controls. We performed the assay according to the manufacturer's protocol. Enriched miRNA was converted to cDNA using an miRNA first strand synthesis kit. First strand was used to perform the miRNA PCR array using a Taqman 7900HT machine. Equal amounts of RNA from both infected and uninfected cells were used for the first strand and assayed as per the manufacturer's protocol.
Gene expression profiling
For total mRNA profiling, we used the Illumina HT-12 array, which targets more than 25,000 annotated genes with more than 48,000 probes covering well-characterized genes, gene candidates, and splice variants. One µg of high-quality total RNA from each sample was used to generate cDNA. Sample labeling, hybridization, and scanning were performed according to the manufacturer's protocols as well as standardized protocols developed by the core laboratory at the University of Pittsburgh. Data analysis was performed using the Illumina software to delineate the false discovery rate (FDR) and differences with statistical significance (p<0.05).
Microarray data analysis
We analyzed the expression of individual miRNA using CT values obtained with a threshold of 0.2. Endogenous controls, RT negative controls, and genomic DNA contamination controls were tested for each array. If a particular miRNA in either the control or the experiment samples showed expression at least three times with a value of 35 or greater, it was excluded from the analysis as undetectable or undetermined. We uploaded values (CT) that passed through these stringent criteria into the SABiosciences software (RT2 Profiler PCR Array Data Analysis) and calculated fold change for each miRNA. Data were further subjected to statistical analysis using the manufacturer's web-based software to define the difference with significant p value (p< = 0.05) between the two groups.
Validation of differentially regulated miRNA and mRNA from the genome-wide array
Based on the data analyses, selected miRNA and mRNA targets were verified by qRT-PCR using specific primers and probes (Applied Biosystems). We used RNA samples (n = 6) from the miRNA microarray profiling to validate the high throughput microarray results. Additional validation was performed by infecting under similar conditions normal donor PBMCs (n = 10) that were not part of the miRNA microarray.
Computational analysis validating the miRNA targets and mRNAs
To identify mRNA targets of miRNAs in the samples, we used GenMIR++ [49], a novel regression-based Bayesian method developed to identify targets of miRNAs by integrating the measured expression data for miRNAs and mRNAs with a database of a potential set of mRNA targets for each miRNA. GenMIR++ takes into account the downregulation of target mRNAs by modeling the expression of each targeted mRNA as a negatively weighted sum of the expressions of multiple miRNAs with some additional Gaussian noise. The method restricts the set of possible targets for each miRNA to a set of candidate mRNAs given a priori. This potential set of targets for each miRNA is derived based on sequence analysis. For this paper, we used predictions from the MicroCosm Targets database [50] to determine this set. For the GenMIR++ analysis, we first used the R package samr [51] to detect differentially expressed mRNAs by using an FDR cutoff of 10%. We next combined these mRNAs with the differentially expressed miRNAs, leading to 2,941 mRNAs and 327 miRNAs that were used as input to GenMIR++. The plots show the top 10% of interactions predicted by GenMIR++ ranked by their p-values.
Pathway analysis
To determine gene interactions and correlation networks, we used Ingenuity Pathway Analysis, STRING, and KEGG. The cutoff values for inclusion in these analyses were differential gene expression, with p-value <0.05 and 2.0 in fold change (based on SAM). Genes identified from miRNA-based predicted targets (score of >70) were also assessed to define the potential networks and pathways.
Results
Infection and miRNA and mRNA quality from PBMC from multiple donors
The goal of our study was to analyze the potential link between miRNA and mRNA in HIV-1 infected cells. We hypothesized that infection of target cells such as PBMCs by HIV-1 may lead to the following scenarios: i) The infection may regulate an identical subset of miRNA in cells derived from genetically diverse individuals, and ii) The changes in miRNA may have an impact on mRNA in genes associated with distinct cellular pathways.
We used PBMCs from multiple donors to validate the differential regulation of miRNA and mRNA profiles by HIV-1 infection. All the donors were healthy and seronegative for HIV-1. To eliminate variability with regard to the virus, we used a well-characterized virus derived from a molecular clone. Infectivity was assessed by qRT-PCR using HIV-1 Gag-specific primers and probes as described [52] employing an equal amount of RNA. The presence of Gag was determined by normalizing with endogenous control and presented as CT values (Figure 1A). Samples with low and high CT values (<12 or >30) were eliminated from further profiling studies (Higher CT value (>30) indicate inefficient virus replication and lower CT value (<12) indicate high infectivity and infection induced cell death). Results indicate that infected cultures of PBMCs showed the presence of Gag with an average CT value of 21.88±1.12, indicating virus replication. Gag CT value was below the level of detection in the uninfected control. Cell death was assessed in the infected and uninfected cultures before RNA isolation (Figure 1B). Cell death in uninfected cultures ranged from 0 to 27% (an average of 8.4±2.2%), whereas infected cultures showed a higher percentage (16–50%, with an average of 29.33±2.4%). To ensure RNA quality, infected cultures with high cell death (>50%) were not included in microarray analysis.
10.1371/journal.pone.0022730.g001Figure 1 Infectivity in and cell death in PBMCs used for analyses.
(A) Normal donor PBMCs were infected with HIV-1 or mock infected for seven days. RNA was isolated and tested for Gag by RT-PCR. Gag CT values were obtained by using the endogenous control for normalization. Figure shows average CT value from 16 independent donors. (B) After seven days, post-infection cell death was assessed by trypan blue dye exclusion method. Dot blot represents percentage of cell death in uninfected (NT) and infected (HIV-1) PBMCs from 16 independent donors.
RNA isolated from these cultures was tested for quality by spectrophotometry and by Taqman assays for endogenous control miRNAs and mRNA species. Each sample was tested for two miRNA and two small RNA controls (Table 1). All but two donors showed CT values (>30) for endogenous controls. These two donors were eliminated from further analyses to avoid bias because the quality of RNA in these samples may not have been ideal for qRT-PCR profiling.
10.1371/journal.pone.0022730.t001Table 1 Selected endogenous controls and their Ct values.
Endogenous controls Control #1 Control #2 Control #3 Control #4 Control #5 Average
SNORD48/RNU48/U48 25.49 25.07 26.8 25.8 24.65 25.56
SNORD47/U47 19.27 21.37 19.52 21.32 22.47 20.79
SNORD44/U44 17 18.9 17.64 19.77 22.69 19.2
RNU6-2/U6-2 20.82 22.35 20.84 21.16 23.35 21.70
Endogenous controls HIV-1#1 HIV-1 #2 HIV-1 #3 HIV-1 #4 HIV-1 #5 Average
SNORD48/RNU48/U48 20.31 24.46 27.57 23.16 25.49 24.198
SNORD47/U47 19.48 17.83 20.72 19.45 24.7 20.436
SNORD44/U44 18.97 15.69 18.53 19.17 21.67 18.806
RNU6-2/U6-2 17.82 18.35 21.35 19.57 24.37 20.292
RNA isolated from infected and uninfected PBMCs was evaluated first for the level of endogenous controls by RT-PCR using specific primers and probes as a measure of RNA quality and quantity before microarray analysis. Five representative donors out of 16 donors are presented here.
HIV infection differentially regulated expression of multiple miRNAs
The expression profile of 704 host cellular miRNAs was assessed in infected and uninfected PBMCs from multiple donors (n = 6). Results revealed that HIV-1 infection differentially regulated expression of several miRNAs (Tables S1 and S2). The level of expression in infected cells was compared with that in uninfected cells, and fold differences were calculated based on normalization with internal controls. The fold differences ranged between 2- and 88-fold for different miRNAs. Among the total 704 miRNAs tested, 208 were upregulated (Table S1) by more than 2-fold compared to controls, and 14 were downregulated (Table S2) compared to controls. Further analyses indicated that among these 222 miRNAs, 41 were upregulated (>4-fold) and five were downregulated (>4-fold).
To further assess the significance of our data, we performed statistical analyses to identify the regulated miRNA in the infected and the uninfected groups, using 2-fold as the cutoff (Figure 2; Table 2). Results indicate that among the 208 upregulated miRNAs, 21 exhibited significant changes across multiple donors (p value of <0.05). It is interesting to note that none of the 14 miRNAs that showed downregulation exhibited significant p value. Heatmap analyses further cluster the miRNA species between the test groups (Figure 2). Together these results indicate that fold differences did not necessarily correlate with the differential regulation of miRNAs in multiple donors.
10.1371/journal.pone.0022730.g002Figure 2 Heatmap and hierarchical clustering of miRNA.
The heatmap represents the result of the two-way hierarchical clustering of miRNA and samples. Each row represents miRNA, and each column represents samples tested. The clustering is represented for the miRNA and samples on top and sides, respectively. Red represents miRNA with an expression level above the mean, and green represents miRNA with an expression level below mean/average.
10.1371/journal.pone.0022730.t002Table 2 Significantly regulated miRNAs in HIV-1 infected cultures compared to uninfected control cells.
Mature ID Fold change p value
#Predicted targets Cellular function based on predicted targets through TargetScan and IPA analysis
miR-593 2.3247 0.0010 67 Amino acid Metabolism
miR-431 4.2845 0.009372 25 Cell growth and proliferation
miR-892a 2.4136 0.013 122 Cellular function and Maintenance
miR-138 2.1364 0.013 44 Cell Morphology
miR-564 4.8795 0.0130 79 7 N/A
miR-628-3p 4.6012 0.017119 60 Cell Signaling
miR-411 8.2454 0.018815 29 Cell Cycle
miR-518f* 3.3215 0.0308 10 DNA Replication, Recombination and Repair
miR-187 2.0631 0.035 4 N/A
miR-188-5p 6.1813 0.037526 72 Cell Cycle
miR-938 3.6903 0.0399 15 Cellular Development
miR-1253 3.1465 0.0419 106 Cellular function and Maintenance
miR-1470 3.317 0.0425 2 N/A
miR-549 5.3005 0.04538 60 Cell Cycle
miR-888* 4.8503 0.0469 153 Cellular function and Maintenance
miR-33b* 2.9726 0.0484 79 Cellular Assembly and Organization
miR-519b-5p 2.8366 0.050 50 Nucleic acid Metabolism
miR-302a 3.6139 0.050 164 DNA Replication, Recombination and Repair
miR-1286 3.3554 0.0523 34 Carbohydrate Metabolism
miR-636 2.5808 0.0548 47 Cell Cycle
miR-760 2.9002 0.0575 42 Cell Signaling
This table lists miRNAs that are significantly regulated (over expressed) with a significance of p<0.05 in HIV-1 infected cultures compared to uninfected control cells. # Column represents targets identified through miRDB having a target score ≥70.
Based on these analyses, we selected the differentially regulated miRNAs with significant p value (<0.05) for further analyses. To understand the significance of these miRNAs in host cellular functions, we identified target genes using TargetScan followed by IPA analysis (Table 2). Our results indicate that HIV-1 infection targets cellular genes primarily involved in cell cycle, proliferation, cellular movement and migration, and cell signaling. Previous studies have reported findings that virus infection and viral antigens modulate these host cellular pathways [53], [54].
Validation of miRNA expression by qRT-PCR
To validate the differentially regulated miRNAs from the microarray results, we randomly selected the nine miRNAs from Table 2 (miRNA with 2-fold change and p<0.05) and the five miRNAs from Tables S1 and S2 (miRNA with 2-fold increase but p>0.05). We tested RNA from the infected and uninfected control pair from multiple donor PBMCs for specific miRNA along with endogenous controls using miRNA specific primers and probes by qRT-PCR (Figure 3). Results indicate that among the nine upregulated miRNAs tested from Table 2, all except miR-628-3p showed upregulation by independent qRT-PCR assay. Although miR-628-3p showed upregulation in miRNA profiling, all donors used in independent validation did not show significant upregulation, thus resulting in no change. This could be due to variations within donors or due to design of primer/probes used in qRT-PCR. Similarly, the single downregulated miRNA, miR-120, showed downregulation in most of the tested samples. This was validated in both the samples that were used to perform the genome-wide microarray (n = 6) and additional PBMCs (n = 10) infected in a similar manner, suggesting that these miRNAs are consistently regulated across multiple donors. Validation of miRNA in both the samples further confirms that the array analyses in our samples are reproducible.
10.1371/journal.pone.0022730.g003Figure 3 Quantitative real time RCR (qRT-PCR) confirmation of randomly selected miRNAs from the microarray analysis.
The expression of selected miRNAs in PBMCs infected with HIV-1 along with the expression of uninfected controls were tested for validation by qRT-PCR using a specific primer and probe for each miRNA. Fold increase/decrease was calculated based on endogenous control normalization. Average fold change for each miRNA represents fold change obtained from 10 independent donors.
HIV-1 induced gene regulation of cellular mRNA transcripts
miRNAs regulate cellular gene expression at the post-transcriptional level, thus silencing and/or downregulating gene expression [55], [56]. To assess whether a direct correlation exists between the expression patterns of miRNA and mRNA, we assessed the expression profile of mRNA transcripts present in HIV-1 infected PBMCs and in uninfected control cells from the same donor. Microarray profiling of mRNA samples was normalized with the internal endogenous control and cross-compared between the two groups.
Among the 48,000 transcripts tested, we found that 444 genes were differentially regulated in the infected culture compared to uninfected controls. Of the 444 differentially regulated genes, 147 were upregulated and 297 were downregulated significantly (FDR corrected with p value of <0.05 with 2-fold regulation). We analyzed these mRNA transcripts to identify the pathways using STRING, IPA, and DAVID databases. To further analyze the interplay between the up- and downregulated mRNA, all the differentially regulated mRNA were combined and evaluated. The results, which are presented in Figure 4, indicate that HIV-1 infection regulates three major clusters including SRC kinases, MAPK, and apoptosis/cell cycle regulators. Similar results were reported by previous studies using transcriptome analysis in HIV-1 infected target cells [45], [46]. Results presented in Figure 4 include both upregulated and downregulated genes. To understand whether HIV-1 targets specific pathways for up- or downregulation, we performed individual pathway analyses (Table 3 & 4). Results from the upregulated genes include clusters of genes that control cell cycle, apoptosis, DNA damage pathways, and several signaling pathways including MAPK, p53, TRL, and T-cell receptor (Table 3). In contrast, analyses of downregulated gene clusters include primarily anti-apoptotic proteins/pathways, NF-kB mediated immune dysregulation, and SRC kinases (Table 4). Additional analyses using various software and databases yielded similar results (data not shown). Collectively, these results indicate that several pathways are regulated by HIV-1 infection including inflammatory response, cell death, cell-to-cell signaling and interaction, and immune function.
10.1371/journal.pone.0022730.g004Figure 4 Predicted interaction networks of genes regulated during HIV-1 infection.
Differentially regulated genes are represented in the links predicted using STRING (http://string.embl.de/). Predicted interactions represent both direct and indirect associations that are derived from four different sources: genomic context, high throughput experiments, conserved co-expression, and previous findings from the literature. The nature of the supporting evidence is indicated by the color lines: green represents neighborhood; red, gene fusion; blue, co-occurrence; black, co-expression; purple, previous experiments; turquoise, database; yellow, text mining; and aqua, homology.
10.1371/journal.pone.0022730.t003Table 3 Genes upregulated in the HIV-1 infected culture compared to uninfected control cells, determined by transcriptome analysis.
Canonical Pathways Count p Value Genes
MAPK signaling 6 1.30E-05 PPP3CC; CACNA1I; PPP3CB; PRKCA; TGFBR2; CDC25B
T-cell receptor signaling 4 5.29E-05 MALT1; PPP3CC; PPP3CB; PRKCQ
Natural killer cell mediated cytotoxicity 4 1.32E-04 PPP3CC; PPP3CB; PRKCA; HCST
Wnt signaling 4 1.91E-04 WNT7A; PPP3CC; PPP3CB; PRKCA
Calcium signaling 4 3.96E-04 PPP3CC; CACNA1I; PPP3CB; PRKCA
B-cell receptor signaling 3 3.78E-04 MALT1; PPP3CC; PPP3CB
Long-term potentiation 3 3.78E-04 PPP3CC; PPP3CB; PRKCA
VEGF signaling 3 3.93E-04 PPP3CC; PPP3CB; PRKCA
Apoptosis 3 6.24E-04 PPP3CC; PPP3CB; ATM
Cell cycle 3 0.00144 CDC14B; ATM; CDC25B
Tight junction 3 0.00211 CLDN18; PRKCA; PRKCQ
10.1371/journal.pone.0022730.t004Table 4 Genes downregulated in the HIV-1 infected culture compared to uninfected control cells determined by transcriptome analysis.
Pathways # p Value Gene
Cell cycle 10 1.80E-10 MCM2; MCM7; PKMYT1; CDK2; GADD45B; MAD2L2; TFDP1; GADD45A; PCNA; ORC1L
Apoptosis 8 7.26E-09 IRAK2; NFKBIA; BCL2L1; TNFRSF10B; BAX; DFFA; EXOG; TNF
Purine metabolism 8 4.74E-07 ADA; ADCY3; ADCY8; POLA1; POLR3K; POLE2; GART; DGUOK
MAPK signaling 8 3.71E-05 MAP3K8; GADD45B; DUSP2; ATF4; GADD45A; DUSP5; TNF; DUSP10
Cytokine-cytokine receptor interaction 7 1.92E-04 TNFSF9; TNFRSF10B; CD70; IL8; TNF; TNFRSF18; TNFSF13B
DNA polymerase 5 5.76E-07 MCM2; MCM7; POLA1; PCNA; POLE2
Base excision repair 5 5.76E-07 PARP2; PCNA; POLE2; APEX2; POLB
Adipocytokine signaling 5 1.32E-05 SOCS3; NFKBIA; TNF; NFKBIE; CPT1A
p53 signaling 5 1.52E-05 CDK2; GADD45B; TNFRSF10B; BAX; GADD45A
Epithelial cell signaling in H.pylori infection 5 1.63E-05 NFKBIA; LYN; IL8; JAM2; SRC
Small cell lung cancer 5 4.44E-05 NFKBIA; BCL2L1; CDK2; TRAF3; TRAF4
Toll-like receptor signaling 5 1.00E-04 NFKBIA; MAP3K8; IL8; TNF; TRAF3
One carbon pool by folate 4 6.80E-07 SHMT2; GART; MTHFS; MTHFD1L
Urea cycle andmetabolism of amino groups 4 7.33E-06 ASS1; ACY1; ALDH7A1; SMS
Aminoacyl-tRNA biosynthesis 4 3.46E-05 WARS; YARS; GARS; RARS
Glycine, serine and threonine metabolism 4 4.19E-05 CBS; PHGDH; SHMT2; GARS
Gap junction 4 8.33E-04 ADCY3; ADCY8; CSNK1D; SRC
Pyrimidine metabolism 4 8.33E-04 UPP1; POLA1; POLR3K; POLE2
GnRH signaling 4 0.00140 ADCY3; ADCY8; ATF4; SRC
T cell receptor signaling 4 0.00150 NFKBIA; MAP3K8; TNF; NFKBIE
Tight junction 4 0.00335 CGN; MYL2; JAM2; SRC
Insulin signaling 4 0.00353 TRIP10; SOCS3; PFKM; PTPN1
Mismatch repair 3 1.46E-04 MSH6; PCNA; EXO1
Arginine and proline metabolism 3 5.66E-04 ASS1; PYCR1; RARS
Nucleotide excision repair 3 0.00102 ERCC1; PCNA; POLE2
Fatty acid metabolism 3 0.00109 ACADVL; ALDH7A1; CPT1A
Glutathione metabolism 3 0.00148 GSTA4; GGCT; SMS
Inositol phosphate metabolism 3 0.001952 IMPA2; INPP1; ITPKA
Amyotrophic lateral sclerosis 3 0.002056 BCL2L1; BAX; TNF
Hedgehog signaling 3 0.002163 CSNK1E; CSNK1D; RAB23
Glycolysis/Gluconeogenesis 3 0.003010 ALDH7A1; PFKM; GCK
Drug metabolism cytochrom.p450 3 0.00419 GSTA4; FMO4; CYP3A5
B-cell receptor signaling 3 0.0047 NFKBIA; LYN; NFKBIE
Chronic myeloid leukemia 3 0.0047 NFKBIA; BCL2L1; STAT5A
Phosphatidylinositol signaling 3 0.0048 IMPA2; INPP1; ITPKA
Prostate cancer 3 0.0073 NFKBIA; CDK2; ATF4
NK cell mediated cytotoxicity 3 0.0243 TNFRSF10B; ULBP1; TNF
Jak-STAT signaling 3 0.0327 SOCS3; BCL2L1; STAT5A
Calcium signaling 3 0.0501 ADCY3; ADCY8; ITPKA
Focal adhesion 3 0.0649 MYL2; SRC; PARVB
Computational analysis validating the miRNA targets and mRNAs
Gene expression is regulated at the transcriptional and the post-transcriptional levels. A single miRNA can potentially regulate multiple mRNA; the opposite is also possible [57]. To further validate whether the observed expression profile of mRNA is directly and/or indirectly regulated by miRNA, we performed a comprehensive computational analysis of mRNA and miRNA expression profiles in HIV-1 infected samples as well as in the uninfected controls. The log ratio of infected to control for both mRNA and miRNA was normalized as suggested by the manufacturers' software (Illumina and SABiosciences) to eliminate the false discovery rate and statistical significance across multiple samples. We used the Significance Analysis of Microarray (SAM) [51] and identified 2,941 differentially expressed genes (mRNAs), which we retained for further studies.
Next, we used GenMIR++ [49] to integrate mRNA, miRNA, and sequence-based predictions into an miRNA-mRNA interaction graph (Figure 5A). GenMIR++ uses predicted miRNA targets (Methods) to generate an initial binary interaction matrix for interactions between miRNA and mRNA (Table 5). Based on this table, a linear Gaussian regression model is created for each mRNA by regressing it on the best subset of miRNAs that can potentially interact with it. Coefficients are constrained to be negative to indicate the anti-correlation between miRNAs and their targets. Using this method, we identified the top 275 interactions involving 196 mRNAs and 118 miRNAs and organized them to represent the set of interactions predicted in this dataset (Figure 5A). The results show several clusters of miRNA-mRNA regulation. For example, miR-144, miR-937, miR-376, miR-519, and miR-548A-3P are shown to regulate a number of mRNAs, and HCK, NFKBIE, IL6, SHMT2, and MCM4 are regulated by several miRNAs.
10.1371/journal.pone.0022730.g005Figure 5 Network of predicted miRNA-mRNA interactions.
(A) Regression-based method was used to predict the miRNAs (green circles) that are actively regulating mRNAs (red diamonds) in our dataset. (B&C) Validation of miRNA-mRNA interaction partners by RT-PCR using independent samples (n = 6) for IL-1ß(B) and IL-6 (C).
10.1371/journal.pone.0022730.t005Table 5 Interplay between miRNA and mRNA based on GenMIR ++ analysis.
miRNA Potential targets (mRNA)
let-7d* APP, CKS1B, DDIT4, IL3RA, NFKBIA, PLSCR1
miR-129-3p KIFC1, NAPSB, ORC1L
miR-130a C13ORF18, DLL1, G0S2, GADD45B, STX11, TNF, TNFAIP6
miR-139-5p BATF, KIF1A, RFC3
miR-144 BARD1, BTG3, CEP55, IDH2, IL6, KHDRBS3, LYN, SGOL1, TRIP6, ZC3H12A
miR-193a-5p ERN1, HCK, SEMA7A, SHMT2
miR-196b ADFP, LOR, MCM4
miR-198 CBS, COL9A2, DYSF
miR-214 MCM4, SLC11A1, TMPRSS6
miR-301b DLL1, RAB34, SLC43A2, TFP1
miR-302c FAIM3, IGF1R, PPA1, RSBN1L
miR-33a ABCA1, INDO, PLTP, RAB9A, SAT1
miR-376a C15ORF48, IL6, MMP7, TFP1, TNFAIP6
miR-501-3p C1ORF57, NFKBIE, SASH1, TIMELESS
miR-502-3p CATSPER1, DTX1, IL17F, SP140
miR-515-5p ENPP3, EXO1, FANCG, HES1, MTHFD1L, POLA2
miR-517a BARD1, GNL3, PTGES, RAD54L
miR-518c KIFC1, NFKBIE, RBBP8, WARS
miR-518d-3p KIFC1, MCM2, NFKBIE
miR-519e CD9, CDC2L5, CYB561D2, EBI2, FBP1, MGAT4A, NINJ2, NPL, PYHIN1, RBL2, SORL1, TBC1D10C, TREM2
miR-520g MCM3, MCM4, MTHFD1L, POLQ, UHRF1
miR-526b ACADVL, OIP5, OXCT2, SLC43A3
miR-532-3p CD70, KIFC1, SLC1A5
miR-548a-3p ANKRD57, MEP1A, POLE2, RAD51AP1, RBBP8, SMPDL3A, TYMS
miR-548b-3p C13ORF18, CD70, DUSP5, IER5, IL1B, KYNU, TMEM106C, WBP5
miR-548d-3p CBS, CD70, DUSP5, GNA11, IL1B, KYNU, L2HGDH, PRPF19, IL6
miR-627 TEX264, TRIM47, TXNIP
miR-632 ASF1B, C17ORF53, DOK3, DYSF,
miR-648 AHI1, BCAR3, HELLS, TK1
miR-659 CXXC5, SHMT2, TRO
miR-744 BID, BRSK1, COL9A2
miR-920 BLR1, CDH1, RBPMS2
miR-935 IER3, MND1, PIF1, SHMT2
miR-937 C15ORF48, CABLES1, FHOD1, GADD45G, GNA15, NEU4, PLEKHA4
miR-938 CDCA5, IRF8, TYMS
These miRNAs were selected on the basis that they target at least three mRNAs with the score of >0.50001.
To validate the predicted target and miRNA combinations, we selected two miRNA-mRNA interaction clusters and assessed the level of their expression in multiple donor samples (n = 7). We selected IL-1ß and IL-6, which were upregulated in the HIV-1 infected culture but not in the uninfected control (Figure 5B). Several studies indicate that these two molecules are known to be upregulated by HIV-1 infection under similar conditions [58], [59], [60], although the role of miRNAs in regulating these cytokines is not well defined. Our interaction analyses indicate that IL-1ß could potentially be regulated by miR-211, miR-578, and miR-548d-5p. Results indicate that the expression of IL-1ß mRNA is upregulated by HIV-1 infection by 12.81±6.05-fold, whereas miR-578 is upregulated by 2.0±0.4-fold, miR-548d-5p does not show any change (Figure 5B), and miR-211 is downregulated by 4.2±0.2-fold. This indicate that among the three miRNAs tested, one showed a significant negative correlation, one showed no change, and one exhibited positive correlation. We also tested the IL-6 mRNA-miRNA interaction (Figure 5C), and results indicate a partial confirmation of the predicted results. Together, results from these confirmation analyses indicate 60–70% validation of the predicted negative correlation of miRNA and mRNA expression.
Discussion
Previous studies have performed comparative global profiling of either mRNA or miRNA using HIV-1 infected cells and lymphoid tissue [1], [44], [61], [62], [63]. This is the first study to measure the miRNA and mRNA expression profile at the same time point in PBMCs infected with the HIV-1 virus, with the exception of one study that assessed miRNA and mRNA profiling in brain tissues from infected individuals with dementia [64]. The in vitro PBMC culture system that we used offers several advantages over the use of cells or tissues derived from HIV-1 infected individuals. The in vitro system allows analysis of miRNA and mRNA at precise points after virus infection. It also enables the use of a virus derived from proviral DNA. In combination with the ability to carry out studies using cells from multiple donors, these features may help to identify potential signature patterns with respect to the regulation of gene expression associated with virus infection.
We identified a number of significantly regulated miRNAs and mRNAs in infected PBMCs compared to uninfected control cells. Among the 704 miRNAs tested, 21 are differentially regulated with statistical significance of <0.05 p value, suggesting that virus infection does alter the miRNA expression profile. It is important to note that the significantly regulated miRNAs showed upregulation ranging from 2.1- to 8.2-fold. Although HIV-1 infection differentially regulated certain miRNAs to a higher degree (>22–88-fold), they did not show statistical significance across multiple donors.
It is worth noting that we did not observe any miRNA that is significantly downregulated by virus infection, whereas Houzet et al. [44] reported downregulation of a number of miRNAs in HIV-1 infected PBMCs obtained from HIV-1 positive subjects. It is not clear whether this difference is due to the assay platforms used, the number of miRNAs assessed, or the methodologies used to evaluate the statistical significance. It is also possible that infection level or viral load/burden could influence the level of miRNA expression. The patient population in the Houzet et al. study had relatively low viral loads compared to the in vitro infected cultures. Our results are in line with those from the study by Tatro et al. [64], which reported the dysregulation of miRNA in brain tissue from HIV-1 positive patients compared to that in uninfected control brain tissue. The authors reported that, of the 19 miRNAs that were dysregulated in HIV-1 positive brain tissue compared to uninfected brain tissue, six were also dysregulated in HIV-1 infected PBMCs compared to uninfected cells. They also identified brain-tissue-specific miRNAs, suggesting that their levels in PBMCs may be undetectable or tissue specific as reported before [65], [66].
Given that many pathogens including viruses depend on host cellular machinery for their replication, survival, and immune evasion, the hypothesis that particular viruses alter cellular miRNAs may not come as a surprise. Many viruses including herpes virus, Epstein-Barr virus, HCV, HHV-8, and retroviruses are known to affect host cellular miRNAs [67], [68], [69]. It is not clear, however, whether all these viruses regulate specific miRNAs for replication, immune evasion, and/or immortalization. Searching the literature to identify the miRNAs that are differentially regulated by HCV, HBV, EVB, and herpes viruses, we found a partial overlap among these viruses. For instance, Li et al. [70] performed miRNA profiling during HBV infection and identified 13 miRNAs that are differentially regulated. When we compared these to HIV-1 mediated miRNA regulation, we found that four miRNAs were similarly altered during HIV-1 infection. We also compared the miRNA profiles in two HCV-infected hepatocyte studies [67], [70] with our results from HIV-1 infected PBMCs. We observed 33% and 40% similarity, respectively, in upregulated miRNAs between our results and those of the two studies. However, we did not observe miRNAs that were similarly downregulated in HBV, HCV, or HIV infection.
It is important to keep in mind that the expression profiles were generated by using PMBCs comprising different cell types in our study. On the other hand, the HCV-mediated miRNA profiling was assessed in hepatoma cell lines, and that for EBV-induced miRNA was performed in transformed BLCL cells [67], [68]. There might be cell-type-specific miRNA and/or altered expression level of miRNAs in different cells that could contribute to the observed differences in miRNA profiling during HCV, HIV, or EBV infection. A recent report by Wu et al. [71] provides further support that the level of miRNA expression might vary among different cell types. Collectively, these studies indicate that host cells might respond to pathogens using similar miRNAs as part of the interactions between the pathogen and the host. Conversely, it is also possible to hypothesize that viruses might target certain miRNAs as part of a general virus-host interaction to aid replication and survival. A well-defined comparative analysis is required to address this. Though this manuscript focuses on HIV-1 induced host cellular miRNAs dysregulation, HIV-1 also codes for viral miRNAs that are known to modulate host cell functions and viral transcription [72], [73], [74]. However, the interplay between the viral and host miRNAs is not well defined.
miRNAs regulate the expression of target mRNAs at the post-transcriptional level [75], [76], [77]. It is now well established that a cluster of miRNAs could regulate a single mRNA and vice versa [78], [79]. Our evaluation of the miRNA and mRNA profile (44,000 probes) from RNA isolated at the same time indicates differential regulation of a number of host cellular genes (444) with the FDR-adjusted p value of <0.05. The differentially regulated genes represent pathways including cell cycle, apoptosis, T-cell receptor signaling, DNA repair, and MAPK signaling. These results are in agreement with previously published studies using CD4+ T cells, monocytes, or macrophages infected with HIV-1 [45], [46]. Although these studies used purified specific cell types, their results indicate similar pathways, suggesting that the interaction between the virus and the target cell might trigger similar gene regulation. It is not clear whether similar genes are targeted by HIV-1 in each cell type or whether similar pathways are targeted using cell-type-specific genes. Collective information from the published studies as well as our results support the latter possibility.
A recent transcriptome-analysis study using monocytes isolated from HIV-1 patients (ART naïve and post-ART therapy) identified several innate factors as upregulated [45]; these factors were in addition to the cell proliferation, apoptotic, and signaling genes, and they were not found in our study. This discrepancy could be due to the use of different viruses. In our study, we used a CXCR4-receptor-utilizing virus (NL4-3), which primarily targets T cells. Different target cells might respond differently to the interaction between the virus and the host cell, although miRNAs that are known to regulate virus replication (e.g., miRNA-198) have been found to be commonly regulated by HIV-1 in both T and monocyte/macrophage targets [80].
In this study, to gain insight into the regulation of mRNA by miRNAs, we performed the combined miRNA and mRNA profile in HIV-1 infected PBMCs. Although previous studies have identified similar host cellular pathways regulated by HIV-1, our approach provides new information regarding their regulation at the post-transcriptional level. We observed a negative correlation between the miRNA and mRNA expression profiles, similar to the observations noted with other viral infections [81]. In conclusion, our results suggest that miRNAs play a role in regulating several host cellular genes during HIV-1 infection, altering the host cell response to the virus. Similar approaches including the role of viral load, immune activation, and HAART will provide useful information regarding biomarkers to predict disease development and the effect of HAART during early stages of the disease.
Supporting Information
Table S1
MicroRNAs over expressed in HIV-1 NL43 infected cultures compared to uninfected control cells. Cut-off was 2 fold change.
(DOCX)
Click here for additional data file.
Table S2
MicroRNAs under expressed in HIV-1 NL43 infected cultures compared to uninfected control cells. Cut-off was 2-fold change.
(DOCX)
Click here for additional data file.
We would also like to thank Dr. Alagarsamy Srinivasan for his critical comments and suggestions.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by ARRA supplement to U01 U0-AI35041 by the NIAID/NIH to VA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 21829529PONE-D-11-0307910.1371/journal.pone.0022836Research ArticleMedicineOncologyBasic Cancer ResearchTumor PhysiologyOncology AgentsUpregulation of HYAL1 Expression in Breast Cancer Promoted Tumor Cell Proliferation, Migration, Invasion and Angiogenesis HYAL1 and Breast CancerTan Jin-Xiang
1
2
Wang Xiao-Yi
1
2
Su Xin-Liang
1
2
Li Hong-Yuan
1
2
Shi Yuan
3
Wang Liang
4
Ren Guo-Sheng
1
2
*
1
Department of Endocrine and Breast Surgery, the First Affiliated Hospital of Chongqing Medical University, Chongqing, China
2
Molecular Oncology and Epigenetics Laboratory, the First Affiliated Hospital of Chongqing Medical University, Chongqing, China
3
Department of Anaesthesiology, Children's Hospital of Chongqing Medical University, Chongqing, China
4
Department of General Surgery, the First Hospital of Jiulongpo District, Chongqing, China
Chammas Roger EditorFaculdade de Medicina, Universidade de São Paulo, Brazil* E-mail: [email protected] and designed the experiments: JXT GSR. Performed the experiments: JXT XYW XLS HYL YS LW. Analyzed the data: JXT XYW XLS HYL GSR. Contributed reagents/materials/analysis tools: JXT XYW YS LW. Wrote the paper: JXT GSR.
2011 28 7 2011 6 7 e2283611 2 2011 29 6 2011 Tan et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Hyaluronic acid (HA) is a component of the Extra-cellular matrix (ECM), it is closely correlated with tumor cell growth, proliferation, metastasis and angiogenesis, etc. Hyaluronidase (HAase) is a HA-degrading endoglycosidase, levels of HAase are elevated in many cancers. Hyaluronidase-1 (HYAL1) is the major tumor-derived HAase. We previously demonstrated that HYAL1 were overexpression in human breast cancer. Breast cancer cells with higher HAase expression, exhibited significantly higher invasion ability through matrigel than those cells with lower HAase expression, and knockdown of HYAL1 expression in breast cancer cells resulted in decreased cell growth, adhesion, invasion and angiogenesis. Here, to further elucidate the function of HYAL1 in breast cancer, we investigated the consequences of forcing HYAL1 expression in breast cancer cells by transfection of expression plasmid. Compared with control, HYAL1 up-regulated cells showed increased the HAase activity, and reduced the expression of HA in vitro. Meantime, upregulation of HYAL1 promoted the cell growth, migration, invasion and angiogenesis in vitro. Moreover, in nude mice model, forcing HYAL1 expression induced breast cancer cell xenograft tumor growth and angiogenesis. Interestingly, the HA expression was upregulated by forcing HYAL1 expression in vivo. These findings suggested that HYAL1-HA system is correlated with the malignant behavior of breast cancer.
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Introduction
Extra-cellular matrix (ECM) is closely correlated with tumor progression. Hyaluronic acid (HA) is a component of the ECM, it is an unsulfated anionic linear glycosaminoglycan polymer comprised of a repeating glucuronic acid and N-acetylglucosamine disaccharide motif [1]. HA keeps tissues hydrated, maintains osmotic balance and cartilage integrity [2]–[3]. HA also actively regulates cell adhesion, migration, and proliferation by interacting with specific cell surface receptors such as CD44 and RHAMM [4]. The concentration of HA is elevated in several inflammatory diseases and various carcinomas, including bladder, prostate, breast, lung, colon, and so forth [5]–[10]. Small fragments of HA, generated by Hyaluronidase (HAase), stimulate angiogenesis [11], [12]. In tumor tissues, it may promote tumor growth and metastasis probably by actively supporting tumor cell migration and offering protection against immune surveillance [13].
HAases are a class of enzymes that predominantly degrade HA, they are endoglycosidases, as they degrade the β-N-acetyl-D-glucosaminidic linkages in the HA polymer [1]. HAase has been shown to alter the expression of CD44 isoforms, which may also be involved in tumor progression [14]. In addition, HAase is associated with increased tumor cell cycling [15]. The HAase levels serve as an accurate marker for detecting prostate and bladder tumors [9]–[10]. In humans, six HAase genes have been identified. Hyaluronidase-1 (HYAL1) was originally purified from human plasma and urine [16]–[17], it is the major tumor-derived HAase expressed in bladder and prostate cancer tissues, and it has characterized expression at the mRNA and protein levels in tumor cells [9], [10]. HYAL1 is a ∼55–60 kDa protein, and it is consisted with 435 amino acids. An elevated level of HYAL1 has been found in prostate, bladder, breast, head and neck cancers, etc [9]–[10], [18]–[20]. HYAL1 was the first HAase to be recognized as being expressed by tumor cells and its expression correlates with their invasive and metastatic potential [9]. No HYAL1 expression is observed in the tumor-associated stroma, although HYAL1 expression appears to correlate and perhaps induce HA production in the tumor-associated stroma [21]–[22].
Among the six HAases, HYAL1 and Hyaluronidase-2 (HYAL2) are widely distributed to degrade high molecular weight (MW) HA [23]. The HYAL2 cleaves high MW HA into ∼20 kDa HA fragments [24], which are transported intracellularly and further digested into low MW HA fragments by HYAL1 [25]. The small angiogenic HA fragments stimulate endothelial cell proliferation, adhesion and migration by activating the focal adhesion kinase and mitogen-activated protein kinase pathways [26]. HYAL1 has been found as an independent predictor of biochemical recurrence [27]. HAase levels also increase in breast cancer cells when they become metastatic [18].
We previously demonstrated that HYAL1 protein and activity were overexpression in breast cancer tissues and cells [19], [28], and breast cancer cells with higher HAase expression, exhibited significantly higher invasion ability through matrigel than those cells with lower HAase expression [28]. Knockdown of HYAL1 expression in breast cancer cells resulted in decreased cell growth, adhesion, invasion and angiogenesis [19], [29]. In this study, to further elucidate the function of HYAL1 in breast cancer, we demonstrated that forcing expression of HYAL1 in breast cancer cells promoted tumor progression in vitro and in vivo. We therefore provided functional evidence that HYAL1 is oncogenic for breast cancer and functional antagonism of HYAL1 constitutes a potential therapeutic strategy for HYAL1 positive breast cancer.
Results
Identification of the recombinant plasmid
The HYAL1 expressing plasmids were constructed using the eukaryotic expression vector pcDNA3.1(+). The positions of the restriction enzyme digestion sites were marked (Fig. 1A). After sequence analysis, transfection of the recombinant plasmid pcDNA3.1-HYAL1 or pcDNA3.1 empty vector into MCF7 cells, respectively. After stably transfected cells were individually selected, the HYAL1, HYAL2 and the housekeeping gene β-actin mRNA levels were measured using RT-PCR. Compared with the control group, the pcDNA3.1-HYAL1 transcripts were sharply up-regulated (Fig. 1B). Accordingly, HYAL1 protein levels were increased in pcDNA3.1-HYAL1 transfected cells too, as indicated by western blotting (Fig. 1C) and immunofluorescence (Fig. 1D) analyses. The pcDNA3.1 empty vector did not substantial affect the endogenous HYAL1 expression, and there was no difference in the HYAL2 expression among groups. These results demonstrated that pcDNA3.1-HYAL1 was up-regulated specifically and effectively.
10.1371/journal.pone.0022836.g001Figure 1 Identification of the recombinant plasmid.
Construction of the recombinant eukaryotic expression vector pcDNA3.1(+) containing the HYAL1 gene used in the studies. The positions of the restriction enzyme digestion sites were marked (A). Transfection of the recombinant plasmid pcDNA3.1-HYAL1 or pcDNA3.1 empty vector into MCF7 cells, respectively. RT-PCR (B), Western blot (C) and immunofluorescence (D) analysis validated the successful transfection and the expression of the HYAL1 mRNA and protein were up-regulated in pcDNA3.1-HYAL1, the pcDNA3.1-HYAL1 did not substantially affect the HYAL2 expession.
Upregulation of HYAL1 in breast cancer cells increased colony formation and enhanced cell proliferation
To determine the functional consequences of forcing HYAL1 expression in human breast cancer cells, we generated the breast cancer cell lines MCF7 and ZR-75-30 with upregulation of HYAL1 by stable transfection with HYAL1 expression vector (termed MCF7-HYAL1 and ZR-HYAL1) or the empty pcDNA3.1 vector (designated MCF7-Vec and ZR-Vec), respectively. Soft agar assays were then performed to determine whether upregulation of HYAL1 could enhance colony formation in an anchorage-independent culture system. In soft agar assays, the number of colonies of MCF7-HYAL1 and ZR-HYAL1 were 42.2±4.6 and 34.9±4.8, however, the number of colonies of MCF7-Vec and ZR-Vec were 31.1±5.4 and 26.2±3.6, respectively. There was a statistically significant increase in the number of colonies of pcDNA3.1-HYAL1 compared to pcDNA3.1-Vec control cells (Fig. 2A; p<0.01). In 3D Matrigel cultures, the size (µm) of colonies of MCF7-HYAL1 (53.2±16.0) and ZR-HYAL1 (48.8±21.5) were bigger than MCF7-Vec (38.3±12.8) and ZR-Vec (39.1±10.9), respectively (Fig. 2B; p<0.05). Then, we examined the proliferation activity of the transfected cells using MTT assay. As shown in Fig. 2C, compared with the controls, the proliferation rates of MCF7-HYAL1 and ZR-HYAL1 transfected cells were increased significantly (p<0.01). These results indicated that HYAL1 is correlated with cell proliferation.
10.1371/journal.pone.0022836.g002Figure 2 Upregulation of HYAL1 in breast cancer cells increased colony formation and enhanced cell proliferation.
MCF7-HYAL1 and ZR-HYAL1 enhanced the number of colony formation in Soft agar (A) and size of colony in 3D Matrigel (B) (p<0.01, p<0.05, respectively), (original magnification ×100). MTT assay showed that MCF7-HYAL1 and ZR-HYAL1 increased cell proliferation rates (C; p<0.01).
Upregulation of HYAL1 expression increased HAase activity, and reduced HA expression
HAase ELISA-like assay was carried out as described previously. As shown in Fig.3A, the amount HAase activity (mU/mg) in MCF7-HYAL1 (14.78±2.59) and ZR-HYAL1 (12.69±2.78) were higher than MCF-Vec (5.34±1.07) and ZR-Vec (4.60±1.09), respectively (p<0.01). However, compare with MCF-Vec (0.50±0.10) and ZR-Vec (0.43±0.11), the HA levels (µg/mg) in MCF7-HYAL1 (0.35±0.09) and ZR-HYAL1 (0.26±0.08) were decreased, respectively (Fig.3B; p<0.05). These results demonstrated that pcDNA3.1-HYAL1 can enhance the HAase activity, and the HYAL1 could degrade HA.
10.1371/journal.pone.0022836.g003Figure 3 Summary of HAase activity and HA expression by ELISA-like assay.
In MCF7 and ZR-75-30 cells, the HAase activity was increased significantly by pcDNA3.1-HYAL1 transfectants (A; p<0.01). However, the HA expression was decreased (B; p<0.05).
Upregulation of HYAL1 expression promoted breast cancer cell cycling
To determine whether HYAL1 regulates cell cycle progression induced by growth factor, Flow cytometry was performed to analyze cell cycle phases. Compared with MCF7-Vec and ZR-Vec, both in MCF7-HYAL1 (Fig. 4A) and ZR-HYAL1 (Fig. 4B) transfectants, the number of cells in G0/G1 phase were decreased, but those in S phase were increased (p<0.05, p<0.01, respectively). No alterations in G2/M phase were observed (p>0.05). These results indicated that HYAL1 is correlated with cell cycle in vitro.
10.1371/journal.pone.0022836.g004Figure 4 Upregulation of HYAL1 effected on cell cycle.
The MCF7-HYAL1 (A) and ZR-HYAL1 (B) transfectants had a higher portion in S phase and a lower portion in G0/G1 phase ((p<0.05, p<0.01, respectively)). No alterations in G2/M phase were observed (p>0.05).
Upregulation of HYAL1 expression enhanced the cell migration, invasion and angiogenesis potential of breast cancer cells
One of the most distinct features of breast cancer cell is the invasive growth pattern and promotion in angiogenesis, which prevents total surgical resection. Therefore, we evaluated the effect of HYAL1 upregulation on cell migration, invasion and angiogenesis properties.
To study the effect of HYAL1 upregulation on the cell migration, wound-healing assays were carried out by allowing the cells to move to the scar region for 24 h and 48 h. We observed that forced expression of HYAL1 in both MCF7 and ZR-75-30 cells did indeed stimulate wound closure compared with their respective vector-transfected cells (Fig. 5A and B; p<0.05, p<0.01, respectively).
10.1371/journal.pone.0022836.g005Figure 5 Upregulation of HYAL1 in breast cancer cells promoted cells migration in vitro.
The MCF7-HYAL1 (A) and ZR-HYAL1 (B) transfectants promoted wound closure compared with their respective empty vector transfected cells (p<0.05, p<0.01, respectively), (original magnification ×100).
Invasion assays were performed using ECM gel-coated transwell culture chambers. After 48 h of incubation, invading cells were counted. We observed that forced expression of HYAL1 in both MCF7 and ZR-75-30 cells did indeed stimulate cell invasion compared their respective vector-transfected cells (Fig. 6A; p<0.01). The potential of promoting angiogenesis was analysed by in vitro angiogenesis assay. CRL-1730 cells formed branch-like or net-like structures when co-cultured with MCF7 and ZR-75-30 cells, which were not seen in the absence of tumor cells. The numbers of branch-like and net-like structures formed by CRL-1730 co-cultured with MCF7 cells were acutely increased in the MCF7-HYAL1. Similarly, in the ZR-75-30 and CRL-1730 co-culture system, compared with ZR-Vec, the numbers of branch-like and net-like structures in ZR-HYAL1 group was increased obviously. (Fig. 6B; p<0.05). These results demonstrated that HYAL1 is correlated with cell migration, invasion and angiogenesis potential of breast cancer cells.
10.1371/journal.pone.0022836.g006Figure 6 Upregulation of HYAL1 in breast cancer cells promoted cell invasion and angiogenic capability in vitro.
Invasion assays (A) were performed using ECM gel-coated transwell culture chambers. 48 h after seeding for both MCF7 and ZR-75-30, the number of invasive cells was increased in pcDNA3.1-HYAL1 transfectants (p<0.01). Angiogenesis assay (B) for CRL-1730 cells formed branch-like or net-like structures through co-culture with MCF7 and ZR-75-30 cells. After 96 h co-culture, the number of vessels developed by CRL-1730 cells was enhanced in pcDNA3.1-HYAL1 transfectants (p<0.05), (original magnification ×100).
Upregulation of HYAL1 expression promoted the tumorigenesis of breast cancer cells in vivo
To evaluate the tumorigenetic capability of HYAL1 upregulation tumor cells in vivo, 20 mice were injected into the mammary fat-pad with 1×107 MCF7 cells, and the tumor growth was monitored for a period of 9 weeks. These mice were randomly divided into 2 groups (MCF7-Vec and MCF7-HYAL1) with 10 mice in each group. All of mice developed tumors at the end of the experiment. However, the mice treated with MCF7-HYAL1 cells showed a significant enhancement of tumor growth compared with those treated with MCF7-Vec cells (Fig. 7A; p<0.01). 9 weeks after inoculation, the average tumor weight in MCF7-HYAL1 (3.18±0.64 g) was heavier than MCF7-Vec (1.91±0.49 g) (Fig. 7B; p<0.01). Western blot (Fig. 7C) and immunohistochemistry (Fig. 7D) analysis confirmed that the HYAL1 protein expression was significant enhanced in the MCF7-HYAL1 group. The HYAL2 was not changed obviously. Ki67 is a molecular maker that is related to proliferation, was located in the nucleus. Compared with MCF7-Vec, the ki67 was highly expressed in MCF7-HYAL1 group (Fig. 7D). CD31 is an endothelial marker, so the microvessel density (MVD) of the tumor tissue was assessed by CD31 immunohistochemistry analysis. Compared with MCF7-Vec, the expression of CD31 in MCF7-HYAL1 was increased obviously (Fig. 7D). In addition, compared with MCF7-Vec (7.26±1.56) group, the HAase activity (mU/mg) in the MCF7-HYAL1 (18.42±3.23) group was enhanced significantly (Fig. 7E; p<0.01). At same time, the HA expression level (µg/mg) in MCF7-HYAL1 (15.69±3.63) was higher than MCF7-Vec (11.75±2.21) (Fig. 7F; p<0.05). These data indicated that HYAL1 could promote tumor proliferation and angiogenesis in vivo, and the HA expression was increased in tumor tissue during enhancing tumor malignant potentiality, although the HYAL1 could degrade HA.
10.1371/journal.pone.0022836.g007Figure 7 Upregulation of HYAL1 increased tumor growth, angiogenesis, HAase activity and HA expression of MCF7 cells in vivo.
Representative mouse bearing tumors, the average tumor volume (A) from 5th weeks and tumor weight (B) in MCF7-HYAL1 group were increased significantly than in MCF7-Vec group (p<0.01). Western blot (C) and immunohistochemistry (D) analysis showed that the expression of HYAL1 protein was enhanced in the MCF7-HYAL1 group obviously, but HYAL2 levels were not altered. Compared with MCF7-Vec, expression of ki67 and CD31 were increased in MCF7-HYAL1 (D). ELISA-like assay measured HAase activity (E) and HA levels (F) present in the tissue extracts, the HAase activity and HA levels in MCF7-HYAL1 were higher than MCF7-Vec (p<0.01, p<0.05, respectively).
Discussion
In this study, the eukaryotic expression plasmid pcDNA3.1-HYAL1 was constructed to force HYAL1 expression in breast cancer cell lines MCF7 and ZR-75-30. Our results showed that upregulation of HYAL1 resulted in cell growth increase in vitro and in vivo (Fig. 2, 7A and 7B). It was also identified that HYAL1 expression in bladder cells regulated tumor gowth [22]. These results suggested that HYAL1 expression in tumor cells is required for cell proliferation. Meanwhile, upregulation of HYAL1 expression enhanced the proportion of cells cycling in S phase (Fig. 4), which is consistent with Lin et al. [15] and our previous researches [19], [29]. Based on the analysis of cell cycle regulators, HYAL1 affects cell proliferation probably by regulating cell cycle.
Our finding that upregulation of HYAL1 in breast cancer cells could enhance the HAase activity significantly (Fig. 3A), and the HA expression was decreased obviously (Fig. 3A) in vitro, these results identified that HYAL1 could degrade HA. Which was according with previous researches [19], [25]. Interestingly, upregulation of HYAL1 expression enhanced the HAase activity (Fig. 7E), at the same time, the HA expression was increased (Fig. 7F) in vivo. Lokeshwar et al. [22] found that high level of HA was expression in tumor-associated stroma of HYAL1-sense tumor specimen, but very low HA expression was observed in the stromal compartment of HYAL1-antisense tumor specimens. Which indicated the HYAL1 could induce the stroma cells of tumor to secrete HA, although it could cleave HA.
In addition to the effect of HYAL1 on tumor growth, its effects on tumor cell migration and invasion are interesting. Our previous researches showed that breast cancer cells with higher HAase expression, exhibit significantly higher invation ability through matrigel than those cells with lower HAase expression [28]. Knockdown of HYAL1 expression in breast cancer cells resulted in decreased cell invasion [19]. HYAL1 was also an independent prognostic indicator for predicting biochemical recurrence in prostate cancer and increased metastatic potential in a prostate cancer model [27]. In the current study, we demonstrated that upregulation of HYAL1 expression in MCF7 and ZR-75-30 cells resulted in high metastasis potential and altered several functions such as cell migration and invasion in vitro (Fig. 5 and 6A). These observations suggested that HYAL1 plays a role in promoting the invasive potential of breast cancer cells. It might also be a marker predicting subsequent development of invasive breast cancer.
One of the well-studied functions of the HA and HAase system is the generation of angiogenic HA fragments [30]. These angiogenic HA fragments have been shown to induce endothelial cell proliferation, migration, and adhesion [26], [31]. The secretion of HAase by tumor cells has been shown to induce angiogenesis [32]. Angiogenic HA fragments are present in the urine of grade 2 and 3 bladder cancer patients, suggesting that the HA and HYAL1 system is active in bladder cancer [33]. The HYAL1 and HYAL2 are widely distributed and degrade high MW HA in collaboration with CD44 [23]. We previously demonstrated that knockdown of HYAL1 expression in breast cancer cells resulted in decreased angiogenesis [19]. In this study, we showed that upregulation of HYAL1 expression induced higher angiogenesis in vitro and in vivo (Fig. 6B and 7D). This was in accordance with previous reports that HYAL1 over-expression increased MVD in rat colon carcinoma xenografts [34], as well as the correlation of HYAL1 with MVD in bladder tumor [22]. Which suggests that HYAL1 promotes tumor angiogenesis might be a general effect. Further studies characterizing this in other cancer models would be interest.
At present, whether HAase is a tumor promoter or a repressor has been controversial. The results presented in this study showed that forcing HYAL1 expression promoted tumor growth, invasion and angiogenesis supporting its role as a tumor promoter. HYAL1 levels in various cancers were associated with high-grade invasive tumors [9]–[10], [20], [27]. However, Jacobson et al. [34] found that the overexpression of HYAL1 by cDNA transfection in a rat colon carcinoma line decreased tumor growth, although the tumors were angiogenic. HYAL1 and HYAL2 have been identified to inhibit lung and renal carcinoma cell growth in vivo but not in vitro [35]. Nykopp TK, et al found that HYAL1 and HYAL2 were coexpressed and significantly downregulated in endometrioid endometrial cancer and correlated with the accumulation of HA [36]. The controversy surrounding HAase as a tumor promoter or a suppressor was recently explained by Lokeshwar et al [1], [21]. Selection of cells for expression of different HYAL1 levels showed that cells expressing amounts found in tumor tissues and cells promote tumor growth, invasion and angiogenesis. In contrast, cells with HAase levels exceeding 100 milliunits/106 cells, (i.e.; levels that are not naturally expressed by tumor cells) exhibit reduced tumor incidence and growth due to induction of apoptosis. Therefore, the function of HAase as a tumor promoter or a suppressor is a concentration dependent phenomenon and levels in genitourinary tumors are consistent with tumor cell derived HAase acting mainly as a tumor promoter.
It is also noteworthy that other proteins related to HA synthase (HAS) and HA-receptor CD44 and RHAMM are also involved in tumor growth and metastasis. For example, overexpression of HAS2, HYAL2 and CD44 is implicated in the invasiveness of breast cancer [37]. Blocking HAS3 expression in prostate cancer cells decreased cell growth in vitro and tumor growth in vivo [38]. Silencing of HAS2 suppressed the malignant phenotype of invasive breast cancer cells [39]. HAS2 expression induced mesenchymal and transformed properties in normal epithelial cells, but interestingly, HAS2 expression in the absence of HAase decreased tumor growth in glioma cells. Moreover, interaction between RHAMM and HA fragments was known to induce the mitogen-activated protein kinase pathway, and over-expression of RHAMM was a useful prognostic indicator for breast cancer [40]. Down-regulated CD44 and HA synthase while upregulating the HAases, suggested that dynamic feedback signalling and complex mechanisms occur in the net deposition of HA [41]. These results showed that the HAS-HA-HAase system is involved in the regulation of tumor growth and invasion.
Summarizing the observations by us and others, we favor the hypothesis that HYAL1 may play a critical role in the longevity of a wide spectrum of breast cancer cells. In our study, upregulation of HYAL1 promoted the cell growth, migration, invasion and angiogenesis. Interestingly, forcing HYAL1 expression induced stoma cells of tumor to secrete HA in vivo, although HYAL1 could cleave HA. To date the expression pattern and function of the HYAL1 gene in human tumors are not completely elucidated. As to the mechanism of how HAS-HA-HAase system influences the biology characteristics of human breast cancer cells, more investigations will be accomplished in the future.
Materials and Methods
Cell lines and cell culture
The human breast cancer cell lines MCF7 and ZR-75-30, mouse embryonal fibroblast cell line HIH-3T3 and human umbilical vein endothelial cell line CRL-1730 were acquired from the cell bank of Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. These cells were maintained with RPMI1640 medium (Gibco BRL), supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco BRL). The medium was replaced every 2 d, cells were passaged every 5 to 6 d, checked routinely and cultivated in a 5% (v/v) CO2 incubator at 37°C.
Plasmid construction and transfection
Total RNA was extracted from breast cancer cells using a RNA extraction kit (Invitrogen). Total RNA (∼1 µg) was subjected to first strand cDNA synthesis using a Superscript™ pre-amplification system and oligo(dT) primers (Invitrogen). The PCR primers for the entire coding region of HYAL1 (611-1918) designed with HYAL1 cDNA sequence (Genebank: U96078), the forward primer was 5′-GAGAAGCTTGCCGCCATGGCAGCCCACCTGCTTCCC-3′, the reverse primer was 5′-CAATTGTCACCACATGCTCTTCCGCTCACACCA-3′. PCR conditions were as follows: 4 min for pre-denaturation at 94°C, then 94°C for 15 s, 55°C for 15 s and 72°C for 30 s for 30 cycles, and a final extension at 72°C for 10 min. The PCR product (1300 bp) was purified and restrictively digested with Hind III and Mfe I, the plasmid pcDNA3.1(+) (Invitrogen) was purified and restrictively digested with Hind III and EcoR I, then cloned the PCR product into pcDNA3.1 to form pcDNA3.1-HYAL1, the pcDNA3.1 vector was treated as a control.
For transfection, MCF7 and ZR-75-30 were seeded in six-well plates at 5×105/well and incubated in 5% CO2 95% air incubator at 37°C. When cells were ∼70% confluence, cells were transfected with Lipofectamine 2000 (Invitrogen) transfection reagent according to the manufacturer's protocol. Recombinant plasmid (4 µg) were mixed with Lipofectamine and pre-incubated for 20 min at room temperature in serum-free and antibiotic-free RPMI1640. MCF7 cells transfected with pcDNA3.1-HYAL1 and pcDNA3.1 vector were named as MCF7-HYAL1 and MCF7-Vec, ZR-75-30 cells transfected with pcDNA3.1-HYAL1 and pcDNA3.1 vector were named as ZR-HYAL1 and ZR-Vec, respectively. G418 (500 µg/µl) was applied to stable screeing and isolating the resistant colonies. At the same time, corresponding empty vector was transfected as control.
Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA extraction was same as before, following manufacturer's instructions. The reverse transcription was carried out with a SuperScript first-strand synthesis system (Invitrogen) using Oligo(dT)12-18 primers. cDNA was amplified by Taq DNA polymerase (Promega, USA). Human β-actin gene was used as an internal control. Each PCR program involved a initial step denaturation 5 min at 94°C, followed by 28 cycles (for HYAL1, HYAL2 and β-actin) at 94°C for 20 s, 62°C for 20 s and 72°C for 30 s, at last 72°C for 10 min. DNA primer sequences were designed as follows: for human HYAL1 gene (Accession No. U96078), Sense: 5′-TGGATGGCAGGCACCCTCCA-3′, and antisense strand: 5′-CACCAGCAGCCACAGCCACA-3′, the amplicon size is 289 bp. For human HYAL2 gene (Accession No. U09577), Sense: 5′-TGGCCCGCAATGACCAGCTG-3′, and antisense strand: 5′-GCCGCACTCTCGCCAATGGT-3′, the amplicon size is 262 bp. For β-actin gene (accession No. NM_001101), sense: 5′-AAATCTGGCACCACACCTTC-3′, reverse primer, 5′-GGGGTGTTGAAGGTCTCAAA-3′, the amplicon size was 139 bp. The PCR amplified products were separated by 1.5% agarose gels electrophoresis, and the bands were visualized by staining with 0.5% Goldview. Gel images were obtained and the densities of PCR products were quantified by using densitometry methods.
Western blot analysis
The cells and fresh tissue specimens (0.5–1 g) were harvested and total protein was extracted with RIPA buffer containing protease inhibitors, protein concentration was determined by the Bradford assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein were subjected to 7.5% SDS–polyacrylamide gel and the resolved proteins were transferred electrophoretically to PVDF membranes (Millipore, Bedford, MA, USA). Membranes were blocked for 1 h with 5% non-fat milk in PBST buffer at room temperature, then were incubated with antibodies against HYAL1 (rabbit polyclonal; 1∶500; Sigma, USA), HYAL2 (rabbit polyclonal; 1∶500; Abcam, UK), and β-actin (rabbit polyclonal; 1∶1000; Santa Cruz, CA) for overnight at 4°C, respectively. The membranes were washed for three times with PBST, and then were incubated with the respective secondary antibodies for 1 h at room temperature. Membranes were incubated with enhanced chemiluminescence (ECL) (Pierce, Rockford, IL, USA), exposed to X-ray film for 1–2 min. The results were analyzed by using the Quantity One 4.6.3 software (Bio-Rad, Hercules, CA).
Immunofluorescence
The transfected cells were cultured on sterile cover slips for 24 h and washed twice with cool PBS. Cells were then fixed with 2% formaldehyde, permeabilized with 0.1% Triton X-100, blocked with 2% BSA for 30 min at room temperature, and incubated with the primary antibody overnight at 4°C. Samples were washed, incubated with the secondary antibody for 30 min. Immunofluorescence was analysed using a confocal microscope.
Soft agar growth assay
pcDNA3.1-Vec or pcDNA3.1-HYAL1 transfected cells (0.5×105 cells/well in 6-well plate) were suspended in 3 ml RPMI1640 containing 10% FBS (pre-warmed to 37°C), and 300 µl of 3% agarose in PBS (pre-warmed to 60°C) was added. Agar suspended cells (1 ml/well in 6-well plates) were plated out in dishes coated with 1 ml of agar-coated dishes (0.6% agarose in RPMI1640). After solidification at room temperature for 20 min, 3 ml complete medium was added to each well and cells were incubated at 37°C in a humidified atmosphere of 5% CO2 (v/v) in air for 2 weeks. After this period, 10 fields were randomly selected and the numbers of colony were counted under a microscope.
3D Matrigel culture
Matrigel basement membrane matrix (BD Biosciences, San Jose, CA) was thawed on ice and coated onto 24-wells cluster plates as a bottom layer. Subsequently, a total of 5,000 cells in 100 µl growth medium resuspended with 100 µl Matrigel was seeded on top of this bottom layer. Growth medium (0.5 ml) was added on top of the Matrigel after the polymerization was completed. Experiments were performed in triplicate. After 10 days, colonies were counted and colony sizes were measured under the stereomicroscope at 100× magnification.
MTT proliferation assay
The capability of cell proliferation was measured by [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] MTT assay. Briefly, cells were plated at 5×103 cells/well in 96-well plates and incubated for 0, 2, 4, 6, 8 and 10 d, respectively. Then cells were incubated with 20 µl MTT (10 mg/ml) for 4 h at 37°C and 100 µl DMSO (Sigma Chemical Co., USA) was pipetted to solubilize the crystal product for 10 min at room temperature. The absorbance (A) of each well was measured with a microplate reader (Bio-Rad) at a wavelength of 490 nm. This experiment was repeated three times.
ELISA-like assay for HAase activity
HAase activity in serum-free conditioned medium of transfectants was assayed using the HAase ELISA-like assay as described previously [19]. Fresh tissue specimens (∼0.5–l.0 g) were suspended in an ice-cold homogenization buffer (5 mM Hepes pH 7.2 and 1 mM benzamidine-HCl) and homogenized for 30 s using the tissue homogenizer. The tissue extracts were clarified by centrifugation at 10,000 rpm for 30 min. The extracts were assayed for protein concentration, HAase activity and HA expression.
ELISA-like assay for HA
The HA ELISA-like assay was described previously [19]. Briefly, ELISA plates coated with HA binding protein were incubated with samples or standards (1 h, room temperature) in triplicate, washed four times with washing buffer, incubated with a solution containing horseradish peroxidase-conjugated HA-binding protein (30 min, room temperature), washed again four times, and incubated with 100 µl of the substrate solution in the dark at room temperature. After 30 min, the reaction was stopped by adding 50 µl stop solution to each well, and the absorbance was measured at a wavelength of 405 nm.
Flow cytometry assay
Different cell cycle phases (G0/G1, S and G2/M phase) are characterized by different DNA contents. The control, pcDNA3.1-Vec and pcDNA3.1-HYAL1 transfected cells were harvested with trypsin-EDTA, washed with chilled PBS twice and fixed with 70% ethanol at −20°C for 2 h, respectively. The fixed cells were pelleted, re-suspended in PI/RNase/PBS (100 µg/ml propidium iodide and 10 µg/ml RNase A) for at least 30 min at 37°C in dark. Cell cycle analysis was performed on flow cytometer. This experiment was repeated three times.
Wound healing assay
Cells were cultured in standard conditions, as described above. Until 100% confluence, the migration potency was determined using scratch wound healing assay. The scratched plates were photographed at the center of the wells using a standard magnification of 100×. The scratched cells were maintained under standard conditions for 24 hr and 48 hr. Plates were washed once again, and then the plates were photographed at the same sites of the wells using the same magnification. The cells migrating into the scratched area from the wound edge per picture were counted. The impact of pcDNA3.1-HYAL1 on cell migration potency was evaluated by comparing the mean of migration width with pcDNA3.1 empty vector.
Cell invasion assay
Invasion capability in vitro was measured in transwell chambers (Costar Inc, USA) according to the protocol of the manufacturer. Briefly, the upper chambers of the transwell inserts were coated with 100 µl diluted ECM gel solution at 37°C for 4 h, and then pretreated with serum-free RPMI1640 medium at 37°C for 1 h before seeding the breast cancer cells at a density of 1×105/well in 100 µl medium with 1% FBS. The lower chambers were filled with 500 µl RPMI1640 with 10% FBS and HIH-3T3 contained medium as a chemoattractant. The transwells were then incubated at 37°C with 5% CO2 for 48 h to allow cells to migrate. At the end of incubation, the cells on the upper side of the insert filter were completely removed by wiping with cotton swab, and the cells that had invaded through the ECM gel-coated filter were fixed in ethanol and stained with H&E. For quantification, the cells were counted under a microscope on 5 random fields at 100×. This experiment was repeated three times.
Angiogenesis assay
Human umbilical vein endothelial cell line CRL-1730 were allowed to grow in transwell chambers coated with ECM gel as described above for the invasion assay (2×105 cells/chamber). At the same time, the breast cancer cells were plated into 6-well plates (2×105 cells/well), and cultured overnight. Then the transwell chambers were set in the 6-well plates where tumor cells were already added. The cells were co-cultured for 96 h, and the medium was changed every 24 h. The transwell chambers were washed three times with PBS, fixed in ethanol and stained with H&E. The membranes were carefully taken out of the chamber, set on glass slides, covered with a coverslip, observed under microscope, and documented with a digital imaging system (Leica MD20, Germany). The degree of angiogenesis was measured by the following method: number of branch points and the total number of branches per point, with the product indicating the degree of angiogenesis. This experiment was repeated three times.
In vivo assay
Balb/c nude mice were purchased from Vital River Lab Animal Technology Co. Ltd, and maintained under specific-pathogen-free conditions in Experimental Animal Department of the Chongqing Medical University (Chongqing, China). The protocols were approved by the Institutional Animal Care and Use Committee in Chongqing Medical University (CQMU-2008-127). Mice were randomly divided into 2 groups (MCF7-Vec and MCF7-HYAL1) with 10 mice in each group. MCF7 cells at 1×107 cells per l00 µl of serum-free medium were injected orthotopically into the mammary fat-pad of 6 weeks old female Balb/C nude mice. Tumor diameters were measured one time per week with a caliper, and the volume of tumors were calculated by the following formula: x × y2/2, where x is the largest diameter of the tumor and y is the shortest diameter. At 9th week after the injection, the tumors were harvested for western blot, immunohistochmistry, HAase activity and HA levels analysis.
Immunohistochemistry staining
The tissue sections (∼5 µm) were de-paraffinized in xylene, rehydrated in graded ethanol solutions, permeabilized in 0.1% Triton X-100 and 0.1% sodium citrate. The endogenous peroxidase activity was quenched by incubation of the sections in 0.3% hydrogen peroxide with methanol. Subsequently, the slides were treated with 1% bovine serum albumin for 30 min to reduce nonspecific binding, followed by incubation overnight with antibody against HYAL1 (rabbit polyclonal; Sigma, USA), HYAL2 (rabbit polyclonal; Abcam, UK), Ki67 (rabbit polyclonal; Abcam, UK) and CD31 (rabbit polyclonal; Abcam, UK) at a dilution of 1∶200, respectively. After washing, the slides were further incubated with HRP-conjugated secondary antibody followed by 3,3-diaminobenzidine solution at 4°C for 30 min. Hematoxylin was used for nucleus counterstaining. For negative controls, the antibody was replaced by normal goat serum. Slides were photographed with microscope attached with CCD camera.
Statistical analysis
Results are represented as means ± standard deviation (SD). Paired and 2-tailed Student's t-tests were used to compare results from the experiments. A p-value of less than 0.05 was considered statistically significant.
We thank Dr Qi Cai for critical reading of the manuscript, and we would like to thank all our colleagues and collaborators who have participated in this work.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by National Natural Science Foundation of China (Number: 30371393). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 21829575PONE-D-11-1117610.1371/journal.pone.0023000Research ArticleMedicineDermatologySkin NeoplasmsMalignant Skin NeoplasmsMelanomasBenign Skin NeoplasmsDermatologic PathologyOncologyBasic Cancer ResearchTumor PhysiologyCancers and NeoplasmsSkin TumorsMalignant MelanomaCancer PreventionCancer TreatmentSilymarin Targets β-Catenin Signaling in Blocking Migration/Invasion of Human Melanoma Cells Silymarin Inhibits Melanoma Cell InvasionVaid Mudit
1
Prasad Ram
1
Sun Qian
1
Katiyar Santosh K.
1
2
3
*
1
Department of Dermatology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America
2
Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama, United States of America
3
Birmingham VA Medical Center, Birmingham, Alabama, United States of America
Zhang Lin EditorUniversity of Pennsylvania School of Medicine, United States of America* E-mail: [email protected] and designed the experiments: SKK MV. Performed the experiments: MV RP QS. Analyzed the data: SKK MV RP QS. Contributed reagents/materials/analysis tools: SKK. Wrote the paper: SKK.
2011 28 7 2011 6 7 e2300022 6 2011 5 7 2011 Vaid et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Metastatic melanoma is a leading cause of death from skin diseases, and is often associated with activation of Wnt/β-catenin signaling pathway. We have examined the inhibitory effect of silymarin, a plant flavanoid from Silybum marianum, on cell migration of metastasis-specific human melanoma cell lines (A375 and Hs294t) and assessed whether Wnt/β-catenin signaling is the target of silymarin. Using an in vitro invasion assay, we found that treatment of human melanoma cell lines with silymarin resulted in concentration-dependent inhibition of cell migration, which was associated with accumulation of cytosolic β-catenin, while reducing the nuclear accumulation of β-catenin (i.e., β-catenin inactivation) and reducing the levels of matrix metalloproteinase (MMP) -2 and MMP-9 which are the down-stream targets of β-catenin. Silymarin enhanced: (i) the levels of casein kinase 1α, glycogen synthase kinase-3β and phosphorylated-β-catenin on critical residues Ser45, Ser33/37 and Thr41, and (ii) the binding of β-transducin repeat-containing proteins (β-TrCP) with phospho forms of β-catenin in melanoma cells. These events play important roles in degradation or inactivation of β-catenin. To verify whether β-catenin is a potent molecular target of silymarin, the effect of silymarin was determined on β-catenin-activated (Mel 1241) and β-catenin-inactivated (Mel 1011) melanoma cells. Treatment of Mel 1241 cells with silymarin or FH535, an inhibitor of Wnt/β-catenin pathway, significantly inhibited cell migration of Mel 1241 cells, which was associated with the elevated levels of casein kinase 1α and glycogen synthase kinase-3β, and decreased accumulation of nuclear β-catenin and inhibition of MMP-2 and MMP-9 levels. However, this effect of silymarin and FH535 was not found in Mel 1011 melanoma cells. These results indicate for the first time that silymarin inhibits melanoma cell migration by targeting β-catenin signaling pathway.
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Introduction
Melanoma is the leading cause of death from skin diseases due to its propensity to metastasize. The overall incidence of melanoma is increasing in US, and is increasing rapidly in children. It accounted for an estimated 114,900 new cases of melanoma which were diagnosed in the US for 2010, out of which 68,130 were invasive and resulted in death of nearly 8,700 individuals [1]. Although, melanoma is less common than other types of skin cancer, however, it causes the majority (75%) of skin cancer-related deaths. Activating mutations of the protooncogene BRAF have been observed in approximately 50% of malignant melanomas. However, BRAF mutations alone are insufficient to cause malignant transformation and other triggering events are needed for melanomagenesis. Once, diagnosed with metastatic melanoma, most patients will ultimately die of their disease within 2 years [2]. Since, melanoma is a highly malignant cancer with a potent capacity to metastasize distantly, an approach that decreases its metastatic ability may facilitate the development of an effective strategy for its treatment and/or prevention.
Phytochemicals offer promising options for the prevention of cancer metastasis. Silymarin is one of them, and this flavanoid is obtained from milk thistle (Silybum marianum L. Gaertn.) plant. Silymarin is composed primarily of silibinin (≈90%) together with small amounts of other silibinin stereoisomers, such as isosilybin, dihydrosilybin, silydianin and silychristin [3]. Because silymarin has been shown to have anti-inflammatory, anti-oxidative and anti-carcinogenic effects [4], [5], it has been tested in various in vitro and in vivo models for its efficacy in prevention of skin carcinogenesis [5]. We previously have shown that topical application of silymarin to sensitive-to-carcinogen (SENCAR) mice resulted in inhibition of 7,12-dimethylbenz(a) anthracene-initiated and 12-O-tetradecanoylphorbol-13-acetate-promoted skin tumorigenesis in terms of tumor incidence, tumor multiplicity and tumor growth [6]. We also have shown that topical application of silymarin inhibits ultraviolet radiation-induced skin carcinogenesis in SKH-1 hairless mice [4]. These studies indicated that silymarin possesses potent anti-skin carcinogenic effects [4]–[6]. Importantly, the chemopreventive effect of silymarin has been studied extensively on non-melanoma skin cancer but its effect on melanoma has not been assessed.
Although the molecular mechanisms underlying the progression of melanoma remain unresolved, various studies have implicated constitutively active Wnt/β-catenin signaling in melanoma progression and metastasis [7], [8]. Non-phosphorylated β-catenin accumulates in the cytoplasm, when activated it enters the nucleus and interacts with T-cell factor transcription factors to control various target genes that are involved in cellular proliferation and migration. Nuclear β-catenin accumulation has been correlated with late stages of tumor progression and metastasis. The presence of mutated β-catenin is associated with aggressive tumor growth and regulates expression of various target genes that mediate cellular processes including proliferation, and migration [9], [10]. In the canonical model of Wnt signaling, β-catenin is phosphorylated at certain key residues by glycogen synthase kinase-3β (GSK-3β) and casein kinase 1 α (CK1α) leading to its ubiquitination and subsequent degradation [11], [12]. Like cancers of other organs, the regulation of β-catenin is lost in melanoma [13]–[15]. This then leads to nuclear accumulation of β-catenin and subsequent stimulation of downstream target genes, which includes the genes of cell proliferation (e.g., cyclins and c-myc) and cell invasion (e.g., matrix metalloproteinases) [16]–[18].
Since metastasis of melanoma is the leading cause of death in humans, in the present study we assessed the chemotherapeutic effects of silymarin on the migration/invasion potential of human melanoma cells. For this purpose, two human metastasis-specific cell lines were selected: A375 which is BRAF-mutated and another Hs294t cell line which is also highly metastasis-specific but not BRAF-mutated. Normal human epidermal melanocytes were used as a control. In this study we assessed whether silymarin inhibits the migration of melanoma cells and whether it is associated with the inactivation of the β-catenin signaling pathway or decreased accumulation of nuclear β-catenin. In order to verify the role of β-catenin in suppression of melanoma cell migration by silymarin, we compared the effect of silymarin on the behavior of two different melanoma cell lines that differ in their states of constitutive activation of Wnt/β-catenin signaling. The cell lines used were: (a) Mel 1241 cells that are characterized by constitutive activation of β-catenin, and (b) Mel 1011 cells which lack constitutively active β-catenin pathway. Here, we present evidence that silymarin inhibits the invasiveness or migratory potential of melanoma cells by inactivation of β-catenin.
Materials and Methods
Cell lines and cell culture conditions
The human melanoma cells lines, A375 and Hs294t, were purchased from the American Type Culture Collection (Manassas, VA), while melanoma cells Mel 1241 and Mel 1011 were a kind gift from Dr. Paul Robbins (Center of Cancer Research, National Cancer Institute, Bethesda, MD). All the cell lines were cultured as monolayers in Dulbecco's modified Eagle's medium, supplemented with 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT), 100 µg/ml penicillin and 100 µg/mL streptomycin and maintained in an incubator with 5% CO2 at 37°C. Normal human epidermal melanocytes (HEMa-LP, Cat. No. C-024-5C) were obtained from Invitrogen (Carlsbad, CA), and were cultured in HMGS-2 medium supplemented with human melanocyte growth supplement provided by the supplier. For the treatment of cells, silymarin was dissolved in a small amount of acetone, which was added to the complete cell culture medium [maximum concentration of acetone, 0.1% (v/v) in media] prior to addition to subconfluent cells (60–70% confluent). Cells treated with acetone alone served as a vehicle control.
Chemicals and antibodies
Purified silymarin was purchased from Sigma Chemical Co. (St Louis, MO). The antibodies specific for β-catenin were purchased from R&D Biosystems (Minneapolis, MN), while antibodies for phospho β-catenin, CK1α, GSK-3β, matrix metalloproteinase (MMP)-2, MMP-9, β-transducin repeat-containing proteins (β-TrCP) and β-actin were obtained from Cell Signaling Technology (Beverly, MA). Antibody specific to β-catenin for immunostaining was obtained from R&D Biosystems (Minneapolis, MN). Respective secondary antibodies (rabbit anti-goat and goat anti-rabbit) conjugated with horseradish peroxidase were purchased from Santa Cruz Biotech (Santa Cruz, CA). Boyden Chambers and polycarbonate membranes (8 µm pore size) for cell migration assays were obtained from Neuroprobe (Gaithersburg, MD).
Cell proliferation assay
The effect of silymarin on the viability of melanoma cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Sigma) or MTT assay, as previously described [19]. A total of 1×104 cells per well in 200 µL complete medium were seeded in a 96-well plate and treated with silymarin as previously described [19]. All treatment concentrations were repeated in six wells.
Matrigel invasion assay
The migration capacity of melanoma cancer cells was determined in vitro using Boyden Chambers in which the two chambers were separated with matrigel coated Millipore membranes (6.5 mm diameter filters, 8 µM pore size), as detailed previously [20]. Briefly, melanoma cells (1.5×104 cells/200 µL serum-reduced medium) were placed in the upper chamber of Boyden chambers. Test agents were added to the upper chamber (200 µL) and the lower chamber contained the medium alone (150 µL). Chambers were assembled and kept in an incubator for 24 h or 8 h. At the desired time point, cells from the upper surface of Millipore membranes were removed with gentle swabbing and the migrant cells on the lower surface of membranes were fixed and stained with crystal violet. Membranes were then washed and mounted onto glass slides. The membranes were examined microscopically and cellular migration per sample was determined by counting the number of stained cells in at least four to five randomly selected fields visualized with an Olympus BX41 microscope. Data are presented as mean number of the migrating cells ± SD per microscopic field per sample. Each cell migration experiment was repeated at least three times. Representative photomicrographs were obtained using a Qcolor5 digital camera system fitted to an Olympus BX41 microscope.
Scratch assay or wound healing assay
Wound healing assay was performed to detect melanoma cell migration, as detailed previously [20]. Briefly, melanoma cells were grown to full confluency in six-well plates and incubated overnight in starvation medium. Cell monolayers were wounded with a sterile 100 µL pipette tip, washed with starvation medium to remove detached cells from the plates. Cells were left either untreated or treated with indicated doses of silymarin in full medium and kept in a CO2 incubator for 24 h. After 24 h, medium was replaced with phosphate-buffered saline (PBS) buffer, the wound gap was observed and cells were photographed using an Olympus BX41 microscope fitted with digital camera.
Immunofluorescent detection of β-catenin
Human melanoma cells (A375 and Hs294t cells) were treated with various concentrations of silymarin (0, 10, 20, and 40 µg/mL) for the desired time period. The cells were then harvested and processed for cytospin preparation (1×105 cells/slide) for immunofluorescent staining and detection of nuclear β-catenin. Briefly, cells were fixed with methanol at −20°C for 10 minutes and non- specific binding sites were blocked with 2% bovine serum albumin (Sigma, St Louis, MO) in PBS for 30 min. Cells were permeabilized with 0.2% Triton X-100 (Sigma Co., St. Louis, MO) in PBS and then incubated with β-catenin-specific antibody for 2 h at room temperature. The cells were washed with PBS buffer and β-catenin was detected by an Alexa fluor 594-conjugated secondary antibody. Cells were mounted with Vectashield mounting medium for fluorescence and stained with DAPI (Vector Laboratories, Burlingame, CA) before they were observed with a fluorescence detection equipped microscope and photographed.
Immunoprecipitation and immunoblotting
Following treatment of melanoma cells with or without silymarin or other agents for the indicated time periods, the cells were harvested, washed with cold PBS buffer and lysed with ice-cold lysis buffer supplemented with protease inhibitors, as detailed previously [20]. Nuclear and cytosolic fractions were also prepared from the cells of different treatment groups following standard protocols, as described earlier [20]. Equal amounts of proteins from each treatment group were resolved on 10% Tris/Glycine gels and transferred onto a nitrocellulose membrane. After blocking the non-specific binding sites, the membrane was incubated with the primary antibody at 4°C overnight. The membrane was then incubated with the appropriate peroxidase-conjugated secondary antibody and the immunoreactive bands were visualized using the enhanced chemiluminescence reagents. To verify equal protein loading, the membrane was stripped and re-probed with anti-β actin antibody. Each experiment was repeated at least three times for western blotting and representative blots are presented.
For β-TrCP binding assay, A375 melanoma cells were treated with vehicle or various concentrations of silymarin for 24 h, washed with ice-cold PBS, and whole cell lysates prepared as described previously [20]. Aliquots containing 200 µg of protein were cleared with protein A/G-plus agarose beads (Santa Cruz, CA). β-TrCP protein was immunoprecipitated from whole cell lysates by overnight incubation with anti-β-TrCP antibody at 4°C followed by the addition of protein A/G-plus agarose beads (50 µL, Santa Cruz, CA) and continued incubation for 2 h. Immunoprecipitates were washed, and subsequently subjected to SDS-PAGE on 10% gels followed by immunoblotting using antibodies specific to phospho forms of β-catenin.
Statistical analysis
For migration assays, the control and silymarin-treatment groups were compared using one-way analysis of variance (ANOVA) followed by post hoc Dunn's test using GraphPad Prism version 4.00 for Windows, GraphPad Software, San Diego, California, USA, www.graphpad.com. All quantitative data for cell migration are shown as mean ± SD/microscopic field, and each experiment was repeated at least 3 times. In each case P<0.05 was considered statistically significant.
Results
Comparative migratory behavior of human melanoma cells and normal human epidermal melanocytes
First the studies were performed to examine the migratory behavior of melanoma cells and normal human epidermal melanocytes under identical conditions. For this purpose, cells were kept in Boyden chambers for invasion assays for 24 h in an incubator to assess their migration capability. As shown in Figure 1A, the cell migration capacity of melanoma cells was significantly higher (P<0.001) than normal human epidermal melanocytes. The migration of A375 cells was greater than Hs294t cells (390±14 cells/microscopic field for A375 vs 340±12 cells/microscopic field for Hs294t). Under identical conditions, migration of normal human epidermal melanocytes was significantly lower (19±4 cells per microscopic field, P<0.001) than melanoma cells, as summarized in Figure 1B.
10.1371/journal.pone.0023000.g001Figure 1 Migration ability of human melanoma cells and normal human epidermal melanocytes.
(A) Equal numbers of human melanoma cells (A375 and Hs294t) and normal human epidermal melanocytes (HEMa) were subjected to cell migration by standard invasion assay using Boyden chambers. Twenty four h later, migratory cells were detected on the membrane after staining the migratory cells with the 0.1% crystal violet dye. Representative photomicrographs are shown from three independent experiments. (B) The migratory cells were counted and the results expressed as the mean number of migratory cells ± SD per microscopic field (n = 3). Significantly less migration of normal human melanocytes versus melanoma cells, *
P<0.001. (C) Chemical structure of silibinin, the major and most biological active component of silymarin.
Silymarin inhibits human melanoma cell migration: wound healing assay
Molecular structure of silibinin is shown in Figure 1C, which is a major (90%) and most active component of silymarin. We first determined whether treatment of A375 and Hs294t human melanoma cells with silymarin inhibited their migration using a wound healing assay, as described in Material and Methods. Before conducting this assay, preliminary screening experiments were performed to determine the effects of lower (low, non-death-inducing) concentrations of silymarin (0–40 µg/mL) that did not induce cell death in melanoma cells. As shown in Figure 2A, relative to untreated control cells, treatment of cells with various concentrations of silymarin (0, 10, 20 and 40 µg/mL) reduced the migration capacity of A375 and Hs294t cells in a concentration-dependent manner after the treatment of cells for 24 h. The major part of gap or wounding space between cell layers after making a wound was occupied by the migrating A375 cells which were not treated with silymarin. However, the healing of the wound or the empty space of the cells was largely not occupied by the migrating cells treated with silymarin and this effect was dose-dependent. The gap or wounding space between the cells is highlighted by broken red lines, as shown in Figure 2A. These observations suggest that silymarin inhibited the migration of melanoma cells. To confirm that the inhibition of cancer cell migration by silymarin was a direct effect on cell migration and not due to a reduction in cell viability, a trypan blue assay was performed using cells that were treated identically to those used in the migration assays. Treatment of A375 and Hs294t cells with various concentrations of silymarin (0, 10, 20 and 40 µg/mL) for 24 h had no significant effect on cell viability or cell death (data not shown).
10.1371/journal.pone.0023000.g002Figure 2 Silymarin inhibits melanoma cell migration and/or invasion in a concentration-dependent manner.
(A) Wound healing assay was performed to assess the effect of silymarin on the migration of A375 and Hs294t human melanoma cells. Incubation of A375 or Hs294t cells with silymarin for 24 h inhibits migration of cells in a concentration-dependent manner compared to non-silymarin-treated control cells. Broken red line indicates the gap without the presence of cells. Assay was repeated three times and representative pictures are shown. (B) Treatment of human melanoma cells with silymarin inhibits migration or invasion ability of cells. Treatment of A375 or Hs294t human melanoma cells with silymarin for 24 h inhibits invasion of cells in a concentration dependent manner. (C) The migratory cells were counted on membrane in at least four to five randomly selected microscopic fields and the results are summarized and expressed as the mean number of migratory cells ± SD per microscopic field. Significant difference versus non-silymarin treated control group, *
P<0.001, **
P<0.01.
Silymarin inhibits melanoma cell invasion: Boyden chamber assay
Since cell invasion is a key step involved in tumor metastasis, inhibition of cell invasion by the use of phytochemicals, such as silymarin, may represent an important strategy to prevent melanoma metastasis. Therefore, we determined whether treatment of A375 and Hs294t human melanoma cells with silymarin inhibited their invasive potential using Boyden chamber cell invasion assay. Again, preliminary screening experiments were performed to determine the effects of lower concentrations of silymarin that did not induce melanoma cell death (data not shown). As shown in Figure 2B, relative to untreated control cells, treatment with silymarin at concentrations of 10, 20 and 40 µg/mL reduced the migratory capacity of A375 and Hs294t cells in a concentration-dependent manner. The density of the migrating cells on the membrane after staining with crystal violet is shown in Figure 2B, and the numbers of migrating cells/microscopic field are summarized in Figure 2C. The cell migration was inhibited by 25 to 75% (P<0.01−0.001) in A375 cells and by 26–67% (P<0.01−0.001) in Hs294t cells in a concentration-dependent manner after treatment with silymarin for 24 h.
Silymarin reduces nuclear accumulation of β-catenin
Activation of β-catenin has been implicated in cancer cell migration. Therefore, we determined the effect of silymarin on the levels of β-catenin protein on both A375 and Hs294t cells using western blot analysis. For this purpose cells were treated with silymarin for 24 h and whole cell lysates, cytosolic and nuclear fractions were prepared. Western blot analysis revealed that treatment of A375 and Hs294t cells with silymarin for 24 h resulted in reduction of β-catenin levels in the nucleus of the cells (Figures 3A and 3D). This change correlated with an increase in cytosolic β-catenin. These observations were further checked and verified in melanoma cells using immunofluorescence staining (Figures 3C and 3F). Magnified cells inside the box clearly show the reduced staining of nuclear β-catenin after the treatment of cells with silymarin. As MMP-2 and MMP-9 are the downstream targets of β-catenin [21]–[23], we also measured the effect of silymarin on the levels of MMP-2 and MMP-9. Consistent with the decreased nuclear localization of β-catenin after treating the cells with silymarin, the expression of MMP-2 and MMP-9 were also found to be decreased in both A375 and Hs294t cells after treatment of the cells with silymarin for 24 h (Figures 3A and 3D).
10.1371/journal.pone.0023000.g003Figure 3 Effect of silymarin on β-catenin and its signaling molecules in melanoma cells.
(A) Effect of silymarin on the cytosolic and nuclear accumulation of β-catenin, and MMP-2 and MMP-9, which are downstream targets of β-catenin, in BRAF-mutated A375 cells. (B) Effect of silymarin on phosphorylation of β-catenin at “critical residues” and on the expression levels of regulatory kinases (GSK-3β, CK1α) implicated in determining nuclear/cytoplasmic accumulation of β-catenin. (C) Immunofluorescence staining shows decrease in nuclear accumulation of β-catenin in A375 cells after the treatment of cells with silymarin for 24 h in a dose-dependent manner. Magnified nuclear staining is shown in the cells inside the box. (D) The effect of silymarin on nuclear and cytosolic levels of β-catenin and its target MMPs proteins important for the cell migration in Hs294t cells after the treatment of cells for 24 h. (E) Effect of silymarin on phosphorylation of β-catenin at “critical residues” and on the expression levels of regulatory kinases (GSK-3β, CK1α) in metastasis-specific Hs294t cells. (F) Immunofluorescence staining showing decrease in nuclear accumulation of β-catenin in Hs294t cells in a dose-dependent manner after treatment of cells with silymarin for 24 h. Magnified nuclear staining is shown in cells inside the box.
Since, nuclear accumulation of β-catenin is inversely correlated with phosphorylation at certain key residues of β-catenin (Ser45, Ser33, Ser37 and Thr41), we checked the effect of silymarin on the levels of β-catenin phosphorylation at these sites. Western blot analysis revealed that treatment of A375 and Hs294t cells with silymarin increased the phosphorylation of β-catenin at Ser45, and Ser33/Ser37/Thr41 in both melanoma cell lines (Figures 3B and 3E). Further, silymarin treatment of melanoma cells resulted in a dose-dependent increase of CK1α and GSK-3β. Both CK1α and GSK-3β are known to target β-catenin for proteasomal degradation via combined phosphorylation at key residues of β-catenin [12].
Silymarin enhances binding of β-TrCP to phospho forms of β-catenin
It has been shown that β-transducin repeat-containing proteins (β-TrCP) are components of the ubiquitin ligase complex targeting β-catenin for proteasomal degradation and are thus a negative regulator of Wnt/β-catenin signaling [24], [25]. Therefore, we were interested to check whether silymarin has any effect on the expression levels or activity of β-TrCP in our melanoma invasion model. For this purpose, A375 melanoma cells were treated with silymarin for 24 h, cell lysates were prepared, and β-TrCP was immunoprecipitated for detection of its binding with the phospho forms of β-catenin. Western blot analysis data revealed that silymarin did not affect the expression levels of β-TrCP after the treatment of cells for 24 h (data not shown). However, treatment of A375 cells with silymarin enhanced the binding of β-TrCP with phospho forms of β-catenin in a dose-dependent manner, as shown in Figure 4. These data suggest that silymarin may have inactivated β-catenin by enhancing the proteasomal degradation of the β-catenin after its binding with β-TrCP.
10.1371/journal.pone.0023000.g004Figure 4 Treatment of melanoma cells with silymarin enhances binding of β-TrCP with phospho forms of β-catenin.
Cells were treated with and without silymarin for 24 h and cell lysates were prepared. In binding assay, β-TrCP was immunoprecipitated using specific antibody from total protein lysates followed by western blot analysis for phospho forms of β-catenin, as detailed in Materials and Methods. IP, immunoprecipitation; IB, immunoblotting.
Specific activation of β-catenin leads to enhanced cell migration
As we found that silymarin exerts a significant inhibitory effect on the migration of A375 and Hs294t cells, and this inhibition was associated with a decrease in nuclear accumulation of β-catenin in both metastasis-specific melanoma cell lines, next we examined the role of β-catenin in melanoma cell invasion. For this purpose we selected two different melanoma cell lines: one was Mel 1241, which possesses constitutively active Wnt/β-catenin signaling and second one was Mel 1011 (lack activated β-catenin) from which Mel 1241 was derived. First the cell migration ability of these two melanoma cell lines was examined. Our preliminary analysis of cell migration indicated that the cell migration ability of Mel 1241 cells after 24 h was exceptionally higher than A375 or Hs294t cells. Therefore, we reduced the incubation period of the cells to 8 h for subsequent measurement of cell migration using the invasion assay. As shown in Figure 5A, the cell migration activity of Mel 1241 cells after 8 h was significantly higher than the cell migration activity of the Mel 1011 cells. The number of migrating cells of Mel 1241 cells was 499±40 cells/microscopic filed whereas the number of migrating cells of Mel 1011 cells were 29±4 cells/microscopic field, as summarized under Figure 5B (n = 3).
10.1371/journal.pone.0023000.g005Figure 5 Silymarin inhibits human melanoma cell migration by targeting β-catenin.
(A), Comparison of invasion ability of two different melanoma cell lines, one has stabilized mutation in β-catenin (Mel 1241) and another possesses wild-type β-catenin (Mel 1011). The migration capacity of Mel 1241 cells after 8 h is significantly higher than the migration capacity of Mel 1011 cells. (B) The migratory cells were counted under microscope and the results are summarized and expressed as the mean number of migratory cells ± SD per microscopic field. Significant difference versus Mel 1241 cells, *
P<0.001. (C), Treatment of Mel 1241 melanoma cells with silymarin for 8 h inhibits migration of Mel 1241 cells in a concentration dependent manner. (D) The migratory cells were counted under microscope and the results are summarized and expressed as the mean number of migratory cells ± SD/microscopic field (n = 3). Significant inhibition versus non-silymarin-treated control, *
P<0.001. (E) The effect of silymarin on the Mel 1011 melanoma cell migration after the treatment for 8 h. (F) The migratory cells were counted under microscope in different treatment groups and the results are summarized and expressed as the mean number of migratory cells ± SD/microscopic field (n = 3). Representative photomicrographs of cell migration are shown from three identical experiments.
Silymarin or FH535, an inhibitor of β-catenin, inhibits melanoma cell invasion ability by targeting β-catenin
To examine whether silymarin inhibits melanoma cell migration by targeting β-catenin, cell migration experiment was conducted with Mel 1241 and Mel 1011 cells with and without the treatment of cells with various concentrations of silymarin (0, 10, 20, and 40 µg/mL) for 8 h. As shown in Figure 5C, treatment of Mel 1241 cells with silymarin significantly inhibited (P<0.001) the migration of Mel 1241 cells in a concentration-dependent manner. Resultant cell migration data are summarized in terms of mean number of migrating cells ±SD/microscopic field for different treatment groups in Figure 5D. In contrast, silymarin did not inhibit the cell migrating ability of Mel 1011 cells, which have inactivated β-catenin (Figure 5E). In other words Mel 1011 cells were resistant to the effect of silymarin on their cell migrating behavior. A summary of migrating cells under different treatment groups is shown in Figure 5F.
Further, in support of these observations, Mel 1241 and Mel 1011 cell lines were treated with various concentrations of FH535 (0, 20, 40 and 60 µM) for 8 h and cell migration was analyzed. FH535 has unique ability to inhibit Wnt/β-catenin pathway [26]. As shown in Figure 6A, treatment of Mel 1241 cells with FH535 inhibited the migration of cells in a dose-dependent manner (40–84%, P<0.001). Mean number of migrating cells per microscopic field± SD in different treatment groups are summarized in Figure 6B (n = 3). In contrast, FH535 did not inhibit the migration of Mel 1011 cells or Mel 1011 cells were resistant to the effect of FH535 on their cell migrating behavior (Figures 6C, 6D). These data along with the data from silymarin treatment suggest that activation of β-catenin stimulates melanoma cell invasion while its inactivation suppresses the migration of melanoma cells.
10.1371/journal.pone.0023000.g006Figure 6 Effect of FH535, an inhibitor of β-catenin, on melanoma cell migration.
(A) Mel 1241 cells were incubated with FH535 for 8 h and cell migration was determined using invasion assay. FH535 inhibits the cell migration of Mel 1241 cells in a dose-dependent manner. (B) The migratory cells were counted on the membrane under microscope and the results are expressed as the mean number of migratory cells ± SD per microscopic field. Significant inhibition versus control, *
P<0.001, **
P<0.01. (C) Treatment of Mel 1011 cells with FH535 for 8 h did not inhibit cell migration compared to non-FH535-treated control. (D) Migratory cells were counted under microscope and the results are expressed as the mean number of migratory cells± SD per microscopic field. Migration assays were repeated three times and representative pictures of cell migration are shown. No statistical significance of difference versus un-treated controls.
In continuation with these studies, the effect of silymarin and FH535 was also determined on the nuclear accumulation of β-catenin, its down-stream targets (MMP-2 and MMP-9) and phosphorylation of β-catenin at various Ser residues using Mel 1241 and Mel 1011 cell lines. For this purpose, cells were treated with and without silymarin or FH535 for 8 h, and cell lysates were prepared for western blot analysis. Western blot analysis revealed that treatment of Mel 1241 cells with both silymarin or FH535 for 8 h resulted in reduced nuclear accumulation of β-catenin and reduced levels of MMP-2 and MMP-9 compared to control cells which were not treated with silymarin or FH535, as shown in Figure 7A. Similarly, the phosphorylation of β-catenin at Ser45, and other target residues (Ser33/Ser37/Thr41), and the levels of CK1α and GSK-3β were increased after the treatment of Mel 1241 cells with silymarin or FH535 (Figure 7B). However, these effects of silymarin and FH535 were not observed in Mel 1011 cell line under identical condition (data not shown) or the Mel 1011 melanoma cells were resistant to the action of silymarin and FH535.
10.1371/journal.pone.0023000.g007Figure 7 Effect of silymarin and FH535 on β-catenin and its signaling molecules in Mel 1241 cells.
(A) Effect of silymarin and FH535 on the cytosolic and nuclear accumulation of β-catenin, and MMP-2 and MMP-9 in Mel 1241 cells. Cells were treated with silymarin or FH535 for 8 h then harvested, nuclear and cytosolic fractions were prepared and subjected to western blot analysis. (B) Effect of silymarin and FH535 on phosphorylation of β-catenin at “critical residues” and on the expression levels of regulatory kinases (GSK-3β, CK1α) implicated in activation of β-catenin. (C) The combined effect of silymarin and FH535 on Mel 1241 cell migration. Cells were treated with the indicated low doses of silymarin and FH535 either alone or in combination for 8 h and cell migration was determined using invasion assay. Cell migration data are expressed as the mean number of migratory cells ±SD per microscopic field (n = 3). Significant inhibition versus untreated control, *
P<0.001, *
P<0.01. Significant inhibition versus either agent alone, ¶
P<0.01.
Combined effect of silymarin and FH535 on melanoma cell (Mel 1241) invasion
We further checked the combined effect of silymarin and FH535 on the invasion ability of Mel 1241 cells and this effect was compared with the individual effect of silymarin and FH535 in these cells using identical cell invasion protocol. As shown in Figure 7C, treatment of Mel 1241 cells with low doses of silymarin (20 µg/mL) and FH535 (20 µM) separately for 8 h inhibited the cell migration respectively by 54% and 40% compared to non-treated control cells. However, the invasion activity of Mel 1241 cells was significantly inhibited (81%, P<0.01) when the cells were treated with silymarin plus FH535 compared with either agent alone, as shown in Figure 7C.
Discussion
The significant findings of the present study are that silymarin inhibits invasion or cell migration ability of melanoma cells in a dose-dependent manner, and that is associated with the inactivation of β-catenin signaling pathway. Based on our observation, cells will go under apoptosis or cell death if melanoma cells are treated with silymarin for more than 24 h time period or at a higher concentration of silymarin (>40 µg/mL). Under these conditions, cell migration will decrease, and this reduction in cell migration could be due to reduced cell viability or cell death and not because of changes in migrating behavior of cells. In our study, cell death or apoptosis is not a reason of silymarin-caused inhibition of melanoma cell migration. Silymarin has been shown to inhibit skin carcinogenesis [4]–[6], and has pleiotropic activities which include the inhibition of cyclooxygenase-2 (COX-2) activity and an inhibitor of polyamine biosynthesis [4], [5]. Traditional non-steroidal anti-inflammatory drugs (NSAIDs), such as sulindac, inhibit COX-2 expression resulting in reduced Wnt-signaling by induced β-catenin degradation, as has been shown in colon cancer [27]. Similar to the function of NSAIDs, silymarin also induced β-catenin degradation in melanoma cells and that is associated with inhibition of melanoma cell migration.
Various studies have implicated the role of constitutively active Wnt/β-catenin signaling in tumor progression. β-catenin is a dual function protein and is an important component of cell-cell adhesion, where it forms a dynamic link between E-cadherin and cytoskeleton [28], [29]. This cell-to-cell adhesion may prevent the migration of cells. However, the breaking of cell-to-cell adhesion due to activation of β-catenin and its nuclear accumulation may increase the migration potential of tumor cells. It can also regulate cell migration via its role as a transcription factor wherein it along with transcription factors of the T-cell factor and lymphoid enhancer factor family regulates expression of various target genes that mediate cellular processes including cell migration [11]. Thus nuclear/cytoplasmic ratio of β-catenin in the cells determines their migration potential. Our results show that silymarin inhibits melanoma cell migration by targeting β-catenin. It has been shown that phosphorylation of β-catenin at critical target residues such as at Ser45, Ser33/37 and Thr41 by GSK-3β and CK1α within the cytosolic destruction complex leads to degradation of β-catenin and thus reduces its nuclear accumulation [12]. In our study, we found that treatment of melanoma cells with silymarin enhances the expression of GSK-3β and CK1α, and β-catenin is phosphorylated at critical target residues. This suggests that silymarin via enhanced expression of GSK-3β and CK1α leads to enhanced phosphorylation of β-catenin at critical residues. This then lead to degradation of β-catenin within the degradation complex resulting in its reduced nuclear accumulation. It thus explains inhibitory effects of silymarin against melanoma cell migration.
Diverse molecular events are integrated in the progression and metastasis of cancer cells. In tumor cells, mechanisms that inhibit GSK-3β-induced phosphorylation of β-catenin block its interaction with the E3 ubiquitin ligase receptor, β-TrCP, which prevents β-catenin ubiquitination and degradation, and ultimately leads to β-catenin activation [24], [25]. Oncogenic activation of β-catenin occurs primarily as a consequence of its stabilization by escaping ubiquitin-mediated proteasomal degradation. A major regulator of β-catenin stability and activity is the β-TrCP. In this study, we sought to determine whether the inactivation of β-catenin in melanoma cells by silymarin is affected by expression of its regulator, the β-TrCP. We found that silymarin enhanced the binding of β-TrCP to phospho forms of β-catenin, which suggests β-TrCP-mediated ubiquitination and degradation/inactivation of β-catenin [25], [30]. Thus, this finding further supports our hypothesis that silymarin inhibits melanoma cell migration by targeting β-catenin.
In an attempt to further verify the role of silymarin on prevention of invasive potential of melanoma cell through inactivation of β-catenin signaling, we used two distinct melanoma cell lines, namely Mel 1241 and Mel 1011. The two cell lines differ in status of constitutive activation of Wnt/β-catenin signaling. Our preliminary data show that Mel 1241 melanoma cells which possess constitutively active Wnt/β-catenin are highly invasive and the capacity of cell migration is multiple-fold higher than A375 and Hs294t cell lines. Treatment of Mel 1241 cells with silymarin resulted in significant inhibition of cell migration which was associated with the reduction in nuclear accumulation of β-catenin and reduction in the levels of MMP-2 and MMP-9 which are the down-stream targets of β-catenin signaling. These observations were supported when treatment of these cells with FH535, an inhibitor β-catenin, also resulted in significant inhibition of Mel 1241 cell migration concomitantly reduced accumulation of nuclear β-catenin and lowering the levels of MMPs. Both silymarin and FH535 elevated the levels of GSK-3β and CK1α and simultaneously enhances the phosphorylation of β-catenin at specific target residues (e.g., Ser45, Ser33/37 and Thr41). Both CK1α and GSK-3β are known to target β-catenin for proteasomal degradation via combined phosphorylation at key residues of β-catenin [12]. Interestingly, under identical experimental conditions, these effects of silymarin and FH535 were not found in the Mel 1011 cell line, which lacks constitutively active β-catenin. Wnt signaling is suggested to inhibit β-catenin phosphorylation, thus inducing the accumulation of cytosolic β-catenin, which associates with the T cell factor/lymphocyte enhancer factor family of transcription factors to activate Wnt/β-catenin-responsive genes [31], [32]. Our study provide evidence that silymarin induced β-catenin phosphorylation degradation in melanoma cells is associated with the up-regulation of CK1α and GSK-3β. Liu et al. [12] have identified CK1α as an essential component that controls β-catenin phosphorylation degradation in Drosophila.
In summary, the outcome of this study suggests that silymarin has the ability to block or inhibit the invasive potential of melanoma cells, and this anti-invasion effect of silymarin is mediated through inactivation of β-catenin, as summarized under Figure 8. Thus intervention strategies targeting key molecules of the Wnt/β-catenin pathway may represent promising approaches to inhibit metastasis of melanoma cells. This new insight into the anti-melanoma cell migration activity of silymarin could serve as the basis for chemoprevention or therapy of malignant melanoma in high risk individuals.
10.1371/journal.pone.0023000.g008Figure 8 Schematic diagram depicts the mechanism of inactivation of β-catenin by silymarin in melanoma cells.
Silymarin blocks β-catenin activation through stimulation of its phosphorylation at different Serine sites and binding with β-TrCP, which subsequently decreases its nuclear accumulation and that results in inhibition of invasive potential of melanoma cells.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by funds from the Veterans Administration Merit Review Award (SKK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Acta Crystallogr Sect E Struct Rep OnlineActa Cryst. EActa Crystallographica Section E: Structure Reports Online1600-5368International Union of Crystallography zl237510.1107/S1600536811022574ACSEBHS1600536811022574Organic PapersOxonium picrate H3O+·C6H2N3O7−Jin Shou-Wen a*Chen Bing-Xia aGe Yu-Shuang aYin Hua-Bing aFang Yu-Ping aa Tianmu College, ZheJiang A & F University, Lin’An 311300, People’s Republic of ChinaCorrespondence e-mail: [email protected] 7 2011 18 6 2011 18 6 2011 67 Pt 7 e110700o1694 o1694 18 5 2011 10 6 2011 © Jin et al. 20112011This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.A full version of this article is available from Crystallography Journals Online.The title compound, H3O+·C6H2N3O7
−, consists of one picrate anion and one oxonium cation. The oxonium cation is located on a crystallographic twofold axis and both its H atoms are disordered, each over two symmetry-equivalent positions with occupancy ratios of 0.75. The picrate anions are also located on twofold axes bisecting the phenolate and p-nitro groups. π–π interactions between the rings of the picrates [centroid-to-centroid distances of 3.324 (2) Å] connect the anions to form stacks along the a-axis direction. The stacks are further joined together by the protonated water molecules through hydrogen bonds to form two-dimensional sheets extending parallel to the ab plane. The sheets are stacked on top of each other along the c-axis direction and connected through C—H⋯O interactions between the CH groups of the benzene rings and the picrate nitro groups, with C⋯O distances of 3.450 (2) Å.
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Related literature
For general background to organic salts of picric acid, see Jin et al. (2010 ▶); Harrison et al. (2007 ▶); Muthamizhchelvan et al. (2005 ▶); Smith et al. (2004 ▶).
Experimental
Crystal data
H3O+·C6H2N3O7
−
M
r = 247.13
Orthorhombic,
a = 7.1510 (6) Å
b = 19.80820 (18) Å
c = 13.50610 (12) Å
V = 1913.12 (16) Å3
Z = 8
Mo Kα radiation
μ = 0.16 mm−1
T = 298 K
0.45 × 0.34 × 0.31 mm
Data collection
Bruker SMART CCD area-detector diffractometer
Absorption correction: multi-scan (SADABS; Bruker, 2002 ▶) T
min = 0.936, T
max = 0.951
3842 measured reflections
848 independent reflections
654 reflections with I > 2σ(I)
R
int = 0.044
Refinement
R[F
2 > 2σ(F
2)] = 0.044
wR(F
2) = 0.140
S = 1.12
848 reflections
89 parameters
2 restraints
H atoms treated by a mixture of independent and constrained refinement
Δρmax = 0.21 e Å−3
Δρmin = −0.41 e Å−3
Data collection: SMART (Bruker, 2002 ▶); cell refinement: SAINT (Bruker, 2002 ▶); data reduction: SAINT; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008 ▶); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008 ▶); molecular graphics: SHELXTL (Sheldrick, 2008 ▶); software used to prepare material for publication: SHELXTL.
Supplementary Material
Crystal structure: contains datablock(s) global, I. DOI: 10.1107/S1600536811022574/zl2375sup1.cif
Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811022574/zl2375Isup2.hkl
Supplementary material file. DOI: 10.1107/S1600536811022574/zl2375Isup3.cml
Additional supplementary materials: crystallographic information; 3D view; checkCIF report
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: ZL2375).
The authors gratefully acknowledge financial support from the Education Office Foundation of Zhejiang Province (project No. Y201017321) and from the Innovation Project of Zhejiang A & F University.
supplementary crystallographic
information
Comment
It is well known that picric acid is used primarily to prepare explosives, and
as an intermediate to manufacture dyes. As a strong organic acid, picric acid
forms salts with many N-containing organic bases (Smith et al.,
2004;
Harrison et al., 2007; Muthamizhchelvan et al.,
2005). As an
extension of our study concerning organic salts based on picric acid (Jin
et al., 2010), we herein report the crystal structure of oxonium
picrate.
The single crystal of the title compound (Fig. 1) with the formula
C6H5N3O8 was obtained by recrystallization of picric acid and
2-chloropyridine from a methanol solution. However the 2-chloropyridine
molecules do not appear in the title compound. X-ray diffraction analysis
indicated that in the title compound there are one protonated water molecule,
and one picrate. The OH group of the picric acid is ionized and the proton is
transferred to the water molecule. In the title compound all of the bond
distances and angles are in the normal range. The oxonium cation is located on
a crystallographic two-fold axis and both its H atoms are disordered over each
two symmetry equivalent positions with occupancy rates of 0.75 each. The
benzene ring of the picrate is almost planar. The ortho-nitro groups
(N1—O2—O3, and N1A—O2A—O3A) deviate from the benzene ring plane and
have a dihedral angle of 25.6 (2)° with the benzene plane, whereas the
para-nitro group lies almost in the benzene plane [with a dihedral angle
of 2.0 (1)° between the N2—O4—O4A group and the benzene ring]. These
structural data are similar to those in other structurally described picrates
(Muthamizhchelvan et al., 2005).
π–π Interactions between the phenyl rings of the picrates (with Cg–Cg
distances of 3.324 (2) Å) connect the picrate anions to form stacks along
the a axis direction. Within one stack molecules alternate and are arranged
in an antiparallel fashion. The one-dimensional picrate stacks are further
linked together by the oxonium ions to form a two-dimensional sheet structure
when it is viewed from the c axis direction (Fig. 2). The sheets are
further stacked along the c axis direction through CH—O interactions
between CH of the benzene rings and the nitro groups of the picrates with C—O
distances of 3.450 (2) Å to form a three-dimensional structure.
Experimental
Crystals of oxonium picrate were formed by slow evaporation of its methanol
solution at room temperature. Picric acid (23 mg, 0.10 mmol) was dissolved in
4 ml of methanol, and 2-chloropyridine (11 mg, 0.10 mmol) was added to the
methanol solution. The solution was then filtered into a test tube and left
standing at room temperature. After about one week yellow block crystals were
obtained.
Refinement
H atoms H5A and H5B bonded to the oxonium O atom were located in a difference
Fourier map and refined isotropically. The oxonium cation is located on a
crystallographic two-fold axis and both H5A and H5B are disordered over each
two symmetry equivalent positions, and both have an occupancy of 0.75. Other H
atoms were positioned geometrically with C—H = 0.93 Å for aromatic H atoms,
and constrained to ride on their parent atoms with Uiso(H) =
1.2Ueq(C).
Figures
Fig. 1. The structure of the title compound, showing the atom-numbering scheme. Displacement ellipsoids are drawn at the 30% probability level. Symmetry code: (i) -x + 1/2, y, -z.
Fig. 2. Two-dimensional sheet structure formed through hydrogen bonds which is viewed along the c axis direction. The blue dashed lines represent O—H···O and π–π interactions.
Crystal data
H3O+·C6H2N3O7− Dx = 1.716 Mg m−3
Mr = 247.13 Mo Kα radiation, λ = 0.71073 Å
Orthorhombic, Ibca Cell parameters from 1867 reflections
a = 7.1510 (6) Å θ = 1.5–25.0°
b = 19.80820 (18) Å µ = 0.16 mm−1
c = 13.50610 (12) Å T = 298 K
V = 1913.12 (16) Å3 Block, yellow
Z = 8 0.45 × 0.34 × 0.31 mm
F(000) = 1008
Data collection
Bruker SMART CCD area-detector diffractometer 848 independent reflections
Radiation source: fine-focus sealed tube 654 reflections with I > 2σ(I)
graphite Rint = 0.044
φ and ω scans θmax = 25.0°, θmin = 2.1°
Absorption correction: multi-scan (SADABS; Bruker, 2002) h = −8→8
Tmin = 0.936, Tmax = 0.951 k = −22→23
3842 measured reflections l = −16→6
Refinement
Refinement on F2 Secondary atom site location: difference Fourier map
Least-squares matrix: full Hydrogen site location: inferred from neighbouring sites
R[F2 > 2σ(F2)] = 0.044 H atoms treated by a mixture of independent and constrained refinement
wR(F2) = 0.140 w = 1/[σ2(Fo2) + (0.0677P)2 + 3.1011P] where P = (Fo2 + 2Fc2)/3
S = 1.12 (Δ/σ)max < 0.001
848 reflections Δρmax = 0.21 e Å−3
89 parameters Δρmin = −0.41 e Å−3
2 restraints Extinction correction: SHELXL97 (Sheldrick, 2008), Fc*=kFc[1+0.001xFc2λ3/sin(2θ)]-1/4
Primary atom site location: structure-invariant direct methods Extinction coefficient: 0.039 (4)
Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes)
are estimated using the full covariance matrix. The cell e.s.d.'s are taken
into account individually in the estimation of e.s.d.'s in distances, angles
and torsion angles; correlations between e.s.d.'s in cell parameters are only
used when they are defined by crystal symmetry. An approximate (isotropic)
treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s.
planes.
Refinement. Refinement of F2 against ALL reflections. The weighted R-factor
wR and goodness of fit S are based on F2, conventional
R-factors R are based on F, with F set to zero for
negative F2. The threshold expression of F2 >
σ(F2) is used only for calculating R-factors(gt) etc.
and is not relevant to the choice of reflections for refinement.
R-factors based on F2 are statistically about twice as large
as those based on F, and R-factors based on ALL data will be
even larger.
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x y z Uiso*/Ueq Occ. (<1)
N1 0.3838 (3) 0.11353 (11) 0.17214 (16) 0.0328 (7)
N2 0.2500 −0.09568 (15) 0.0000 0.0361 (8)
O1 0.2500 0.18513 (12) 0.0000 0.0369 (8)
O2 0.3327 (3) 0.17048 (10) 0.19325 (15) 0.0502 (7)
O3 0.4947 (3) 0.08131 (11) 0.22328 (15) 0.0497 (7)
O4 0.3100 (4) −0.12491 (10) 0.07276 (19) 0.0630 (8)
O5 0.0000 0.2500 0.1338 (2) 0.0489 (9)
H5A 0.089 (6) 0.233 (3) 0.091 (3) 0.073* 0.75
H5B 0.052 (7) 0.279 (2) 0.177 (3) 0.073* 0.75
C1 0.2500 0.12179 (17) 0.0000 0.0270 (8)
C2 0.3107 (4) 0.08102 (13) 0.08289 (17) 0.0270 (7)
C3 0.3134 (4) 0.01147 (13) 0.08305 (17) 0.0286 (7)
H3 0.3569 −0.0122 0.1378 0.034*
C4 0.2500 −0.02233 (17) 0.0000 0.0282 (8)
Atomic displacement parameters (Å2)
U11 U22 U33 U12 U13 U23
N1 0.0376 (14) 0.0358 (13) 0.0250 (12) −0.0067 (10) −0.0014 (10) 0.0001 (9)
N2 0.0350 (19) 0.0269 (16) 0.046 (2) 0.000 0.0059 (15) 0.000
O1 0.0587 (19) 0.0226 (13) 0.0295 (14) 0.000 0.0085 (13) 0.000
O2 0.0748 (17) 0.0372 (12) 0.0387 (12) 0.0047 (10) −0.0128 (11) −0.0126 (9)
O3 0.0569 (15) 0.0538 (13) 0.0385 (12) 0.0006 (11) −0.0193 (10) 0.0019 (9)
O4 0.096 (2) 0.0300 (12) 0.0629 (15) 0.0117 (11) −0.0140 (14) 0.0089 (10)
O5 0.061 (2) 0.0414 (17) 0.0443 (18) −0.0008 (15) 0.000 0.000
C1 0.0273 (19) 0.0275 (18) 0.0260 (18) 0.000 0.0050 (14) 0.000
C2 0.0287 (14) 0.0305 (14) 0.0217 (13) −0.0032 (10) 0.0009 (10) −0.0014 (9)
C3 0.0279 (14) 0.0303 (14) 0.0275 (13) 0.0010 (11) −0.0001 (11) 0.0051 (10)
C4 0.0262 (19) 0.0248 (17) 0.0337 (19) 0.000 0.0041 (15) 0.000
Geometric parameters (Å, °)
N1—O2 1.220 (3) O5—H5B 0.91 (2)
N1—O3 1.230 (3) C1—C2 1.447 (3)
N1—C2 1.463 (3) C1—C2i 1.447 (3)
N2—O4 1.219 (3) C2—C3 1.378 (4)
N2—O4i 1.219 (3) C3—C4 1.383 (3)
N2—C4 1.453 (5) C3—H3 0.9300
O1—C1 1.255 (4) C4—C3i 1.383 (3)
O5—H5A 0.92 (2)
O2—N1—O3 122.8 (2) C3—C2—C1 124.3 (2)
O2—N1—C2 119.5 (2) C3—C2—N1 115.7 (2)
O3—N1—C2 117.7 (2) C1—C2—N1 119.9 (2)
O4—N2—O4i 123.3 (3) C2—C3—C4 118.6 (2)
O4—N2—C4 118.37 (17) C2—C3—H3 120.7
O4i—N2—C4 118.37 (17) C4—C3—H3 120.7
H5A—O5—H5B 111 (5) C3i—C4—C3 122.1 (3)
O1—C1—C2 123.92 (15) C3i—C4—N2 118.97 (16)
O1—C1—C2i 123.92 (15) C3—C4—N2 118.97 (16)
C2—C1—C2i 112.2 (3)
O1—C1—C2—C3 −179.13 (18) C1—C2—C3—C4 −1.7 (3)
C2i—C1—C2—C3 0.87 (18) N1—C2—C3—C4 −178.59 (19)
O1—C1—C2—N1 −2.4 (3) C2—C3—C4—C3i 0.81 (17)
C2i—C1—C2—N1 177.6 (3) C2—C3—C4—N2 −179.19 (17)
O2—N1—C2—C3 −155.8 (3) O4—N2—C4—C3i 178.42 (19)
O3—N1—C2—C3 23.8 (3) O4i—N2—C4—C3i −1.58 (19)
O2—N1—C2—C1 27.2 (3) O4—N2—C4—C3 −1.58 (19)
O3—N1—C2—C1 −153.3 (2) O4i—N2—C4—C3 178.42 (19)
Symmetry codes: (i) −x+1/2, y, −z.
Hydrogen-bond geometry (Å, °)
D—H···A D—H H···A D···A D—H···A
O5—H5B···O2ii 0.91 (2) 2.17 (2) 3.061 (3) 166 (5)
O5—H5A···O1 0.92 (2) 1.93 (2) 2.848 (2) 172 (5)
C3—H3···O3iii 0.93 2.52 3.450 (2) 175
Symmetry codes: (ii) −x+1/2, −y+1/2, −z+1/2; (iii) x, −y, −z+1/2.
Table 1 Hydrogen-bond geometry (Å, °)
D—H⋯A D—H H⋯A D⋯A D—H⋯A
O5—H5B⋯O2i 0.91 (2) 2.17 (2) 3.061 (3) 166 (5)
O5—H5A⋯O1 0.92 (2) 1.93 (2) 2.848 (2) 172 (5)
C3—H3⋯O3ii 0.93 2.52 3.450 (2) 175
Symmetry codes: (i) ; (ii) .
==== Refs
References
Bruker (2002). SADABS, SMART and SAINT Bruker AXS Inc., Madison, Wisconsin, USA.
Harrison, W. T. A., Swamy, M. T., Nagaraja, P., Yathirajan, H. S. & Narayana, B. (2007). Acta Cryst. E63, o3892.
Jin, S. W., Zhang, W. B., Liu, L., Gao, H. F., Wang, D. Q., Chen, R. P. & Xu, X. L. (2010). J. Mol. Struct.
975, 128–136.
Muthamizhchelvan, C., Saminathan, K., Fraanje, J., Peschar, R. & Sivakamar, K. (2005). Anal. Sci.
21, x61–x62.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122.
Smith, G., Wermuth, U. D. & Healy, P. C. (2004). Acta Cryst. E60, o1800–o1803. | 21837091 | PMC3151795 | CC BY | 2021-01-04 19:56:02 | yes | Acta Crystallogr Sect E Struct Rep Online. 2011 Jun 18; 67(Pt 7):o1694 |
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World J Surg OncolWorld Journal of Surgical Oncology1477-7819BioMed Central 1477-7819-9-732175226510.1186/1477-7819-9-73Case ReportMetastatic collecting duct carcinoma of the kidney treated with sunitinib Tazi El Mehdi [email protected] Ismail [email protected] Mohamed Fadl [email protected] Youness [email protected]'rabti Hind [email protected] Hassan [email protected] Department of Medical Oncology, National Institute of Oncology, Rabat, Morocco2 Department of Urology, CHU Hassan II, Fez, Morocco2011 13 7 2011 9 73 73 4 1 2011 13 7 2011 Copyright ©2011 Tazi et al; licensee BioMed Central Ltd.2011Tazi et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Collecting duct carcinoma (CDC) of the kidney is a rare and aggressive malignant tumor arising from the distal collecting tubules which has been shown to have a poor response to several kinds of systemic therapy. We present a case of metastatic CDC that responded favorably to a multiple tyrosine kinase inhibitor, sunitinib, achieving a partial response in both lung and skeletal metastases. To our knowledge, this is the first report showing therapeutic activity of sunitinib against CDC. Considering these findings, it would be worthwhile prospectively investigating the role of multiple tyrosine kinase inhibitors, particularly sunitinib, in the management of metastatic CDC.
Collecting duct carcinomaSunitinibMetastasis
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Introduction
Collecting duct carcinoma (CDC) of the kidney, also known as Bellini duct carcinoma, is a rare variant of renal cell carcinoma (RCC) arising from the epithelium of the distal collecting ducts; it accounts for 2% of all RCCs [1]. Clinically, CDC is characterized by an extremely aggressive phenotype, accompanying metastatic diseases at presentation in most reported cases; the prognosis ofCDC is therefore poor, with approximately 70% of patients dying of disease progression within 2 years after diagnosis. In fact, several systemic therapies, including cytokine therapy and cytotoxic chemotherapy, have failed to achieve favorable response to metastatic CDC except for very limited cases [2-7]. Sunitinib is an orally available inhibitor of multiple receptor tyrosine kinases, including vascular endothelial growth factor receptor, platelet-derived growth factor receptor, and others, with direct antitumor and antiangiogenic activity. Based on impressive outcomes in several clinical trials, sunitinib has been approved worldwide for treatment of RCC patients with clear cell histology [8]. Furthermore, significant therapeutic activities of sunitinib against non-clear cell RCCs, for example papillary and chromophobe carcinomas, have also been reported in recent studies [8,9]; however, it remains unknown whether sunitinib has a therapeutic impact on CDC of the kidney. Here, we report the first case of a patient with metastatic CDC of the kidney who had a favorable response to sunitinib treatment.
Case report
A 47-year-old man with a 14.1 cm left renal mass extending into the renal vein and metastases involving the bilateral lungs and retroperitoneal lymph nodes were referred to our institution.
Radical left nephrectomy combined with lymphadenectomy was performed. Pathological examination resulted in diagnosis of this case as CDC with tubulopapillary architecture consisting of tumor cells with eosinophilic cytoplasm and high-grade nuclei (Figure 1). In addition, immunohistochemical staining was characteristic of CDC; that is, tumor cells were positive for Ulex Europaeus agglutinin (Figure 2), cytokeratin 19, 34bE12, epithelial membrane antigen and focally positive with vimentin [1]. Following radical nephrectomy, this case was treated with sunitinib rather than systemic chemotherapy, considering her poor performance status. After 4 courses of sunitinib therapy with 37,5 mg daily schedule, metastases to the lungs and left rib decreased by more than 30% compared with findings before sunitinib treatment (Figure 3). Despite the favorable effects of sunitinib on metastatic diseases, pleural effusion appeared to be remarkable after the administration of sunitinib; however, cytological examination showed no malignant cells in the pleural fluid. In addition to pleural effusion, several adverse events associated with sunitinib treatment, including appetite loss, thrombocytopenias, and hypothyroidism, were observed. Thereafter, disease progression occurred 10 months after the initiation of sunitinib, and the patient died.
Figure 1 Hematoxylin and eosin staining of tissue sections from the nephrectomy specimens demonstrating collecting duct carcinoma (× 400).
Figure 2 Tumor showed positive staining for Ulex Europaeus agglutinin.
Figure 3 Metastatic lesion to the right lung and left sixth rib before (A) and after (B) 4 courses of sunitinib treatment.
Discussion
Because CDC is an uncommon and aggressive disease with extremely poor prognosis, accumulated information about CDC is very limited. Therefore, no established therapy for CDC exists except for surgical resection of localized diseases. To date, however, there have been 14 reported cases of metastatic CDC showing response to systemic therapy, consisting 9, 2, 1, and 1 who were treated by gemcitabine plus cisplatin or carboplatin, paclitaxel plus carboplatin, gemcitabine plus doxorubicin, and interferon-a, respectively [2-7]. Considering these findings in addition to the characteristics of CDC similar to those of urothelial cancer, chemotherapy is the currently favored approach for patients with metastatic CDC.
In the case presented, because of her poor performance status associated with skeletal metastases, it was judged to be difficult to perform intensive systemic chemotherapy. Accordingly, she was treated with sunitinib, which has been regarded as one of the most powerful agents against metastatic RCC [8], and showed a partial response to this drug. Recently, the favorable clinical activity of sunitinib against non-clear cell carcinomas, including papillary and chromophobe carcinomas, has also been reported [8,9]; however, this is the first reported case demonstrating a therapeutic response of metastatic CDC to sunitinib. Furthermore, a recent report presented a case of metastatic CDC showing response to sorafenib [10].
Conclusion
Although the precise molecular mechanism involved in the antitumor activity of multiple tyrosine kinase inhibitors against CDC remains largely unknown, these findings suggest that the efficacy of these agents, for example sunitinib and sorafenib, against metastatic CDC needs to be prospectively evaluated.
Consent
Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
EMT, IE, MFT and YA analyzed, interpreted the patient data regarding its oncological features, and has been involved in drafting the manuscript; HM and HE has given final approval of the version to be published. All authors read and approved the final manuscript.
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Srigley JR Eble JN Collecting duct carcinoma of kidney Semin Diagn Pathol 1998 15 54 67 9503506
Gollob JA Upton MP DeWolf WC Atkins MB Long-term remission in a patient with metastatic collecting duct carcinoma treated with taxol/carboplatin and surgery Urology 2001 58 1058 11744492
Milowsky MI Rosmarin A Tickoo SK Papanicolaou N Nanus DM Active chemotherapy for collecting duct carcinoma of the kidney: a case report and review of the literature Cancer 2002 94 111 116 10.1002/cncr.10204 11815966
Peyromaure M Thiounn N Scotté F Vieillefond A Debré B Oudard S Collecting duct carcinoma of the kidney: a clinicopathological study of 9 cases J Urol 2003 170 1138 1140 10.1097/01.ju.0000086616.40603.ad 14501710
Tokuda N Naito S Matsuzaki O Nagashima Y Ozono S Igarashi T Collecting duct (Bellini duct) renal cell carcinoma: a nationwide survey in Japan J Urol 2006 176 40 43 10.1016/S0022-5347(06)00502-7 16753362
Oudard S Banu E Vieillefond A Fournier L Medioni J Banu A Duclos B Rolland F Escudier B Arekelyan N Culine S Prospective multicenter phase II study of gemcitabine plus platinum salt for metastatic collecting duct carcinoma: results of a GETUG (Groupe d'Etudes des Tumeurs Uro-Génitales) study J Urol 2007 177 1698 1702 10.1016/j.juro.2007.01.063 17437788
Chao D Zisman A Pantuck AJ Collecting duct renal cell carcinoma: clinical study of a rare tumor J Urol 2002 167 71 74 10.1016/S0022-5347(05)65385-2 11743278
Rini BI Flaherty K Clinical effect and future considerations for molecularly-targeted therapy in renal cell carcinoma Urol Oncol 2008 26 543 549 10.1016/j.urolonc.2008.03.012 18774471
Choueiri TK Plantade A Elson P Negrier S Ravaud A Oudard S Zhou M Rini BI Bukowski RM Escudier B Efficacy of sunitinib and sorafenib in metastatic papillary and chromophobe renal cell carcinoma J Clin Oncol 2008 26 127 131 10.1200/JCO.2007.13.3223 18165647
Ansari J Fatima A Chaudhri S Bhatt RI Wallace M James ND Sorafenib induces therapeutic response in a patient with metastatic collecting duct carcinoma of kidney Onkologie 2009 32 44 46 10.1159/000183736 19209019 | 21752265 | PMC3152528 | CC BY | 2021-01-04 19:56:36 | yes | World J Surg Oncol. 2011 Jul 13; 9:73 |
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 21887248PONE-D-11-0879110.1371/journal.pone.0023395Research ArticleBiologyModel OrganismsAnimal ModelsMouseMolecular Cell BiologyCell DeathCell GrowthResveratrol and Black Tea Polyphenol Combination Synergistically Suppress Mouse Skin Tumors Growth by Inhibition of Activated MAPKs and p53 Combined Resveratrol and BTP Inhibit Skin TumorsGeorge Jasmine Singh Madhulika Srivastava Amit Kumar Bhui Kulpreet Roy Preeti Chaturvedi Pranav Kumar Shukla Yogeshwer
*
Proteomics Laboratory, Indian Institute of Toxicology Research, Council of Scientific and Industrial Research (CSIR), Uttar Pradesh, India
Kashanchi Fatah EditorGeorge Mason University, United States of America* E-mail: [email protected] and designed the experiments: JG YS. Performed the experiments: JG MS AKS KB PR PKC. Analyzed the data: JG MS KB. Contributed reagents/materials/analysis tools: YS. Wrote the paper: JG MS YS.
2011 26 8 2011 6 8 e2339518 5 2011 14 7 2011 George et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Cancer chemoprevention by natural dietary agents has received considerable importance because of their cost-effectiveness and wide safety margin. However, single agent intervention has failed to bring the expected outcome in clinical trials; therefore, combinations of chemopreventive agents are gaining increasing popularity. The present study aims to evaluate the combinatorial chemopreventive effects of resveratrol and black tea polyphenol (BTP) in suppressing two-stage mouse skin carcinogenesis induced by DMBA and TPA. Resveratrol/BTP alone treatment decreased tumor incidence by ∼67% and ∼75%, while combination of both at low doses synergistically decreased tumor incidence even more significantly by ∼89% (p<0.01). This combination also significantly regressed tumor volume and number (p<0.01). Mechanistic studies revealed that this combinatorial inhibition was associated with decreased expression of phosphorylated mitogen-activated protein kinase family proteins: extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase 1/2, p38 and increased in total p53 and phospho p53 (Ser 15) in skin tissue/tumor. Treatment with combinations of resveratrol and BTP also decreased expression of proliferating cell nuclear antigen in mouse skin tissues/tumors than their solitary treatments as determined by immunohistochemistry. In addition, histological and cell death analysis also confirmed that resveratrol and BTP treatment together inhibits cellular proliferation and markedly induces apoptosis. Taken together, our results for the first time lucidly illustrate that resveratrol and BTP in combination impart better suppressive activity than either of these agents alone and accentuate that development of novel combination therapies/chemoprevention using dietary agents will be more beneficial against cancer. This promising combination should be examined in therapeutic trials of skin and possibly other cancers.
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Introduction
Since early in the history of medicine, an association between diet and cancer has persisted. The most consistent findings on diet as a determinant of several types of cancers risk prevention is the association with consumption of fruits and vegetables [1]. To date, hundreds of natural or synthetic compounds have been found to possess promising cancer chemopreventive actions. When reviewing the literature on the effects of several dietary agents in animal and in vitro studies, there is ample evidence that specific antioxidants and other phytochemicals present in foods of plant origin protect against genotoxicity and other cancer-initiating or -promoting processes [2], [3].
The concept of using a combination of agents for cancer chemoprevention has recently received much attention. Considerable evidence from laboratories studies suggests that combinations of chemopreventive agents can be more effective for the prevention of cancer than any single constituent. Recently, Xu et al. [4] showed that combination treatment of curcumin and green tea catechins prevent dimethylhydrazine-induced colon carcinogenesis rat model more potently than each of the compounds alone. In another recent study, genistein-selenium combination significantly inhibited growth of LNCaP and PC3 cells in a dose- and time-dependent manner by decreasing matrix metalloproteins-2 levels [5]. Zhou et al. [6] identified the possible chemopreventive effects of soy and tea components on prostate tumor progression in in vivo. The combination of both synergistically inhibited final tumor weight and metastasis and significantly reduced serum concentrations of testosterone and dihydrotestosterone. Our laboratory is actively investigating the hypothesis that combinations of food-based cancer prevention strategies will be a highly effective strategy for the reduction of carcinogenesis. During this course we have investigated that combination of pomegranate fruit extract and diallyl sulfide synergistically inhibited mouse skin tumor growth through reduce proliferation, inhibition of mitogen-activated protein kinase (MAPKs) and nuclear factor- kappa B (NF-κB) signaling and induction of apoptotic cell death [7]. Now, we have chosen to focus our experimental efforts on resveratrol and black tea polyphenol (BTP), two foods frequently cited to protect humans from skin carcinogenesis [8], [9].
Resveratrol (3,4,5-trihydroxy-trans-stilbene), a natural plant polyphenol is widely present in foods such as grapes, wine and peanuts. One of the most striking biological activities of resveratrol intensely investigated during the last years has been its anti-cancer and anti-inflammation properties. These properties were first appreciated when Jang et al. [10] demonstrated that resveratrol possesses cancer-chemopreventive and cytostatic properties via the three major stages of carcinogenesis, i.e. initiation, promotion and progression. Since then, there has been a flurry of papers reporting the implication of resveratrol in cancer chemoprevention through a wide range of actions [11]. Most of the cancer chemopreventive evidence for resveratrol is well documented in various cancers such as those of hepatocellular, lung, skin and prostate by multiple regulatory mechanisms [12], [13], [14], [15]. Kundu et al. [16] showed that resveratrol exert anti-tumor promoting in the 12-O-tetradecanoylphorbol 13-acetate (TPA)- induced mouse skin carcinogenesis model suppression of cyclooxygenase-2 expression by blocking the activation of MAPKs and activator protein. Previously study from our laboratory have reported that resveratrol-induces apoptosis in 7, 12-dimethylbenz[a]anthracene (DMBA)-initiated and TPA promoted, mouse skin tumors through cell cycle arrest, activation of p53 activity and alteration of apoptosis-related proteins [17].
Tea, the most widely consumed beverage has received a great deal of attention because of its polyphenolic constituents known to have strong antioxidants and inhibitory activity against tumorigenesis [18]. Many studies have demonstrated the anti-inflammatory and anti-tumor effects of BTP; it can inhibit proliferation and metastasis and induce apoptosis in various malignant tumors, including skin cancer, by modulating several different signal pathways [19], [20], [21]. Epidemiological studies on black tea and cancer are limited, but several investigators have demonstrated positive correlations between black tea consumption and a lower incidence of breast [22] and ovarian [23] cancer. BTP treatment has been shown to induce apoptosis in prostate cancer cells by induction of p53, down-regulation of NF-κB activity, causing a change in the ratio of pro-and anti-apoptotic proteins and inhibiting expression of activated MAPKs [24].
It is clear that resveratrol and BTP independently have biological activities for cancer prevention purposes, however, productive application of these two compounds in combination to exert synergistic effects against cancer inflammation and growth of tumor warrants further studies. Therefore, multiple properties of resveratrol and BTP in cancer prevention lead us to assume a more benefit in combining these two agents rather than the single agents for cancer prevention and therapy. Thus the present study was designed to investigate whether resveratrol in combination with BTP at low doses synergistically suppress the DMBA-initiated, TPA- promoted two-stage mouse skin tumors. Our results clearly demonstrate that a combination of resveratrol and BTP can synergistically inhibit the development and progression of mouse skin tumors then their solitary treatment.
Materials and Methods
Materials
DMBA, TPA, resveratrol, β-actin (clone AC-74) and propidium iodide (PI) were purchased from Sigma Chemical Co. (St. Louis, USA). Annexin-V and PI fluorescein isothiocyanate (FITC) detection Kit was purchased from BD (San Jose, CA, USA). Purified BTP (>98% pure) was kindly gifted by M/S Indfrag company, Bangalore, India. Hydroxylapatite and N, N-dimethylformamide was purchased from Sisco Research Laboratory (Mumbai, India). The mouse monoclonal total and phospho specific antibodies for p38, p53, c-Jun N-terminal kinase (JNK1/2) (Thr183/Tyr185), p44/42 MAPK (Thr202/Tyr204) i.e extracellular signal-regulated kinase (ERK1/2) were purchased from Cell Signaling Technology (Beverly, USA). Proliferating cell nuclear antigen (PCNA) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The rabbit anti-mouse or goat anti-rabbit horse radish peroxidase (HRP) conjugate secondary antibodies were obtained from Bangalore Genei (Bangalore, India). The poly(vinylidene) fluoride (PVDF) membranes were obtained from Millipore (Billerica, MA, USA). Other used chemicals were of analytical grade and procured locally.
Animals and treatments
Male, Balb/c mice (15–18 g body weight [b. wt.]) were obtained from the Indian Institute of Toxicology Research (Lucknow, India) animal breeding colony. Prior ethical approval for the experiments was obtained from institutional ethical committee. All animals were quarantined in polypropylene cages under standard laboratory conditions (temperature 23±2°C, relative humidity 55±5%, 12/12 h light/dark cycle) and were fed solid pellet diet (Ashirwad, Chandigarh, India) and water ad libitum. After a week of acclimatization animals were used for further experiments.
Long term animal bioassay: For treatment, animals were randomly divided into 6 groups consisting 25 animals in each. Animals of all groups were carefully shaved on dorsal skin in the interscapular region of 2 cm2. DMBA (tumor initiator), TPA (tumor promoter) and resveratrol were dissolved in acetone. In brief, treatment was given as described below for 18 weeks of duration to attain the entire period of skin tumorigenicity:
Group I- No treatment was given to the animals and served as untreated controls.
Group II- DMBA+TPA (animals were treated with single topical application of DMBA (52 µg/animal), one week later followed by topical application of TPA (5 µg/animal) thrice in a week throughout the experimental period and served as positive controls).
Group III- Acetone+Resveratrol+BTP (200 µL acetone was applied topically followed by topical treatment of 50 µM/animal resveratrol to each animal and 0.2% BTP was given as sole source of drinking fluid thrice a week).
Group IV- DMBA+TPA+resveratrol (50 µM/animal resveratrol was given topically to the animals as mentioned in group III, rest of the treatments was same as in group II).
Group V- DMBA+TPA+BTP (treatment of DMBA was similar as in group II after a week followed by TPA application and 0.2% BTP supplementation as in group II and III, respectively).
Group VI- DMBA+TPA+resveratrol+BTP (DMBA and TPA application was same as in group II, resveratrol (25 µM/animal) application and BTP (0.1%) supplementation as in group III).
During treatment period animals were carefully observed for any change in b. wt., fluid/food intake and development of skin tumor. Tumors >1 mm in diameter were considered in the cumulative number if they persist for 2 weeks or more. After completion of 18 weeks of treatments duration, the regression of pattern of tumors in terms of both number and volume were recorded up to 26 weeks in the animals of groups IV, V and VI (bearing tumor or not). These were divided into 3 subgroups and treatment schedule was as follow:
Group IV A - VI A: comprises animals' without tumor and no further treatment was given to these groups.
Group IV B and C- comprises tumor bearing animals' and treatment of TPA+resveratrol and only TPA was withdrawn, respectively.
Group V B and C- comprises tumor bearing animals' and treatment of TPA+BTP and only TPA was withdrawn, respectively.
Group VI B and C- comprises tumor bearing animals' and treatment of TPA+resveratrol+BTP and only TPA was withdrawn, respectively.
After completion of the study period (26 weeks), all the animals of groups IV–VI (A–C) were first examined for tumor volume/number regression and thereafter sacrificed. Tumor volume was calculated per mouse in each group using formula V = D×d2×π/6, where D = bigger dimension and d = smaller dimension. Skin from the painted area (with or without tumors) was excised out, cleaned, snap frozen in liquid nitrogen, and stored at −80°C until further use.
Synergy between resveratrol and BTP combination
The nature of the combined effects of resveratrol and BTP was determined using the method described by Zhou et al. [6], based on the principles described by Chou and Talalay [25]. In brief, the expected value of combination effect between agent 1 and agent 2 is calculated as [(observed agent 1 value)/(control value)]×[(observed agent 2 value)/(control value)]×(control value); and the ratio is calculated as (expected value)/(observed value). A ratio of >1 indicates a synergistic effect, and a ratio of <1 indicates a less than additive effect.
Short term animal bioassay: Animals were divided into 6 groups of 5 in each and doses for selected treatments was same as detailed in long term study section for 2 weeks period. Briefly, groups and treatments are as below:
Group I: Untreated control (No treatment).
Group II: DMBA+TPA (single dose of DMBA was applied topically one week later followed by TPA application 4 times in a week).
Group III: Acetone+resveratrol+BTP (animals were treated with acetone and 50 µM resveratrol topically, and 0.2% BTP as sole source of drinking fluid for 4 times in a week).
Group IV: DMBA+TPA+resveratrol (DMBA and TPA application as in Gr. II and resveratrol treatment as in group III).
Group V: DMBA+TPA+BTP (DMBA and TPA application as in Gr. II and BTP supplementation as in group III).
Group VI: DMBA+TPA+resveratrol+BTP (DMBA and TPA application as in Gr. II and, resveratrol (25 µM/animal) and BTP (0.1%) treatment as in group III).
At the end of the experimental period all the animals were sacrificed and skin from the painted area was excised out, cleaned, snap frozen in liquid nitrogen, and stored at −80°C until further use.
Preparation of lysates
Whole skin tissue/tumor homogenates (10%) were made as described by Kataoka et al. [26], in both, the long term as well as the short term study. The supernatants were collected and stored at −80°C till use.
Western blotting
Western blotting was carried out as described earlier [27]. Protein concentration was measured following standard protocols [28]. Proteins (60 µg) were resolved on 10–12% SDS PAGE and electroblotted on PVDF membrane. The blots were blocked overnight with 5% non-fat dry milk and probed with antibodies at dilutions recommended by the suppliers. Immunoblots were detected through chemiluminescence using kit of Millipore (Billerica, MA, USA).
Histopathological analysis
Paraffin sections (5 µm) of the skin tissues/tumors were stained with haematoxylin and eosin for histopathological analysis. Histopathological observations were made according to Bogovaski [29].
Immunohistochemical (IHC) staining
Buffered formalin fixed and paraffin embedded skin/tumor tissues were cut into sections (5 µm thick), which were subsequently de-waxed and hydrated. Then endogenous peroxidase activity was quenched and epitope retrieval was performed. This was followed by blocking of non-specific binding of primary antibody to epitopes by a preincubation step with normal serum. Sections were then incubated overnight with the primary monoclonal anti-PCNA (1∶100) antibody. After incubation, the sections were again incubated with normal serum and then with HRP-conjugated secondary antibody. The colour was developed using substrate chromogen system diaminobenzidine (Dako, CA, USA). For the negative control, phosphate-buffered saline was used in place of the primary antibody. The immunostained slides were analyzed under microscope (Leica, Wetzler, Germany) attached with charge coupled device camera (JVC).
Annexin-V and PI dual staining
Annexin-V and PI FITC detection Kit (BD, San Jose, CA, USA) was used for the differentiation between apoptotic and necrotic cell population. The single cell suspensions of treated and untreated skin tissues (from short term study) were prepared using Medimachine (Beckton Dickinson, San Jose, USA). For each sample, Annexin-V/PI fluorescence was analyzed, wherein fluorescence of cells was gated and counted using ‘Cell Quest 3.1 software’.
DNA alkaline unwinding assay
Strand breaks in cellular DNA were quantitated by alkaline unwinding assay using hydroxyapatite batch procedure as described previously [30]. In brief, 100 µg of DNA from treated and untreated skin tissue samples (from short term study) was subjected to alkaline unwinding and the relative amount of duplex and single stranded DNA present at the end of the alkaline unwinding was quantified.
Statistical analysis
For the statistical analysis of skin tumor appearance dynamics, the Kaplan-Meier method of tumor-free survival estimation was applied. Statistically significant differences were determined between control and treatment groups using one-way ANOVA (GraphPad Prism software) followed by Dunnett post hoc test. Values with p<0.05 were considered significant.
Results
Effects of resveratrol and/or BTP on tumorigenicity rate
The b. wt. of each animal was recorded every week during the entire period of the experiment. Compared with the untreated control (group I), mice on DMBA and TPA treatment (group II) showed a significant reduction in b. wt. However, there was an increasing percentage of change in the b. wt. gain of the animal of the experimental treatments (groups III–VI) (data not shown). The first day of tumor incidence in the positive control (DMBA and TPA) group II was 48th day; however, it was 63th day in resveratrol treated group IV, 58th day in BTP treated group V and 84th day in the resveratrol and BTP treated group VI. There was no tumor induction in group III. The chemopreventive potential of resveratrol and BTP was also evident by significant (p<0.01) increase in tumor free survival of animals. 36%, 48% and 72% of animals of group IV, V and VI, respectively, remained tumor-free till the termination of the experiment. However, in positive control group (II) 100% animals were observed with tumors by the end of 18th week (Figure 1A). The expected effect of the resveratrol/BTP combination on tumorigenicity rate (33%) was greater than the observed combination effect (28%) with a ratio of 1.17, suggesting that the resveratrol/BTP combination had a synergistic inhibitory effect on tumorigenicity (Table 1).
10.1371/journal.pone.0023395.g001Figure 1 Effect of resveratrol and/or BTP on DMBA and TPA-induced mouse skin tumors.
(A) Percentage of tumor-free survival, (B) cumulative number of tumors (CNT) per group, (C) reduction in average no. of tumors/mouse and (c) reduction in tumor volume/mouse (mm3). The data showed are of surviving animals upto 18th weeks in (A), (B) and (C) however, mean±SD of surviving animals from 18th to 26th weeks in (D). * indicates significant reduction over DMBA and TPA treated group II at 18th week (p<0.01). ** indicates significant reduction over resveratrol and BTP alone treated groups at 18th week (p<0.01). *** indicates significant reduction after 26th weeks (p<0.01).
10.1371/journal.pone.0023395.t001Table 1 Possible synergistic chemopreventive effect of the interaction between Res and BTP against DMBA and TPA-induced mouse skin tumorigenesis.
Treatment (Groups) Tumorigenicity (%) CNT (%) ANT (%) ATV (%)
O Ea
Rb
O Ea
Rb
O Ea
Rb
O Ea
Rb
Untreated control (I)
DMBA+TPA (II)
100.0 100.0 100.0 100.0
Acetone+Res+BTP (III)
DMBA+TPA+Res (IV)
64.0 30.9 47.8 40.5
DMBA+TPA+BTP (V)
52.0 38.7 62.0 48.6
DMBA+TPA+Res+BTP (VI)
28.0 33.0 1.17 10.9 11.9 1.09 27.1 30.0 1.08 18.9 19.7 1.03
All the data are statistically significant, p<0.01.
a Expected value of Res and BTP combination = [(observed value of Res)/(control value)]×[(observed value of BTP)/(control value)]×(control value).
b Ratio = (expected value/observed value). A ratio of>1 indicates a synergistic effect, and a ratio of <1 indicates a less than additive effect.
O, observed; E, expected; R, ratio; Res, resveratrol; CNT, cumulative number of tumors; ANT, average number of tumors/mouse; ATV, average tumor volume/tumor bearing mouse.
Protection was also seen in terms of reduction in tumor volume. The tumor volume was 130.0 mm3/mouse in DMBA and TPA group, which reduced to 52.6, 63.2 and 24.6 mm3/mouse in group IV, V and VI, respectively. The expected effect of the combined resveratrol and BTP on average tumor volume (19.7%) was higher than the observed combined effect (18.9%) with a ratio of 1.05, suggesting that resveratrol and BTP combination also had synergistic effect in reducing the tumor volume (Table 1).
Protection afforded by resveratrol and BTP was also evident in terms of reduction in the cumulative number of tumors (CNT). While the positive control group showed 230 CNT, group IV, V and VI showed 71, 89, and 25 CNT, respectively, at 18th week (Figure 1B). The observed effect (10.9%) in combination group VI was less than the expected value (11.9%), with ratio of 1.09, suggesting that resveratrol and BTP combination synergistically reduces CNT (Table 1).
When these tumor data were considered in terms of number of tumors per mice, at the termination of the experiment at 18 weeks on test, compared with 9.2 tumors per mouse in DMBA and TPA treated group, 4.4, 5.7 and only 2.5 tumors per mouse in group was recorded in group IV, V and VI, respectively (Figure 1C). Compared with the solitary resveratrol (75%) and BTP (67%) treated groups, decrease in the number of tumor per mouse in combination-treated group corresponded to 89% inhibition. The observed effect (27.2%) in combination group VI was less than the expected value (29.6%), with ratio of 1.08, suggesting that resveratrol and BTP combination also synergistically reduces CNT (Table 1).
Effects of resveratrol and/or BTP on tumor growth and regression
We further extended the work to observe regression offered by combined doses of resveratrol and BTP in tumor volume and number if any. At 19th week, TPA treatment was ceased and morphological changes of the tumors were recorded in group IV B, IV C, V B, V C, VI B and VI C. Among these groups, in Gr. VI C, major commencement of regression was recorded from 22nd week onwards (p<0.01). Apparently, group VI B from which resveratrol and BTP were withdrawn showed regression in terms of both tumor volume and number (Table 2). The tumor volume at 26th week reduced to 105.4 mm3/mouse in DMBA and TPA group II, 21.8 mm3/mouse in group VI B and 18.9 mm3/mouse in group VI C (Table 2, Figure 1D). CNT reduced to 198, 10 and 6 in group II, VI B and VI C, respectively (Table 2). Changes in both tumor number and volume were also observed in solitary resveratrol and BTP treated groups (IV B, IV C, V B and V C) but the extent was lesser than combination (Table 2, Figure 1D). Moreover, animals of group VI A did not show any morphological changes as well as development of tumors till the termination of the experiment (Table 2). Thus, combined supplementation of resveratrol and BTP resulted in regression in both tumor volume and number and imparts better efficacy in terms of tumor regression then either of these agents alone.
10.1371/journal.pone.0023395.t002Table 2 Combinatorial chemopreventive effect of Res and/or BTP on tumor regression.
Groups Treatment Number of animals with tumors CNT ANT(Mean ± SD) ATV(Mean ± SD)
18th 26th 18th 26th 18th 26th 18th 26th
II
DMBA+TPA 25/25 25/25 230 198 9.2±2.6 7.92±2.1 130.0±10.2 105.4±10.4
IV A
(−)Res-Non-tumor bearing 0/12 0/12 - - - - - -
IV B
(−)Res-tumor bearing 6/6 6/6 34a
28a
5.66±1.9a
5.66±2.0a
56.1±7.2a
51.8±6.0a
IV C
(+)Res-tumor bearing 7/7 7/7 35a
26a
5.00±1.1a
3.71±2.5a
49.1±6.1a
40.3±5.5a
V A
(−)BTP-Non-tumor bearing 0/9 0/9 - - - - - -
V B
(−)BTP-tumor bearing 8/8 8/8 42a
36a
5.25±2.1a
4.5±2.2a
59.3±6.2a
50.8±6.6a
V C
(+)BTP-tumor bearing 8/8 8/8 46a
34a
5.75±2.1a
4.2±2.6a
67.1±5.4a
53.6±7.5a
VI A
(−)Res+BTP-Non-tumor bearing 0/13 0/13 - - - - - -
VI B
(−)Res+BTP-tumor bearing 5/5 5/5 13b
10b
2.6±2.0b
2.0±2.0c
24.2±5.4b
21.8±5.6c
VI C
(+)Res+BTP-tumor bearing 5/5 3/5 12b
6b
2.4±1.6b
2.0±1.6c
23.9±7.4b
18.9±8.6c
a significant reduction over DMBA and TPA treated group II at 18th week (p<0.01).
b significant reduction over BTP and Res alone treated groups IV B–C and V B–C at 18th week (p<0.01).
c significant reduction at 26th week (p<0.01).
(−) indicates without treatment, (+) indicates with treatment.
Res, resveratrol; CNT, cumulative number of tumors; ANT, average number of tumors/mouse; ATV, average tumor volume/tumor bearing mouse.
Effect of resveratrol and/or BTP on phosphorylated MAPKs
It has been reported that topical application of TPA in mouse skin results in a marked increase in the phosphorylated form of MAPKs [31], affecting fundamental cellular processes like proliferation, differentiation, and survival, thus we further evaluated the expression levels of MAPK family proteins [32]. In the present study, western blot analysis showed that the activation of MAPKs (ERK1/2 , JNK1/2 and p38) was gradually increased during progression of TPA-induced papillomagenesis in DMBA-initiated mouse skin over untreated control group (p<0.05), in both long and short term studies (Figure 2A and B). However, supplementation of combined and solitary doses of resveratrol and BTP in DMBA and TPA treated animals, significantly (p<0.05) down-regulated the expression levels of ERK1/2, JNK1/2 and p38 through inhibition of their phosphorylation as compared to animals of group II, in both experimental sets (Figure 2A and B). There was no effect on the total amount of ERK1/2, JNK1/2 and p38 proteins after DMBA and TPA treatment (Figure 2A and B).
10.1371/journal.pone.0023395.g002Figure 2 Western blots showing the inhibitory effect of resveratrol and/or BTP on the expression levels of total and phospho-ERK1/2, JNK1/2 and p38 in mouse skin tumors.
(A) Long and (B) short term studies. Details for groups and treatments are described in Materials and methods section. The bands shown here are from a representative experiment repeated three times with similar results. Equal loading was confirmed by stripping the immunoblot and reprobing it for β-actin. The pixel density of the specific immunoreactive bands was quantified by densitometry and expressed as a fold difference against β-actin. * more than corresponding value of untreated control group I (p<0.05). **less than corresponding value of DMBA and TPA group II (p<0.05). # less than corresponding value of BTP and Res alone treated groups IV and V (p<0.05).
Effect of resveratrol and/or BTP on histopathology of skin/tumor
Histologically skin tumor sections from DMBA and TPA applied group II exhibited varying degrees of structural and cytological changes as compared to untreated control group I (Figure 3A). Tumors section of group II animals exhibit focal proliferation of squamous cells, presence of some necrotic cells and keratinization (Figure 3A). This clearly indicates the benign nature of tumors initiated and promoted by DMBA and TPA, respectively. However, skin sections from animals treated with resveratrol/BTP (group III), did not reveal any histopathological abnormalities (Figure 3A). The administration of both resveratrol and BTP led to suppression of DMBA and TPA induced skin tumorigenesis which was evident by skin/tumor sections (Figure 3A). Skin tumor sections of group VI showed chances towards normalization of skin as compared to group II (Figure 3A).
10.1371/journal.pone.0023395.g003Figure 3 Histological study of mouse skin/tumors.
(A) Long and (B) short term experimental groups. Details for groups and treatments are described in Materials and methods section.
Similarly, in short term experimental groups' haematoxylin and eosin stained sections were showing pronounced preventive effects of resveratrol and BTP treatment when given in combination than alone over DMBA and TPA -induced changes in animals skin (Figure 3B). In group II, disorganization of epithelium, presence of necrotic cells and focal proliferative area were noticed as compared to group I (Figure 3B).
Effect of resveratrol and/or BTP on proliferation marker
IHC analysis of PCNA was used to assess the proliferation activity during tumor promotion (Figure 3A). PCNA reactivity is associated with S phase of DNA replication [33]. It was observed that PCNA labelling indices were higher in animals treated with DMBA and TPA as compared to other groups. A characteristic intense staining and higher number of PCNA positive cells were observed as shown in Figure 4A. The representative untreated control group did not show significant positive staining (Figure 4A). Further, treatment of resveratrol and BTP resulted in low expression levels of PCNA positive cells (p<0.01) as compared to group II revealing that effects of combinational treatment of resveratrol and BTP were superior to single treatment (Figure 4A).
10.1371/journal.pone.0023395.g004Figure 4 Effects of resveratrol and BTP treatment on modulation of PCNA expression.
(A) Long and (B) short term experiments. Details are described in Materials and methods section. The brown colour nuclei mark the reactivity with PCNA indicated by arrows. Bar diagram shows mean ± SD of three independent sets analysis. *significantly increased in comparison of group I (p<0.01), **significantly decreased when compared with group II (p<0.01). #significantly decreased when compared with groups IV and V (p<0.01).
Similarly, PCNA staining were analyzed to determine the proliferative status of skin after treatment with DMBA and TPA and the respective preventive treatments in short term groups (Figure 4B). It was observed that the number of PCNA positive cells were higher in animals of group II as compared to untreated animals (Figure 4B). The skin sections from untreated animals did not show significant positive staining for PCNA (p<0.01). Treatment with either resveratrol or BTP or both in combination in DMBA and TPA applied animals resulted in significantly low level of PCNA positive cells (p<0.01) (Figure 4B).
Effect of resveratrol and/or BTP on phosphorylation and stabilization of tumor suppressor protein, p53
p53 in response to toxic insults to DNA, triggers a chain of cell cycle regulatory events to check the proliferation of altered cells to repair or minimize the damage [34], [35] and Ser 15 phosphorylation is essential for stabilization and activation of p53 [36]. To determine whether p53 is phosphorylated at Ser15 in mouse skin tumors (long term study) treated with resveratrol, BTP or their combination, we used a phospho-specific antibody against p53 at Ser15 to do Western blot analysis. Our data showed that the level of p53 phosphorylation at Ser15 was increased in group IV and V upon treatment with resveratrol and BTP as compared with DMBA and TPA group II, however, significant increase in their expression was observed in combination group VI (Figure 5). Immunoblotting showed that the increased level of wild-type p53 protein corresponded well with the increased level of p53 phosphorylation at Ser15. These results are in agreement with earlier reports from our laboratory showing inhibitory effects of resveratrol and BTP individually in DMBA induced mouse skin tumors via enhancement of wild-type p53 [17], [21]. These results suggest that the combination of resveratrol and BTP more efficiently stabilized p53 and induced its phosphorylation at ser15 than either single agent.
10.1371/journal.pone.0023395.g005Figure 5 Effects of resveratrol and BTP treatment on modulation of p53 expression and p53 phosphorylation and stabilization.
Details for groups and treatments are described in Materials and methods section. The bands shown here are from a representative experiment repeated three times with similar results. Equal loading was confirmed by stripping the immunoblot and reprobing it for β-actin. The pixel density of the specific immunoreactive bands was quantified by densitometry and expressed as a fold difference against β-actin. ** less than corresponding value of untreated control group I (p<0.05). * more than corresponding value of DMBA and TPA group III (p<0.05). # more than corresponding value of BTP and Res alone treated groups IV and V (p<0.05).
Effect of resveratrol and/or BTP on apoptosis
Apoptosis is the most potent defence mechanism against cancer [37]. We next quantified the extent of apoptosis by flow cytometric analysis of the skin cells labelled with Annexin V/PI. Results showed significant (p<0.05) percent increase in apoptotic population by either resveratrol/BTP or the two in combination (6.12±0.34, 5.23±0.52 and 14.00±0.20, respectively) over untreated (2.10±0.36) and DMBA and TPA group II which recorded 1.40±0.15 apoptotic populations (Figure 6).
10.1371/journal.pone.0023395.g006Figure 6 Representative figure of flow cytometric analysis of apoptosis in mouse skin on treatment with resveratrol and BTP.
The data is representative of three independent experiments (p<0.05). Details are described in Materials and methods section. Figure showing cells in the upper right (UR), upper left (UL) and lower right (LR) portions portion of the picture indicates late apoptotic cells, necrotic and pre-apoptotic cells, respectively whereas cells present in lower left (LL) portion of the picture indicate percentage of live cells.
Effect of resveratrol and/or BTP on DMBA and TPA-induced nick formation
Based on the amount of duplex DNA remaining after alkali treatment, the number of strand breaks generated per unit DNA was determined in mouse skin tissues obtained from short term study. DMBA and TPA caused a significant DNA damage (n = 1.50±0.17) over untreated (n = 0.007±0.001) in terms of strand breaks (p<0.01). Inhibition of DMBA and TPA-induced DNA alkylation damage was recorded by either resveratrol or BTP or the two in combination and prevention percentage was found to be 45.79%, 40.52% and 60.53%, respectively (Table 3).
10.1371/journal.pone.0023395.t003Table 3 Number of DNA strand breaks inhibited by resveratrol or/and BTP against DMBA and TPA- induced DNA alkylation damage.
Groups Treatment Number of DNA strand breaks (n)
II
DMBA+TPA 1.50±0.17
IV
DMBA+TPA+resveratrol 0.81±0.18a (45.79%)
V
DMBA+TPA+BTP 0.90±0.19a (40.52%)
VI
DMBA+TPA+resveratrol+BTP 0.60±0.14b (60.53%)
Data are expressed as mean ± SD of five animals. ‘n’ represents number of strand breaks of duplex DNA over untreated control group.
a significant prevention over DMBA and TPA- induced DNA strand breaks (p<0.01).
b significant prevention over BTP and Res alone treated groups IV and V(p<0.01).
Values in parenthesis represents the percentage prevention offered by resveratrol or/and BTP against DMBA and TPA- induced DNA strand breaks.
Discussion
Chemoprevention by using dietary agents in combination is gaining the attention of researchers and consumers as a plausible approach for the management of various neoplasia. In this study, employing two-stage mouse skin carcinogenesis protocol, we are able to show that combined treatment of resveratrol (25 µM/animal) and BTP (0.1%) synergistically suppressed the skin tumors more efficiently than either of these solitary agents (Figure 1). Administration of resveratrol and BTP in combination was found to be highly effective in decreasing the CNT and tumor volume (Table 2). Earlier studies from our laboratory have demonstrated the chemopreventive potential of both the agents against mouse skin carcinogenesis, respectively [17], [21]. The central finding of the study is that combined doses of resveratrol and BTP also resulted in significant regression of tumors in regard to both tumor volume and number. Also, no increase in tumor volume or occurrence of tumors was observed in groups (tumor bearing and non tumor-bearing) after withdrawal of the combined treatment. Hence, we observed that resveratrol and BTP (in combination) had superior chemopreventive effects as compared to either resveratrol or BTP.
MAPKs, comprising a family of serine and threonine kinases of ERK, JNK, and p38, are important signaling components which convert external stimuli into a wide range of cellular responses, such as proliferation, survival, differentiation and migration [32]. MAPK signalling cascades are highly relevant in the process of tumor promotion and progression induced by chemical carcinogens in mouse skin carcinogenesis, affecting not only cell proliferation, but also apoptosis by promoting the survival of the tumor cell [38], [39]. Activation of the MAPKs pathway occurs in response to integrin-mediated cellular adhesion to the extracellular matrix, which plays a critical role in both tumor metastasis and angiogenesis [40], [41]. In the present study, we found that topical application of DMBA and TPA (Gr. II) resulted in a marked increase in the phosphorylated form of ERK1/2, JNK1/2 and p38 protein expression; however, resveratrol and/or BTP treatment inhibited this phosphorylation. Importantly, the combined administration of both resveratrol and BTP was found to be more potent in inhibiting phosphorylation of MAPKs (Figure 2). These findings suggest a possibility that as an initial response resveratrol and BTP (in combination) modulate MAPKs activation further leads to inhibition of tumor growth and regression by apoptotic cell death.
In the present study, we further investigated the solitary and combined effect of resveratrol and BTP on expression of proliferation marker PCNA. Enhanced expression of PCNA, a 36 kDa co-factor of DNA polymerase δ, is one of the downstream effects of the activation of MAPK/ERK1/2 signalling and well correlated to the status of cellular proliferation [42]. Although single administration of resveratrol and BTP resulted in growth inhibition of DMBA and TPA-induced skin tumors, their combined treatment markedly inhibited the growth accompanying significant decrease in PCNA immunoexpression in tumor sections. Besides this, histologic analysis of the skin tissues of untreated animals displays major epithelial proliferation. In contrast the skin/tumor sections of mice given resveratrol and/or BTP display no indication of neoplasia. Therefore, inhibition of ERK1/2, JNK1/2 and p38 activation, suppression of PCNA upon resveratrol and BTP supplementation in combination, rather than alone treatments, suggest the superiority in synergistic effects of the two. This finding is quite intriguing, and it is tempting to speculate that a combination of resveratrol and BTP might have efficiently more influenced the cell death–related signaling pathways, ultimately leading to the suppression of well-established DMBA and TPA induced tumor in vivo, which might not possibly be achieved by a single administration of resveratrol or BTP.
To dissect the possible mechanisms in which the apoptotic and/or anti-proliferation signaling were triggered, we also examined the expression levels of the tumor suppressor p53 protein. p53 plays a crucial role in controlling the cell cycle, apoptosis, genomic integrity and DNA repair in response to various forms of stress [35]. As aforementioned, post-translational modifications such as phosphorylation and acetylation are critical for stabilization and activation of p53 [36] our data showed that phosphorylation of p53 (Ser15) increased in mouse skin tumors treated with resveratrol and BTP, but the combination induced more efficient stabilization and phosphorylation of p53 protein at (Figure 5). Consistent with the findings of our previous reports [17], [21], wild-type p53 activity also increased more in these groups (Figure 5).Thus, indicate that in mouse skin tumors, growth inhibition induced by resveratrol and/or BTP is p53-dependent and induced p53 phosphorylation at Ser15 may be attributed to stabilization of p53.
Apoptosis is a selective process of physiological cell deletion that plays an important role in the balance between cellular replication and death. Furthermore, it has been suggested that some cancer chemotherapeutics and chemopreventives exert their effects by triggering either apoptotic cell death or cell cycle transition, and accordingly, the induction of tumor cell apoptosis is used to predict tumor treatment response [43], [44]. Therefore, we quantified the extent of apoptosis by flow-cytometric analysis of skin cells labelled with Annexin-V-PI. The results showed that combinatorial effect of resveratrol and BTP, leads to enhanced cell death than either of them alone. In addition, it is further noted that the administration of resveratrol and/or BTP inhibited the DMBA and TPA- induced DNA damage as revealed by the reduction in strand breaks. But combination doses exerted better effects than either of these two agents alone. This might be occurring because both of them are known to have ability to repair DNA damage [45], [46].
In recent years, emerging evidence suggests that cancer-preventive agents might be combined for more effective treatment of cancer [47]. They can react together to give synergistic action to intervene cancer stages by different signaling pathways or to compensate for the opposite properties in cancer cell proliferation or apoptosis. Thus, we conclude that both resveratrol and BTP at low doses in combination synergistically inhibit established skin tumor growth than either of these agents alone accompanied by decrease in tumor volume and number. This is attributable to reduction in nick formation associated with a decrease in proliferation marker and tumor suppressor protein p53 together with an inhibition of MAPKs signaling and induction of apoptotic cell death in skin cells. Further pre-clinical and clinical trials are warranted to characterize the efficacy of dietary agents in combination with existing therapeutics for chemoprevention and chemotherapy of cancer.
The authors thank all their colleagues who were involved in the study for their excellent assistance. The authors are grateful to Director, Indian Institute of Toxicology Research (CSIR) Lucknow, India for his keen interest in the study. They are thankful to Neeraj Mathur, Scientist for statistical analysis of all the data. Authors are thankful to B. P. Chaudhari for all support of histopathological and IHC analysis. Authors also acknowledge Mr. SHN Naqvi for helping in animal experimentation.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by a research grant from Network Project (NWP-017), Council of Scientific & Industrial Research (CSIR), New Delhi, India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
==== Refs
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J Med LifeJ Med LifeJMedLifeJournal of Medicine and Life1844-122X1844-3117Carol Davila University Press Romania 22567051JMedLife-04-275General ArticleHighlights in the minimally invasive treatment of SUI in women
Surcel C *Chibelean C *Iordache A *Mirvald C *Gîngu C *Margaritis S *Stoica R *Codoiu C *Savu C *Marksteiner R **Sinescu I ** ‘Fundeni’ Clinic of Urology and Renal Transplantation, BucharestRomania
** Life Science Center Biotechnologie InnsbruckAustria
Correspondence to:Assist.Prof. C. Surcel, MD, PhD, ‘Fundeni’ Clinic of Urology and Renal Transplantation, Bucharest, Romania, Telephone: +40 744 963 035
,e-mail
[email protected] 8 2011 25 8 2011 4 3 275 279 25 2 2011 25 6 2011 ©Carol Davila University Press
2011This is
an open-access article distributed under the terms of the Creative
Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work
is properly cited.Objective: Treatment of stress urinary incontinence consists of a wide range of options, from conservative therapies like lifestyle changes, medication, pelvic floor muscles exercises, electro-stimulation, to minimally invasive procedures- injection of collagen, suburethral slings TVT / TOT and last but not least, invasive surgical treatment reserved for recurrent and complex cases. Among the latest minimally invasive procedures reported in literature, the injection of intra-and perisphincterian of autologous stem cell (mioblasts and /or mature fibroblasts grown and multiplied in the laboratory from biopsy samples taken from the pectoralis muscles).
Material and method: On October 18, 2010, in ‘Fundeni’ Clinical Institute of Uronephrology and Renal Transplantation was performed the first stem cell implantation procedure in the urethral sphincter, in Romania.
Results: Assessment at 6 weeks, the quality of life questionnaires, micturition diary and clinical examination revealed a stunning decrease of urine loss from 6 pads / day at one per day, which significantly improved the patient's quality of life.
Conclusions: Stem–cell–mioblasts therapy may represent in the future an every–day intervention in the urologist's armamentarium. The effectiveness of this treatment can change the course of therapy and last but not least, the accessibility to urological evaluation of patients with stress urinary incontinence. Clinical and urodynamic evaluations will continue and will be future scientific topics
stress urinary incontinencestem cellsurethral sphincterminimal invasive treatment
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Introduction
Stress urinary incontinence is a symptom/sign/condition that is defined by involuntary loss of urine that occurs during physical activity, with the effort of coughing, sneezing, laughing, prolonged standing, sexual activity, etc. [1]. Although not life threatening, it is certainly a public health problem, affecting the quality of life, mainly of the female population. The prevalence of this disorder reaches alarming rates, about 20% of total female population being affected, percentages increasing to 35% for those aged over 60 years [2, 3].
Urinary continence and the act of micturition depend on the normal functioning of the lower urinary tract and of the nervous system. Two muscular structures are mainly involved in controlling the act of micturition:
the urethral sphincter, which must be fully functional in order to facilitate continence and micturition;
the detrusor, the bladder muscle layer, which should gradually relax to allow the filling of the bladder and to contract efficiently in order to eliminate the urine.
Whenever the pelvic floor structures are impaired, the base of the bladder and the urethra would weaken, with the appearance of urinary incontinence due to the increasing of the abdominal pressure during efforts (coughing, laughing, sneezing, exercise). In addition, it has been described an entity in which the components of the pelvic floor are not affected, still the urine loss persists, the mechanism being described as intrinsic sphincter deficiency [1].
Among the most important risk factors reported in the occurrence of stress urinary incontinence (SUI) are mentioned: female sex, multiparity, obstetric history, lifestyle, chronic cough (chronic bronchitis, asthma), advanced age, estrogen status, obesity and history of pelvic surgery [4, 5, 6, 7, 8].
The treatment of SUI consists in a wide range of options, from conservative therapies including lifestyle changes, medication, pelvic floor muscles exercises, electrostimulation to minimally invasive – injection of collagen, suburethral slings TVT/TOT and invasive surgical treatment reserved for complex, recurrent cases [9].
Stress urinary incontinence is still a ‘battlefield’ for many minimally invasive therapies but, unfortunately, few can restore the anatomical and functional background of this disorder. Intrinsec urethral sphincter deficiency emerged as a key mechanism underlying the occurrence of this disease, along with other pathogenic theories, such as hipermobility of the urethra. Moreover, recently, according to the ‘trampoline’ theory, any structural defect in the pelvic ligaments, bones, fascial structures may contribute to the impairment of the pelvic muscle cybernetic system [10]. However, clinical experience has shown that not all lesions have a proportional role in the development of SUI and the mechanism of urethral sealing– mainly muscular, contributes fundamentally to the achievement of urinary continence. It is not a lower density of skeletal muscle fibers in the structure of the urethral sphincter involved in the appearance of SUI? The literature is contradictory, but many studies show a reduction in muscle fibers density in a category of patients from which we had excluded all other pelvic pathology [11, 12]. Thus, theoretically an augmentation of the number of muscle fibers in the structure of the urethral sphincter could represent an innovative solution.
Methods
Among the latest minimally invasive procedures includes the injection of autologous stem cell intra and around the intrinsec sphincter as seen in Figure 1 (mioblasts and/or mature fibroblasts multiplied in the laboratory from biopsy samples taken from the pectoral muscles). This method has proven clearly superior to the injection of collagen, not being associated with major side effects, with minimal morbidity, reduced mean hospitalization time and even if it is still in an experimental stage, it stands to be a promising procedure in the near future.
Figure 1 Theoretical scheme of stem cells implantation
(With the permission of Prof. Marksteiner)
Given the significant clinical experience in renal transplantation activity performed in ‘Fundeni’ Clinic of Urology and Renal Transplantation of over 30 years, coupled with the uroginecologic expertise made by the pioneers of urodynamic evaluation and surgical treatment of pelvic static disorders in Romania, our Center actively participates in the clinical research of female pelvic disorders. In addition, the expertise gained in the most important Renal Transplantation Center from Central and Eastern Europe – the ‘Fundeni’ Clinic allows and provides the infrastructure needed for the development of this project.
In fact, selecting those patients with stress urinary incontinence, in which the main pathogenic mechanism is represented by urethral sphincter deficiency, is not an easy task.
Urodynamic evaluation in conjunction with physical examination and medical history are the necessary steps in gathering the patients included in the study group which later, in time, will be compared to a control group to whom a standard minimally invasive is performed according to the guidelines of the Romanian Association of Urology–European Association of Urology and International Society of Continence. Finding well balanced groups regarding age, performance and estrogen status, medical and surgical history, the severity of SUI, previous therapies both medical and surgical performed for the treatment of pelvic disorders, are becoming reasons for concern to bring into daily practice such procedures in the future. Based on the clinical evidence and the results obtained from follow–up, randomized, multicenter, well–managed studies, SUI will be designed. The implantation of stem cells (mioblasts) implies a paradigm shift in the current treatment of SUI which is currently using synthetic materials such as polypropylene which, although well tolerated, they can never replace the auto/allografts in terms of biomaterials quality, and organic compatibility. In addition, the anatomical restoration of the defects mean a return to the ‘restitutio ad integrum’ principle and not just a simple adjustment in order to resolve a pelvic static problem.
From a technical standpoint, the procedure involves four major stages:
Selecting patients with SUI with intrinsic sphincter insufficiency.
The collection of biopsy material (mioblasts) from the pectoris major muscle. It is a maneuver easy to perform, with a short learning curve.
Isolation of stem cells (mioblasts) and multiplying them in cell cultures at the Center of Excellence in Cell and Tissue Research in Innsbruck, Austria. (Figure 2, Figure 3)
The surgical maneuver of stem cells implantation in the urethral sphincter
Figure 2 Mioblast growth in cell culture –200x, antiDesmine staining
Figure 3 Mioblasts fusion to the miotubuli in cell culture –200x, antiDesmnine staining (with the permission of
Prof. Marksteiner).
It involves the use of a special biopsy device – Sonoject– which contains a central piece through which the biopsy is being performed, that provides an adapter to a syringe containing the cell suspension and a channel for a 20MHz circular ultrasound arm that is used to locate the external urethral sphincter and to guide the injections. The device is fixed to a metal arm that is attached to the surgical table (similar to that used in brachytherapy) together with a metal cylinder that slides manually, on which the –Sonoject– is locked on.
The patient is placed in lithotomy position under general anesthesia. The pubic region, internal thighs and perineal area are disinfected and draped properly. Before the procedure, the device is assembled and tested in a saline solution or sterile water. Thus we can calibrate the circular ultrasound in order to detect the needle and its signal.
Afterwards, we put the device in the bladder after it has been previously filled with 200 cc of saline. The system is armed so it does not move during the maneuver in order to identify the bladder neck, urethra and urethral sphincter (Figure 4). Once established the injection site, we introduce the cell suspension solution on the anterior side of the sphincter, in two different semi–circular quadrants. The maneuver stops after we punctured in 20 different sites with 100 μL of solution. At the end of the procedure, the device is withdrawn from the urethra and disassembled.
Figure 4 Urethral sphincter ultrasound – intraoperative aspect
On October 18th 2010, in ‘Fundeni’ Clinic of Urology and Renal Transplantation was performed the first stem cell implantation procedure in the urethral sphincter in Romania. The team was led by Professor Dr. Ioanel Sinescu and was made out of Dr. Cristian Surcel, Dr. Alexandru Iordache, Dr. Calin Chibelean, Dr. Cristian Mirvald, Dr. Carmen Savu, Nurse Liviu Andrei and Professor Rainer Marksteiner. The procedure followed the standard protocol without incidents, the patient being discharged after 24 hours.
Results
Assessment at 6 weeks, containing a clinical examination, QoL questionnaires and voiding diaries, revealed a stunning improvement, the loss of urine being reduced from 6 pads/day to one per day, with a significantly improvement in the patients' quality of life. Clinical and urodynamic evaluations will continue, being the topics of future scientific research.
Discussions
The pelvic floor contains structures whose pathology is treated by at least three surgical specialties. Posterior compartment prolapse and anal incontinence are evaluated by the general surgeon or proctologist, uterine and vaginal prolapse, dyspareunia by the gynecologist and cystocele and low urinary tract symptoms by the urologist. Urinary incontinence and pelvic floor prolapse are two pathological entities that occur as a result of structural damage to several components of the urogenital diaphragm. Thus, according to the first symptoms that appear, the first presentation to the doctor, the outcome of treatment, follow–up are carried out by the specialist who treated only a symptom for which the patient initially presented, though as we mentioned above, the pathogenesis is multifactorial and the symptoms appear after the breakdown of the structures with a high grade of fragility. For this reason, many patients receive an incomplete treatment, which often worsens the clinical background or trigger other symptoms caused by other structures which, at the time of presentation, were compensated.
Suburethral slings, inserted transobturatory, were introduced in Europe several years ago. This procedure was carried out by urogynecologist despite the absence of long–term data regarding efficiency and the rate of healing. The same thing happened with TVT's when they were introduced and, although the medium and long term data were lacking, they were adopted and became today's gold–standard treatment for SUI in women. Abdel–Fattah has recently published a series of reports [14, 15] that assessed physicians' preferences for minimally invasive treatment of stress urinary incontinence. The results were encouraging and emphasized that one third of respondents considered that TVT–O was a procedure up–to–date and must be applied immediately, while others expect the medium and long term statistics.
Treatment is tailored to the patient's suffering and not just treats the loss of urine. In other words, a successful therapy includes the main objective parameters (dry/wet) and the subjective quality of lifwhich is assessed by questionnaires [16]. However, understanding the ‘results’ and the statistical methods used in their quantification are not homogeneous and sufficiently clear in order to remove any controversy. Before we compare and decide which is the most effective procedure, we should reach a consensus on the definition of ‘results’, how they should be measured, follow–up intervals, etc. Until these issues are clarified, the urologist will continue to choose one of the many existing procedure and will remain autonomous in his selection. Thus, the need for new therapeutic methods, that can restore as close as possible the integrity of the pelvic structures, is urgently required.
Conclusions
Stem–cell– mioblasts therapy may represent in the future an every–day intervention in the urologist's armamentarium. At least for this group of patients, to whom, from the pathogenic point of view, the deficiency is limited to the urethral sphincter, part of the excretory system, we believe that the urologist's interest should be maximal. The effectiveness of this treatment can change the course of therapy and last but not least, the accessibility to urological evaluation of patients with stress urinary incontinence.
The Centers of Excellence in Urology must develop research programs and become partners in multicenter studies in order to obtain solid long term data. Thus, new standards will be created that will be approved by urologists everywhere.
==== Refs
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 21949767PONE-D-11-0676910.1371/journal.pone.0024849Research ArticleBiologyBiochemistryImmunologyMolecular Cell BiologyCell DeathSignal TransductionMedicineHematologyHematologic Cancers and Related DisordersOncologyBasic Cancer ResearchCancer TreatmentSimultaneous Inhibition of mTOR-Containing Complex 1 (mTORC1) and MNK Induces Apoptosis of Cutaneous T-Cell Lymphoma (CTCL) Cells Joint mTORC1 and MNK Inhibition Induces ApoptosisMarzec Michal
1
Liu Xiaobin
1
Wysocka Maria
2
Rook Alain H.
2
Odum Niels
3
Wasik Mariusz A.
1
*
1
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America
2
Department of Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America
3
Institute of Molecular Biology, University of Copenhagen, Copenhagen, Denmark
Ulasov Ilya EditorUniversity of Chicago, United States of America* E-mail: [email protected] and designed the experiments: MM MAW. Performed the experiments: MM XL. Analyzed the data: MM XL NO MAW. Contributed reagents/materials/analysis tools: MW AHR. Wrote the paper: MAW.
2011 16 9 2011 6 9 e2484911 4 2011 19 8 2011 Marzec et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
mTOR kinase forms the mTORC1 complex by associating with raptor and other proteins and affects a number of key cell functions. mTORC1 activates p70S6kinase 1 (p70S6K1) and inhibits 4E-binding protein 1 (4E-BP1). In turn, p70S6K1 phosphorylates a S6 protein of the 40S ribosomal subunit (S6rp) and 4E-BP1, with the latter negatively regulating eukaryotic initiation factor 4E (eIF-4E). MNK1 and MNK2 kinases phosphorylate and augment activity of eIF4E. Rapamycin and its analogs are highly specific, potent, and relatively non-toxic inhibitors of mTORC1. Although mTORC1 activation is present in many types of malignancies, rapamycin-type inhibitors shows relatively limited clinical efficacy as single agents. Initially usually indolent, CTCL displays a tendency to progress to the aggressive forms with limited response to therapy and poor prognosis. Our previous study (M. Marzec et al. 2008) has demonstrated that CTCL cells display mTORC1 activation and short-term treatment of CTCL-derived cells with rapamycin suppressed their proliferation and had little effect on the cell survival.
Methods
Cells derived from CTCL were treated with mTORC1 inhibitor rapamycin and MNK inhibitor and evaluated for inhibition of the mTORC1 signaling pathway and cell growth and survival.
Results
Whereas the treatment with rapamycin persistently inhibited mTORC1 signaling, it suppressed only partially the cell growth. MNK kinase mediated the eIF4E phosphorylation and inhibition or depletion of MNK markedly suppressed proliferation of the CTCL cells when combined with the rapamycin-mediated inhibition of mTORC1. While MNK inhibition alone mildly suppressed the CTCL cell growth, the combined MNK and mTORC1 inhibition totally abrogated the growth. Similarly, MNK inhibitor alone displayed a minimal pro-apoptotic effect; in combination with rapamycin it triggered profound cell apoptosis.
Conclusions
These findings indicate that the combined inhibition of mTORC1 and MNK may prove beneficial in the treatment of CTCL and other malignancies.
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Introduction
mTOR (mammalian target of rapamycin) is a ubiquitously expressed serine/threonine kinase. mTOR associates with either protein called raptor or another named rictor and other proteins to form the mTORC1 and mTORC2 complexes, respectively. The function and signaling pathways activated by mTORC1 have thus far been much better characterized [1], [2]. Accordingly, TORC1 affects a number of key cell functions such as cell size, proliferation, protein synthesis, and angiogenesis. mTORC1 acts by phosphorylating and activating p70S6kinase 1 (p70S6K1) and inhibiting 4E-binding protein 1 (4E-BP1). p70S6K1 is a serine/threonine kinase that phosphorylates a S6 protein of the 40S ribosomal subunit (S6rp) at several sites including serines 235 and 236. In turn, 4E-BP1 is a translational repressor that negatively regulates eukaryotic initiation factor 4E (eIF-4E). Two related kinases MNK1 and, to the lesser degree, MNK2 phosphorylate eIF4E at serine 209 (S209) augmenting its activity [3]. Rapamycin and its analogs are highly specific, potent, and relatively non-toxic inhibitors of mTORC1 [1], [2].
CTCL is the most frequent type of T-cell lymphoma. Although initially usually indolent, it displays a tendency to progress to the aggressive forms with limited response to therapy and poor prognosis [4]. Sezary Syndrome (SS) is a leukemic form of CTCL in which the malignant (Sezary) T cells sometimes comprise a vast majority of the peripheral blood lymphocytes. Our recent study has demonstrated that CTCL cells display mTORC1 activation in the lymphoma stage-related fashion with the highest percentage of positive cells identified at the late, clinically aggressive stage of the large cell transformation [5]. Short-term treatment of CTCL-derived cells with the mTORC1 inhibitor rapamycin partially suppressed the cell proliferation and had little effect on their survival [5].
Materials and Methods
CTCL cell lines and primary cells
The MyLa2059 and MyLa3675 derived from skin lesions of advanced CTCL and the IL-2-dependent Sez-4 cell line was derived from peripheral blood, leukemic (Sezary) CTCL cells [5]. The leukemic cells used in the study were from CTCL patients with a high lymphocytosis and were >90% pure as determined by the CD4∶CD8 ratio and CD7 and/or CD26 loss by the CD4+ T cells. Cell lines and primary malignant cells were cultured at 37°C and 5% CO2 in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin/Fungizone mixture, and 2 mM L-glutamine at 37°C and, in the case of Sez-4 cells, 100 U/mL of IL-2. To obtain primed cells, leukemic CTCL cells were cultured for 7 days in the presence of a mitogen PHA-L (Sigma-Aldrich, St Louis, MO) used at 10 µg/mL.
Kinase Inhibitors
Inhibitors of MNK (MNKi) and mTORC1 (rapamycin) were purchased from Calbiochem and used at the indicated doses. MNK inhibitor, 4-Amino-5-(4-fluoroanilino)-pyrazolo[3,4-d]pyrimidine, inhibits MNK1 with IC 50 of 2.2 µM in vitro and 3 µM in vivo. It has no inhibitory activity against p38, JNK1, ERK1/2, PKC, or Src-like kinases.
Western blot
The cells were washed in phosphate-buffered saline (PBS), centrifuged and lysed in radioimmunoprecipitation assay buffer supplemented with 0.5 mM phenylmethylsulfonyl fluoride, phosphatase inhibitor cocktails I and II from Sigma (St Louis, MO, USA) and protease inhibitor cocktail from Roche (Basel, Switzerland) as described previously [5], [6]. For normalization of gel loading, the protein extracts were assayed using the Lowry method (Bio-Rad, Hercules, CA, USA). Typically, 5–50 mg of the protein per lane was loaded. To examine protein phosphorylation, the membranes were incubated overnight with antibodies specific for S6rp S235/236, eIF4E S209, 4E-BP1 T37/46, 4E-BP1 T70, MNK1, MNK1 T197/202, MCL-1, and BcL-xL (Cell Signaling) and actin (Santa Cruz). Next, the membranes were incubated with the appropriate secondary, peroxidase-conjugated antibodies. The blots were developed using the ECL Plus System from Amersham.
siRNA Assay
Mixture of four siRNA specific for MNK 1 and 2 oreIF4E or non-targeting siRNA (all purchased from Dharmacon) was introduced into cells at 1 µM by co-incubation in transfection media (Dharmacon Acell) for 48 h. The cells were cultured for additional 24 h, harvested and extend of the protein knock-down was examined by Western blot and RT-PCR.
Cell proliferation (BrdU incorporation) assay
After cell culture for 1 to 10 days in the presence of inhibitors or siRNA, cell proliferation was evaluated in bromodeoxyuridine (BrdU) incorporation assay using the commercially available kit Cell Proliferation enzyme-linked immunoabsorbent assay (ELISA; Roche) according to the manufacturer's protocol. In brief, cells were seeded in 96-well plates (Corning, Corning, NY) at a concentration of 104 cells/well in RPMI medium supplemented with 10% FBS and labeled with BrdU (Roche) for 4 hours. After the plate centrifugation (10 minutes at 300 g), supernatant removal, and plate drying, the cells were fixed, and the DNA was denaturated by the addition of 200 mL FixDenat reagent. The amount of incorporated BrdU was determined by incubation with a specific antibody conjugated with peroxidase followed by colorimetric conversion of the substrate and OD evaluation using the ELISA plate reader.
Cell Growth: Proliferation and Survival (MTT Enzymatic Conversion) Assay
Cell lines were seeded in 96-well plates at 1×104 cells/well in RPMI medium supplemented with 10% FBS. After 6 h culture, the cells were exposed in triplicates to serial dilutions of the inhibitors. After 1–14 days, the relative number of viable cells was determined by the use of MTT reduction colorimetric assay (Promega).
Cell apoptosis (terminal dUTP nick-end labeling: tunel) assay
We used the ApoAlert DNA Fragmentation Assay Kit from BD Biosciences (San Jose, CA) according to the manufacturer's protocol. In brief, cells were cultured at 0.5×106 cells/mL for 3, 7 or 14 days with inhibitors. The cells were collected, washed twice in PBS, and fixed with 1% formaldehyde/PBS. After the wash, cells were permeabilized with 70% ice-cold ethanol for at least 2 hours, washed, and incubated in TdT incubation buffer for 1 hour at 37°C. The reaction was stopped by adding 20 mM EDTA, and the cells were washed twice in 0.1% Triton X 100/BSA/PBS. Finally, samples were resuspended in 0.5 mL of PI/RNAse/PBS, collected, and analyzed by flow cytometry (FACSort; Becton Dickinson, Franklin Lakes, NJ) using the CellQuest PRO software.
Statistics
The significance of difference between controls and different treatment conditions in BrdU, MTT and terminal dUTP labeling assay was evaluated using Student's t-test. P value of <0.05 was considered to be statistically significant. All presented results are calculated as mean +/− S.D. of three or four separate experiments.
Results
Rapamycin partially inhibits growth of CTCL cells and promotes eIF4E phosphorylation at serine 209
To evaluate impact of the extended exposure of malignant T cells to mTORC1 inhibition, we treated two CTCL-derived cell lines, MyLa2059 and MyLa3675, with rapamycin for up to 10 days. As shown in Fig. 1A, rapamycin, used at the predetermined saturating dose [5] was able to suppress CTCL cell proliferation as determined by the BrdU incorporation assay (p<0.05 for days 2–10 as compared to the untreated control). However, the suppression was only partial despite the very effective and persistent inhibition of the mTORC1 signaling as determined by the marked, sustained dephosphorylation of S6rp and 4E-BP1 with the latter dephophorylation appearing less pronounced in MyLa2059 (Fig. 1B). . Of note, mTORC1 inhibition enhanced phosphorylation of eIF4E at S209 (Fig. 1B) suggesting that phospho-eIF4E may be involved in supporting the residual cell growth.
10.1371/journal.pone.0024849.g001Figure 1 Rapamycin partially inhibits proliferation of T-cell lymphoma cells and promotes eIF4E S209 phosphorylation.
The CTCL-derived cell lines MyLa2059 and MyLa3675 were treated in triplicates with 200 nM of rapamycin (mTORC1i) for up to 10 days and (A) labeled with BrdU for the last 4 hr of the culture and examined using the EIA plate reader (the result shows mean value of four separate experiments) or (B) lysed and analyzed by Western blotting with antibodies against the depicted phosphorylated and total proteins with detection of actin serving as the loading control.
MNK mediates eIF4E S209 phosphorylation
Because MNK 1 and 2 kinases have been reported to target the eIF4E S209 site [3], [7], we examined next the effect of MNK inhibition on the eIF4E S209 phosphorylation status. A selective, small molecule MNK inhibitor profoundly suppressed eIF4E S209 phosphorylation in the dose-dependent manner without affecting phosphorylation of Akt-T308, used as control of the inhibitor's specificity (Fig. 2A). The inhibitor suppressed not only the spontaneous, but also the rapamycin-enhanced eIF4E S209 phosphorylation (Fig. 2B), indicating that MNK1/2 is the sole kinase of the S209 site responsible for its baseline as well as the mTORC1 inhibition-augmented phosphorylation. MNK phosphorylation at T197/202 as well as the expression of two anti-apoptotic proteins MCL-1 and BcL-xL remained unaffected indicating that MNK does not self-phosphorylate itself at this key site, on one hand, and that MCL-1 and BcL-xL are not affected by the MNK and mTORC1 inhibition in the CTCL cells. To confirm the MNK's role in the eIF4E S209 phosphorylation, we inhibited MNK expression using siRNA active against both MNK1 and MNK2. The MNK1/2 depletion resulted in the proportionate decrease in the amount of S209-phosphorylated eIF4E protein that matched the decrease achieved by the depletion of eIF4E itself using the eIF4E-specific siRNA (Fig. 2C).
10.1371/journal.pone.0024849.g002Figure 2 MNK inhibition suppresses eIF4E S209 phosphorylation.
A: effect of MNK1/2 inhibitor (MNKi) used at various concentrations on eIF4E S209 phosphorylation in MyLa2059 and MyLa3675 cell lines with Akt T308 phosphorylation serving as a control. B: effect of simultaneous application of MNKi (5 µM) and mTORC1i (200 nM) on phosphorylation of eIF4E S209, S6rp S235/236, and MNK T197/202 and expression of the anti-apoptotic proteins Mcl-1 and BcL-xL. C: effect of siRNA-mediated MNK1 and MNK2 depletion on eIF4E S209 phosphorylation in the presence or absence of rapamycin. D: effect of simultaneous exposure of IL-2-dependent CTCL cell line Sez-4 to MNKi and mTORC1i on phosphorylation and expression the depicted proteins. E: effect of simultaneous treatment of patient-derived, primed Sezary CTCL cells to MNKi and mTORC1i on phosphorylation and expression the depicted proteins.
To determine if eIF4E is phosphorylated by MNK also in other types of CTCL cells, we examined leukemic (Sezary) cells, both in the form of an established IL-2-dependent cell line Sez-4 and primary cells from two CTCL patients. Our previous study [5] has established that, in contrast to the CTCL cell lines and tissues, native leukemic CTCL cells are quiescent and require both mitogen priming and IL-2 stimulation to activate mTORC1 as well as PI3K/Akt and MEK/ERK signaling pathways. As shown in Fig. 2D, Sez-4 cell line displayed essentially the same pattern of response to mTORC1 and/or MNK inhibition as MyLa2059 and MyLa3675 cells, including the inhibition of eIF4E S209 phosphorylation by the MNK inhibitor and enhancement of this phosphorylation secondary to mTORC1 inhibition. While the native leukemic CTCL also displayed eIF4E S209 phosphorylation that was suppressed by the MNK inhibitor (Fig. 2E), the rapamycin-induced enhancement of the phosphorylation was not definitive, mostly likely reflecting overall less malignant phenotype of these cells. Regardless, the presence of the MNK-mediated eIF4E phosphorylation in all types of CTCL cells examined, strongly suggests that it may contribute to their malignant phenotype.
eIF4E S209 phosphorylation is independent of MEK, p38 MAPK, PKC, PKA, and PI3K
Previous studies have implicated MEK1/2-ERK1/2 and p38 MAPK-MSK pathways, separately and in combination in MNK activation in some cell systems [8]. However, inhibition of MEK1/2, p38 MAPK (separately or in combination), PKC, PKA (Fig. 3A) or PI3K (Fig. 3B) did not suppress eIF4E phosphorylation indicating involvement of a different kinase that remains to be identified. Of note, activation of MNK independent of ERK1/2 and p38 MAPK has also been described by others [8].
10.1371/journal.pone.0024849.g003Figure 3 Lack of effect on eIF4E phosphorylation of several kinase inhibitors.
A and B: Expression of eIF4E S209 was examined after treatment of MyLa 2059 cells with inhibitor of p38 MAPK, MEK1/2 (U0126), PKC (Go6983), PKA (Bisindolylmaleimide I) and PI3K (Wortmannin and LY294002). Expression of phospho-Akt T308 and -ERK1/2 T202/Y204 and actin and cell treatment with MNKi served as controls.
Inhibition of MNK suppresses growth of CTCL cells
To determine if MNK controls proliferation of CTCL cells, we treated MyLa2059 and MyLa3675 cells with the MNK inhibitor, applied either as a single compound or together with rapamycin. While MNK inhibition alone suppressed the BrdU uptake, this effect was more pronounced in the presence of rapamycin indicating an additive effect of the compounds (Fig. 4A). Similarly, combination of the siRNA-mediated MNK depletion and rapamycin resulted in the decreased CTCL cell proliferation (Fig. 4B). Of note, siRNA-triggered depletion of eIF4E markedly affected the BrdU uptake, in particular when applied jointly with rapamycin, highlighting the importance of eIF4E in promoting cell proliferation.
10.1371/journal.pone.0024849.g004Figure 4 Simultaneous mTORC1 and MNK inhibition suppresses growth and induces apoptosis of CTCL-derived cells.
Combined inhibition of mTORC1 and MNK induces apoptotic cell death of CTCL cells
To determine if MNK affect long-term growth of CTCL cells, we exposed MyLa2059 and MyLa3675 cells for up to 14 days to the MNK inhibitor in the presence or absence of rapamycin (Fig. 4C). While each of the drugs alone exerted a moderate (rapamycin: p<0.05 as compared to the control) or mild (MNKi) inhibitory effect on the cell growth as determined by the MTT enzymatic conversion, their combination profoundly affected the growth in the time-dependent manner (p<0.01 vs. control). As shown in Fig. 4D, the leukemic cells from two CTCL patients yielded very similar result (p<0.01) further supporting the notion that the mTORC1 and MNK inhibitor combination may be therapeutically effective.
To directly examine MNK effect on the cell survival, we performed the DNA fragmentation (tunel) assay in the MNK inhibitor- and/or rapamycin-treated MyLa2059 and MyLa3675 cells. Whereas MNK inhibitor alone induced very limited cell death, its combination with rapamycin resulted in profound apoptosis of the CTCL cells (Fig. 4E; p<0.01 at day 7 and 14 vs. control).
Discussion
Given the critical role of mTORC1 signaling in cell biology and carcinogenesis as well as the availability of very potent and highly specific mTORC1 inhibitors from the rapamycin family, there has been an immense interest in inhibiting the pathway in patients with various malignancies and other ailments [1]. In fact, numerous clinical trials have been conducted with mTORC1 inhibitors in the large spectrum of cancer types [1], [9]. The drugs used as single agents showed substantial efficacy in several different tumors including transplant-associated lymphoma [10], [11] and Kaposi sarcoma [12], [13], tuberous sclerosis-related astrocytoma [14], and mantle cell and other non-Hodgkin lymphomas [15]. Recently, two mTORC1 inhibitors gained FDA approval for treatment of the renal cell carcinoma [16], [17]. However, the treatment with rapamycin-type compounds typically leads to the clinically stable disease or partial remission rather than the tumor elimination [9]. This suboptimal drug effect is likely due at least in part to the cytostatic rather than cytotoxic properties of the mTORC1 inhibitors. Therefore, there is a great need for the drug combination therapy that ideally would result in the complete remissions and cancer cures. However, most of the attempts to combine mTORC1 inhibitors with other drugs, typically the standard chemotherapeutic agents targeting DNA replication, have so far been rather disappointing, on occasion leading to the drug antagonism, although some combinations, e.g. with cis-platin [18] or methotrexate [19] seem promising based on the preclinical studies.
Here we report that the extended exposure of CTCL cells to the combination of mTORC1 and MNK inhibitors essentially abolishes the cell growth by triggering extensive apoptotic cell death. MNK inhibition eliminates S209 phosphorylation of the indirect target mTORC1 target eIF4E, not only basal but also rapamycin-enhanced, the latter noted in CTCL cell lines. Although we cannot exclude that other MNK targets unrelated directly to the protein synthesis also contribute to cell survival, several observations indicate that the effect of MNK on the eIF4E-containing complex and protein translation are critical in this regard. First, MNK inhibition alone has very little effect on the cell viability; only the combined inhibition of MNK and mTORC1 triggers the apoptosis. Second, recent studies indicate that the inhibition of the eIF4E complex by mTORC1 inhibitors is incomplete due to the relatively ineffective suppression of the 4E-BP1 phosphorylation [20]. The addition of MNK inhibitor appears to correct this deficiency and the MNK-mTORC1 inhibitor combination fully inhibits eIF4E leading to not only suppression of CTCL cell proliferation but, strikingly, also to induction of cell death. The study by Furic et al. [21] showed that the substitution in eIF4E of serine with alanine at position 209 (S209A) inhibited the development of prostatic carcinoma initiated by the prostate-specific loss of PTEN. This finding supports the notion that the MNK-mediated eIF4E S209 phosphorylation plays a key role in the biology of malignant cells, although MNK seems to modify also other members of the translation initiation complex [8], [22]. Finally, the other key MNK targets such as hnRNP A1 and Spry2 are involved in generating anti-growth signals by inducing expression of TNFa and enhancing ERK signaling, respectively. Therefore, their inhibition should not induce cell apoptosis. However, the specific proteins responsible for initiating the apoptosis induced by the joint mTORC1-MNK inhibition remain to be identified. Although MCL-1 expression has been reported as translationally regulated by mTORC1 and MNK in some cell types [7], inhibition of neither of them affected expression of MCL-1 and of another anti-apoptotic protein BcL-xL (Fig. 2). Regardless, our findings strongly suggest that the simultaneous inhibition of mTORC1 and MNK1/2 kinases, both targeting eIF4E activity, may prove highly efficacious in treatment of CTCL and, in all likelihood, many other malignancies. Furthermore, since mTORC1 is activated by a large spectrum of oncogenic cell-surface receptors and intracellular kinases [1], combined inhibition of MNK and the relevant oncogenic kinase upstream of mTORC1 may also prove therapeutically beneficial. The highly effective combination of BCR-ABL inhibitor with MNK inhibitor [23], suggests that this indeed may be the case.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported in part by grants from the National Cancer Institute and Leukemia and Lymphoma Society. No additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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J NeuroinflammationJournal of Neuroinflammation1742-2094BioMed Central 1742-2094-8-1012184638410.1186/1742-2094-8-101ResearchIncreased neuroinflammatory and arachidonic acid cascade markers, and reduced synaptic proteins, in brain of HIV-1 transgenic rats Rao Jagadeesh Sridhara [email protected] Hyung-Wook [email protected] Matthew [email protected] Dede [email protected] Mei [email protected] Andrew David [email protected] Gaylia Jean [email protected] Stanley Isaac [email protected] Mireille [email protected] Brain Physiology and Metabolism Section, National Institute on Aging, Bethesda, MD, 20892, USA2 National Institute of Mental Health, National Institutes of Health, Bethesda, MD, 20892, USA3 Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, 27709, USA2011 16 8 2011 8 101 101 9 3 2011 16 8 2011 Copyright ©2011 Rao et al; licensee BioMed Central Ltd.2011Rao et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
Cognitive impairment has been reported in human immune deficiency virus-1- (HIV-1-) infected patients as well as in HIV-1 transgenic (Tg) rats. This impairment has been linked to neuroinflammation, disturbed brain arachidonic acid (AA) metabolism, and synapto-dendritic injury. We recently reported upregulated brain AA metabolism in 7- to 9-month-old HIV-1 Tg rats. We hypothesized that these HIV-1 Tg rats also would show upregulated brain inflammatory and AA cascade markers and a deficit of synaptic proteins.
Methods
We measured protein and mRNA levels of markers of neuroinflammation and the AA cascade, as well as pro-apoptotic factors and synaptic proteins, in brains from 7- to 9-month-old HIV-1 Tg and control rats.
Results
Compared with control brain, HIV-1 Tg rat brain showed immunoreactivity to glycoprotein 120 and tat HIV-1 viral proteins, and significantly higher protein and mRNA levels of (1) the inflammatory cytokines interleukin-1β and tumor necrosis factor α, (2) the activated microglial/macrophage marker CD11b, (3) AA cascade enzymes: AA-selective Ca2+-dependent cytosolic phospholipase A2 (cPLA2)-IVA, secretory sPLA2-IIA, cyclooxygenase (COX)-2, membrane prostaglandin E2 synthase, 5-lipoxygenase (LOX) and 15-LOX, cytochrome p450 epoxygenase, and (4) transcription factor NF-κBp50 DNA binding activity. HIV-1 Tg rat brain also exhibited signs of cell injury, including significantly decreased levels of brain-derived neurotrophic factor (BDNF) and drebrin, a marker of post-synaptic excitatory dendritic spines. Expression of Ca2+-independent iPLA2-VIA and COX-1 was unchanged.
Conclusions
HIV-1 Tg rats show elevated brain markers of neuroinflammation and AA metabolism, with a deficit in several synaptic proteins. These changes are associated with viral proteins and may contribute to cognitive impairment. The HIV-1 Tg rat may be a useful model for understanding progression and treatment of cognitive impairment in HIV-1 patients.
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Background
Despite improved survival rates for human immunodeficiency virus (HIV-1)-infected patients due to antiretroviral therapy, HIV-1-associated neurocognitive disorders remain a significant public health burden [1,2]. Among HIV-1-infected patients, cognitive impairment is a serious complication of HIV-1-infection, and occurs in a substantial (15-50%) proportion of patients [2]. Indeed, a pilot study revealed high rates of asymptomatic neurocognitive impairment in perinatally infected HIV-positive young adults (67%) when compared with older subjects (19%) [3]. Another study highlighted that the prevalence of HIV-associated neurocognitive disorders is high even among long-standing aviremic HIV-positive patients [4].
Deficits in spatial learning also have been demonstrated in aged HIV-1 transgenic (Tg) rats [5,6]. The HIV-Tg rat contains the HIV-1 virus in its genome, but is not infectious because it lacks the gag and pol replication genes of the virus [7]. HIV-1 Tg rats express the functional viral envelope proteins glycoprotein (gp) 120 and trans-activator of transcription (Tat) in brain and circulating white cells [7]. It has been proposed that these rats can be used to examine effects of these envelope proteins in the absence of infection (viral replication), which may mimic the condition in patients given highly active antiretroviral therapy, who have limited (controlled) viral replication but persistent HIV-1 infection [8]. HIV-1 Tg rats demonstrate reduced spatial learning at 5 months of age, and by 7-9 months show neuroinflammation and upregulated brain arachidonic acid (AA) metabolic rates [5,6,9].
Synapto-dendritic injury, a likely cause of cognitive impairment in HIV-1 patients [10-12], can be exacerbated by a neuroinflammatory microenvironment [13]. During inflammation, AA is released from membrane phospholipids by AA-selective Ca2+-dependent cytosolic phospholipase A2 (cPLA2) and secretory sPLA2. This process is associated with increased production of cytokines (e.g., tumor necrosis factor alpha (TNFα) and interleukin (IL)-1β and nitric oxide from activated microglia. Released TNFα and IL-1β can continue to activate AA cascade metabolism by activating transcription factor NF-κB [14-17]. Further, the released AA can be converted into pro-inflammatory lipid mediators, such as prostaglandin (PG) H2, leukotrienes, and related compounds by the action of cyclooxygenase (COX), lipoxygenase (LOX) and thromboxane synthase (TXS) enzymes. PGH2 is converted to PGE2 by membrane prostaglandin E synthase (mPGES) or cytosolic PGES (cPGES), or by TXS to TXA2. HIV-1 patients show increased concentrations of PGE2, PGF2 and TXB2 in their cerebrospinal fluid [18], consistent with in vivo and in vitro studies [19-21].
A relation of AA and its pro-inflammatory metabolites to neuronal apoptosis and synapse loss has been demonstrated in vivo and in vitro [22-26]. Furthermore, reduced dendritic spine density and complexity have been associated with deficits in learning, memory, and general cognitive function [12]. Neuronal loss also may result from insufficient trophic factors, including brain-derived neurotrophic factor (BDNF) [27]. The post-synaptic dendritic proteins, drebrin and neurofilament light chain (L), are abundantly expressed in neurons [28-31], and changes in their expression have been used to evaluate neuronal damage [32,33]. Loss of drebrin has been associated with cognitive impairment in Alzheimer disease and mild cognitive impairment patients [32,34-37]. However, an association between synapse loss and upregulation of the AA cascade has not been identified in vivo. In the current study we used 7- to 9-month-old HIV-1 Tg rats to characterize the brain pro-inflammatory microenvironment and synaptic integrity (determined by levels of drebrin and neurofilament-L). We now show upregulated levels of AA cascade markers and of IL-1β and TNFα in the brain of these HIV-1 Tg rats, in association with lower levels of BDNF, drebrin and neurofilament-L.
Methods
Animals
This protocol was approved by the Animal Care and Use Committee of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, and followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication 86-23). Seven- to 9-month-old male, specific pathogen-free, Fischer 344/Hsd HIV-1 Tg rats (n = 6) and age-matched parental wild-type inbred Fischer 344/Hsd non-Tg control rats (n = 6) were purchased from Harlan Laboratories (Indianapolis, IN) and housed in an animal facility with controlled temperature, humidity, and 12-h light/dark cycle. Food (Teklad global 18% protein diet, 2018S (sterilized) for controls and 2918 (irradiated) for HIV-1 Tg rats (Harlan) [9] and water were provided ad libitum. After three days of acclimation, rats were anaesthetized with an overdose of CO2 and decapitated. Their brain was rapidly excised, sagittally cut into four sections from the left and right hemispheres, frozen in 2-methylbutane at -50°C, and stored at -80°C until studied. One section from the left hemisphere from each rat was used to isolate the cytosolic fraction, a corresponding section from the right hemisphere was used for total RNA extraction, and remaining sections from both hemispheres were used to prepare nuclear extracts.
Preparation of cytosolic fractions
Cytosolic brain fractions were prepared as reported [38]. One section from each brain was homogenized in a buffer containing 20 mM Tris-HCl (pH 7.4), 2 mM EGTA, 5 mM EDTA, 1.5 mM pepstatin, 2 mM leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, 0.2 U/ml aprotinin, and 2 mM dithiothreitol, using a Polytron homogenizer. The homogenate was centrifuged at 100,000 g for 60 min at 4°C, and the resulting supernatant (cytosolic fraction) collected. Protein concentrations were determined using Bio-Rad Protein Reagent (Bio-Rad, Hercules, CA).
Total RNA isolation and real time RT-PCR
Brain tissue was homogenized in Qiagen® lysis solution and total RNA was isolated by phenol-chloroform extraction using a RNeasy® lipid tissue mini kit (Qiagen, Valencia, CA). Complementary DNA was prepared from total RNA using a high-capacity cDNA Archive kit (Applied Biosystems, Foster City, CA). mRNA levels (IL-1β, TNFα, GFAP, CD11b, cPLA2-IVA, sPLA2-IIA, iPLA2-VIA, COX-1, COX-2, mPGES, cPGES, 5-, 12-, 15-LOX, TXS, cytochrome p450 epoxygenase, drebrin and neurofilament-L) were measured by quantitative RT-PCR, using an ABI PRISM 7000 sequence detection system (Applied Biosystems). Specific primers and probes for cPLA2-IVA B, sPLA2-IIA B, iPLA2-VIA, COX-1, COX-2, mPGES, cPGES, 5-, 12-, 15-LOX, TXS, cytochrome p450 epoxygenase, drebrin and neurofilament-L were purchased from TaqManR gene expression assays (Applied Biosystems), and consisted of a 20× mix of unlabeled PCR primers and Taqman minor groove binder (MGB) probe (FAM dye-labeled). The fold-change in gene expression was determined by the ΔΔCT method [39]. Data are expressed as the relative level of the target gene (IL-1β, TNFα, GFAP, CD11b, cPLA2-IVA B, sPLA2-IIA, iPLA2-VIA, COX-1, COX-2, mPGES, cPGES, 5-, 12-, and 15-LOX, TXS, cytochrome p450 epoxygenase, drebrin or neurofilament-L) in the brain of the HIV-1 Tg rat normalized to the endogenous control (β-globulin) and relative to the control (calibrator). All experiments were carried out in triplicate from each control and HIV-1 Tg rat brain (n = 6).
Western blot for protein levels
Proteins from the cytosolic fraction (65 μg) were separated on 4-20% SDS-polyacrylamide gels (PAGE) (Bio-Rad), and electrophoretically transferred to a nitrocellulose membrane (Bio-Rad). Cytosolic protein blots were incubated overnight in Tris-buffered-saline containing 5% nonfat dried milk and 0.1% Tween-20, with specific primary antibodies for proinflammatory cytokines: IL-1β (1:500), TNFα (1:500); astrocytes: glial fibrillary acidic protein (GFAP) (1:1000); CD11b (1:1000); AA cascade proteins: cPLA2-IVA, sPLA2-IIA, iPLA2-VIA, COX-1 (1:1000), COX-2 (1:1000), cytochrome p450 epoxygenase, TXS, 5-, 12-, 15-LOX, mPGES, cPGES (1:1000); gp120 (1:100); tat (1:100); drebrin (1:1000), BDNF (1:1000) (Santa Cruz, Santa Cruz, CA);); neurofilament-L (1:500) (Cell Signaling Technology, Danvers, MA) and β-actin (1:10,000) (Sigma Aldrich, St. Louis, MO). The cytosolic blots were incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Bio-Rad), and were visualized using a chemiluminescence reaction (Amersham, Piscataway, NJ). Optical densities of immunoblot bands were measured using Alpha Innotech Software (Alpha Innotech, San Leandro, CA) and were normalized to β-actin. All experiments were conducted on 6 independent samples. Values are expressed as percent of control.
Transcription factor NF-κBp50 and NF-κBp65 activities
Nuclear extracts were prepared as reported [40,41] and protein concentrations were determined using Bio-Rad Protein Reagent (Bio-Rad). NF-κBp50 and NF-κBp65 activities were measured according to the manufacturer's instructions (Panomics, Freemont, CA), using nuclear extracts obtained from the control and HIV-1 Tg rats. Briefly, 10 μg of nuclear extract from each sample was preincubated with biotin-labeled NF-κBp50 or p65 oligonucleotides in a separate vial for 60 min. The labeled oligonucleotide-nuclear protein complexes were immobilized on a streptavidin-coated 96-well plate. The bound oligonucleotide nuclear protein complex was detected by adding NF-κBp50 or p65 antibody to the respective NF-κBp50 or p65 complex, followed by addition of secondary antibody conjugated to HRP. Color was developed with tetramethylbenzidine substrate and optical densities were measured at 450 nm. Values are expressed as percent of control. All experiments were conducted on 6 independent samples.
Measurement of active caspase-3 protein
Active caspase-3 protein was measured according to the manufacturer's instructions (Cell Signaling, Danvers, MA), using cytosolic brain fractions from the control and HIV-1 Tg rats Briefly, 100 μl (100 μg) of cytosolic fraction was incubated with pre-coated capture antibody in a microwell plate overnight at 4°C. After incubation, the target protein was captured by coated antibody. Following extensive washing, an HRP-linked secondary antibody was added to recognize the bound antibody complex. Color was developed with tetramethylbenzidine substrate and optical densities were measured at 450 nm. Values are expressed as percent of control. All experiments were conducted on 6 independent samples.
Immunohistochemistry
In a separate cohort of animals, astrocyte and microglia morphology was analyzed by immunohistochemistry. Following CO2 anesthesia, the brain (n = 4) was rapidly excised, cut in the midsagittal plane, and the individual hemispheres immersion-fixed in 4% paraformaldehyde/phosphate buffer (pH 7.2) for 18 h, followed by cryoprotection. Fifty- μm free-floating coronal serial cryosections of the forebrain were stored in solution (FD Neurotechnologies, Baltimore, MD) at -20°C. Sections (between +1.0 and 0.4 mm from bregma) were washed with phosphate buffered saline (PBS), equilibrated to room temperature (RT), transferred to 10 mM citrate buffer containing 0.05% Tween-20 and incubated 30 min at 80°C. Sections were then rinsed in PBS and incubated 2 h in blocking solution (2% goat serum, 1% bovine serum albumin, 0.1% Triton X-100 in automation buffer (Biomedia, Foster City, CA). Sections were incubated with anti-GFAP or ionized calcium binding adopter molecule 1 (Iba-1, 1:500, Dako, Glostrup, Denmark) in blocking solution for 18 h at 4°C, re-equilibrated to RT, washed with PBS, and incubated with Alexa Fluor antibody conjugates (1:250, Invitrogen, Carlsbad, CA) in blocking solution without Triton X-100 for 2 h at RT. Digital images of immunostaining in the somatosensory cortex and the dentate gyrus of the hippocampus were collected using a LSM 410 inverted confocal laser-scanning microscope (Carl Zeiss, Oberkochen, Germany) equipped with argon, HeNe, and iFlex 2000 PSU lasers. Image stacks were collected at 1.5 mm steps (20×) or 1.0 mm steps (63×) and displayed as a single image using 3D maximum projection.
Statistics
Data are expressed as mean ± SEM. We used t-tests for independent samples for group comparisons. We further tested significance using the false discovery rate (FDR) to correct for multiple comparisons. We set alpha = 0.01 to reduce type one error risk. An alpha = 0.01 and an n of 15 markers per mRNA and protein assays would give a 14% chance of at least one false positive for each mRNA and protein assay using the following formula 1-(1-.01)e15. A p value less than 0.01 and 0.001 is represented by ** and *** respectively.
Results
Gp120 and tat proteins and neuroinflammatory markers in HIV-1 Tg rats
Gp120 and tat protein levels were detectable in cytosolic brain fractions of HIV-1 Tg but not of control rats (Figure 1A). Brain mRNA and protein levels for the astrocyte structural protein, GFAP, were not significantly altered in the HIV-1 Tg rats compared to controls (Figure 1B-C). As a molecular marker for activated microglia and macrophages [42], mRNA level of the CD11b was elevated significantly by 7.1-fold in the HIV-1 Tg compared with control brain (Figure 1D), corresponding to a significant 190% elevation in CD11b protein (Figure 1E) (p < 0.001).
Figure 1 (A) Representative immunoblot of gp120 and tat protein in HIV-1 Tg rat brain (A), detected as described in Methods. mRNA levels of brain GFAP (B) and CD11b (D) in control and HIV-1 Tg rat brain, measured using real time RT-PCR, normalized to β-globulin and relative to control level (calibrator) using the ΔΔCT method. Representative immunoblots of (C) GFAP and (E) CD11b protein in control and HIV-1 Tg rat brain. Bar graphs are ratios of optical densities of individual protein bands to β-actin, expressed as percent of control. Data represent mean ± SEM, statistical significance: **p < 0.01, ***p < 0.001 as determined by unpaired t-test.
To characterize regional specificity of the changes, we measured GFAP immunoreactivity and microglial markers in the hippocampus and somatosensory cortex. In contrast to the initial report on the HIV-1 Tg rat [7], histological examination of the somatosensory cortex (Figures 2 A-B) and of the hippocampus (Figures 2 C-D) did not indicate increased GFAP immunoreactivity in the HIV-1 Tg rats, as their cells maintained a normal thin process-bearing morphology and there was no evidence of astrocyte hypertrophy (Figures 2 E-H). When we examined the morphological phenotype of microglia within various brain regions using Iba-1+ to label diverse phenotypes, minimal differences from control were noted in the HIV-1 Tg rats (Figures 3 A-H). In the somatosensory cortex, microglia maintained a normal appearance with fine ramified processes and had no prominent evidence of activation or of a phagocytic phenotype (Figures 3 A-B). When these immunopositive cells were examined at higher magnification (Figures 3 E-F), the Iba-1+ cells displayed decreased arbor complexity. Given previous reports of deficits in a hippocampal-dependent spatial memory task in HIV-1 Tg rats, we further examined the morphological phenotype of microglia within the dentate gyrus of the hippocampus. Overall labeling of Iba-1+ microglia was not significantly different in the HIV-1 Tg compared to control rats, with no evidence of overt microglia activation or amoeboid phenotype (Figures 3 C-D). At higher magnification, Iba-1+ microglia displayed fine processes and complicated arborization in the control brain (Figure 3G). A distinct difference was noted in the Iba-1+ microglia in the HIV-1 Tg rat hippocampus, with the cells displaying diminished arbor complexity and approximately 50% shortened processes (p < 0.05 by t-test) as determined by a modified Sholl analysis (Figure 3H), but with no evidence of amoeboid phagocytic microglia.
Figure 2 Representative immunofluorescence (gray scale) for GFAP+ astrocytes (white) in layers IV-V of somatosensory cortex of control (A) and HIV-1 Tg rats (B) and in the hippocampus dentate gyrus of control (C) and HIV-1 Tg rats (D) at 7 months of age. Scale bar = 50 microns. In 3-5 sections obtained from each of 4 animals per group, there was no evidence of astrocyte hypertrophy as represented in the higher magnification image of the astrocyte morphology in the (E, F) somatosensory cortex or (G, H) hippocampus. Images represent compiled z-stack images collected through a 50 micron section. Scale bar = 4 microns.
Figure 3 Representative immunofluorescence (gray scale) for Iba-1+ microglia (white) in layers IV-V of the somatosensory cortex of control (A, C) and HIV-1 Tg rats (B, D) and within the dentate gyrus of the hippocampus of control (E, G) and HIV-1 Tg (F, H) rats at 7 months of age. Images represent compiled z-stack images collected throughout a 50 micron section. Higher magnification of individual representative cells demonstrates diminished arborization of Iba-1+ microglia primarily within the hippocampus. Microglia within defined regional areas were randomly selected (10/section/animal) and the projection distance of the processes was determined using a modified Sholl analysis. In the control brain, 90% (± 10%) of the processes projected past the 4th Sholl while in the HIV-1 Tg rat this was decreased to only 40% (± 18%). Estimates of complexity of the dendritic branching were generated by counting the number of processes originating at cell body. The number of processes was not statistically different from the number in the HIV-1 Tg rat hippocampus, ranging between 5 and 6 in Tg rats and between 7 and 8 in controls, although complexity and secondary branching appeared lower.
Increased proinflammatory cytokine response in HIV-1Tg brains
HIV-1 Tg rats showed significantly increased mRNA levels of inflammatory cytokines IL-1β (9.6-fold) (p < 0.001) and TNFα (3.5-fold) (p < 0.01) respectively (Figures 4A, B). These elevations corresponded to elevated brain protein levels of IL-1β (59%) and TNFα (45%) as compared to controls (Figures 4C, D) (p < 0.01). There was a 73% increase in NF-κBp50 activity in HIV-1 Tg compared to control rat brain (Figure 4E) (p < 0.01). However, NF-κBp65 activity did not differ significantly between groups (Figure 4F).
Figure 4 mRNA levels of brain IL-1β (A) and TNFα (B) in control and HIV-1 Tg rats, measured using real time RT-PCR. Data are levels of brain IL-1β and TNFα in the HIV-1 Tg rat normalized to β-globulin and represented relative to control level (calibrator) using the ΔΔCT method. Representative immunoblots of (C) IL-1β and (D) TNFα protein in control and HIV-1 Tg rat brain. Bar graphs are ratios of optical densities of immunoblots to β-actin, expressed as percent of control (mean ± SEM). Representative brain transcription factor binding activities (DNA-protein complex) of NF-κBp50 (E) and NF-κBp65 (F) in control and HIV-1 Tg rats. DNA binding activity was measured in brain nuclear extracts as described in Methods. Data represent mean ± SEM. Statistical significance: **p < 0.01, ***p < 0.001 as determined by unpaired t-test.
Upregulation of arachidonic cascade enzymes in HIV-1 Tg rat brain
Brain protein and mRNA levels of a number of AA cascade markers were elevated significantly in HIV-1 Tg rats relative to controls. Mean mRNA levels of cPLA2-IVA, sPLA2-IIA and COX-2 were increased (p < 0.01) in HIV-1 Tg compared to control rats by 5-fold, 9-fold and 4.5 fold respectively (Figures 5A-C), but the iPLA2-VIA mRNA level did not differ between groups (HIV-1 Tg 0.92 ± 0.12 vs. control 1.00 ± 0.30). Mean mRNA levels of mPGES (Figure 5D), COX-1 (HIV-1 Tg 0.87 ± 0.20 vs. control 1.00 ± 0.20) and cPGES (HIV-1 Tg 0.97 ± 0.20 vs. control 1.00 ± 0.20) were not significantly different between groups.
Figure 5 mRNA levels of brain cPLA2-VIA (A), sPLA2-II (B), COX-2 (C), and mPGES (D) in control and HIV-1 Tg rats, determined using real time TaqMan RT-PCR. Data are levels of brain cPLA2-VIA, sPLA2-II, COX-2 and mPGES in the HIV-1 Tg rat normalized to the endogenous control (β-globulin) and relative to control level (calibrator) using the ΔΔCT method. Representative immunoblots of (E) cPLA2-VIA, (F) sPLA2- IIA (G) COX-2, and (H) mPGES protein in control and HIV-1 Tg rats. Bar graphs represent ratios of optical densities of each individual protein band relative to β-actin, expressed as percent of control mean ± SEM. Mean ± SEM. Data were analyzed by individual unpaired t-tests, statistical significance: **p < 0.01, ***p < 0.001.
The mean protein level of cPLA2-VIA was increased by 119% (p < 0.01), whereas sPLA2 IIA protein was not changed significantly, as the increase was only at p < 0.05 (Figures 5E, F). The mean iPLA2--VIA protein level also did not differ significantly between groups (HIV-1 Tg 114 ± 6.8 vs. control 100 ± 15). The mean protein level of COX-2 was increased significantly by 42% (p < 0.01) (Figure 5G), but the mean mPGES protein level was not (Figure 5H). COX-1 and cPGES protein levels did not differ significantly between groups (COX-1, HIV-1 Tg 118 ± 17 vs. control 100 ± 15; cPGES, HIV-1 Tg 101 ± 11.2 vs. control 100 ± 11).
5-LOX, 15-LOX and p450 epoxygenase expression in HIV-1 Tg rat brain
There were statistically significant increases in mean brain mRNA levels of 5-LOX, 15-LOX and cytochrome p450 expoxygenase in HIV-1 Tg relative to control rats by 2.9-fold (Figure 6A) (p < 0.001), 4.6-fold (Figure 6B) (p < 0.01) and 4.4-fold (Figure 6C), respectively. Upregulation of these was unaccompanied by significant elevations in the respective mean protein levels, whose increases in each case were only at p < 0.05 (Figures 6D-F). Further, there was no significant difference in 12-LOX or TXS protein between groups (data not shown).
Figure 6 mRNA levels of brain 5-LOX BB(A), 15-LOX (B) and cytochrome p450 epoxygenase (C) in control and HIV-1 Tg rats, measured using real time TaqMan RT-PCR. Data represent individual transcript levels normalized to β-globulin, in HIV-1 Tg rat brain relative to control level (calibrator) using the ΔΔCT method. Representative immunoblots of (D) 5-LOX, (E) 15-LOX, and (F) cytochrome p450 epoxygenase protein in control and HIV-1 Tg rats. Bar graphs display ratios of optical densities of individual protein bands to β-actin, expressed as percent of control. Mean ± SEM, statistical significance: **p < 0.01, ***p < 0.001 as determined by an unpaired t-test.
Indications of neuronal damage and loss in HIV-1 Tg rat
The active caspase 3 protein level (Figure 7A), and levels of neurofilament-L mRNA and protein (Figures 7C, D) did not differ significantly between HIV-1 Tg and control rats, as the former mean decreased at p < 0.05 and the values for neurofilament-L increased only at p < 0.05. BDNF protein (Figure 7B) and drebrin mRNA and protein (Figures 7E, F) were significantly less in HIV Tg than control rats (p < 0.01).
Figure 7 (A) Representative brain active caspase-3 level in control and HIV-1 Tg rats. The active caspase-3 level was measured in brain cytosolic fractions as described in Methods. Bar graphs are relative to control and were compared using an unpaired t-test, mean ± SEM. Representative immunoblots of BDNF (B), neurofilament-L (D) and drebrin (F) protein levels in control and HIV-1 Tg rats. Bar graphs display mean ± SEM optical densities of individual protein bands relative to β-actin, expressed as percent of control. mRNA levels of neurofilament-L (C) and drebrin (E) in control and HIV-1 Tg rat brain, measured using real time TaqMan RT-PCR. Data represent mean ± SEM mRNA levels in the HIV-1 Tg rat brain normalized to β-globulin, relative to control level (calibrator) using the ΔΔCT method. statistical significance: **p < 0.01, ***p < 0.001 as determined by an unpaired t-test.
Discussion
Direct effects of viral gp120 and tat proteins or secondary effects due to neuroinflammatory factors have been associated with HIV-1 infection and HIV-1 related cognitive impairment. HIV-1 Tg rats aged 7-9 months showed gp120 and tat protein in brain, accompanied by significantly elevated AA cascade markers. These differences were accompanied by significant (p < 0.01) elevations in mRNA levels of neuroinflammatory cytokines TNFα and IL-1β, and of the microglial marker CD11b, and reductions in mRNA and protein levels for the synaptic marker, drebrin. These changes occurred in the absence of significantly increased expression of GFAP protein, a marker of astrogliosis.
Elevations in the neuroinflammatory and the AA signaling cascade in HIV-1 Tg rats
We have reported increased cPLA2-IV and sPLA2-IIA activities in the brain of 7- to 9-month-old HIV-1 Tg rats [9]. Consistent with these findings, HIV-1 Tg rat brain in the present study showed elevated protein and mRNA levels (p < 0.01) of cPLA2-IVA and an elevated mRNA level of sPLA2-IIA, without a significant change in iPLA2-VIA or sPLA2-IIA protein levels. COX-2 mRNA and protein levels were significantly higher in HIV-1 Tg rats than controls, whereas COX-1, cPGES or TXS did not differ significantly, consistent with our report of an increased brain concentration of PGE2 but not of TXB2 in HIV-1 Tg rat brain [9]. mPGES protein and mRNA levels were also were not increased in HIV-1 Tg rats.
Our earlier study also showed elevated levels of leukotriene B4, a product of 5-LOX and leukotriene A4 hydrolase, in the brain of HIV-1 Tg rats [9]. Consistent with that report, HIV-1 Tg brain in the present study showed significantly increased 5-LOX mRNA without a significant change in 12-LOX expression. This change was accompanied by increased mRNA levels of cytochrome p450 expoxygenase and 5-LOX. Given that epoxyeicosatrienoic acid produced by cytochrome p450 expoxygenase can be neuroprotective [43,44], the elevated brain mRNA level of cytochrome p450 epoxygenase in HIV-1 Tg may reflect a compensatory neuroprotective process. While elevations in protein levels of 5-LOX, 15-LOX and cytochrome p450 expoxygenase did not reach significance because of our requirement for multiple comparisons, in each case changes were in the same direction as elevations of the respective mRNA at p < 0.05.
The changes in the AA cascade markers noted in HIV-1 Tg rat brain may be related to microglial activation, with release of proinflammatory cytokines and activation of the NF-κB transcription factor. NF-κB binding sites are present on the promoter region of the gene transcripts of the AA cascade markers, cPLA2-IVA, sPLA2-IIA and COX-2 [45-47]. Cell culture studies have shown that IL-1β or TNFα can induce transcription of cPLA2, sPLA2 and COX-2 genes in an NF-κB-dependent manner [14-17,48]. NF-κBp50 is known to regulate transcription of pro-inflammatory genes [49,50] and can influence HIV-1 gene expression [51]. Elevated DNA binding activity of NF-κBp50 in the HIV-1 Tg rat suggests that the elevated AA cascade markers in the current study may be related to increased levels of IL-1β and TNFα and increased NF-κBp50 DNA binding activity, but are independent of NF-κBp65.
In the absence of HIV-1 replication, the presence of gp120 and tat proteins in HIV-1 Tg rat brain likely account for microglial activation and the increased level of CD11b. In vitro, gp120 directly stimulates microglia and increases expression of CD11b [52]. However, microglia did show retraction of their processes and diminished complexity of arborization, which suggests an early reactive response. As we did not examine animals younger than 7 months, we cannot conclude that these changes were age-related. The altered microglial morphology in the hippocampus is of interest, given the role of the hippocampus in spatial learning tasks and the proposed involvement of microglia during synapse stripping and remodeling [53]. CD11b and Iba-1 cannot be used to distinguish between resident microglia and infiltrating blood borne monocytes. Thus, while we did not observe amoeboid brain macrophages, we cannot rule out a contribution of monocytes from the circulation, especially since gp120 can compromise the blood-brain barrier [54].
In vitro, gp120 can stimulate AA release and PGE2 formation in glial cells [19-21] and elevate levels of IL-1β in co-cultures of primary hippocampal neurons and astrocytes [19]. In the initial characterization of the HIV-1 Tg rat, a response of astrocytes was suggested by an increase in GFAP immunoreactivity [7]. In the current study, we did not find an astrocytic response. Consistent with no change in astroglial morphology, protein and mRNA levels of astroglial marker GFAP were not significantly altered. Further studies are needed to understand the role of astrocytes in HIV-1 infection. Similar to gp120, tat protein is also known to stimulate AA release and COX-2 expression in rat brain [55-57]. The changes observed with neuroinflammatory and AA cascade markers in HIV-1Tg rats could be due to the presence of tat protein in the HIV-1Tg brain. Altogether, viral proteins can induce neuroinflammatory and AA cascade markers in brain. Despite altered protein levels of the AA cascade enzymes 5-LOX, 12-LOX and p450 epoxygenase a p < 0.05 in HIV-1 Tg brain, these changes did not reach statistical significance at p < 0.01. This may be due to the small sample size; further studies are required to understand changes in HIV-1 Tg brain.
HIV-1 Tg rats show subtle changes in synaptic marker
Neuropathological features of human HIV-1 infection include cortical atrophy, altered dendritic arborization of neurons, and decreased synaptic density [10-12,58]. Neurons are vulnerable to both gp120 and the HIV-1 virus protein, tat [59,60]. Gp120 and tat are reported to induce apoptosis of neurons in vitro and in vivo [61-63] by activating caspases, particularly caspase-3 [59]. The current study did not show a statistically significant increase in the protein level of active caspase-3 in HIV-1 Tg rats. Damage and apoptosis of neurons would be manifest as a loss of neuronal and related markers. Within this framework, we now report a significantly lower mRNA and protein levels of the post-synaptic dendritic marker drebrin, and a reduced protein level of BDNF (p < 0.01). Protein and mRNA levels of neurofilament-L were reduced only at p < 0.05 in the HIV-1 Tg rats. Reduced BDNF is consistent with a report that gp120 reduces BDNF in rat brain in association with neuronal death [59]. A lifelong presence of gp120 in brain may impair neuronal development by reducing neurofilament and microtubule expression [32]. A significantly reduced level of drebrin suggests that altered synaptic structure contributes to cognitive-behavioral defects reported in the HIV-1 Tg rat [5,6].
In brain, microglia are the primary source of TNFα [64,65], and its release is implicated in neurotoxicity [66]. In HIV-1 Tg rats, elevated levels of IL-1β and TNFα and increased expression of AA cascade enzymes, have been implicated in neuronal damage [67] and cognitive-behavioral impairment [68-73]. A recent study indicates that similar changes could contribute to cognitive impairment in HIV-1 infected patients despite antiretroviral therapy [74]. An association of increased expression of AA cascade enzymes with neurocognitive/neurodegeneration also has been suggested for Alzheimer disease and vascular dementia [75,76]. In this regard, cPLA2 inhibition or deletion improved learning and memory performance in a transgenic mouse model of Alzheimer disease [72]. Treatment with lithium or sodium valproate also was beneficial in HIV-1 associated dementia patients [77,78], possibly by attenuating neuroinflammation and an upregulated brain AA cascade [79,80]. Collectively, these observations suggest that neuroinflammation associated with increased AA metabolism can contribute to cognitive impairment, and that attenuation of AA release by inhibiting cPLA2 may be beneficial.
The significant changes observed with AA cascade and neuroinflammation markers in HIV-1 Tg rats must be interpreted with caution because potential contamination of brain tissue by peripheral cells during cytosolic or total RNA isolation would give higher background levels for measured proteins, except for the neuron-specific marker drebrin. However, such changes are unlikely because we areported increased global brain AA incorporation from plasma in awake HIV-1 Tg rats [9]. Further examination is required to study the extent of activation of neuroinflammation and AA cascade markers in peripheral cells of HIV-1 Tg rats.
While differences between HIV-1 Tg and controls rates in several brain measures at the p < 0.05 level were not considered statistically significant because of the constraint of multiple comparisons (see Methods), they should be given some weight for several reasons, and might be reconsidered in the future with larger samples. This study was exploratory, and was focused on generating hypotheses that could be tested more discretely in the future. Importantly, many of the p < 0.05 changes in a protein occurred with a significant change at p < 0.01 in the respective mRNA, and vice versa, making the p < 0.05 change more credible.
Conclusion
Multiple markers of neuroinflammation and the AA cascade are upregulated, and levels of the postsynaptic markers drebrin and BDNF are reduced, in brain of 7- to 9-month-old HIV-1 Tg rats compared with control rats. These changes may contribute to cognitive impairment in these rats, and likely are related to the presence of viral proteins that trigger activation of several pathways. Our study provides additional critical characterization of neuropathological changes in the mature HIV-1 Tg rat, further establishing this rat as a potentially useful animal model to examine disease progression and effects of therapeutic intervention that can impact treatment and understanding of cognitive and behavioral changes in HIV-1 infected patients.
Abbreviations
AA: arachidonic acid; cPGES: cytosolic prostaglandin E synthase; cPLA2: calcium-dependent cytosolic phospholipase A2; COX: cyclooxygenase; GFAP: glial fibrillary acidic protein; gp120: glycoprotein 120; HIV: human immunodeficiency virus; IL-1β: interleukin-1β; iPLA2: calcium-independent phospholipase A2; mPGES: membrane prostaglandin E synthase; LOX: lipoxygenase; NF-κB: nuclear factor-kappa B; PG: prostaglandin; sPLA2: secretory phospholipase A2; TNFα: tumor necrosis factor α; Tg: transgenic; TX: thromboxane; TXS: thromboxane synthase.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
RJS, BM, and RSI conceived the project and designed experiments; RJS, KHW, KM, MC, HGJ, and KAD conducted experiments. RJS, HGJ, BM, and RSI prepared the manuscript. All authors have read and approved the final manuscript.
Acknowledgements
This research was entirely supported by the Intramural Research Programs of the National Institute on Aging and the National Institute of Mental Health National Institutes of Health, Bethesda, MD, and the National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC. We thank the NIH Fellows Editorial Board for editing the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 21966488PONE-D-11-1526910.1371/journal.pone.0025295Research ArticleMedicineClinical ImmunologyImmunologic SubspecialtiesTransplantationGastroenterology and HepatologyGastrointestinal CancersOncologyCancers and NeoplasmsGastrointestinal TumorsHepatocellular CarcinomaCancer Risk FactorsSurgerySurgical OncologyA Scoring Model Based on Neutrophil to Lymphocyte Ratio Predicts Recurrence of HBV-Associated Hepatocellular Carcinoma after Liver Transplantation A Score Model Predicts HCC Recurrence after LTWang Guo-Ying
1
Yang Yang
1
Li Hua
1
Zhang Jian
1
Jiang Nan
1
Li Min-Ru
1
Zhu Huan-Bing
1
Zhang Qi
2
*
Chen Gui-Hua
1
2
*
1
Liver Transplantation Center, Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China
2
Guangdong Provincial Key Laboratory of Liver Disease Research, Guangzhou, Guangdong, China
Guan Xin-yuan EditorThe University of Hong Kong, China* E-mail: [email protected] (GHC); [email protected] (QZ)Conceived and designed the experiments: G-YW QZ G-HC. Performed the experiments: G-YW H-BZ. Analyzed the data: G-YW YY HL JZ NJ. Contributed reagents/materials/analysis tools: M-RL H-BZ. Wrote the paper: G-YW QZ.
2011 26 9 2011 6 9 e252958 8 2011 31 8 2011 Wang et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
Neutrophil to lymphocyte ratio (NLR) has been proposed to predict prognosis of hepatocellular carcinoma (HCC). However, the cut-off values are empirical. We determined the optimal cut-off value to predict HCC recurrence after liver transplantation (LT) and further established a scoring model based on NLR.
Methodology/Principal Findings
We analyzed the outcome of 101 HBV-associated HCC patients undergoing LT. Preoperative risk factors for tumor recurrence were evaluated by univariate analysis. By using ROC analysis, NLR≥3 was considered elevated. The disease-free survival (DFS) and overall survival (OS) for patients with high NLR was significantly worse than that for patients with normal NLR (the 5-year DFS and OS of 28.5% and 19.5% vs. 64.9% and 61.8%, respectively; P<0.001). Univariate analysis revealed that tumor size >5 cm, tumor number >3, macrovascular invasion, AFP≥400 µg/L, NLR≥3, and HBV-DNA level >5 log10 copies/mL were preoperative predictors of DFS. Cox regression analysis showed macrovascular invasion, tumor number, and high NLR were independent prognostic factors. We then established a preoperative prognostic score based on multivariate analysis. Each factor was given a score of 1. Area under the ROC curve of the score was 0.781. All nine patients with score 3 developed recurrence within 6 months after LT. Of 71 patients without vascular invasion, three patients with both tumor number >3 and NLR≥3 developed recurrence within 14 months after LT while the 5-year DFS and OS for patients with a score of 0 or 1 were 68.1% and 62.8%, respectively.
Conclusions/Significance
Preoperative elevated NLR significantly increases the risk of recurrence in patients underwent LT for HCC. Patients with both NLR≥3 and tumor number >3 are not a good indication for LT. Our score model may aid in the selection of patients that would most benefit from transplantation for HCC.
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Introduction
Hepatocellular carcinoma (HCC) is one of the most prevalent cancers in the world, particularly in China. Chronic hepatitis B virus (HBV) infection is the leading risk factor for HCC in China. Liver transplantation (LT) appears to be an ideal treatment for unresectable HCC, which accounts for about 40% of the indications for LT in China [1]. In the absence of metastases and macroscopic vascular invasion, LT is the best available curative treatment option for patients with HCC on cirrhosis, since it not only removes the tumor completely but also effectively treats the underlying liver disease. However, before the introduction of Milan criteria in 1996 (single nodule ≤5 cm or two to three nodules ≤3 cm) [2], survival after transplantation was disappointing due to high rate of HCC recurrence. Milan criteria have significantly improved the outcome of LT for HCC and have been adopted by the United Network for Organ Sharing (UNOS) to guide patient selection. Many transplant centers confirmed the prognostic value of Milan criteria and established LT as therapy for HCC patients with cirrhosis [3]–[6]. However, the favorable outcomes have raised the question of whether selection criteria should be expanded. Over the last 10 years, expanded criteria have been most thoroughly investigated in an attempt to select suitable candidates beyond Milan criteria [3], [4], [7]–[9]. However, both Milan and the University of California San Francisco (UCSF) criteria (single nodule ≤6.5 cm, or two to three nodules with the largest nodule ≤4.5 cm and the total tumor burden ≤8 cm) solely rely on preoperative imaging findings including tumor number, size, and macrovascular invasion. Unfortunately, inaccuracy of radiological imaging remains a problem, particularly in cirrhosis [10]. Furthermore, radiological imaging can not detect microvascular invasion which has been associated with an increased risk of tumor recurrence. Although tumor size is a surrogate parameter for vascular invasion and poor differentiation [11], there are at least two reasons to explain why about 20% of patients who meet Milan criteria still develop tumor recurrence after LT. The first one is the inadequacy of preoperative imaging studies in assessing vascular invasion and tumor grade. Second, a proportion of patients tend to have some types of tumors which are more aggressive than others although the size of tumor was small. In addition, a proportion of patients with large tumors can achieved excellent outcomes after LT. Therefore, to identify the risk factors for recurrence is very important to limit or expand the indications for LT.
Numerous clinical and experimental data have widely developed the concept that inflammation is a critical component of tumor progression [12], [13]. It is now accepted that the tumor microenvironment contributions to the development of angiogenesis. Several inflammatory markers such as C reactive protein have been suggested as surrogate markers for HCC [14]. One such a simple and effective marker of inflammation that has been linked with several gastroenterological malignancies is the neutrophil-lymphocyte ratio (NLR). An elevated NLR has been shown to be an indictor of poor outcome in patients undergoing hepatic resection for colorectal liver metastasis [15], [16], and curative resection for HCC [17]. More recently, two studies have demonstrated the efficacy of the NLR in predicting outcome in patients undergoing LT for HCC [18], [19]. Elevated NLR significantly increases the risk for tumor recurrence after LT. However, in above all these studies, the cut-off value for NLR of 5 has been set empirically. The number of patients in these studies who had NLR more than 5 was small.
The aim of this study was to determine the optimal cut-off value for preoperative NLR in HCC patients undergoing LT and evaluate whether the new cut-off point for NLR correlates with tumor recurrence. Furthermore, we established a simple preoperative prognostic score model that may aid in the selection of patients that would most benefit from transplantation for HCC.
Methods
Patient selection
One hundred and one HBV-associated HCC patients treated with LT in our hospital (Third Affiliated Hospital, Sun Yat-sen University, Guangdong, China) during the 6-year period from October 2003 to June 2009 were enrolled in this study. Demographic, clinical, and laboratory data, including patient age, gender, white cell and differential counts within 7 days before surgery, serum alpha-fetoprotein (AFP) level, preoperative imaging data based on abdominal computed tomography or magnetic resonance imaging (tumor size, number, and macrovascular invasion), the history of pretransplant tumor therapy, HBV infection, HBV-DNA level, and explant pathology were recorded. Pretransplant tumor therapies included radiofrequency ablation, liver resection, ethanol ablation, transarterial chemoembolization, local radiotherapy and systemic chemotherapy. NLR was defined as the absolute neutrophil count divided by the absolute lymphocyte count. Final diagnosis of HCC was made by pathological examination of the explanted liver. The eligibility criteria for the patients studied are as follows: (a) all patients were HBV infected and none of them were hepatitis C virus-positive; (b) all patients were adults (more than 18 years of age); (c) complete clinical and laboratory data such as AFP level, tumor size, and tumor number were available. (d) any patients missing blood results within 7 days before surgery or missing preoperative imaging data within 1 month before surgery were excluded; (e) any patients demonstrating signs of preoperative sepsis were excluded; (f) none of the patients occurred gastrointestinal hemorrhage, undergoing pretransplant tumor therapy, or given hematopoietic agents such as G-CSF within 1 month before surgery as these conditions can result in a falsely elevated NLR; (g) any patients on high-dose steroids before transplantation were excluded; and (h) patients had no HCC on pathology or no any follow-up data were also excluded. This study was approved by the Institutional Review Board of Key Lab of Liver Disease Research in Guangdong Province. Informed written consent was obtained according to the Declaration of Helsinki.
Surgery and postoperative management
Liver transplants are performed using standard techniques without the use of venovenous bypass. The piggyback's technique with suprahepatic venacavaplasty of retrohepatic cava extention for LT was performed in most patients. Generally, our transplant center's postoperative immunosuppression regimen consisted of calcineurin inhibitors and steroids. Steroids were discontinued within 3 months.
Follow-up
After LT, patients were regularly followed up at the outpatient clinics. Abdominal and chest CT were monitored every 3 months in the first two postoperative years, followed by every 6 months in the third year. AFP measurement is performed every month in the first year and then every 3 months for the next two years. In the following years, an annual abdominal CT scan was performed. CT or MR imaging of the abdomen pelvis, chest and bone scan was monitored to define suspicious lesions demonstrated on CT or raised AFP level.
Statistical analysis
SPSS for Windows program (version 13.0) and MedCalc statistical software v11.3.0.0 were used to analyze the data. Patients were censored at last follow-up if still alive or lost to follow-up. A suitable cut-off value for elevated NLR was selected using the receiver operating characteristic (ROC) curve analysis. The independent samples t test, and Pearson's chi-square (χ2) test were used to analyze differences in clinicopathological features between patients with high and normal NLR. The clinicopathologic factors that were significant differences between patients with high and normal NLR were entered into a stepwise forward logistic regression model to determine their independent risk factors. Survival curves were determined by the Kaplan–Meier method. Potential predictors of patient outcomes and disease-free survival were entered into univariate Kaplan–Meier models and tested by the log-rank statistic. Preoperative clinicopathologic factors that had significant impact on disease-free survival in the univariate analysis were entered into a multivariate Cox regression model (stepwise forward method) to determine their independent effect. A preoperative prognostic score model was established to predict tumor recurrence based on preoperative factors that were significant on multivariate analysis. Univariate Cox proportional hazards regression analysis was applied to estimate the hazard ratio for the risk of tumor recurrence. By using MedCalc statistical software, the area under the ROC curve (AUC) for the score model was measured and then compared with the Milan, UCSF, and HangZhou criteria (tumor <8 cm, or ≥8 cm but with well differentiation and serum AFP≤400 ng/mL) [20]. Variables with P<0.05 were considered statistically significant.
Results
Patient demographics and outcomes
Ninety-two patients (91.1%) were men and 9 (8.9%) women. The mean age of patients at transplant was 48.4 years (range: 27–72 years). Thirty-five patients (34.7%) received pretransplant tumor therapy. Fifty-one patients died during follow-up and 4 died within one month post-transplantation. Of 42 patients (41.2%) developed tumor recurrence, 28 (66.7%) developed recurrence within 1 year and 38 (90.5%) developed recurrence within 2 years after LT. Mean follow-up time was 2.85 years (range: 0.38–6.22 years) with 7 patients lost to follow-up. The 1-, 3-, and 5-year (yr) overall survival (OS) and disease-free survival (DFS) rates for all patients included in this study were 80.2%, 55.4%, and 47.6% and 70.7%, 55.6%, and 53.6%, respectively (Figure S1).
An optimal cut-off value for elevated NLR
By using ROC curve analysis, we determined the optimal cut-off value for elevated NLR. The area under the receiver operating characteristic curves was 0.667 with a 95% confidence interval (95% CI) for the area being between 0.557 and 0.777 (Figure S2). A cut-off value of 2.48 presented a sensitivity of 59.5% and a specificity of 71.2%. When the cut-off point was increased to 2.99, the sensitivity was 50% and the specificity was 79.7%. Therefore, the cut-off value of 3.0 was used in this study.
Risk factors for recurrence of hepatocellular carcinoma after LT
Univariate analysis of factors affecting disease-free survival was shown in Table 1. More than three tumor nodules, size of largest tumor >5 cm, macrovascular invasion, AFP ≥400 ng/mL, NLR≥3, and HBV-DNA level >5 log10 copies/mL were all preoperative prognostic predictors of poorer DFS. The NLR was elevated at ≥3 in 34 patients (33.3%). A significant difference in DFS existed between patients with normal and elevated NLR. The 5-yr DFS of 64.9% in patients with a normal NLR compared with 28.5% in patients with an elevated NLR, respectively, P<0.001, Figure 1A). Of 30 patients with macrovascular invasion, 3 died within 1 month after transplantation; 2 died from biliary infection and multiple organ dysfunction syndrome 5 and 7 months after transplantation, respectively; 4 had no tumor recurrence during 5 years of follow-up; tumor recurrence were detected in all the other 21 patients during 18 months of follow-up. Of 4 patients without tumor recurrence, 3 had no macroscopic tumor thrombus in the explant liver and histopathologic analysis revealed no evidence of microvascular invasion. Of 71 patients without macrovascular invasion on preoperative imaging, 16 patients with high NLR had slightly lower DFS than patients with normal NLR but no statistically significant difference was found (5-yr DFS of 70.8% vs. 53.0%, respectively, P = 0.106, Figure S3A).
10.1371/journal.pone.0025295.g001Figure 1 Kaplan-Meier survival curves for patients with high or normal NLR.
There was a significant difference in DFS and OS between patients with low and high NLR. The 5-yr DFS (A) and OS (B) for patients with normal or high NLR were 64.9%, 28.5%, and 61.8%, 19.5%, respectively (both P<0.001).
10.1371/journal.pone.0025295.t001Table 1 Preoperative factors affecting disease-free survival and overall survival.
Category Subcategory (n) Disease-free survival Overall survival
5 years Univariate analysis Multivariate analysis HR (95%CI) 5 years Univariate analysis Multivariate analysis HR (95%CI)
Gender Male (92) 52.0% 0.271 – 46.4% 0.360 –
Female (9) 72.9% 58.3%
Age <60 years (87) 72.9% 0.189 – 46.7% 0.703 –
≥60 years (14) 63.5% 50.0%
Preoperative tumor therapy Yes (35) 63.5% 0.189 – 42.2% 0.171 –
No (66) 47.9% 57.8%
Size of largest tumor ≤3 cm (26) 69.9% 0.002 NS 66.7% <0.001 NS
≤5 cm (34) 61.5% 62.8%
≤8 cm (14) 40.8% 27.8%
>8 cm (27) 31.1% 20.0%
Tumor numbers ≤3 (68) 67.9% <0.001 <0.001 4.117 (2.087–8.123) 58.0% <0.001 <0.001 3.013 (1.644–5.524)
>3 (33) 24.5% 26.4%
Vascular invasion No (71) 66.8% <0.001 <0.001 3.908 (1.965–7.772) 61.6% <0.001 <0.001 3.253 (1.734–7.772)
Yes (30) 17.2% 15.0%
AFP <400 ng/mL (61) 65.9% <0.001 NS 62.6% <0.001 NS
≥400 ng/mL (40) 32.1% 24.6%
NLR <3 (68) 64.9% <0.001 <0.001 3.665 (1.799–7.466) 61.8% <0.001 <0.001 2.654 (1.419–4.964)
≥3 (33) 28.5% 19.5%
HBV-DNA level >5 log10 copies/mL No (88) 60.8% 0.016 NS 51.9% 0.021 NS
Yes (13) 10.4% 19.2%
Abbreviation: CI, confidence interval; HR, hazard ratio; NS, not significant.
Multivariate regression analysis was performed on all 6 preoperative factors that were statistically significant difference in DFS by univariate analysis. The results revealed that the presence of macrovascular invasion, tumor number >3, NLR ≥3 were the independent prognostic predictors of poor DFS (Table 1).
With regards to overall survival, there was a significant difference in overall survival between patients with low and high NLR (5-yr OS of 61.8% vs. 19.5%, respectively, P<0.001, Figure 1B). Again, on multivariate analysis, the presence of macrovascular invasion, tumor number >3, NLR≥3 were the independent prognostic predictors of overall survival (Table 1). Of patients without macrovascular invasion, patients with high NLR had significant lower OS than patients with normal NLR (5-yr OS of 69.6% vs. 36.7%, respectively, P = 0.037, Figure S3B).
The analysis of clinicopathologic characters in patients with elevated NLR
The analysis of clinicopathologic characters in patients with normal or elevated NLR is shown in Table 2. Size of largest tumor >5 cm, macrovascular invasion, AFP≥400 ng/mL, outside Milan, outside UCSF, and outside HangZhou criteria were significant differences between patients with normal and high NLR based on univariate analysis. Furthermore, multivariate logistic regression analysis using all three parameters, eg, size of largest tumor, macrovascular invasion, and AFP, revealed that only macrovascular invasion remained associated with high NLR (P = 0.001, relative risk = 4.495, 95% CI = 1.086–11.188).
10.1371/journal.pone.0025295.t002Table 2 Comparison of clinicopathological features of patients with elevated and normal NLR.
Factors NLR<3 (n = 68) NLR≥3 (n = 33)
t or χ2
P
Age (mean) 48.8 years 47.5 years 0.62 0.536
Male 61 (89.7%) 31 (93.9%) 0.108 0.743
Preoperative tumor therapy 27 (39.7%) 8 (24.2%) 2.346 0.126
Size of largest tumor >5 cm 21 (30.9%) 20 (60.6%) 8.140 0.004
Tumor numbers >3 21 (30.9%) 12 (36.4%) 0.303 0.582
Vascular invasion 13 (19.1%) 17 (51.5%) 11.168 0.001
AFP≥400 ng/mL 20 (29.4%) 20 (60.6%) 9.039 0.003
HBV-DNA level >5 log10 copies/mL 9 (13.2%) 4 (12.1%) 0.000 1.000
Outside Milan criteria 36 (52.9%) 29 (87.9%) 11.823 0.001
Outside UCSF criteria 31 (45.6%) 25 (75.6%) 8.186 0.004
Outside HangZhou criteria 18 (26.5%) 21 (63.6%) 12.947 <0.001
Abbreviation: NLR, neutrophil-lymphocyte ratio.
Classification of patients according to NLR and the different criteria
In this study, 36 patients (35.6%) were within Milan criteria. Of the patients who were outside Milan, 9 and 26 were classified within UCSF and HangZhou criteria, respectively. The DFS of patients who were inside Milan criteria did not significantly differ from those patients outside Milan but within UCSF criteria (log rank test, P = 0.07), and similarly, patients outside Milan but within UCSF did not significantly differ from patients outside UCSF but within HangZhou criteria. However, patients within Milan had significantly better DFS than patients outside UCSF but within HangZhou criteria (log rank test, P = 0.001), and patients outside UCSF but within HangZhou criteria had significantly better DFS than patients outside HangZhou criteria (log rank test, P = 0.025), as shown in Figure 2A. Hangzhou criteria significantly expand the indications for liver transplantation with acceptable rates of 5-yr DFS and OS (70.3% and 65.3%, respectively, Figure S4).
10.1371/journal.pone.0025295.g002Figure 2 Disease-free survival for patients classified according to NLR and the different criteria.
(A) The Kaplan-Meier curves for patients classified according to the Milan, UCSF and HangZhou criteria showed patients within Milan had significantly better DFS than patients outside UCSF but within HangZhou criteria (log rank test, P = 0.001). (B) Among patients beyond Milan criteria, a significant difference in DFS existed between patients with normal and elevated NLR (log rank test, P = 0.015).
Four patients who met Milan criteria had an elevated NLR. Of 32 patients with normal NLR and within Milan criteria, only three patients have shown tumor recurrence within 1 year and have died within 2 years after OLT. No tumor recurrence was found in all the other 29 patients during follow-up. There was no significant difference in DFS between patients with normal and high NLR of patients who met Milan criteria. However, of the patients beyond the criteria, a significant difference in DFS existed between patients with normal and elevated NLR; 29 patients with a high NLR having the 5-yr DFS of 21.5% compared with 42.2% in 36 patients with a normal NLR, respectively (P = 0.015, Figure 2B). The similar results were obtained when patients were reclassified according to UCSF and HangZhou criteria. Of 56 patients beyond UCSF criteria, 25 patients with a high NLR having a 5-yr DFS of 10.7% compared with 41.2% in 31 patients with a normal NLR (P = 0.001).
A preoperative prognostic scoring model
Based on the preoperative recurrence score recently published by Halazun et al. [18] for tumor recurrence in patients transplanted for HCC, we established a preoperative prognostic score model, using the 3 preoperative prognostic factors found to be significant on multivariate analysis, namely, vascular invasion, tumor number, and NLR. Each factor was given a score of 1 and then patients were divided into 4 categories. Differences in DFS stratified according to the preoperative prognostic scores are shown in Figure 3A. The 5-yr DFS for 43 patients with a score of 0 was 81.9%, compared with 53.6% for 30 patients with a score of 1 (P = 0.018). The DFS in 19 patients with a score of 2 decreased sharply (the 1- and 2-yr survivals of 32.7% and 6.5%, respectively). Nine patients with a score of 3 had the worst DFS of all groups. All these patients showed tumor recurrence within 6 months and died within 17 months after LT. The median DFS for patients with a score of 2 and 3 were 3.8 and 8.5 months, respectively, compared with 5 years for patients with a score of 1. Patients with a score of 0 had minimal recurrence, with 36 patients (83.7%) being disease free at 5 years. Clearly, therefore, the DFS for patients with a score of 2 or 3 was significantly worse than that for patients with a score of 0 or 1 (the 1- and 3-yr DFS, 23.8% and 4.8% vs. 86.0% and 71.7%, respectively, P<0.001, Figure 3B).
10.1371/journal.pone.0025295.g003Figure 3 Disease-free survival for patients classified according to the preoperative prognostic score.
(A) The Kaplan-Meier curves showed there was a significant difference in DFS among four groups of patients with score 0 to 3. (B) A significant difference in DFS existed when patients were reclassified into two groups (score 0 or 1, and 2 or 3).
Of 30 patients with macrovascular invasion on preoperative imaging, one of 3 patients with a score of 1 had no macroscopic tumor thrombus in the explant liver and histopathologic analysis revealed no evidence of microvascular invasion. He had no tumor recurrence during 23 months of follow-up. The other 27 patients had 2 or 3 scores. These results indicated that patients with a score of 2 or 3 were not a good indication for liver transplantation but patients with a score of 0 or 1 can achieve an acceptable survival after LT with a 5-yr DFS and OS of 69.1%, 63.8%, respectively.
The predictive value of the preoperative prognostic score model, as well as the value of Milan, UCSF and HangZhou criteria was assessed using univariate Cox proportional hazards regression analysis (Table 3). The preoperative prognostic score was superior to the Milan, UCSF and HangZhou criteria, with scores of 1, 2, and 3 having hazard ratios of 2.912, 15.533, and 48.715, respectively. ROC curve analysis of the preoperative prognostic score versus that of Milan, UCSF and HangZhou criteria is shown in Table 4 and Figure S5 by using MedCalc statistical software. The preoperative prognostic score was the most accurate at predicting recurrence with the AUC of 0.781 though no statistically significant difference was observed.
10.1371/journal.pone.0025295.t003Table 3 Univariate Cox regression analysis of preoperative score model as well as Milan, UCSF and HangZhou criteria.
P
Hazard Ratio 95% CI
Preoperative prognostic score = 0 — — —
Preoperative prognostic score = 1 0.023 2.912 1.161–7.305
Preoperative prognostic score = 2 <0.001 15.533 6.097–39.568
Preoperative prognostic score = 3 <0.001 48.715 13.974–169.825
Milan criteria <0.001 8.528 3.035–23.960
UCSF criteria <0.001 6.615 2.926–14.956
HangZhou criteria <0.001 5.240 2.772–9.903
10.1371/journal.pone.0025295.t004Table 4 ROC curve analysis of preoperative prognostic score versus Milan, UCSF and HangZhou criteria.
AUC 95% CI
Milan criteria 0.724 0.624–0.823
UCSF criteria 0.739 0.640–0.838
HangZhou criteria 0.699 0.593–0.806
Preoperative prognostic score 0.781 0.688–0.875
Abbreviation: AUC, the area under the ROC curve; CI, confidence interval; ROC, the receiver operating characteristic.
Preoperative prognostic scores predict tumor recurrence in patients without macrovascular invasion
Because patients with gross vascular invasion of the portal vein, hepatic veins, or vena cava were not considered a good indication for LT due to the high rate of tumor recurrence, we next analyzed the prognostic value of our score model for patients without vascular invasion detected by preoperative imaging. Patients were divided into 3 categories. The results showed all 3 patients with a score of 2 got tumor recurrence within 14 months and died within 23 months after LT while the 5-yr DFS and OS for patients with a score of 0 and 1 were 81.4%, 74.2%, and 54.6%, 53.7%, respectively (Figure 4A and B). The DFS and OS for patients with a score of 2 was significantly worse than patients with a score of 1 (P<0.001 and = 0.001, respectively). Of 26 patients with a score of 1, 13 patients with high NLR had slightly lower DFS rates than patients with more than 3 tumor nodules but no statistically significant difference was found (the 3- and 5-yr survivals, 68.4% and 45.6% vs. 76.9% and 59.8%, respectively, P = 0.756). Patients with scores of 1 and 2 had hazard ratios of 2.615 and 31.810, respectively. Patients with a score of 0 or 1 can achieve long-term survival after LT with a 5-yr DFS of 69.9% and OS of 64.4%, respectively. The preoperative prognostic score model for patients without vascular invasion revealed the strong associations of tumor recurrence risk with tumor numbers and high NLR with the AUC of 0.705 with the 95% CI of 0.565–0.844. These results indicated that patients without vascular invasion but with both elevated NLR and more than 3 tumor nodules were also not a good indication for LT. Our preoperative prognostic score significantly expand the indications for liver transplantation.
10.1371/journal.pone.0025295.g004Figure 4 The prognostic value of the preoperative prognostic score model for patients without vascular invasion.
The Kaplan-Meier curves showed there was a significant difference in DFS (A) and OS (B) between patients with a score of 2 and patients with a score of 0 or 1.
Discussion
Among appropriately selected candidates, LT for HCC provides excellent outcomes with 5-yr survival rates similar to patients undergoing LT for liver cirrhosis without HCC. However, about 20% of patients with Milan criteria still develop tumor recurrence after LT [2]. There remains controversy about expanding the criteria for selection of HCC patients for LT for a proportion of patients with tumor burden beyond Milan criteria may potentially benefit from LT. Lack of available liver donors is the main restricting factor for LT and contributes to prolonged waiting time, which is associated with increased drop out rates. However, expansion of selection criteria increases not only the risk of tumor recurrence, but also the need for donor organs, and further lengthens waiting time. Early experience demonstrated that tumor size was an important predictor of recurrence and survival for patients undergoing LT for HCC. But the preoperative tumor size can only be assessed by preoperative radiological imaging, which underestimates tumor stage in about 30% of cases, especially in patients with tumors beyond Milan criteria [8]. For all these reasons, there is an urgent need to develop new non-invasive biomarkers predicting patients at high risk of recurrence after hepatic resection or transplantation.
Consistent lines of evidence have suggested that there is a close relationship between the development of cancer and inflammation. Inflammation contributes to the development of at least 15% of all cancers, in particular, for the digestive system cancers [21]. Patients with HBV infections experience chronic inflammation which increases risk of liver cancer. Neutrophil to lymphocyte ratio (NLR), a simple and effective marker of inflammation, is easily calculated from routinely available data. During the past five years, some studies have demonstrated that an elevated NLR is an important prognostic factor in patients with a variety of digestive system malignancies including esophageal cancer [22], gastric cancer [23], [24], colorectal cancer [25], [26], colorectal liver metastases [15], [16], [27], pancreatic adenocarcinoma [28], intrahepatic cholangiocarcinoma [29], and HCC [17], [18]. However, in all these studies, the cut-off value for NLR of 5 has been set empirically except for one study of gastric cancer with the cut-off level of 4 based on Kaplan-Meier analysis. To our knowledge, this is the first report discussing the appropriate cut-off point of NLR in predicting prognosis in patients with HCC. First, we determined the optimal cut-off point for preoperative NLR to predict HCC patients with high risk of tumor recurrence after LT. By using ROC curve analysis, we found that the cut-off value of 3.0 had a relatively high specificity. Although patients with NLR values between 3.0 and 5.0 were classified as having an elevated NLR based on our new cut-off value, our results showed that patients with NLR<3 showed significantly better DFS and better OS than those of patients with NLR≥3. The results of multivariate regression analysis revealed that NLR≥3 was the independent prognostic predictor of poor DFS. This is consistent with the above studies. Although the relationship between elevated NLR and increased risk for early recurrence and poor prognosis is largely unclear, there are several possible mechanisms explaining the predictive role of preoperative elevated NLR. The systemic and local inflammatory response to tumor may provide a favorable environment for tumor invasion and metastases [13]. Furthermore, except for chronic inflammation caused by HBV and tumor itself, high expressions of granulocyte colony-stimulating factor in tumor tissue and macrophage colony-stimulating factor in peritumoral tissue are also associated with the elevated circulating neutrophils and poor prognosis [30]–[32]. Circulating elevated levels of vascular endothelial growth factor secreted mainly by circulating neutrophils have been associated with increased risk of recurrence in patients with HCC [33]. On the other hand, reduced lymphocyte infiltration, reflecting an impaired host immune response, has been shown to predict recurrence in HCC patients following LT [34]. NLR reflects an immune microenvironment that both favors tumor vascular invasion and suppresses the host immune surveillance. In addition, NLR can not only predict tumor recurrence but also be used for diagnosis of tumor. A recent study has showed that NLR can be a useful tool for preoperative diagnosis in patients with uterine sarcomas [35].
Although univariate analysis in this study showed that tumor size, AFP level, and HBV-DNA level were preoperative prognostic predictors of poorer DFS, none of these factors were identified as independent predictors on multivariate analysis. However, this result did not mean that these factors were not associated with recurrence. In fact, previous studies have showed that tumor size >3 cm on imaging is an independent predictor of microvascular invasion [36], [37]. Our results showed a significant association between the elevated NLR and the tumor size, AFP level, and macrovascular invasion. Taken together, these results indicated that preoperative elevated NLR can indirectly reflect tumor burden, malignancy, invasion, and metastasis.
We found that more than 3 tumor nodules were an independent predictor of recurrence. The result was concomitant with some studies [37], [38] but not the others [20], [39], [40]. Multiple tumor nodules can be categorized into two types: multifocal occurrence and intrahepatic metastasis. HCC patient with intrahepatic metastasis has a poorer prognosis than those with multifocal occurrence. Although fast developed imaging techniques can detect small intrahepatic metastasis, we can not accurately distinguish intrahepatic metastasis from multifocal occurrence based on preoperative imaging. However, in our current study including 27 patients with tumor size >8 cm, we notified that multiple small satellite nodules seemed to surround a large main tumor. The different background of hepatitis may also partly explain that our results were different from that of other publications from Japan or Europe. HCC patients with HCV-associated cirrhosis have a higher incidence of multifocal occurrence than patients with HBV-related cirrhosis [41].
Based on multivariate analysis, we have therefore established a simple preoperative prognostic score model that is superior to the Milan, UCSF and HangZhou criteria at predicting recurrence, with an AUC of 0.781. All patients with a score of 3 showed tumor recurrence within 6 months after LT. The median DFS for patients with a score of 2 and 3 were 3.8 and 8.5 months, respectively. Clearly, LT for these patients is futile. As for patients without macrovascular invasion on radiological findings, all 3 patients with scores of 2 (both NLR≥3 and tumor nodules >3), who got recurrence within 14 months after LT, had hazard ratios of 31.810 with an AUC of 0.705. Whereas patients with a score of 0 or 1 may achieve similar survival outcomes as patients within HangZhou criteria, but including more patients (69 and 62 patients, respectively). These results indicated that patients without vascular invasion but with both elevated NLR and more than 3 tumor nodules were also not a good indication for LT. A similar study has demonstrated that a scoring model based on NLR before treatment offers a very informative method for predicting prognosis of gastric cancer [42].
Although we demonstrated the prognostic value of NLR in predicting recurrence, it would not be appropriate to conclude that LT should not be considered only because preoperative NLR is high. There are many other factors affecting NLR, such as an acute undetected infection, which affects the accuracy of prognostic prediction. In addition, all patients enrolled had a history of hepatitis B, which may bias the study since hepatitis C is the most common predisposing factor to HCC development in Western countries and Japan. In addition, our study was limited by the retrospective nature of the analysis and the relatively small number of patients was included in the report. Clearly, further prospective studies are needed to confirm and update our preoperative prognostic score model for the prediction of post-transplant tumor recurrence in patients with HCC.
Supporting Information
Figure S1 Disease-free survival (A) and overall survival (B) rates for all patients included in this study.
(TIF)
Click here for additional data file.
Figure S2 Receiver operating characteristic curve for NLR predicting tumor recurrence.
(TIF)
Click here for additional data file.
Figure S3 Kaplan-Meier survival curves for patients without macrovascular invasion and with high or normal NLR. Of patients without macrovascular invasion, the impact of high NLR on DFS was not statistically significant (A), but patients with high NLR had significant lower OS than patients with normal NLR (B).
(TIF)
Click here for additional data file.
Figure S4 Kaplan-Meier DFS (A) and OS (B) curves for patients with or beyond Hangzhou criteria.
(TIF)
Click here for additional data file.
Figure S5 Receiver operating characteristic curve for the different criteria predicting tumor recurrence.
(TIF)
Click here for additional data file.
The authors thank Xiao-Cui Fang for her assistance in the follow-up data collection.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by the Major State Basic Research Development Program of China (973 Program) (No. 2009CB522404, http://www.most.gov.cn/kjjh/), the National Natural Science Foundation of China (No. 30972914, No. 81000190, and No. U0932006, http://www.nsfc.gov.cn/), and State Key Projects on Infection Diseases of China (2008ZX10002-025 and 026, http://www.most.gov.cn/kjjh/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 21980374PONE-D-11-1069410.1371/journal.pone.0025019Research ArticleBiologyMolecular Cell BiologySignal TransductionMembrane Receptor SignalingNeurotransmitter Receptor SignalingNeuroscienceDevelopmental NeuroscienceSynaptic PlasticityMolecular NeuroscienceSignaling PathwaysNeurochemistryNeurochemicalsGlutamateNeurotransmittersMedicineDrugs and DevicesBehavioral PharmacologyRecreational Drug UsePrenatal Cocaine Exposure Increases Synaptic Localization of a Neuronal RasGEF, GRASP-1 via Hyperphosphorylation of AMPAR Anchoring Protein, GRIP Prenatal Cocaine Elevates Synaptic GRASP-1Bakshi Kalindi
1
2
3
Kosciuk Mary
4
Nagele Robert G.
4
Friedman Eitan
1
2
Wang Hoau-Yan
1
2
*
1
Departments of Physiology, Pharmacology and Neuroscience, Sophie Davis School of Biomedical Education, The City University of New York Medical School, New York, New York, United States of America
2
Department of Biology & Neuroscience, Graduate Center of the City University of New York, New York, New York, United States of America
3
Center for Developmental Neuroscience/Institute for Basic Research/City University of New York Graduate School, Staten Island, New York, United States of America
4
New Jersey Institute for Successful Aging, University of Medicine and Dentistry New Jersey-School of Osteopathic Medicine, Stratford, New Jersey, United States of America
Mouillet-Richard Sophie EditorINSERM, UMR-S747, France* E-mail: [email protected] and designed the experiments: H-YW RN EF. Performed the experiments: KB MK. Analyzed the data: H-YW KB. Wrote the paper: H-YW KB EF.
2011 27 9 2011 6 9 e2501913 6 2011 23 8 2011 Bakshi et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Prenatal cocaine exposure causes sustained phosphorylation of the synaptic anchoring protein, glutamate receptor interacting protein (GRIP1/2), preventing synaptic targeting of the GluR2/3-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs; J. Neurosci. 29: 6308–6319, 2009). Because overexpression of GRIP-associated neuronal rasGEF protein (GRASP-1) specifically reduces the synaptic targeting of AMPARs, we hypothesized that prenatal cocaine exposure enhances GRASP-1 synaptic membrane localization leading to hyper-activation of ras family proteins and heightened actin polymerization. Our results show a markedly increased GRIP1-associated GRASP-1 content with approximately 40% reduction in its rasGEF activity in frontal cortices (FCX) of 21-day-old (P21) prenatal cocaine-exposed rats. This cocaine effect is the result of a persistent protein kinase C (PKC)- and downstream Src tyrosine kinase-mediated GRIP phosphorylation. The hyperactivated PKC also increased membrane-associated GRASP-1 and activated small G-proteins RhoA, cdc42/Rac1 and Rap1 as well as filamentous actin (F-actin) levels without an effect on the phosphorylation state of actin. Since increased F-actin facilitates protein transport, our results suggest that increased GRASP-1 synaptic localization in prenatal cocaine-exposed brains is an adaptive response to restoring the synaptic expression of AMPA-GluR2/3. Our earlier data demonstrated that persistent PKC-mediated GRIP phosphorylation reduces GluR2/3 synaptic targeting in prenatal cocaine-exposed brains, we now show that the increased GRIP-associated GRASP-1 may contribute to the reduction in GluR2/3 synaptic expression and AMPAR signaling defects.
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Introduction
Prenatal cocaine exposure results in long-lasting changes in synaptic plasticity that may be responsible for the cognitive deficits in humans and animal models [1], [2], [3]. Synaptic plasticity, such as long-term potentiation (LTP) and depression (LTD) are regulated in part by AMPARs [4], [5]. The notion that AMPARs are sensitive to cocaine is supported by our earlier findings that prenatal cocaine exposure reduces GluR2- and GluR3-AMPAR synaptic expression and attenuates AMPAR-mediated LTD [6]. We demonstrated that reduced GluR2-, GluR3-containing AMPARs on the synaptic membrane is the result of a deficient interaction between AMPARs and synaptic anchoring protein GRIP resulting from sustained PKC-mediated GRIP phosphorylation [6]. However, there are no data discerning how this cocaine-induced effect influences other GRIP-associated proteins such as GRASP-1, which is known to affect GluR2/3-GRIP interaction [7] and regulate AMPAR trafficking to the synaptic membrane.
GRASP-1 is a neuronal RasGEF (guanine nucleotide exchange factor) and a neuron- specific effector for monomeric guanine nucleotide-binding proteins (G proteins) such as Rab4 which have been implicated in the regulation of membrane protein trafficking [7], [8], AMPAR targeting, and JNK signaling [7], [9]. Through its RasGEF domain in the N terminal region, GRASP-1 associates with GRIP1 by binding to the seventh PDZ domain of GRIP located at the C terminal region that is distinct from the GluR2/3 binding site on GRIP [7]. This GRIP-GRASP-1 association enables the formation of the GRASP-1/GRIP/GluR2 complex which activates monomeric G proteins via GRASP-1's RasGEF activity. In addition, the level of GRIP associated AMPAR(s) is reduced when GRASP-1 is overexpressed [7].
Monomeric G proteins are prominent regulators of the actin network that has profound influence on the trafficking of surface proteins including GluR2/3-AMPARs. It is highly likely that hyperphosphorylation of GRIP in prenatal cocaine exposed brains noted in our earlier work [6] could affect the GRIP-GRASP-1 association, and thus the GRASP-1 cellular distribution and/or activity. The altered GRIP-GRASP-1 complex level in prenatal cocaine-exposed brain could in turn influence GluR2/3 trafficking via modulating the activity of monomeric G proteins.
In accordance with this hypothesis, monomeric G proteins such as Ras and Rap regulate AMPAR trafficking during LTP and LTD [10], and an increase in active Rap1 is associated with reduced synaptic targeting of GluR2 [11]. Monomeric G protein Rac1/cdc42, clusters AMPARs during spinogenesis [12] and regulates synaptic structure and function [13], whereas RhoA regulates actin polymerization in dendritic spines and modulates spine length and density [14], [15]. Altogether, these data suggest strongly that alteration of monomeric G protein levels and/or activity could influence AMPAR trafficking and consequently the synaptic function.
We have previously demonstrated that in prenatal cocaine-exposed brain PKC- and Src-mediated persistent phosphorylation of GRIP reduces GluR2/3 synaptic expression [6]. Here, we show that hyperphosphorylated GRIP resulting from persistently activated PKC also increases GRASP-1 association with GRIP, leading to higher activated RhoA, cdc42/Rac1, and Rap1, as well as F-actin levels in FCX of prenatal cocaine-exposed rats. Their relevance to AMPAR trafficking is discussed.
Results
Prenatal cocaine exposure markedly increases GRASP-1 localization and GRASP-1 - GRIP interaction in the synaptic membrane
The GRIP binding partner GRASP-1 can regulate AMPAR targeting by forming a complex with GluR2/3-AMPARs via its association with GRIP [7]. To test directly whether prenatal cocaine exposure changes the expression and/or cellular distribution of GRASP-1, the level of GRASP-1 was measured in total post-mitochondrial FCX lysate as well as in cytosolic and membrane fractions of FCX synaptosomes prepared from 21-day-old (P21) prenatal cocaine- and saline-exposed rats. Prenatal cocaine exposure did not alter the expression of GRASP-1 (Fig. 1a,b). GRASP-1 localizes predominantly in the cytosol of FCX synaptosomes of saline-exposed brains, corresponding to 82.9±1.7% of overall synaptosomal GRASP-1 expression. Prenatal cocaine exposure dramatically increased the membrane-associated GRASP-1 level to 63.7±2.6% of the overall synaptosomal GRASP-1 expression level (Fig. 1c,d). A higher level of membrane-localized GRASP-1 in prenatal cocaine-treated brain was also evidenced immunohistochemically by the increased GRASP-1 immunoreactive puncta on membrane (Fig. 1e).
10.1371/journal.pone.0025019.g001Figure 1 Prenatal cocaine exposure did not alter the expression but markedly increases the synaptic membrane distribution of GRASP-1.
(a) Expression levels of GRASP-1 in the post-mitochondrial synaptosome-enriched fraction prepared from frontal cortices (FCX) of P21 prenatal saline- and cocaine-exposed rats were compared by Western blotting. Blots were stripped and re-probed with anti-β-actin to validate equal loading. Blots are representative for each treatment group. (b) Densitometric quantification of GRASP-1 expression levels in FCX of P21 prenatal cocaine- vs. saline-exposed rats showed no significant differences. n = 4. Data are means ± s.e.m. of the ratios of the indicated protein to loading control. (c) Membranous (M) and cytosolic (Cy) distribution of GRASP-1 in the FCX of P21 prenatal cocaine- and saline-exposed rats. Solubilized proteins (50 µg) were size-fractionated on 7.5% SDS-PAGE and Western blots were performed with antibody against GRASP-1. Blots were stripped and sequentially re-probed with anti- GRIP1 (2618±177 and 2405±159 optical intensity in saline and cocaine, respectively) and -Caspase3 (1159±131 and 1210±118 optical intensity in saline and cocaine, respectively) antibodies to validate equal loading in M and Cy extracts. Blots are representative for each treatment group. (d) Densitometric quantification of GRASP-1 cellular distribution blots. n = 4. Data are means ± s.e.m. of the optical intensity. *p<0.01 compared the level of GRASP-1 in cytosolic and membrane fractions in the FCX of P21 prenatal cocaine- vs. saline-exposed rats. (e). Immunohistochemical detection of GRASP-1 confirms a higher membrane-localized GRASP-1 in the FCX of P21 prenatal cocaine- vs. saline-exposed rats. The representative sections show more GRASP-1 puncta in membrane of cocaine-treated rats. Scale bar = 50 µm. n = 4 for each group.
Because GRASP-1 also associates with GRIP, we next examined whether an increase in GRASP-1 membrane localization is mediated by the heightened association between GRASP-1 and GRIP1. Using an in vitro association assay with individually purified GRASP-1 and GRIP1 from FCX of P21 prenatal saline- and cocaine-exposed rats, we demonstrate that prenatal cocaine exposure markedly increases GRASP-1 and GRIP1 association (Fig. 2a,b). Since GRASP-1 overexpression reduces AMPAR synaptic expression [7], the increase in membrane-localized GRASP-1 observed in prenatal cocaine-exposed brain suggests that the heightened GRIP-associated GRASP-1 may contribute to the reduced GluR2/3-AMPARs synaptic targeting observed here and previously [6].
10.1371/journal.pone.0025019.g002Figure 2 PKC- and Src-mediated phosphorylation of GRIP1 heightens GRASP-1 - GRIP1 coupling in prenatal cocaine-exposed P21 rats.
(a) Immunoaffinity-purified native GRIP1 from frontal cortices of either prenatal saline (S) - or cocaine (C) -exposed rats was treated with vehicle or 100 µg/ml of alkaline phosphatase. Following completion of dephosphorylation by addition of inhibitors, phosphate-free GRIP1 was phosphorylated by recombinant PKC or Src in the presence of ATP. The reaction was terminated by specific PKC or Src inhibitors and GRIP1 was analyzed for phospho-serine and -tyrosine by Western blotting. The interaction between GRASP-1 and GRIP1 with different phosphorylation states was assessed in vitro by incubation of purified GRASP-1 derived from either prenatal saline- or cocaine-exposed rats with purified, phosphorylated GRIP1. GRASP-1 - GRIP1 complexes were isolated along with GRIP1 by immunoprecipitation and levels of GRASP-1 associated with GRIP1 were determined by Western blotting. (b) Densitometric quantification of the top blot shown in a. n = 4 for each group. Data are means ± s.e.m. of the optical intensity. The statistical significance was evaluated by Newman-Keuls multiple comparisons that followed one-way ANOVA. *p<0.01 compared to native GRIP1 from saline-treated group. #p<0.01 compared with dephosphorylated GRIP from both saline- and cocaine-treated groups. +p<0.01 compared with native GRIP1 from cocaine-treated group.
PKC- and Src-mediated phosphorylation of GRIP1 increases GRASP-1 and GRIP1 interaction in prenatal cocaine-exposed brain
Because hyper-phosphorylation of GRIP1 mediates reduced GluR2/3 synaptic expression in prenatal cocaine-exposed brains [6], we examined whether hyper-phosphorylation of GRIP also promotes GRIP-GRASP-1 coupling. Confirming our previous observation, an increased GRIP1 phosphorylation on serine and tyrosine but not threonine residues was noted in FCX from prenatal cocaine-exposed rats (Fig. 2a). Whether hyperphosphorylation of GRIP1 increases the GRASP-1 – GRIP1 interaction was then assessed following treatment of GRIP1 with alkaline phosphatase: dephosphorylation sharply reduced GRIP1-associated GRASP-1 (Fig. 2a,b). Further, increases in GRIP1 phosphorylation on serine and tyrosine, respectively by PKC and Src, heightened GRIP1-coupled GRASP-1 levels (Fig. 2a, b). Together with our earlier work, these results support the conclusion that PKC and Src-mediated phosphorylation of GRIP1 increases GRASP-1 coupling but decreases GluR2/3 coupling to GRIP1.
Prenatal cocaine exposure reduces GRASP-1 RasGEF activity
Since GRASP-1 contains the catalytic domain of guanine nucleotide exchange factors (GEF) for the ras family of G proteins [7], we compared whether prenatal cocaine affects the RasGEF activity of GRASP-1. The kinetics and capacity of GRASP-1, immunoaffinity-purified from FCX of prenatal cocaine- and saline-exposed rats, were analyzed using a time-course and the level of [3H]GDP release from [3H]GDP pre-loaded immunopurified RhoA and Rap1. Our results show that although 76.7±3.3% and 81.3±5.2% of [3H]GDP was released from RhoA and Rap1, respectively, within 5 min following exposure to purified GRASP-1 obtained from prenatal saline-exposed brains, GRASP-1 from prenatal cocaine-exposed brains only reduced 62.0±1.5% and 69.7±3.2% of [3H]GDP from RhoA and Rap1, respectively in 5 min (Fig. 3). Further analysis revealed that prenatal cocaine exposure reduces GRASP-1 GEF activity on RhoA and Rap1, respectively, by approximately 40% from 0.87±0.07 and 0.69±0.08 to 0.46±0.03 and 0.42±0.03 nmol/nmol/min (Fig. 3).
10.1371/journal.pone.0025019.g003Figure 3 Prenatal cocaine exposure reduces RasGEF activity of GRASP-1 in the FCX.
(a) RhoA and (b) Rap1 purified from frontal cortices (FCX) of naïve rats by immunoaffinity column were loaded with [3H]GDP and incubated with purified GRASP-1 from the FCX of prenatal cocaine- (dashed line) and saline- (bold line) treated P21 rat FCX. Aliquots of these two reactions were taken out every 30 sec up to 5 min and were filtered on HA filters. The [3H]GDP that remained on RhoA and Rap1 was measured by liquid scintillation counting. In GRASP-1 obtained from saline-treated FCX, [3H]GDP-loaded RhoA and Rap1 lost 76.7–81.3% of its radioactivity within 5 min, whereas only 62–69.7% of [3H]GDP was dissociated from RhoA and Rap1 by the GRASP-1 from prenatal cocaine-treated FCX. (c) The calculated GRASP-1 specific activity using RhoA and Rap1 as the substrate in FCX of prenatal saline- (blank bar) and cocaine- (solid bar) exposed P21 rats. *p<0.01 compared to saline-treated group using the respective substrate.
Prenatal cocaine exposure increases activated RhoA, Rap1 and Rac1/Cdc42 levels
To investigate the impact of increased levels of GRIP-associated GRASP-1 and reduced RasGEF activity in prenatal cocaine-exposed brains, we compared levels of activated RhoA, Rac1/Cdc42 and Rap1, all of which modulate AMPAR trafficking through their effects on the actin-dependent cytoskeleton [10], [11], [12]. Using GST-conjugated Rhotekin-, RalGDS- and GST-Pak1-RBD beads to isolate GTP-bound (activated) RhoA, Rap1 and Rac1/cdc42 respectively, we found that prenatal cocaine exposure markedly increased activated RhoA, Rap1 and Rac1/cdc42 without a detectable difference in their expression levels (Fig. 4a,b).
10.1371/journal.pone.0025019.g004Figure 4 Prenatal cocaine exposure increases activated ras-like G proteins, RhoA, cdc42/Rac1 and Rap1 levels.
(a) Synaptosomes were prepared from the frontal cortices (FCX) of P21 prenatal cocaine- and saline-treated rats. GTP-bound RhoA, Rac1/Cdc42 and Rap1 were isolated from the synaptosomes by a GST fusion protein pull down method and their levels determined by Western blotting. In addition, the expression levels of RhoA, Cdc42/Rac1 and Rap1 were also measured by Western blotting in post-mitochondrial FCX synaptosome-enriched extracts from prenatally cocaine- and saline-exposed P21 rats. Blots were stripped and re-probed with anti-β-actin to validate equal loading (not shown). (b) Densitometric quantification of blots. n = 4. Data are means ± s.e.m. of the optical intensity. *p<0.01 compared to respective protein from saline-treated group.
To determine whether the increased activated RhoA, Rap1 and Rac1/cdc42 in the prenatal cocaine-exposed brain is also related to the hyper-activated PKC [6], organotypic FCX slice cultures were treated with combination of γ and ζ-PKC specific pseudosubstrate inhibitor peptides (GIP and ZIP) or control scrambled peptides (GIP SC and ZIP SC). The data summarized in Fig. 5a, b indicate that GIP and ZIP robustly reduce the active RhoA, Rap1 and Rac1/cdc42 levels to that of saline-treated animals. Together with the data showing that a blockade of PKC γ/ζ by a combination of GIP and ZIP normalizes GRASP-1 and GluR2 cellular distribution (Fig. 6), these data indicate that the increased active RhoA, Rap1 and Rac1/cdc42 in prenatal cocaine brains originated from a persistent activation of PKC. These data further support the hypothesis that prenatal cocaine exposure alters GluR2/3 trafficking by increasing the relative abundance of polymerized (filamentous) actin.
10.1371/journal.pone.0025019.g005Figure 5 Blockade of PKCγ and PKC/Mζ with isozyme-specific pseudosubstrate inhibitors abolishes prenatal cocaine exposure induced increases in activated monomeric G proteins.
(a) Organotypic FCX slice cultures were serum-depleted and treated with combination of myristoylated PKCγ- and PKC/Mζ-specific pseudosubstrate inhibitors (GIP and ZIP), 10 µM each or 10 µM control peptide, GIP/ZIP scrambled [GIP/ZIP SC] for 4 hr. Following homogenization and solubilization of the slices, GTP-bound RhoA, Rac1/Cdc42 and Rap1 as well as β-actin in the resultant lysate were either isolated by a GST fusion protein pull down method or immunoprecipitated with anti-actin. Their levels were then determined by Western blotting using specific antibodies. (b) Densitometric quantification of blots. n = 4. Data are means ± s.e.m. of the optical intensity. *p<0.01, **p<0.05 compared to respective protein in the respective GIP/ZIP SC-treated group. #p<0.01 compared to respective protein in the saline-treated group.
10.1371/journal.pone.0025019.g006Figure 6 Blockade of PKCγ and PKC/Mζ with isozyme-specific pseudosubstrate inhibitors abolishes prenatal cocaine exposure induced increase and decrease in synaptic membrane associated GRASP-1 and GluR2, respectively.
(a) Organotypic FCX slice cultures were serum-depleted and treated with combination of myristoylated PKCγ- and PKC/Mζ specific pseudosubstrate inhibitors (GIP and ZIP), 10 µM each or 10 µM control peptide, GIP/ZIP scrambled [GIP/ZIP SC] for 4 hr. The slices were homogenized to yield synaptosomes and the resultant synaptosomes were hypotonically lyzed to yield synaptic membranes (M) and cytosol (Cy). The levels of GRASP-1 and GluR2 were then determined sequentially using Western blotting. The blots were stripped and re-probed sequentially to measure the exclusive cytosolic and membranous markers, caspase-3 and GRIP to illustrate equal loading. (b) Densitometric quantification of blots. n = 4. Data are means ± s.e.m. of the optical intensity. *p<0.01 compared to respective protein in the respective GIP/ZIP SC-treated group. #p<0.01 compared to respective protein in the saline-treated group.
Prenatal cocaine exposure increases F-actin levels
Toda et al (2006) indicate that withdrawal from repeated cocaine in the adult rat increases actin cycling and actin polymerization in the nucleus accumbens, suggesting that actin cycling and polymerization may be altered by in utero cocaine exposure [16]. To test this hypothesis, we measured F-actin levels in synaptosomes prepared from FCX of prenatal cocaine- and saline-exposed rats using rhodamine-conjugated phalloidin, a toxin which binds tightly to F-actin. The data summarized in Fig. 7a shows a 2.2-fold increase in F-actin level in FCX synaptosomes from P21 prenatal cocaine-exposed rats. This prenatal cocaine-induced effect was not caused by a heightened actin phosphorylation since serine-, threonine- and tyrosine-phosphorylated actin levels were comparable in both FCX and prefrontal cortex between P21 prenatal saline- and cocaine-exposed rats (Fig. 7b). Since treatment of synaptosomes with alkaline phosphatase normalized F-actin levels, this result indicates that phosphorylation of the upstream actin regulators such as GRIP by PKC is pivotal in determining the F-actin level, (Fig. 7a).
10.1371/journal.pone.0025019.g007Figure 7 Prenatal cocaine exposure induces an increased F-actin level which is normalized by protein phosphatase treatment without alteration in phosphorylation state of actin.
(a) Synaptosomes obtained from the frontal cortices (FCX) of P21, prenatal cocaine- or saline-treated rats were treated with alkaline phosphatases, alkaline phosphatases (1 mg/ml) plus phosphatase inhibitors or cytochalasin D (10 µM) in vitro. The reactions were terminated, synaptosomes solubilized, and actin-containing proteins extracted using biotin-conjugated anti-actin antibodies and placed into streptavidin-coated plates. The level of F-actin was determined by rhodamine-conjugated phalloidin and the fluorescence intensity of phalloidin was measured using Beckman multimode plate reader, DX880. n = 6. Data are means ± s.e.m. of the fluorescence intensity. The statistical significance was evaluated by Newman-Keuls multiple comparisons that followed one-way ANOVA. *p<0.01 compared to native GRIP1 from saline-treated group. #p<0.01 compared to dephosphorylated GRIP in cocaine-treated group. +p<0.01 compared with native GRIP1 from respective group. (b) The phosphorylation state of actin was evaluated in synaptosomes derived from the FCX of P21, prenatal cocaine- or saline-treated rats. Total actin was purified by immunoprecipitation with anti-actin and the levels of phosphor-serine (pS), -threonine (pT) and -tyrosine (pY) in the anti-actin immunoprecipitate was determined by Western blotting using phosphoepitope-specific antibodies. n = 4. Data are means ± s.e.m. of the ratios of pS-, pT- or pY-actin to total actin optical intensities. There was no discernible difference noted in any of the actin phosphoepitopes in FCX of the prenatal cocaine- comparing to saline-exposed rats.
Discussion
Cocaine exposure in utero can modify synaptic plasticity at excitatory synapses resulting in long-lasting deficits in brain function and altered cognitive and psychological development. Our previous findings show that prenatal cocaine exposure attenuates AMPAR-mediated LTD and reduces AMPARs the in synaptic membrane resulting from a disrupted GluR2/3 – GRIP interaction [6]. The reduced GluR2/3 – GRIP association in prenatal cocaine-exposed brains is the result of a sustained PKC-mediated phosphorylation of the AMPAR scaffolding protein GRIP [6]. Resonating with our finding of altered GRIP in prenatal cocaine-exposed brain, chronic cocaine exposure of adult brains also changes the expression or function of glutamate receptor scaffolding proteins such as PSD-95 and Homer [17]–[20]. Collectively, these data suggest that scaffolding proteins for the glutamatergic receptors such as AMPARs in the postsynaptic density are prominent targets of cocaine.
In addition to serving as a synaptic anchor for AMPARs, GRIP also interacts with other signaling molecules including GRASP-1 [7], liprin-α [21], ephrin B receptors [22], and matrix metalloproteinase 5 [23]. Although the precise mechanism through which GRIP-interacting signaling molecules contribute to the reduced GluR2/3 synaptic membrane localization in prenatal cocaine-exposed brain remains ambiguous, a previous demonstration that overexpression of GRASP-1 in cultured hippocampal neurons can reduce AMPAR synaptic targeting suggests that an overly active or abundant GRASP-1 may hinder GluR2/3-GRIP interaction [7]. This hypothesis is supported by our current data showing a markedly higher synaptic localization of GRASP-1 (and compensatory reduction in cytosolic GRASP-1 level) in FCX of prenatal cocaine-exposed rats. This prenatal cocaine-induced GRASP-1 synaptic localization is caused by a greater coupling of GRASP-1 to GRIP resulting from sustained PKC and Src-mediated phosphorylation of GRIP that we previously showed to reduce GluR2/3-GRIP association [6]. An increased GRASP-1 - GRIP association may present a physical hindrance preventing efficient GluR2/3 - GRIP1 binding, although GRASP-1 and GluR2/3 bind to different PDZ domains on GRIP. Alternatively, an increased GRASP-1 and GRIP interaction may alter GRIP conformation rendering the GluR2/3 binding sites on GRIP inaccessible.
In addition to a greater GRASP-1 synaptic localization, prenatal cocaine exposure slows enzymatic kinetics without altering GRASP-1 RasGEF capacity but dramatically increases the level of active monomeric G proteins such as RhoA, cdc42/Rac1 and Rap1. These data suggest that the tremendous increase in membrane-associated GRASP-1 through interaction with GRIP overcomes slower enzymatic kinetics to promote monomeric G protein activation that may ultimately influence the AMPAR-regulated synaptic transmission. The GRASP-1 having rasGEF activity observed herein agrees with the earlier finding by Ye et al. (2000) but sharply contrasts to a recent report indicating that GRASP-1 lacks enzymatic activity [10]. While the precise reason for the discrepancy in whether GRASP-1 possesses GEF activity remains elusive, there are clear methodological and species differences in these three studies that may have contributed to the opposite findings.
In agreement with the notion that increased active monomeric G proteins contribute to AMPAR synaptic transmission regulation, Rap mediates NMDA receptor-dependent, activity-induced LTD by removing GluR2/3-containing AMPARs from the synaptic membrane [10] and suppresses synaptic transmission by reducing GluR2 surface expression [11]. Hence, increased active Rap1 observed here may contribute to the previously observed reduction in synaptic targeting of GluR2/3 containing AMPARs and LTD in prenatal cocaine-exposed brains [6]. In contrast to Rap1, activated cdc42/Rac1 induces clustering of AMPARs in dendritic spines [24]. Thus, an elevated active Rac1 level in prenatal cocaine-exposed brains may compensate for the reduced AMPAR synaptic transmission by enabling higher transportation and clustering of AMPARs. RhoA is localized in the postsynaptic density and is associated with excitatory glutamatergic receptors at the spine plasma membrane [25]. Since NMDAR and AMPAR activation dampens RhoA activity and destabilizes actin networks [25], the reduced glutamatergic NMDAR (unpublished findings) and AMPAR activity together with the elevated association of GRIP and GRASP-1 should afford a more stable actin cytoskeleton in the prenatal cocaine-exposed brain. This notion of a more stable actin network is supported by our demonstration that F-actin levels are higher in FCX synaptosomes of prenatal cocaine-exposed rats. The Rho family of small GTPases also plays a pivotal role in regulating spine architecture and synaptic plasticity [26], [27]. Hence, an abnormally upregulated RhoA activity in prenatal cocaine-exposed brains may adversely influence neuronal development leading to cognitive deficits [28], [29].
The increased F-actin level in FCX synaptosomes of prenatal cocaine-exposed rat may promote GluR2/3 trafficking since latruculin A, an actin-depolymerizing agent, reduces AMPAR-containing spines in cultured hippocampal pyramidal neurons [30]. By contrast, Rac1 and RhoA activation reduces dendritic pruning in hippocampal pyramidal neurons [31]. Interestingly, increased activated Rac1 was shown to attenuate synaptic and cognitive functions such as learning and memory [32]. Altogether, these data indicate that excessive RhoA and Rac1 activation in prenatal cocaine-exposed brain may have facilitated GluR2/3 transport to membrane to compensate for GluR2/3-GRIP interaction blockade and altered dendritic morphology. The latter agrees with our findings that cocaine exposure in utero increases dendritic spine density in rats [33], [34], dendritic length in rabbits [35], as well as dendritic length, volume, and extension in mouse FCX [36]. Since three weeks of withdrawal from repeated cocaine exposure increased F-actin levels [16], [37], it is also possible the increased F-actin levels in prenatal cocaine-exposed rats could be caused by extended cocaine abstinence. In contrast to prenatal cocaine affects on F-actin but not overall actin level shown here, a 24-hr exposure of the human fetal cortical cells derived at 20-week gestation to 100 µM cocaine results in down-regulation of the cytoskeleton-related genes [38]. Such discrepancy may be related to different experimental systems used, including rodent vs. human cortices, 24-hr constant exposure vs. in vivo administration, and cytoskeletal protein vs. gene levels.
Most importantly, we show here that the persistently increased membrane localization of the activated PKC in prenatal cocaine exposed brain reported previously [6] is the primary mechanism underlying excessive GRASP-1 association with GRIP that leads to the elevated monomeric G proteins. Our data showing that pseudosubstrate PKC inhibitors targeting PKCγ and PKC/Mζ normalize the monomeric G protein activity in prenatal cocaine exposed rats further demonstrates the critical role of membrane localized, activated PKC. Additional support can also be drawn from previous reports showing that PKC activation in cultured hippocampal neurons induces the formation of dendritic lamellae in a Rho/Rac-dependent manner [39], [40]. Given that aberrant PKC overactivation leads to abnormal dendritic spine density, morphology and function [41], [42], the abnormally hyperactivated PKC and monomeric G proteins may act in tandem to promote AMPAR synaptic transmission and dendritic abnormalities observed in prenatal cocaine-exposed brains [6], [33], [34]. Such drastic functional and structural defects most likely play an important role in mediating the eventual cognitive changes, including impaired reward processing in animal models [43]–[46], [34] and in humans [47]. Future experiments are needed to determine whether the observed increase in GRASP-1 membrane localization in brains from P21 prenatal cocaine-exposed rats is persistent or simply a transient modification of synaptic plasticity during early development. Given that a sustained PKC activation, indicated by an overwhelming presence of synaptic membrane-associated multiple PKC isoforms, and a markedly reduced phorbol ester-induced PKC translocation were observed in adult rabbit brains exposed to cocaine in utero
[48], it is highly likely that the elevated GRASP-1 membrane localization persists into adulthood. Previous studies conducted by us and others in rabbits also indicate that such synaptic plasticity changes last well into adulthood [48], [49], [45].
In summary, our results indicate that increased GRASP-1 membrane localization resulting from sustained PKC- and Src-mediated phosphorylation of GRIP plays a significant role in mediating AMPAR dysfunction and dendritic abnormalities in the prenatal cocaine-exposed brains observed previously [6], [33] (Fig. 8). AMPAR signaling is governed by their synaptic localization and association with scaffolding proteins. The scaffolding proteins in turn recruit proteins that regulate actin-dependent movement of subunits to and from the synaptic membrane. Therefore, alteration in the functional state of AMPAR scaffolding proteins can result in deficits in excitatory synaptic transmission [6]. Excessive PKC activation markedly impairs prefrontal cortex-mediated cognitive function and increases distractibility [50]. The above findings therefore suggest that preventing further PKC activation such as blocking PKC cytosol-to-membrane translocation may reduce the protracted PKC-mediated deficits and restore AMPAR-regulated neurotransmission in prenatal cocaine-exposed brains. In this regard, mood stabilizers such as valproate that block PKC translocation without interfering with the enzymatic activity [51] may help attenuate prenatal cocaine-induced synaptic plasticity and dendritic structural defects leading to AMPAR-related brain dysfunction.
10.1371/journal.pone.0025019.g008Figure 8 Schematic illustration of the effect of prenatal cocaine on the scaffolding and signaling molecules involved in the AMPA-GluR2 mediated synaptic long-term depression (LTD).
Based on the results of this and our earlier studies (Bakshi et al., 2009), prenatal cocaine exposure enhances membrane localization and activation of PKC (especially γ and ζ isoforms) leading to hyper-phosphorylation of GRIP. The heightened GRIP phosphorylation results in increased GRASP-1 and diminished GluR2 association with GRIP (synaptic targeting) and consequent reduction and elevation of the cytosolic GRASP-1 and GluR2, respectively. By virtue of its neuronal ras-GEF activity, the increased GRASP-1 presence in the membrane of prenatal cocaine-exposed brain elevates the level of activated monomeric G proteins, RhoA, Rap1 and cdc42/Rac1 and eventual increase in actin polymer (F-actin) content. The increased F-actin level should then promote the trafficking of GluR2 in an attempt to restore the synaptic GluR2 content and LTD. The size of blocks and thickness of arrows indicates the level of each protein in the prenatal saline- and cocaine-exposed brains. The stripped or dotted patterns symbolize modification made to the proteins such as increased phosphorylation of GRIP1 and reduced neuronal ras-GEF activity in GRASP-1.
Materials and Methods
Materials and Chemicals
Soybean trypsin inhibitor, phenylmethylsulfonyl fluoride [PMSF], 2-mercaptoethanol, NaF, Na2VO4, Digitonin, protein phosphatase inhibitor I & II cocktails, recombinant γPKC, alkaline phosphatase, phorbol 12-myristate, 13-acetate (PMA), anti-phosphoserine (P3430), anti-phosphothreonine (P3555) were purchased from Sigma (St. Louis, MO). Leupeptin and aprotinin were from Peptide International (Louisville, KY). Recombinant Src, celestrine and PP1 were from Cal-Biochem (La Jolla, CA). Antibodies against GRASP-1 (SC-15568 and SC-15569), phosphotyrosine (SC-508), caspase-3 (SC-7272), β-actin (SC-47778), RhoA (SC-32039, SC-418), Rap1 (SC-28197), cdc42/Rac1 (SC-217) and actin (SC-1616R) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Seize-X immunoprecipitation kit, antigen elution buffer, EZ-link Biotiniylation kit and West pico chemiluminescent reagents were purchased from Pierce-Endogen (Rockford, IL). Bradford reagent, SDS-PAGE reagents, and pre-stained molecular weight markers were purchased from Bio-Rad (Hercules, CA). 10-KDa cut-off filters were obtained from Cole-Palmer (Vernon Hills, IL). The antibody against β-tubulin (MAB3408), GST-Rhotekin, GST-RalGDS and GST-Pak1 were from Upstate Biotechnology/Chemicon (Temecula, CA). Target Buffer was purchased from Dako (Carpenturia, CA). Avidin-peroxidase-labeled biotin complex (ABC) was from Vector Labs (Foster City, CA). 3-3-diaminobenzidine-4 HCl (DAB)/H2O2 was from Biomeda (Foster City, CA). Cell permeable (myristoylated) pseudosubstrate inhibitors for PKCγ [GIP] and PKC/Mζ [ZIP] were custom synthesized by Peptide2 Inc. (Chantilly, VA). Guanosine 5′-diphosphate, trisodium salt [8,5′-3H] ([3H]GDP, 27 Ci/mmol, PerkinElmer, Boston, MA).
Animal treatment
Pathogen-free, 10-week-old male and female Sprague-Dawley rats weighing approximately 200–215 g (Taconic, Germantown, NY) were housed individually in a 12-hr light/dark cycle with free access to food and water. All animal procedures were in compliance with the National Institutes of Health Guide for Care Use of Laboratory Animals and were approved by the City College of New York Animal Care and Use Committee. The animal treatment was performed as described previously [6]. Briefly, pregnant dams were assigned to receive daily intraperitoneal (ip) injections from GD 8–20 of either cocaine HCl, 30 mg/kg in 0.9% saline or saline, 2 ml/kg. The animals were injected daily between 9–10 AM. Following each injection, these pregnant rats were observed for 1 hr and behavioral abnormalities recorded. There were no discernible differences in litter size (between 7–13 pups) and body weight of the pups at 21-day of age (48.9±2.5 and 50.7±2.8 g for cocaine and saline, respectively; n = 40 each) and gender distribution (23 males/17 females and 19 males/21 females for cocaine and saline groups, respectively). Importantly, the dose of cocaine used in this study did not induce seizure or fatality.
The progenies were cross-fostered to a naïve mother until sacrificed at 21 days of age (P21). They were subjected to the minimum handling associated with routine animal husbandry. Importantly, we did not find gender differences in our previous studies conducted in rabbit and rats [52], [53], [6], [35], both sexes from separate litters were employed in these experiments. Pups were sacrificed by rapid decapitation, the brains removed immediately on ice, and coronal cuts at optic chiasm and +4 mm rostral to optic chiasm were made to dissect out the frontal cortex and prefrontal cortex.
Rat cortical slice organotypic cultures
Rat brain FCX from P21 prenatal cocaine- and saline-exposed pups were chopped coronally into 200 m slices using a Mcllwain chopper (Brinkman Instruments) and suspended in 10 ml of ice-cold oxygenated K-R.
The rat brain slice organotypic culture was performed with a modified procedure [54]. Rat FCX slices were transferred to sterile, porous 0.4 µm Millicell-CM insert, 2 slices per insert per well containing 2 ml medium: 50% MEM with Earl's salts, 2 mM L-glutamine, 25% Earl's balanced salt solution, 6.5 g/l D-glucose, 20% fetal bovine serum (FBS), 5% horse serum, 25 mM HEPES buffer, pH 7.2, and 50 mg/ml streptomycin and 50 mg/ml penicillin. Cultures were kept in an incubator for 2 days at 36°C in 5% CO2. On the day of experiment, medium was removed, the brain slices rinsed and incubated in 0.1% FBS-containing medium for 4 hr at 36°C in 5% CO2. Brain slices were then incubated with 10 µM of cell permeable (myristoylated) pseudosubstrate inhibitors for PKCγ [GIP] and PKC/Mζ [ZIP] or control peptides, GIP/ZIP scrambled [GIP/ZIP SC] in fresh 0.1% FBS-containing medium for 4 hr. The effect of GIP/ZIP on the active RhoA, cdc42/Rac1 and Rap1 levels was determined as described below in the Affinity precipitation of GTP RhoA, Rap1 and Rac1/Cdc42 section.
Preparation of synaptosomes and fractionation
Synaptosomes (P2 fraction) were prepared from frontal cortices as previously described with a few modifications [54], [55], [56], [6]. To further purify synaptosomal fractions, the synaptosome-enriched P2 fraction was washed twice in 5 ml of ice-cold Kreb's-Ringer solution (25 mM HEPES, pH 7.4; 118 mM NaCl, 4.8 mM KCl, 25 mM NaHCO3, 1.3 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 10 mM glucose, 100 µM ascorbic acid, 50 µg/ml leupeptin, 10 µg/ml aprotinin, 2 µg/ml soybean trypsin inhibitor, 0.04 mM PMSF, 0.1 mM 2-mercaptoethanol, 10 mM NaF, 1 mM Na2VO4 and 0.5 µl/ml protein phosphatase inhibitor I & II cocktails). To obtain cytosolic and membranous fractions of the synaptosomes, the washed synaptosomes were sonicated for 10 sec on ice in 0.5 ml hypotonic homogenization solution (25 mM HEPES, pH 7.4; 120 mM NaCl, 4.8 mM KCl, 25 mM NaHCO3, 1.3 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 10 mM glucose, 100 µM ascorbic acid, 50 µg/ml leupeptin, 10 µg/ml aprotinin, 2 µg/ml soybean trypsin inhibitor, 0.04 mM PMSF and 0.1 mM 2-mercaptoethanol, 10 mM NaF, 1 mM Na2VO4 and 0.5 µl/ml protein phosphatase inhibitor I & II cocktails). Samples were centrifuged at 50,000× g for 30 min. The resulting supernatant was reserved as the cytosolic fraction and the synaptic membrane pellet was resuspended in 0.5 ml of hypotonic solution. Protein concentrations of the synaptic membranes were determined using the Bradford method before solubilization by adding 6× SDS-PAGE sample preparation buffer and boiled for 5 min. Since GRASP-1 was shown to elicit endocytosis of AMPARs [8], we measured transferrin receptor level in our synaptosomal fraction to rule out endosomal contamination.
Immunoaffinity purification of native GRASP-1, GRIP1 and GRIP2
The immunoaffinity purification was performed as described in Bakshi et al (2009) [6] with some modifications. To isolate native GRASP-1 and GRIP1, frontal cortices of P21 prenatal cocaine- or saline-exposed rats were homogenized in hypo-tonic homogenization solution described above. The obtained homogenates were solubilized using 0.5% digitonin, 0.2% sodium cholate, 0.5% NP-40 and 0.2% SDS in the presence of cocktails of protease and protein phosphatase inhibitors for 20 min at 25°C followed by 60 min at 4°C with end-over-end constant shaking. Following centrifugation to remove insoluble debris, the obtained brain lysate was diluted 5-fold and GRIP1 and GRASP-1 were individually purified using immunoaffinity columns (Seize-X immunoprecipitation kit, Pierce-Endogen) with covalently immobilized antibodies directed against GRASP-1 and GRIP1. GRASP-1 and GRIP1 were each eluted twice with 90 µl antigen elution buffer. The resulting eluates were neutralized immediately with 20 µl 1.5 M Tris, pH 8.8 and concentrated to 100 µl by passing through 10-KDa cut-off filter. Protein concentrations were determined using the Bradford method. The purity of each protein was validated by Western blotting. In each case, the purified protein yielded a single protein band with apparent molecular weight identical to that found using rat brain lysate.
Immunoprecipitation of native actin for measurement of phosphorylated actin
To measure the level of serine-, threonine- and tyrosine-phosphorylated actin, total actin was purified by immunoprecipitation with the method described in Bakshi et al (2009) with some modifications [6]. FCX of P21 from prenatal cocaine- or saline-exposed rats were homogenized in hypo-tonic homogenization solution described above. The obtained brain lysate was diluted 5-fold and protein concentration was measured by the Bradford method. Total actin in the 200 µg FCX lysate was immunoprecipitated using covalently immobilized anti-actin conjugated protein A-agarose beads overnight at 4°C. The resultant anti-actin immunoprecipitate was centrifuged, washed 3 times with 1 ml phosphate-buffered saline, solubilized by boiling in 100 µl SDS-PAGE sample preparation buffer (62.5 mM Tris-HCl, pH 6.8; 10% glycerol, 2% SDS; 5% 2-mercaptoethanol, 0.1% bromophenol blue) and used for measurement of phosphorylated actin levels by Western blotting. The obtained blots were screened for phosphoserine first (anti-phosphoserine), stripped and re-probed twice sequentially with anti-phosphotyrosine and anti-phosphothreonine. The signals were detected using a chemiluminescent method and visualized by exposure to x-ray film.
In vitro determination of GRASP-1 – GRIP1 interaction and immunoprecipitation
To control the GRIP1 phosphorylation state, native GRIP1 proteins (10 µg) purified from frontal cortices of P21 prenatal saline- and cocaine-exposed rats were incubated with 100 µg/ml alkaline phosphatase in Tris, pH 8.0, 130 mM NaCl and protease inhibitors at 30°C for 20 min (total incubation volume 100 µl) as described in Bakshi et al (2009) [6]. The phosphatase activity was terminated by adding 10 mM NaF/1 mM Na3VO4 and specific PKC- and src-mediated phosphorylation was induced by incubation with 0.5 µg/ml recombinant γPKC, 20 µg phosphotidylserine and 100 nM PMA or 10 µg/ml recombinant Src in the presence of 30 µM ATP in Kreb's-Ringer at 30°C for 10 min (total incubation volume 125 µl). The actions of PKC and Src were terminated by addition of 1 µM celestrine and PP1, respectively. One-half of the GRIP1 solution (containing 5 µg) was immediately solubilized by adding 6× SDS-PAGE sample preparation buffer and boiled for 5 min for analysis of phospho-serine and -threonine and -tyrosine levels by Western blotting. To determine the influence of GRIP1 phosphorylation state on the interaction between GRIP1 and GRASP-1, purified brain GRASP-1 (5 µg) from gestational saline- and cocaine-exposed rats were individually added to 5 µg of GRIP1 with different phosphorylation states and incubated in 100 µg/ml brain phospholipids, 1% BSA-containing Kreb's-Ringer at 30°C for 30 min with constant end-over-end shaking. The GRIP1-associated GRASP-1 was isolated along with GRIP1 by 20 µl immobilized anti-GRIP1 conjugated protein A-agarose beads and measured using Western blot with anti-GluR2. The obtained blots were screened for phosphoserine first (anti-phosphoserine), stripped and re-probed twice sequentially with anti-phosphotyrosine and anti-GRIP1. The signals were detected using a chemiluminescent method and visualized by exposure to x-ray film.
Western blotting
To determine cellular distribution or the interaction between GRASP-1 and GRIP1, cytosolic and membranous fractions of frontal cortices or anti-GRIP1 immunoprecipitates were boiled for 5 minutes in 100 µl SDS-PAGE sample preparation buffer and then size fractionated on 7.5 or 10% SDS-PAGE based on the molecular mass of the protein. Proteins were electrophorectically transferred to nitrocellulose membrane and Western blotting was performed with antibodies for GRASP-1, phosphotyrosine, phosphoserine, phosphothreonine. The blots were stripped and re-probed with anti-GRIP1 or caspase-3 to assess the level of sample loading.
To determine the expression level of GRASP-1 and GRIP1, protein extracts of the synaptosome-enriched P2 fractions (50 µg) were size fractionated on 7.5% SDS-PAGE and Western blotting was performed using specific antibodies. In some cases, the blots were stripped and re-probed with anti-β-actin, β-tubulin and GRIP1.
Immunoreactivity was detected by reacting with chemiluminescent reagents for exactly 5 min and visualized by immediately exposing to X-ray film for 10–30 sec. Specific protein bands were quantified by densitometric scanning (GS-800 calibrated densitometer, Bio-Rad Laboratories).
Immunohistochemistry
Immunohistochemical analyses were performed using antibodies directed against GRASP-1 were carried out on paraffin-embedded tissues as described previously [57]. Briefly, after removal of paraffin with xylene and rehydration through a graded series of decreasing concentrations of ethanol, protein antigenicity was enhanced by microwaving sections in Target Buffer for 2 min. Following 30-min incubation in 0.3% H2O2, sections were treated for 30 min in normal blocking serum and then incubated with primary antibodies at appropriate dilutions for 1 h at room temperature. Following a thorough rinse in PBS, a secondary biotin-labeled antibody was applied for 30 min. Immunoreactions were treated with the avidin-peroxidase-labeled biotin complex (ABC) and visualized by treatment of sections with 3-3-diaminobenzidine-4 HCl (DAB)/H2O2. Sections were lightly counterstained with hematoxylin, dehydrated through a graded series of increasing concentrations of ethanols, cleared in xylene and mounted in Permount. Controls consisted of comparable sections treated with non-immune serum, pre-absorbed antibody or omission of the primary antibody. Specimens were examined and photographed with a Nikon FXA microscope, and digital images were recorded using a Nikon DXM1200F digital camera and processed using Image Pro Plus (Phase 3 Imaging, Glen Mills, PA) imaging software.
GDP Dissociation Assay to assess GRASP-1 rasGEF activity
GRASP-1 rasGEF activity in the FCX of P21 in utero cocaine- and saline- exposed rats was measured using purified GRASP-1, by the method described by Ye et al. (2000) with some modifications [7]. To load RhoA or Rap1 with [3H]GDP, 0.5 µg immunopurified RhoA or Rap1 (linked to covalently immobilized agarose-protein A, PIERCE) was incubated with 10 µCi [3H]GDP in 50 µl nucleotide loading buffer (50 mM Tris with 10 mM EDTA, 5 mM MgCl2, 1 mM DTT, and 1 mg/ml BSA) at 37°C. Twenty minutes later, 60 µl of nucleotide loading stopping buffer (50 mM Tris-HCl [pH 7.4], with 5 mM MgCl2, 1 mM DTT and 1 mg/ml BSA) was added. The 110 µl sample was divided into two, and each sample was added to 500 µl dissociation reaction buffer (25 mM Tris-HCL [pH 7.4] with 2 mM MgCl2, 1 mM DTT, 1 mg/ml BSA, and 0.1 mM GDP) containing 5 µg of immunopurified GRASP-1 from frontal cortical synaptosomes of prenatally cocaine- and saline-treated rats, respectively. The two reactions were incubated at 25°C. A sample was then removed from each mixture every 30 sec up to 5 min and mixed with 200 µl ice-cold dissociation reaction stopping buffer (50 mM Tris-HCL [pH 7.4] with 10 mM MgCl2). Rapid filtration was performed using GF/C filters under vacuum. The filter was washed twice with 5 ml ice-cold stopping solution, and the radioactivity was measured by liquid scintillation counting after air drying. The data were expressed as the percent of [3H]GDP bound at each time point comparing to input [3H]GDP level (at time 0). The specific activity of the RhoA and Rap1 was calculated using Prism. Data points are means and vertical bars are the s.e.m. derived from 5 independent rats in each treatment group.
Affinity precipitation of GTP RhoA, Rap1 and Rac1/Cdc42
GTP-bound RhoA, Rap1 and Rac-1/Cdc42 were affinity-purified from the synaptosome-enriched fraction of the FCX region from prenatal cocaine- and saline-treated rats. Precipitation of active Rho was performed using the fusion protein GST-Rhotekin, which specifically recognizes the active GTP-bound form of RhoA. Similarly, GTP-Rap1 was precipitated using fusion protein GST-RalGDS and Rac1/Cdc42 from GST-Pak1, respectively. Synaptosomes were ruptured by sonicating in 0.25 ml of immunoprecipitation buffer and solubilized using 0.5% digitonin, 0.2% sodium cholate and 0.5% NP-50 at 4°C for 1 hr. Following dilution with 0.75 ml of immunoprecipitation buffer and centrifugation, the GTP-bound RhoA, Rap1 and cdc42/Rac1 in the resultant synaptosomal lysates were purified by incubating at 4°C for 1 hour with Rhotekein-RBD beads (10 µg) (Upstate Technologies), GST-RalGDS (10 µg) (Upstate Technologies) and GST-Pak beads (10 µg) (Upstate Technologies), respectively with end-over-end rotation. Following centrifugation, the beads were washed twice with 1 ml of ice-cold 25 mM Tris-HCl, pH 7.4, 20 mM MgCl2 containing protease and protein phosphatase inhibitors. To determine the levels of GTP-RhoA, GTP-Rac1/Cdc42, and GTP-Rap-1 the proteins were size-fractionated on 12% SDS-PAGE and Western blot analysis was performed using antibodies specific to RhoA, Rac-1/Cdc42, and Rap1 (Santa Cruz). Signals were detected using a chemiluminescent method (Pierce) and visualized by exposing to x-ray film.
Determination of F-actin levels
To measure the level of F-actin, synaptosomes were prepared from the FCX of prenatal cocaine- and saline-treated P21 rats by the method described above. Synaptsomes (500 µg) were treated with vehicle, alkaline phosphatases (1 mg/ml) or alkaline phosphatases (1 mg/ml) plus phosphatase inhibitors (10 mM NaF/1 mM Na3VO4) or cytochalasin D (10 µM) in vitro for 20 min at 30°C in 50 mM Tris HCl, pH 8.0, 100 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, protease inhibitors (total incubation volume: 100 µl). Following termination of the reaction by diluting with 300 µl immunoprecipitation buffer, the synaptosomes were solubilized in 0.5% digitonin, 0.2% sodium cholate, and 0.5% NP-40. Actin-containing proteins were extracted by incubating with 2 µg of biotin-conjugated anti-actin antibodies for 1 hr at 4°C and the anti-actin-linked actin proteins were then immobilized by loading 50% of the reaction mixture into each well of the streptavidin-coated 96-well plate (Pierce) and incubating at 4°C for 1 hr. The solution was then removed and the plate was washed twice with 25 mM Tris HCl, pH 7.4; 100 mM NaCl (200 µl/well). The level of F-actin was determined using rhodamine-conjugated phalloidin (0.5 µl/well) (Molecular Probes/Invitrogen). After two washes with 100 µl 25 mM Tris, [pH 7.4] containing 100 mM NaCl, the fluorescence intensity of phalloidin was measured using Beckman multimode plate reader, DX880.
Data Analysis and statistical evaluation
Statistical differences between cocaine and saline groups were assessed using the two-tailed Student's t test. Differences between in vitro dose-response relations were analyzed by ANOVA followed by Newman-Keuls multiple comparisons.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work is supported by public service grant, Minority Institutions' Drug Abuse Research Program R24-DA018055 from the National Institute on Drug Abuse (EF, H-YW), The City University of New York (CUNY) collaborative grant (H-YW) and CUNY Doctoral Student Research Grant (KB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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J Allergy (Cairo)J Allergy (Cairo)JAJournal of Allergy1687-97831687-9791Hindawi Publishing Corporation 2196629510.1155/2011/785983Review ArticleImmunopathology and Immunogenetics of Allergic Bronchopulmonary Aspergillosis Knutsen Alan P.
* Departments of Pediatrics, Division of Allergy and Immunology, Saint Louis University, 1465 S. Grand Boulevard, St. Louis, MO 63104, USA*Alan P. Knutsen: [email protected] Editor: Prescott Woodruff
2011 28 9 2011 2011 7859833 5 2011 14 7 2011 Copyright © 2011 Alan P. Knutsen.2011This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Allergic bronchopulmonary aspergillosis (ABPA) is a Th2 hypersensitivity lung disease in response to Aspergillus fumigatus that affects asthmatic and cystic fibrosis (CF) patients. Sensitization to A. fumigatus is common in both atopic asthmatic and CF patients, yet only 1%–2% of asthmatic and 7%–9% of CF patients develop ABPA. ABPA is characterized by wheezing and pulmonary infiltrates which may lead to pulmonary fibrosis and/or bronchiectasis. The inflammatory response is characterized by Th2 responses to Aspergillus allergens, increased serum IgE, and eosinophilia. A number of genetic risks have recently been identified in the development of ABPA. These include HLA-DR and HLA-DQ, IL-4 receptor alpha chain (IL-4RA) polymorphisms, IL-10 −1082GA promoter polymorphisms, surfactant protein A2 (SP-A2) polymorphisms, and cystic fibrosis transmembrane conductance regulator gene (CFTR) mutations. The studies indicate that ABPA patients are genetically at risk to develop skewed and heightened Th2 responses to A. fumigatus antigens. These genetic risk studies and their consequences of elevated biologic markers may aid in identifying asthmatic and CF patients who are at risk to the development of ABPA. Furthermore, these studies suggest that immune modulation with medications such as anti-IgE, anti-IL-4, and/or IL-13 monoclonal antibodies may be helpful in the treatment of ABPA.
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1. Introduction
Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity lung disease due to bronchial colonization by Aspergillus fumigatus that occurs in susceptible patients with asthma and cystic fibrosis (CF). The first published description of ABPA as an entity came from the United Kingdom in 1952 [1], while the first cases in the United States were reported a decade later [2, 3]. ABPA affects approximately 1%–2% of asthmatic patients and 7%–9% of CF patients [4–6]. If unrecognized or poorly treated, ABPA leads to airway destruction, bronchiectasis, and/or pulmonary fibrosis, resulting in significant morbidity and mortality.
The diagnosis of ABPA is based on clinical and immunologic reactivity to A. fumigatus. The minimal criteria required for the diagnosis of ABPA are: (1) asthma or cystic fibrosis with deterioration of lung function, for example, wheezing, (2) immediate Aspergillus skin test reactivity, (3) total serum IgE ≥ 1000 IU/mL, (4) elevated Aspergillus specific IgE and IgG antibodies, and (5) chest radiographic infiltrates. Additional criteria may include peripheral blood eosinophilia, Aspergillus serum precipitating antibodies, central bronchiectasis, and Aspergillus containing mucus plug production [7–11]. The designation of ABPA-seropositive (ABPA-S) may be used to classify asthmatic patients who meet the required criteria but lack the proximal or central bronchiectasis (ABPA-CB). High-resolution computed tomography (HRCT) may demonstrate central bronchiectasis in the inner two thirds of the field even in the absence of chest radiograph lesions. The clinician should note that the development of ABPA is not dependent on asthma severity. The diagnosis of ABPA in CF is more complicated and disagreement exists in the literature regarding the diagnostic criteria. The difficulty lies in the fact that the usual criteria for ABPA and the common signs and symptoms of CF overlap. The most recent Cystic Fibrosis Foundation Consensus Conference proposed the following diagnostic criteria: (1) acute or subacute pulmonary deterioration not attributable to another etiology, (2) total serum IgE >1000 IU/mL, (3) immediate cutaneous reactivity to Aspergillus or in vitro specific IgE antibodies to Aspergillus, and (4) one of the following: Aspergillus serum precipitins, elevated specific IgG anti-Aspergillus antibodies, new or recent chest radiographic, or chest CT abnormalities that have not cleared with antibiotics and chest physiotherapy [12].
1.1. Radiographic and Laboratory Investigations
There are several characteristic radiographic abnormalities associated with ABPA [7–11]. The most common lesion is a large, homogeneous shadow in one of the upper lobes with no change in volume. The shadow may be triangular, lobar, or patchy, and it frequently moves to another site. “Tram-line” shadows are fine parallel lines radiating from the hila that represent inflammation of airway walls. Mucoid impaction causes toothpaste shadows or gloved-finger shadows, which can be seen on plain radiograph.
Adult patients have been reported with normal chest radiographs so radiographic abnormalities are not invariably present. In these individuals, HRCT scan may reveal central cylindrical bronchiectasis even in the absence of chest radiograph abnormalities. Sometimes, “tree-in-bud pattern” may been seen on HRCT scan that indicates some degree of airway mucus plugging. It is more commonly considered evidence of atypical mycobacterial infection and may be seen in cystic fibrosis. However, central bronchiectasis is a common complication and finding in all CF patients.
Laboratory tests that support the diagnosis of ABPA are those that demonstrate allergy to the A. fumigatus, such as elevated specific IgE anti-Aspergillus antibodies and positive Aspergillus precipitins [7–11]. Culture of A. fumigatus from the sputum is only a secondary criterion for the diagnosis of ABPA, because a large proportion of individuals with CF without ABPA have Aspergillus on sputum cultures. Some normal individuals and many individuals with lung diseases have small numbers of spores in their sputum; these are probably present because of passive inhalation. The presence of hyphae is more specific, and the presence of eosinophils in association with hyphal elements is suggestive of the diagnosis. At the time of radiographic exacerbation, the presence of eosinophilia in sputum or blood is suggestive of ABPA in asthmatics and is a primary diagnostic criterion. The peripheral blood eosinophil count is usually greater than 1000/mm3, and values greater than 3000/mm3 are common. Eosinophilia is not a diagnostic criteria of ABPA in CF patients. In the authors' experience, eosinophilia is an uncommon finding in CF ABPA patients.
An increased total serum IgE level is very characteristic of ABPA, and values may reach as high as 30,000 IU/mL. Usually, the level is greater than 1000 IU/mL. Much of the IgE is not specific to Aspergillus but is the result of polyclonal B-cell activation. The IgE level is a very useful marker of disease activity, and it can be used to follow outpatients for “flares”. The simple skin-prick test is a useful screening test, as ABPA is very unlikely in patients with a negative reaction. A dual-reaction skin test with an immediate (10–15 minutes) and a late (4–8 hours) reaction may occur but is uncommon in ABPA. Alternatively, serum may be measured for the presence of specific IgE and IgG antibodies. Patients with Aspergillus-sensitive asthma will generally have elevated Aspergillus-specific IgE antibodies, but patients with ABPA will have much higher Aspergillus-specific IgE levels. Hemmann et al. [13] reported that ABPA and Aspergillus-sensitive patients have elevated IgE antibodies to recombinant Aspergillus Asp f1, Asp f3, Asp f4, and Asp f6 allergens and that IgE levels to Asp f4 and Asp f6 is highly specific for ABPA in CF patients.
Differentiating between a bacterial flare versus an ABPA flare in CF patients may be difficult. A useful serum biologic marker may be thymus and activation-regulated chemokine (TARC) or CCL17. Latzin et al. [14] and Hartl et al. [15] reported that TARC was elevated in CF patients with ABPA and was further elevated during acute flares of ABPA. TARC is a chemokine whose ligand is CCR4 receptor on CD4+ Th2 cells.
In ABPA, immunoelectrophoresis generally shows one to three precipitin lines, often to only one extract [7–11]. Patients with aspergilloma will have multiple precipitin lines to all antigen extracts. Extracts of A. fumigatus contain a complex mixture of proteins that are mainly derived from the hyphae. Antigenic composition varies between batches according to the culture conditions even within the same laboratory. There is, therefore, a lack of standardization that makes it difficult to compare results between laboratories. There are 22 recognized recombinant allergens by the International Union of Immunological Societies. With purification of these major antigenic components, this may lead to improved diagnosis.
1.2. Pulmonary Pathology of ABPA
The gross pathology of ABPA demonstrates cylindrical bronchiectasis of the central airways, particularly those to the upper lobes [7–11]. These airways may be occluded by “mucoid impaction,” a condition in which large airways are occluded by impacted mucus and hyphae. Airway occlusion may lead to atelectasis of a segment or lobe and, if the atelectasis is long-standing, saccular bronchiectasis may result. Typically, ABPA is worse in the upper lobes than in the lower lobes. Microscopic examination of the airways shows infiltration of the airway wall with eosinophils, lymphocytes, and plasma cells. The airway lumen may be occluded by mucus containing hyphal elements and inflammatory cells, especially eosinophils. Squamous metaplasia of the bronchial mucosa commonly develops, and granulomas may form. Rarely, bronchiolitis obliterans or bronchocentric granulomatosis develops.
2. Immunopathogenesis of ABPA
As seen in Figure 1, the pathogenesis of ABPA in susceptible persons begins with the inhalation of A. fumigatus spores that germinate into hyphae deep within the bronchi. Fragments of hyphae have also been found within the lung parenchyma, potentially resulting in high concentrations of Aspergillus allergens exposed to the respiratory epithelium and immune system [16–19]. These allergens are processed by HLA-DR2 or HLA-DR5 bearing antigen presenting cells (APCs) and presented to T cells within bronchoalveolar lymphoid tissue (BALT). The resulting CD4+ T cell responses to Aspergillus are skewed toward Th2 response with the production of IL-4, IL-5, and IL-13 cytokines.
2.1. Effect of Aspergillus on Bronchial Epithelium
A. fumigatus spores 3 to 5 μm in size are inhaled and germinate deep within the bronchi into hyphae [17]. In addition, fragments of the hyphae can be identified within the interstitial of the pulmonary parenchyma. The implication of this is that there is the potential for high concentrations of A. fumigatus allergens exposed to the respiratory epithelium and immune system. A. fumigatus releases a variety of proteins, including superoxide dismutases, catalases, proteases, ribotoxin, phospholipases, hemolysin, gliotoxin, phthioic acid, and other toxins. The first line of defense against Aspergillus colonization in the lungs is macrophage and neutrophil killing of the conidia and the hyphae. In the development of ABPA, Kauffman's group proposed that Aspergillus proteins have a direct effect on the pulmonary epithelia and macrophage inflammation [20, 21]. They demonstrated that Aspergillus proteases induce epithelial cell detachment. In addition, protease-containing culture filtrates of Aspergillus induce human bronchial cell lines to produce proinflammatory chemokines and cytokines, such as IL-8, IL-6, and MCP-1. Thus, various Aspergillus proteins have significant biologic activity that disrupts the epithelial integrity and induces a monokine inflammatory response. This protease activity is thought to allow for enhanced allergen exposure to the bronchoalveolar lymphoid tissue immune system. This is evident by the bronchoalveolar lymphoid tissue synthesis of Aspergillus-specific IgE and IgA antibodies.
An important pathogenic feature of Aspergillus and other microbes is their ability to interact with epithelial cells on the mucosal surface. Macrophage and neutrophil killing of the conidia and hyphae is the first line of defense against colonization in the lungs [12, 22–24]. This is evidenced by an increased susceptibility to invasive pulmonary aspergillosis in patients with chronic granulomatous disease, a disorder of phagocyte killing. A. fumigatus has several virulence factors, including proteolytic enzymes that can interfere with humoral and cellular defense in the airways [25, 26]. Proteases from Aspergillus and other fungi, including Alternaria and Cladosporium, have been shown to cause epithelial cell detachment though Aspergillus proteases demonstrated more activity at lower concentrations [25–28].
In addition to damaging the integrity of the epithelial cell layer, Kauffman's group demonstrated that protease containing culture infiltrates of A. fumigatus induced human bronchial cell lines to produce proinflammatory chemokines and cytokines, such as monocyte chemoattractant protein (MCP)-1, IL-8, and IL-6 [20]. MCP-1 has been implicated in directly stimulating the development of Th2 cells [29]. The cytokine-release activity could be ascribed to the proteolytic activities of these extracts [20, 27]. These observations suggested that proteolytic enzymes released by Aspergillus, growing on and between epithelial cells, were responsible for the induction of chemoattractive cytokines by epithelial cells and the corresponding inflammation. It was proposed that the induction of the severe inflammatory responses by the direct activation of epithelial cells may cause additional harm to the epithelial cell layer [25]. Destruction of the epithelial cell barrier either by fungal proteases or eosinophilic and neutrophilic inflammation was followed by repair mechanisms, resulting in the influx of serum proteins and extracellular matrix proteins to the luminal side of the epithelium [30]. Because spores and mycelium of A. fumigatus have surface structures that are able to interact with extracellular matrix molecules, damage and repair mechanisms of the airway mucosa may facilitate the binding of Aspergillus to the damaged sites of the airways. The enhanced release of proteolytic enzymes and allergens on the epithelial surface would induce a continuous inflammatory response and mast cell degranulation, resulting in severe and long-lasting periods of exacerbations of ABPA.
2.2. Aspergillus-Specific Th2 Cells
The immune response to Aspergillus antigens in ABPA patients, as well as allergic asthmatic and CF patients, is characterized by a Th2 CD4+ T lymphocyte response [17, 31–35]. Skin test reactivity to Aspergillus is found in 20%–25% of asthmatic patients [5, 36, 37] and 31%–59% of CF patients [13, 38]. Although sensitization is common in these populations, only a small percentage of patients develop ABPA.
Several groups have observed T cell lymphoproliferative responses to crude Aspergillus extracts [31, 39–41]. Subsequently, Aspergillus-specific T cell responses were examined and shown to enhance B cell IgE synthesis [41]. In addition, Asp f1 T cell lines were generated, and the phenotypes were found to be CD4+ CD25+ T cells with the cytokine profile IL-4+ and IFNγ
−, indicating Th2 CD4+ T cells [4]. Chauhan et al. [42] subsequently developed T cell clones from asthmatic ABPA patients and demonstrated either Th2 (IL-4+, IFN-γ
−) or Th0 (IL-4+, IFN-γ
+) patterns. We demonstrated that ABPA subjects have increased frequency of IL-4+ CD3+ T cells from Asp f2/f3/f4-stimulated peripheral blood lymphocytes compared to Aspergillus sensitive non-ABPA subjects [4]. IL-4 produced by T lymphocytes binds to the IL-4 receptor (IL-4R) on B cells and in association with the CD40L/CD40 signals, results in IgE isotype switching and B cell proliferation [43]. IL-4 also increases the expression of CD86, which has been linked to eosinophilic airway inflammation and airway hyperresponsiveness after allergen challenge. A central question then is how ABPA patients differ from Aspergillus-sensitive atopic asthmatic and CF patients. We hypothesize that ABPA develops in genetically susceptible individuals with asthma and CF because of increased frequency and/or activity of A. fumigatus-specific Th2 CD4+ cells. We further propose that polymorphisms of the interleukin-4 receptor alpha chain (IL-4RA) subunit and HLA-DR2/DR5 are the genetic susceptibility risk factors responsible for the development of ABPA.
2.3. IL-4 Responses in ABPA
Human studies and murine models have shown that CD4+ Th2 cells and their cytokines are central to the development of ABPA [4, 32–35, 39, 44]. In particular, IL-4 has a key role in the allergic inflammatory response with effects on various cell populations. Its functions include increasing VCAM-1 expression on endothelial cells, which enhances the recruitment of other immune cells, particularly eosinophils, stimulating proliferation of fibroblasts, important in airway remodeling, and increasing Th2 differentiation while decreasing Th1 differentiation and the production of IFN-γ [45, 46]. IL-4 also has a myriad of effects on B lymphocytes including the stimulation of growth and activation, increasing HLA-DR class II expression important for antigen presentation and inducing cell surface expression of CD23 and soluble CD23. This cell surface molecule is the low affinity IgE receptor (FcεRII) and an activation marker present on a number of cells including B cells, activated T cells, monocytes, and eosinophils. CD23 plays a role in augmenting B cell IgE synthesis through its interactions with CD21 [47, 48]. Recently, in 2003, anti-CD23 monoclonal antibody was administered to atopic asthmatic subjects and resulted in decreased serum IgE levels [49]. In addition, IL-4 has a more direct role in IgE isotype switching by B-cells. It should be noted that IL-13 may also stimulate the synthesis of IgE and is the only other cytokine that has this capability [50–52]. Recently, in 2000 and 2004, increased sensitivity to in vitro IL-4 stimulation as measured by enhanced expression of the low-affinity IgE receptor (CD23) on B cells was observed in ABPA patients [32, 33]. This was associated with single-nucleotide polymorphisms of the IL-4 receptor alpha chain (IL-4RA) in 92% of ABPA subjects, principally the IL-4-binding single-nucleotide polymorphism ile75val [19, 34, 35]. This increased sensitivity to IL-4 is demonstrated by increased expression of CD23 and CD86 on B cells of ABPA subjects and increased CD23 expression during flares of ABPA [19]. CD23 is expressed on a variety of cells, including B cells, natural killer cells, subpopulations of T cells, and a subpopulation of dendritic cells. T-cell CD23 and B-cell CD21 form a costimulatory pathway. T-cell CD28, B-cells CD80, and CD86 costimulatory pathways activate both T and B cells, and CD28:CD86 is important in IgE synthesis. CD86 is also found on dendritic cells that have the histamine receptor 2, which skews antigen-specific T cells to a Th2 response. We have also observed increased CD86 expression on monocyte-derived dendritic cells of ABPA subjects. Thus, antigen-presenting cells such as monocytes and dendritic cells bearing HLA-DR2 and/or HLA-DR5 and increased sensitivity to IL-4 stimulation probably play a critical role in skewing A. fumigatus-specific Th2 responses in ABPA.
3. Immunogenetics of ABPA
3.1. HLA-DR and HLA-DQ
HLA-DR restriction has been shown to be a risk factor for the development of ABPA (Table 1). Chauhan et al. [42, 53] observed that asthmatic and CF patients who expressed HLA-DR2 and/or DR5 but lacked HLA-DQ2 were at increased risk for ABPA after exposure to A. fumigatus. Within HLA-DR2 and HLA-DR5, there are restricted genotypes. In particular, HLA-DR2 HLA-DRB1*1501 and HLA-DRB1*1503 genotypes were reported to provide high relative risk. On the other hand, 40% to 44% of non-ABPA atopic Aspergillus-sensitive individuals have the HLA-DR2 and/or DR5 type. Further studies indicated that the presence of HLA-DQ2, especially DQB1*0201, provided protection from the development of ABPA. Furthermore, Chauhan et al. [42] demonstrated that Asp f l allergen has a low-affinity of binding to HLA-DR. This is consistent with Th2 T cell response previously reported by others in that strong antigen HLA-DR-Ag-TCR affinity binding favored a Th1 cellular response, whereas low affinity binding favored a Th2 humoral response [54–58]. Four major Vβ chains, Vβ 3, 6, 13, and 14, reacted to Asp f1.
3.2. IL-4 Alpha Chain Receptor (IL-4RA) Polymorphisms
The IL-4 receptor is a type I cytokine receptor and exists as a heterodimer that shares a subunit, IL-4 receptor alpha chain (IL-4RA), with the IL-13 receptor alpha (IL-13RA) [59]. There are two types of IL-4 receptors. Type I receptors, found on all lymphohematopoietic cells, are composed of the IL-4RA and the common gamma chain (γC), which is also a component of IL-2, IL-7, IL-9, IL-15, and IL-21 cytokine receptors [60]. IL-4 receptor type II, also known as the IL-13 receptor, is formed by the association of IL-4RA with the IL-13RA subunits and is located on immune cells, bronchial epithelium, and vascular endothelium. IL-4 stimulates both type I and type II receptors, while IL-13 signals through type II receptors.
A potential gain-of-function in the IL-4RA subunit may be responsible for B cell hyperreactivity in ABPA. As a consequence of increased IL-4R activity, proinflammatory cytokines skew T cell responses to a dominant Th2 pattern which ultimately contributes to the pathophysiology and progression of ABPA. There are eight naturally occurring single nucleotide polymorphisms (SNPs) of the IL4RA gene: ile75val, glu400ala, cys431arg, ser436leu, ser503pro, gln576arg, ser752ala, and ser786pro reported thus far [61–71]. Chromosome 16, which has been associated with asthma, contains the IL-4RA gene [66]. Studies have identified a number of these SNPs to be associated with atopy prevalence and asthma severity. In 1997, Hershey et al. [62] initially reported on a high prevalence of atopy and a gain-of-function in the IL-4RA as measured by increased CD23 expression in patients with gln576arg and a later study found that this allele correlated with asthma severity [68]. Hershey's group found that the presence of two variants, val75 and arg576 together, resulted in elevated IL-4 dependent CD23 expression which was not observed when these SNPs were present alone [71]. In our studies, the presence of the val75 allele, located within the IL-4 binding region, was found in 87.5% of ABPA subjects examined, while the cytoplasmic SNPs were present much less frequently at 27.3% for ala400, 27.3% pro503, 27.3% arg576, and 9.1% arg431. Although these alleles, particularly val75, appear to be common in the general population, their high prevalence in ABPA suggests that they may be a risk factor in the development of the disease (Table 1).
3.3. IL-10 Polymorphisms
Brouard and coworkers [72] recently in 2005 reported another genetic risk, the association of the −1082GG genotype of the IL-10 promoter with colonization with A. fumigatus and the development of ABPA in CF (Table 1). The −1082GG polymorphism has been associated with increased IL-10 synthesis, whereas the −1082A allele has lower IL-10 synthesis. Thus, dendritic cells expressing HLA-DR2/DR5, increased IL-10 synthesis and increased sensitivity to IL-4 stimulation due to IL-4RA polymorphisms, may be responsible for skewing Aspergillus-specific Th2 responses in ABPA.
3.4. Surfactant Protein A2 (SP-A2) Polymorphisms
Recently, in 2003, Saxena et al. [73] reported that ABPA patients with polymorphisms (ala91pro and arg94arg) in the collagen region of pulmonary surfactant protein A2 (SP-A2) had more elevated total IgE levels and higher percentages of eosinophilia than observed in those patients who lacked the SNPs (Table 1). They also found that 80% of patients carrying both alleles had ABPA (P = 0.0079, OR = 10.4), while only 50% and 60% of patients carrying each allele, individually, were ABPA subjects, suggesting an additive effect. How these SNPs affect SP-A has not yet been elucidated, but the collagen region spanning both SNPs has been shown to associate with receptors of alveolar macrophages [74], which are important in protecting against Aspergillus colonization [22]. It is theorized that changes in conformation or affinity of SP-A2 may decrease these interactions and compromise host defense.
3.5. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene Mutations
Because ABPA is found in highest incidence among atopic patients with CF, Miller et al. [75] examined mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) in subjects without CF (Table 1). Their group reported that mutations were present at a higher frequency in asthmatic patients who developed ABPA, 6 of 21 (28.5%), versus control asthmatics, 2 of 43 (4.6%). These ABPA patients were heterozygous for the mutations (1 patient was compound heterozygote and reclassified as atypical CF), did not have a clinical diagnosis of CF, and had sweat chlorides <60 mEq/L. Although the abnormal airway mucus in CF is thought to be a susceptibility factor for ABPA due to enhanced trapping of Aspergillus spores, it is unclear what effect heterozygous CFTR mutations may have on mucus quality in asthmatic airways.
3.6. Toll-Like Receptor (TLR) Polymorphisms
Carvalho et al. [76] examined Toll-like receptor (TLR) polymorphisms of TLR2, TLR4, and TLR9 in cavitary pulmonary aspergillosis (CCPA) and severe asthma associated with fungal sensitization (SAFS). TLR-4 is among the major receptors for Aspergillus hyphae and plays an important part in innate host defense as TLR-4-deficient mice have increased susceptibility to invasive aspergillosis [77]. In CCPA patients, there was significantly increased frequency of the G allele of TLR4 on asp299gly. ABPA patients had increased frequency of allele C for the TLR9 T-1237C polymorphism compared to control patients. However, in SAFS patients who are predominantly Aspergillus sensitive, there was no association of polymorphisms of TLR2, TLR4, or TLR9. TLR-9 is a receptor that recognizes CpG motifs prevalent in bacterial and viral DNA. Aspergillus hyphae and conidia do signal through TLR-9 on murine neutrophils [78]. TLR-9-deficient mice demonstrate greater conidial and hyphal damage. In addition, Lazarus et al. [79] reported that TLR9 polymorphisms have been associated with increased risk of asthma. Novak et al. [80] reported that the TLR9 C allele of T-1237C decreases expression. Thus, decreased TLR-9 protective function may be an underlying susceptibility in the development of ABPA.
4. Conclusions
The prognosis of ABPA is good if the disease is detected early and treatment started promptly. It is important that the diagnosis is made and treatment commenced before there is permanent lung damage from bronchiectasis. In such patients, there should be no progression of the disease, although relapses can occur many years later, and long-term followup is recommended. In children with CF, the relapses seem to be more frequent than they are in patients with asthma, and careful surveillance is necessary to ensure resolution of the disease process. In some CF patients, it is difficult to wean the steroids without an increase in symptoms, such as dyspnea and wheezing, whether this is due to the underlying CF lung disease or due to patients going from stage II to stage III ABPA on withdrawal of steroids is unclear. Adjunctive treatment with antifungal therapy to Aspergillus should be considered. Symptoms are not a reliable guide to therapy; therefore, it is important to reevaluate the chest radiograph and the serum IgE at regular intervals until a long-term remission is established.
ABPA occurs with a worldwide distribution in a significant number of patients with CF and less frequently in those with asthma. Early diagnosis and treatment are essential in preventing end-stage progression. The development of ABPA is probably the combination of many genetic susceptibility factors, gene-gene interactions, and environmental exposure which work together. Understanding of the genetic risks and immunopathogenesis of ABPA hopefully will lead to improved early diagnosis and improved treatment of ABPA.
Abbreviations
Af:
Aspergillusfumigatus
Asp fx:
Aspergillus fumigatus proteins
APC:Antigen presenting cell
MBP:Major basic protein
ECP:Eosinophil cationic protein
EDN:Eosinophil derived neurotoxin
VLA:Very late activation antigen
VCAM:Vascular cell adhesion molecule
CxCR and CCR:Chemokines receptors
MCP:Monocyte chemotactic protein
sCD23:Soluble CD23
cyst-LT:Cysteinyl leukotriene
ABPA:Allergic bronchopulmonary aspergillosis
CFTR:Cystic fibrosis transmembrane conductance regulator
IL-4RA:IL-4 receptor alpha chain
TARC:Thymus and activation-regulated chemokine
SP-A2:Surfactant protein A2 polymorphisms.
Figure 1 Proposed immunopathogenesis of ABPA. In the pathogenesis of ABPA, A. fumigatus proteases have a direct effect on bronchial epithelia causing epithelial cell damage with subsequent stimulation of cytokines and chemokines. Aspergillus proteins are processed via HLA-DR2/DR5 bearing dendritic cells that skew the Th0 response to a Th2 response. Th2 cytokines stimulate IgE synthesis and eosinophil activation. This leads to an eosinophilic inflammatory in the bronchial airways.
Table 1 Genetic risk factors in the development of allergic bronchopulmonary aspergillosis.
(i) HLA-DR restriction and HLA-DQ protection
(a) HLA-DR2 restriction
HLA-DRB1*1501 and *HLA-DRB1*1503
(b) HLA-DR5
HLA-DRB1*1104
(c) HLA-DQ2 protective, decreased in ABPA
DQB1*0201
(ii) IL-4RA polymorphisms
(a) IL-4RA ile75val
(iii) IL-10 polymorphisms
(a) Promoter −1082 GG genotype
(iv) Surfactant protein A2 (SP-A2) polymorphisms
(a) SP-A2 ala91pro
(v) Cystic fibrosis transmembrane conductance regulator (CFTR) mutations
(a) Heterozygous CFTR mutations in asthmatic patients with ABPA
(vi) Toll-like receptor (TLR) polymorphisms
(a) TLR9 T-1237C polymorphism
==== Refs
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 21991300PONE-D-10-0585010.1371/journal.pone.0022800Research ArticleBiologyGeneticsCancer GeneticsGene NetworksMolecular Cell BiologySignal TransductionSignaling CascadesCell DivisionCell GrowthA Regulatory Mechanism Involving TBP-1/Tat-Binding Protein 1 and Akt/PKB in the Control of Cell Proliferation TBP-1 and Akt in the Control of Cell ProliferationSepe Maria
1
Festa Luisa
1
Tolino Fabio
1
2
Bellucci Luca
1
Sisto Luca
1
Alfano Daniela
3
Ragno Pia
3
Calabrò Viola
1
de Franciscis Vittorio
2
La Mantia Girolama
1
Pollice Alessandra
1
*
1
Dipartimento di Biologia Strutturale e Funzionale, Università di Napoli “Federico II”, Naples, Italy
2
Istituto per l'Endocrinologia e l'Oncologia Sperimentale del Consiglio Nazionale delle Ricerche (CNR) “G. Salvatore”, Naples, Italy
3
Dipartimento di Chimica, Università degli Studi di Salerno, Salerno, Italy
Agoulnik Irina EditorFlorida International University, United States of America* E-mail: [email protected] and designed the experiments: AP VdF PR GLM. Performed the experiments: MS LF FT LB LS DA. Analyzed the data: MS LF FT LB LS DA PR VC VdF GLM AP. Contributed reagents/materials/analysis tools: PR VdF. Wrote the paper: AP GLM VdF. MS and LF equally contributed to this work.
2011 4 10 2011 6 10 e2280030 11 2010 6 7 2011 Sepe et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.TBP-1 /Tat-Binding Protein 1 (also named Rpt-5, S6a or PSMC3) is a multifunctional protein, originally identified as a regulator of HIV-1-Tat mediated transcription. It is an AAA-ATPase component of the 19S regulative subunit of the proteasome and, as other members of this protein family, fulfils different cellular functions including proteolysis and transcriptional regulation. We and others reported that over expression of TBP-1 diminishes cell proliferation in different cellular contexts with mechanisms yet to be defined. Accordingly, we demonstrated that TBP-1 binds to and stabilizes the p14ARF oncosuppressor increasing its anti-oncogenic functions. However, TBP-1 restrains cell proliferation also in the absence of ARF, raising the question of what are the molecular pathways involved. Herein we demonstrate that stable knock-down of TBP-1 in human immortalized fibroblasts increases cell proliferation, migration and resistance to apoptosis induced by serum deprivation. We observe that TBP-1 silencing causes activation of the Akt/PKB kinase and that in turn TBP-1, itself, is a downstream target of Akt/PKB. Moreover, MDM2, a known Akt target, plays a major role in this regulation. Altogether, our data suggest the existence of a negative feedback loop involving Akt/PKB that might act as a sensor to modulate TBP-1 levels in proliferating cells.
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Introduction
TBP1/Tat-Binding Protein 1 (also named Rpt-5, S6a or PSMC3) is a member of a large highly conserved gene family of ATPases (ATPAses Associated to a variety of cellular Activities) whose key feature is a highly conserved module of 230 aa consisting of an ATPase and a DNA/RNA helicase motif. This protein family fulfils a large diversity of cellular functions including cell cycle regulation, gene expression, vesicle mediated transport, peroxisome assembly and proteasome function [1]. Indeed, as other members of the family, TBP-1 is associated with the 19S regulatory subunit of the proteasome, the chief site of protein destruction in eukaryotic cells [2]. The last 10 years have highlighted the essential role of proteolysis in governing cell physiology. Protein breakdown is required not only for removal of abnormal or aged proteins, but also to control most biological pathways through the regulated degradation of key cellular factors. Moreover, abnormal proteasome expression levels have been described in many tumor cells and proteasome plasma levels appear elevated in neoplastic patients, underlying the involvement of the proteasome in cancer development [3], [4]. Consistent with the role in protein destruction, TBP-1 has been shown to bind the tumour suppressor VHL (Von-Hippel-Landau) gene product [5] contributing to its E3-ubiquitin ligase function towards the Hif1-a factor, thus acting as a bona fide tumor suppressor.
On the other hand, 19S protein components (TBP-1 among them) behave as multifaceted proteins, being implicated in different cellular events that do not require proteolysis like transcriptional initiation and elongation, [6], [7], [8] Nucleotide Excision Repair [9] and regulation of mitosis [10].
We and others have reported that TBP-1 may function as a negative regulator of cell proliferation: inhibition of the oncogenic phenotype of erb-B transformed cells was accompanied by an increase of TBP-1 intracellular levels and, accordingly, its overexpression in erb-B transformed cells strongly inhibited tumour formation in athymic mice [11]; furthermore, TBP-1 overexpression in different cellular contexts diminished cell proliferation [11], [12]. Our reported results [12], [13] showing that TBP-1 enhances the levels of the p14ARF oncosuppressor well fit with TBP-1 proposed antioncogenic role [11]. On the other hand, the observation that TBP-1 overexpression can inhibit cell proliferation also in ARF minus contexts [11], [12] suggests an ARF-independent role of TBP-1, raising the question of what molecular pathways may be involved.
In this paper, we address the role of TBP-1 in the control of cell proliferation. To this aim we used, as model, a primary human fibroblast cell line immortalized by h-TERT (human telomerase) expression where p14ARF levels are undetectable and in which we have silenced the expression of TBP-1. Our results show that cellular levels of TBP-1 are critical in the control of cell proliferation pointing to a functional relationship between TBP-1 and the Akt/PKB serine-threonine kinase, one of the major transducers of growth signals mediating proliferative and pro-survival effects.
Results
TBP-1 depletion determines an increase in the growth properties
We decided to first study the effects of long term silencing of TBP-1 in an immortalized human fibroblast cell line (T11hT). To this purpose, by retrovirus infection, we generated stable T11hT-derived cell clones that constitutively express a sh-RNA specifically designed to silence TBP-1 expression (see Materials and Methods). As shown in Figure 1A, TBP-1 is efficiently silenced in six stable clones analyzed, with an extent of silencing ranging from 80% to 48%. To exclude that reduced expression of TBP-1 may have altered proteasome assembly and function [14], we analyzed intracellular levels of proteasome subunits other than TBP-1 in three of the silenced clones (T1, T10E and T10C). In all cases we observed that the levels of expression of three different proteasome subunits (Rpt-6, Rpt-1 of the 19S subunit and C8 of the 20S subunit) do not change significantly as compared to parental T11hT (Figure 1B). Furthermore, we didn't observe any variation of the in vitro proteasome activity of cell extracts obtained from TBP-1-silenced clones and parental cells on two different peptide substrates (data not shown).
10.1371/journal.pone.0022800.g001Figure 1 Characterization of TBP-1 silenced clones.
A, B: Cells stably transfected with TBP1 sh-RNA plasmid or control cells (wt T11hT, Human Primary Fibroblasts Immortalized by hTERT) were cultured in DMEM+10%FBS for 24 hrs. Levels of TBP-1 expression was evaluated by Western Blot with anti-TBP-1 on whole protein lysates. B: As control, protein levels of other proteasome components (two 19S-ATPases, Rpt-1 and Rpt-6, and a 20S component, C8) was evaluated in the clones T1, T10C and T10E. Bands intensity was evaluated by ImageQuant analysis on at least two different expositions to assure the linearity of each acquisition, each normalised for the respective actin values. Asterisk, fold value is expressed relative to the reference point (i.e. TBP-1 levels in T11hT cells), arbitrarily set to 1. Representative of at least four independent experiments.
We then measured the growth rate of the T1 and T10C clones as compared to that of the T11hT cells. Figure 2A shows that both the TBP-1 silenced clones analysed proliferate at higher rate respect to the parental T11hT cell line. In particular, the T1 clone, expressing very low TBP-1 levels (see Figure 1), grows at a rate that is roughly twice that of the parental cell line. Moreover, serum deprivation doesn't appreciably alter the growth rate of the silenced clones (Figure 2B). To exclude any clonal secondary effect due to the selection process, we also generated, by stable transfection, T11hT cell pools either containing the sh-TBP-1 vector or the empty vector. As it is shown in Figure 2C and D, TBP-1 silenced cells, although in a less pronounced way respect to single clones, display the same growth profile, both in presence and absence of serum.
10.1371/journal.pone.0022800.g002Figure 2 TBP-1 knockdown determines an increase in the growth properties.
A, B: Cells from the T1, T10C and control cells (wt T11hT) were cultured in DMEM either in the presence (A) or in absence (B) of 10% FBS. Cells were collected at the time points indicated and counted in a Burker chamber. The values are the mean ± SE of three experiments performed in triplicate. C, D: wt T11hT cells, cells from control cell pool or from the sh-TBP-1 cell pool were cultured in DMEM either in the presence (C) or in absence (D) of 10% FBS. Cells were collected at the time points indicated and counted in a Burker chamber. The values are the mean ± SE of three experiments performed in triplicate. E, F: Cells from the T1 clone were transfected by electroporation with empty vector (indicated just as T1) or TBP-1 expression plasmid (indicated as T1+TBP-1); cells were then cultured either in the presence (E) or in absence (F) of 10%FBS and collected at the time points indicated (being T0 the time at 24 hours after transfection). Cells from each time point have been counted in a Burker chamber. Values are mean ± SE of two experiments performed in triplicate and are indicated as values relative to the reference point (T0). E, F lower panels: TBP-1 expression and Akt activation have been evaluated by Western Blot with anti-TBP-1, anti-Phospho-Akt Ser473, anti-Akt and anti-actin, as loading control, on whole protein lysates of cells collected at each time point, as indicated.
The enhanced growth rate in TBP-1 silenced cells seems to be dependent on TBP-1 silencing. In fact, transient expression of TBP-1 in the faster proliferating T1 clone dramatically reduces its proliferation rate, both in presence (Figure 2E) and absence (Figure 2F) of serum; however, after 48 hrs, when the expression of exogenous TBP-1 was greatly reduced (see Figure 2E and F, lower panels), cells start to proliferate faster suggesting that slow proliferating TBP-overexpressing cells were selected against.
Consistently with the ability of TBP-1 silenced clones to actively proliferate even in the absence of serum, the cell viability of the T1 clone, measured after 6 hrs of serum withdrawal, remains high. In particular, T11hT cell viability was reduced from 60% to 10% (depending on cell density), while that of the T1 clone is reduced only up to 60% at the lowest cell density (Figure 3A and B). We thus investigated whether TBP-1 silencing may increase cell resistance to serum withdrawal-induced apoptosis. As shown in Figure 3C and D, the T1 clone behaves more resistant to serum-deprivation, respect to the parental cells, as assessed by the very faint amounts of both Caspase-3 and PARP-1 cleavage. Accordingly, flow cytometry analysis indicates that serum starvation only slightly affects the percentage of T1 cells in S phase (8% reduction), while more drastically reduces that of the parental cell line (55% reduction) (Figure 3E). Furthermore, the increase of the sub-G1 population (of around 1.8 fold for the T1 clone and 3,14 fold for the parental cell line) is consistent with PARP-1 and Capase-3 cleavage data (Figure 3E and see 3C and D).
10.1371/journal.pone.0022800.g003Figure 3 TBP-1 knockdown reduces sensitivity to serum starvation.
A, B: Cells from the T1 clone and control cells (wt T11hT) were plated at different cell densities as indicated, either in the presence or absence of 10% FBS. After six hrs from plating, cell viability was measured by MTS assay. In the histograms, cell viability is expressed as relative to controls, arbitrarily set to 100 (%). The values are the mean ± SE of three experiments performed in quintuplicate. C, D: 1.8×105 cells/35 mm plates from the T1 clone and control cells (wt T11hT) were grown for 24 hrs, in the presence or absence of 10% FBS. Apoptosis was checked by detection of Caspase-3 (C) and PARP-1 (D) cleavage in Western Blot. Detection with anti-actin was included for control of equal loading. Bands Intensity was measured by ImageQuant analysis on at least two different expositions to assure the linearity of each acquisition. Representative of at least four independent experiments. E: T11hT and T1 cells were counted and seeded at 2×105cells/35 mm plate. At 24 hrs cells were collected and treated for analysis of cellular DNA content by flow cytometry. Percentages of cells in the SubG1, G0–G1, S and G2–M phases were quantified with Summit 4.1 software. Representative of three different experiments. The numerical ratios reported on the right highlight the different behaviour of T1 cells when grown in absence or presence of serum. Table 1 provides the mean values (and standard deviations in parentheses) relative to this analysis.
10.1371/journal.pone.0022800.t001Table 1 Mean values (and standard deviations in parentheses) relative to the flow cytometry analysis described in Figure 3.
T11hT+serum T11hT−serum T1+serum T1−serum
Sub-G1
12,69 (+/−2,35)
39,79 (+/−5,02)
13,02 (+/−1,8)
29,05 (+/−3,12)
S
14,87 (+/−1,2)
6,44 (+/−0,9)
13,94 (+/−1,75)
14,13 (+/−2,49)
Next, we analyzed the invading capability of the T1 clone respect to control cells by a chemoinvasion assay in which cells were plated on Matrigel coated filters and allowed to migrate. As shown in Figure 4A, as compared to parental cells, T1 cells possess a moderate but significant higher ability to migrate through Matrigel. Interestingly, similar results are also obtained when cells were allowed to migrate toward a generic chemoattractant as EGF (Epidermal Growth Factor) (Figure 4B). To further prove that the difference in invasion ability could be ascribed to the reduction of TBP-1 protein levels and not to any clonal secondary effects, making use of a specific siRNA, we transiently silenced TBP-1 in parental T11hT cells. Consistently, transient silencing of TBP1 is even more effective than stable silencing in T1 cells in inducing a high per cent of migrating cells (Figure 4C). Thus, the difference in Matrigel invasion was likely due to an increased invading capability of TBP1 silenced cells, as also suggested by the fact that we don't observe any difference both in the expression and activation status of the EGF receptor (not shown).
10.1371/journal.pone.0022800.g004Figure 4 Silencing of TBP-1 determines an increase of the invading capability.
A: Cells from the T1 clone or control cells (wt T11hT) were plated in Boyden chambers and allowed to migrate on filters coated with Matrigel. The values are the mean ± SE of three experiments performed in triplicate. (*) p = 0.046 as determined by the Student's t test. B: Cells from the T1 clone or control cells (wt T11hT) were plated in Boyden chambers and allowed to migrate toward EGF on Matrigel filters. 100% values represent cell migration in the absence of chemoattractants. The values are the mean ± SE of three experiments performed in triplicate. (*) p = 0.027 as determined by the Student's t test. C: Cells transiently transfected with TBP1 si-RNA or with the control si-RNA (si-Luc) were plated in Boyden chambers and allowed to migrate toward EGF on filters coated with Matrigel. 100% values represent cell migration in the absence of chemoattractants. The values are the mean±SE of three experiments performed in triplicate. (*) p = 0.016 as determined by the Student's t test.
Taken together these data show that TBP-1 sensitizes cells to apoptosis induced by serum withdrawal and interferes with cell growth and migration.
TBP-1 inhibits Akt/PKB activation
The observation that TBP-1 depletion allows cells to grow in a serum-independent manner, prompted us to ask whether TBP-1 expression levels may control, in some way, the activity of the Akt/PKB serine-threonine kinase, one of the major transducers of growth signals, critical for cell proliferation and apoptosis. We thus evaluated the levels of phospho-Akt in our TBP-1 depleted clones, under actively growth conditions (i.e. in the presence of serum). As shown in Figure 5A, pAkt/PKB levels are inversely correlated to the extent of silencing of TBP-1, being the lowest in the parental T11hT and the highest in the T1 clone. Consistently, we observed an increase in the extent of phosphorylation of GSK3β, a well characterized Akt/PKB direct target. TBP-1 reduction appears to specifically affect Akt activation but not that of other important transducers of growth signals, like ERK1/2. Furthermore, in agreement with the observed higher proliferation rate of the clones, we observed a reduction of phospho-cyclin D1 protein levels (data not shown). Both in parental cells and in TBP-1 silenced clones, Akt activation appears to be dependent on the upstream phosphatidylinositol 3-kinase activity (PI3K) as evidenced by Wortmannin and LY294002 treatment that block PI3K activity (Figure 5B).
10.1371/journal.pone.0022800.g005Figure 5 TBP-1 knockdown determines activation of the Akt/PKB kinase.
A: Cells from the T1, T10C and control cells (wt T11hT) were cultured in DMEM+10%FBS for 24 hrs. Activation of Akt/PKB was revealed by Western Blot with anti-Phospho-Akt Ser473 antibody. As control, extracts were also probed with anti-Akt, anti-Phospho-GSK-3β/pSer219, anti-pERK1/2, anti-ERK1/2 and anti-actin antibodies. Bands Intensity was measured by ImageQuant analysis on at least two different expositions to assure the linearity of each acquisition, each normalised for the respective actin values. Asterisk, fold value is expressed relative to the reference point, arbitrarily set to 1. Representative of at least four independent experiments. B: Cells from the T1 clone or control cells (wt T11hT) were plated at the cell density of 2.5×105 in DMEM+10%FBS in six wells. After 24 hrs, either DMSO (/) or with Wortmannin or LY294002, where indicated, were added to the cells at the concentrations indicated and left for either 1 hour (with Wortmannin) or 15′(with LY294002). Extracts were then probed in Western Blot with antibodies against Akt, Phospho-Akt Ser473 and actin. C, D: T11hT cells (C) or U2OS cells (D) were transfected with an siRNA directed against TBP-1 or Luciferase. Extracts were probed with antibodies against Phospho-Akt Ser473, Akt and actin. E: Cells from the T1 clone were transfected with empty vector (first lane) or increasing amounts of TBP-1 expression plasmid. Activation of Akt/PKB was evaluated by Western Blot on whole protein lysates probed with anti-Phospho-Akt Ser473 and, as control, with anti-Akt and anti-actin. F: U2OS cells were transfected with empty vector (lanes 1–4) or TBP-1 expression plasmid (lanes 5, 6). After 24 hrs cells were starved for 4 hrs and treated with 10 ng/ml insulin for 10′ where indicated. Activation of Akt/PKB was evaluated by Western Blot on whole protein lysates probed with anti-Phospho-Akt Ser473. Extracts were also probed with anti-Akt, anti-actin and anti-Xpress (to reveal transfected TBP-1).
Importantly, we could reproduce, in T11hT cells, the same effect after transient reduction of TBP-1 levels by siRNA: silencing of TBP-1 was accompanied by a concomitant increase in the steady-state level of pAkt, suggesting the existence of a causal relationship between TBP-1 intracellular levels and Akt activation (Figure 5C). This effect was not cell-specific since we could reproduce it in the U2OS osteosarcoma-derived cells (Figure 5D).
Further, we set up a rescue experiment in which we re-established high TBP-1 levels in the T1 clone by transient overexpression. In these conditions we observed a strong reduction of pAkt levels (Figure 5E and see also Figure 2E, F). Consistently, insulin-mediated activation of Akt in a different cellular context (U2OS cells) is counteracted by TBP-1 overexpression (Figure 5F). Altogether these data suggest that TBP-1 levels modulate the extent of Akt/PKB activation.
TBP-1 is a downstream target of Akt activation
The new insights into the role of TBP-1 in the control of cell growth prompted us to investigate whether TBP-1 protein levels are sensitive to acute growth factors stimulation. We thus stimulated either T11hT or U2OS osteosarcoma cells by insulin treatment for the indicated time periods and analyzed protein lysates by Western Blots with anti-TBP-1 antibodies. Figure 6A clearly shows that insulin treatment results in a rapid, transient drop of TBP-1 intracellular levels; indeed, TBP-1 levels are reduced of around two times in 5 min and remain low up to 40 minutes with a kinetic that mirrors that of the activation of Akt/PKB (Figure 6A, left panel). On the other hand, other proteasome subunits (Rpt-6 and C8) protein levels remain almost stable or, at least, slightly increased, following insulin treatment (Figure 6A). To test the effects of inhibition of the PI3K/Akt pathway on TBP-1 protein levels, U2OS cells or T11hT cells were treated for the indicated time periods with PI3K inhibitors and protein lysates analyzed by Western Blots. As shown in Figure 6B, inhibition of the PI3K/Akt pathway determines a slight though reproducible increase in TBP-1 endogenous levels, suggesting that they are either directly or indirectly regulated by PI3K activity. Again, in these conditions, other proteasome subunits (Rpt-6 and C8) protein levels remain stable (Figure 6B, left panel). To further confirm these observations, we transiently transfected increasing amounts of a constitutively active mutant of the Akt kinase (CA-Akt) in U2OS cells. Overexpression of CA-Akt was accompanied by a reduction of endogenous TBP-1 levels, while other proteasome subunits protein levels remain unchanged (Figure 6C). Taken together, these data strongly indicate that TBP-1 protein levels are modulated by the Akt/PKB activity.
10.1371/journal.pone.0022800.g006Figure 6 TBP-1 is a downstream target of Akt activation.
A: U2OS cells or T11hT cells were starved for 4 hrs and then treated with 10 ng/ml insulin for the times indicated. Activation of Akt/PKB was evaluated by Western Blot on whole protein lysates probed with anti-Phospho-Akt Ser473 and anti-Akt. Levels of endogenous TBP-1 and of two proteasome components (C8 and Rpt-6) were analyzed where indicated. TBP-1 bands intensity was measured by ImageQuant analysis on two different expositions to assure the linearity of each acquisition, each normalised for the respective actin values. Asterisk, fold value is expressed relative to the reference point, (i.e. TBP-1 levels in starved cells) arbitrarily set to 1. Representative of three independent experiments. B: U2OS cells or T11hT cells were treated, 24 hrs after plating, either with DMSO (/) or with 200 nM Wortmannin or 50 mM LY294002 for the times indicated. Cells were then lysed and Western Blot analysis was performed by using specific antibodies against Phospho-Akt Ser473, anti-Akt, anti-TBP-1, anti-C8 and anti-Rpt-6. TBP-1 bands intensity was calculated as in A. Representative of three independent experiments. C: U2OS cells were transfected with empty vector (lane 1) or increasing amounts of the constitutive active mutant of the Akt kinase (CA-Akt). After 24 hrs cells were lysed and whole cell lysates probed with anti-Phospho-Akt Ser473, anti-Akt, anti-TBP-1, anti-Rpt-1, anti-Rpt-6, and anti-phospho-GSK3b. D: U2OS cells were transfected with a siRNA directed against MDM2 or Luciferase, as control, at the final concentration of 10 nM. After 24 hrs, cells were starved for 4 hrs and then treated with 10 ng/ml insulin for the times indicated. Cells were then lysed and Western Blot analysis was performed by using specific antibodies against Phospho-Akt Ser473, TBP-1, MDM2, Akt, and actin. E: U2OS cells were transfected with a siRNA directed against MDM2 or Luciferase, as control. After 48 hrs, either DMSO (/) or 200 nM Wortmannin was added to the cells and left for the times indicated. Cells were then lysed and Western Blot analysis was performed by using specific antibodies against Phospho-Akt Ser473, TBP-1, MDM2, Akt and actin.
On the other hand, by immunoprecipitation experiments we were unable to observe any physical interaction between TBP-1 and Akt/PKB (data not shown), suggesting that TBP-1 levels are indirectly modulated by Akt activation. We thus wondered which could be the mediator of Akt/PKB action on TBP-1.
Among the known Akt/PKB effector is the MDM2 protein that, following phosphorylation by Akt/PKB, increases its activity [15], [16], [17]. We thus determined whether MDM2 mediates the functional relationship between TBP-1 and Akt/PKB. In order to obtain Akt activation, we treated with insulin U2OS cells that were previously either treated with a MDM2 specific siRNA or, as control, with a luciferase siRNA (Figure 6D). Interestingly, MDM2 silencing prevented the reduction of TBP-1 intracellular levels following treatment with insulin, although has no effects on TBP-1 basal levels. Consistently, the increase in TBP-1 levels following treatment with PI3K inhibitors, is prevented in cells in which MDM2 is silenced (Figure 6E), suggesting that, indeed, silencing of MDM2 renders Akt activation/inactivation ineffective on TBP-1 levels. These data strongly implicate MDM2 to be needed, even not sufficient, for TBP-1 regulation by Akt/PKB. Involvement of MDM2 is supported by co-immunoprecipitation experiments in U2OS cells. As shown in Figure 7A and 7B we found TBP-1 in complex both with endogenous and with transfected MDM2. Furthermore, we observed that overexpression of MDM2 causes a decrease of TBP-1 intracellular levels (Figure 7A and B, see input). We confirmed this observation transfecting U2OS cells with fixed concentration of pcDNA-TBP-1 and increasing amounts of the MDM2 expression plasmid (Figure 7C). Since the observed effect occurs both on the endogenous and on the exogenous protein, it is likely that MDM2 acts on TBP-1 at the post-transcriptional level. Moreover, treatment of U2OS cells with the proteasome inhibitor MG132 counteracts the MDM2 effect on TBP-1, indicating the proteasome as the final effector of the MDM2 action on TBP-1 (Figure 7D).
10.1371/journal.pone.0022800.g007Figure 7 TBP-1 is a downstream target of MDM2 activation.
A: U2OS cells were either transfected (lanes +) or untransfected (lanes −) with the MDM2 expression plasmid. 24 hrs after transfection cell extract was prepared and subjected either to immunoprecipitation with anti-TBP-1 antibody where indicated or, with anti-GFP antibody as negative control. Cell extracts were also incubated with protein A-agarose as control, where indicated. Immunoprecipitated extracts were analyzed by Western Blot with anti-MDM2 or anti-TBP-1 antibody. Aliquots of cell extracts were analyzed by Western Blot before immunoprecipitation (input). B: U2OS cells were either transfected (lanes +) or untransfected (lanes −) with the MDM2 expression plasmid. 24 hrs after transfection cell extract was prepared and subjected either to immunoprecipitation with anti-MDM2 antibody where indicated or, with anti-Flag antibody as negative control. Cell extracts were also incubated with protein A-agarose as control, where indicated. Immunoprecipitated extracts were analyzed by Western Blot with anti-MDM2 or anti-TBP-1 antibody. Aliquots of cell extracts were analyzed by Western Blot before immunoprecpitation (input). C: U2OS cells were transfected with TBP-1 expression plasmid and increasing amounts of MDM2 expression plasmid. After 24 hrs, cells were lysed and whole cell extracts probed with anti-TBP-1, anti-MDM2, and anti-actin, for loading control. D: U2OS cells were transfected with TBP-1 expression plasmid and increasing amounts of MDM2 expression plasmid. After 24 hrs cells were treated either with DMSO (first four lanes) or with 10 µM MG132 where indicated. Cell extracts were analyzed by Western Blot with anti-Xpress (to reveal transfected TBP-1), anti-MDM2, and anti-actin, for control. E: U2OS cells were transfected with TBP-1 expression plasmid and increasing amounts of either MDM2wt, MDM2S166A or MDM2S166A/S186A expression plasmids. After 24 hrs cells were lysed and whole cell extracts were analyzed by Western Blot with anti-Xpress (to reveal transfected TBP-1), anti-MDM2, and anti-actin, for control. F: U2OS cells were transfected with TBP-1 expression plasmid and increasing amounts of either MDM2wt, MDM21–441 or MDM2Δ150–230 expression plasmids. After 24 hrs cells were lysed and whole cell lysates analyzed by Western Blot with anti-Xpress (to reveal transfected TBP-1), anti-MDM2, and anti-actin, for control.
We thus asked if mutations in MDM2 that render it less responsive to Akt/PKB stimulation [15], [16], [17], [18] reduces, as well, its ability to downregulate TBP-1 levels. The MDM2S166A and MDM2S166A/186A mutants appear almost unable to mediate TBP-1 degradation (Figure 7E), indicating that only a functionally Akt-responsive MDM2 molecule, could regulate TBP-1 levels. Accordingly, a MDM2 deletion mutant that lacks all the Akt target sites in MDM2 (MDM2Δ150–230) [19] appear unable to act on TBP-1 levels (Figure 7F). Interestingly, a MDM2 mutant, lacking the ring finger domain (MDM21–441), is still able to act on TBP-1 (Figure 7F), indicating that MDM2 is not acting on TBP-1 levels through its ubiquitination activity.
These data provide clear evidence that TBP-1 is a downstream target of the Akt/PKB-MDM2 axis, even though the molecular mechanisms through which MDM2 acts on TBP-1 remain to be elucidated.
Discussion
Herein we report data showing that reduction of TBP-1 intracellular levels affects cell proliferation, invading capabilities and resistance to apoptosis of human fibroblasts immortalized by h-TERT expression. Interestingly, unlike the parental cells, proliferation of TBP-1 silenced clones appears to be serum-independent. Our data indicate that TBP-1 modulates the extent of activation of the Akt/PKB kinase, a critical effector of intracellular signaling. In fact, we demonstrate that reduction of TBP-1 intracellular levels causes the activation of the Akt signaling pathway. It has to be underlined that this can be directly ascribed to TBP-1 depletion rather than to clonal secondary effects as it also occurs after transient silencing of TBP-1 and irrespective of the cell type. Remarkably, transient expression of TBP-1 in one of the silenced clones restores phospho-Akt basal levels and drastically reduces the proliferation rate. Furthermore, TBP-1 overexpression in other cellular systems prevents Akt/PKB activation thus confirming that TBP-1 can act upstream of Akt.
Activation of the Akt/PKB pathway plays a central role in tumorigenesis. Indeed, Akt is overexpressed in many different tumour cell types, with a burgeoning list of substrates implicated in oncogenesis [20]. In principle, the increase of Akt/PKB activity could account for all the changes induced by TBP-1 silencing (i.e. proliferation, cell viability, escape from apoptosis, migration capabilities) [21], [22], [23], [24]. On the other hand, the acquisition of a transformed phenotype is a quite complex stepwise accumulation of genetic changes [25], [26]. In this context, it seems plausible to predict that, by acting on Akt/PKB, down-modulation of TBP-1 intracellular levels might contribute to the acquisition of a transformed phenotype thus cooperating with other genetic lesions. Since TBP-1 silenced clones are normal fibroblasts that only bear h-TERT overexpression to guarantee immortalization, an intriguing possibility to explore is the introduction of “key” cellular lesions to cause cell transformation in these clones.
The mechanism by which TBP-1 prevents Akt/PKB activation remains an open question. Even though, like the other AAA-ATPases of the 19S base of the proteasome, TBP-1 is supposed to act by conferring specificity to the proteasome [27], [28] various observations suggest that TBP-1 may act, as well, in a proteasome independent manner [6], [7], [8], [12], [13], [29]. Indeed, the proteasome seems very unlikely involved in the modulation of the Akt/PKB activity by TBP-1. In fact, an increase in the proliferation rate is frequently associated to an increase of proteasome levels needed to guarantee high metabolic activity. Here we show that TBP-1 silenced clones don't display a significant alteration in proteasome composition and activity. Furthermore, unlike other proteasome components (C8 and Rpt-6), TBP-1 responds to acute insulin stimulation with a decrease of its intracellular levels. In a different context, other proteasome subunits respond to growth factor stimulation with an increase of intracellular levels [30].
It has to be underlined that we have already observed that TBP-1 stabilizes p14ARF [12], [13] avoiding ARF entrance into the proteasome. We retain that TBP-1 could play a role in ARF folding, rendering it a poor substrate for degradation by the 20S as well as by the 20S/11S proteasome [31], [32]. The existence of a similar mechanism that permits to TBP-1 to increase the intracellular levels of proteins that regulate Akt/PKB activity is the subject of further studies.
Furthermore, our results reveal the existence of a reciprocal regulatory loop where Akt/PKB activation leads to TBP-1 reduction and, in turn, TBP-1 overexpression prevents Akt/PKB activation. In this scenario, the Akt/PKB kinase thus might act as a sensor that modulates TBP-1 levels in actively duplicating cells. On the other hand, based on the fact that the PI3K/Akt signaling effect on TBP-1 is prevented in cells in which MDM2 is silenced, we propose, as mediator of the PI3K/Akt signaling on TBP-1, the MDM2 protein, one of the main direct targets of Akt/PKB activation [15], [16], [17], [18]. Actually, MDM2 can bind to TBP-1 and its overexpression causes a reduction of TBP-1 intracellular levels. Strikingly, the MDM2S166A/S186A mutant and the MDM2Δ150–230, lacking Akt responsive sites, are unable to act on TBP-1 protein levels, likely placing TBP-1 downstream of the Akt/PKB-MDM2 axis.
Even though the specific mechanism for MDM2-dependent depression of TBP-1 levels remains to be understood, it has to be noted that MDM2 has multifaceted roles in protein degradation. In fact, aside its well-described role as E3-ubiquitin ligase, under appropriate stimuli, MDM2 can shuttle p63 to the cytoplasm mediating its interaction with proteins specifically involved in its turnover [33]. Moreover, MDM2 has been shown to mediate proteasome-dependent but ubiquitin-independent degradation of p21Waf1/Cip1
[19] and of Retinoblastoma Protein [34] through direct binding with the C8 subunit of the 20S proteasome. On the other hand, it has very recently been reported that MDM2 interacts with components of the 19S proteasome in a ubiquitylation independent manner [35] claiming a wider view of its mechanism of action.
Interestingly, the MDM2Δ150–230 mutant was described to be unable to shuttle between the nucleus and the cytoplasm, displaying a predominant cytoplasmic localization [19]. This could imply that the MDM2 action on TBP-1 levels requires its nuclear localization that, indeed is described to occur following phosphorylation by Akt [15], [18].
Moreover, the fact that the MDM21–441 deletion mutant, that lacks the ring finger domain, is still able to act on TBP-1 (Figure 7F), indicates that MDM2 is not acting on TBP-1 levels through its ubiquitination activity, supporting the possibility that it rather acts as a molecular cargo and should plausibly act in concert with other pAkt effector molecule(s) needed to direct TBP-1 for degradation.
Altogether our observations provide further insights on the proposed antiproliferative role of TBP-1 [11], [12], [13], indicating the involvement of the Akt/PKB kinase. Indeed, we could speculate that, under standard growing conditions, TBP-1 contributes to balance Akt/PKB up-regulation; whilst, under growth factor acute stimulation, activation of the Akt/PKB signaling pathway lowers TBP-1 levels and initiate a feedback loop. Further, it's interesting to underline that the human oncosuppressor p14ARF that is stabilized by TBP-1 overexpression [12], [13] is itself able to antagonize the activity of Akt/PKB [36] with yet unknown mechanism. On the other hand, other reports [37] underline an in vivo requirement of ARF for full activation of PTEN, one of the major negative regulators of Akt activity.
In conclusion, our data well support a role for TBP-1 in the attenuation of Akt/PKB activity and place this protein with a key role in the control of cell proliferation. Even though, further studies are necessary to understand the potential cross-talks linking TBP-1 action on p14ARF and on Akt/PKB regulation.
Materials and Methods
Cell cultures, viral infection, transfections
T11hT (human primary fibroblasts immortalized by constitutive expression of the telomerase catalytic subunit h-TERT) human cell line was kindly provided by dr. Eric Gilson. T1, T10C and T10E (TBP-1 silenced clones) derived by retroviral infection of T11hT: briefly, 3×106 HEK 293-LinX packaging cells (kindly provided by Prof. Nicola Zambrano) were transfected with ARREST-IN (Open Biosystems, Huntsville, AL, USA) with pSUPERIOR.shTBP-1. 24 hrs after transfection, virus containing supernatant was filtrated through 0,45 µm cellulose acetate syringe filter, supplemented with 5 µg/ml polybrene, and used to infect recipient T11hT cells, previously plated at 50% confluence. Twenty-four hours following infection, 1 mg/ml G418 was applied to select stably infected cells. After three weeks, 23 individual single G418 resistant clones were picked up and expanded. Six neomycin resistant colonies from 5 different plates, were screened by Western Blot with anti-TBP-1.
Both T11hT and TBP-1 silenced clones were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine serum and 1 mg/ml puromycin (to maintain selection for h-TERT). U2OS cells were grown in Dulbecco's Modified Eagle Medium supplemented with 10% Fetal Bovine serum.
To obtain sh-TBP-1 pool and control pool, 2×106 T11hT cells were transfected by electroporation by making use of a Microporator MP-100 (Digital Bio Technology) either with 3 µg of pSUPERIOR.shTBP-1 or 3 µg of pSUPERIOR.retro.neo; twenty-four hours following transfection, 1 mg/ml G418 was applied to select cells. After four weeks, resistant cells were collected, expanded and analyzed.
Transfection by Lipofectamine2000 were performed as described [12].
Transfection of the T1 clone was performed by the use of a Microporator MP-100 (Digital Bio Technology) using either 2×106 cells with 2 µg of either pcDNA empty vector or pcDNATBP-1 (rescue of cell proliferation, Figure 2E and F) or 1×106 cells with either pcDNA empty vector or pcDNATBP-1 (0.3 or 0.6 µg) (rescue of Akt activation, Figure 5 E). Cells were then plated in DMEM+10% FBS for 24 hrs at 37°C or DMEM without FBS at 37°C and collected for subsequent analysis.
For transient silencing experiments, the duplex siRNA oligomer designed to target human TBP-1 is described in [12]; the duplex siRNA oligomer targeting human MDM2 has the following sequence: 5′- AAGCCAUUGCUUUUGAAGUUA-3′ and was designed as described in [19]. siRNA were all synthesized by MWG Biotech, Germany. Either U2OS, T11hT or T1 cells were transfected by Hyperfect (Quiagen, GmBH, Germany) according to the manufacturer's instructions.
Cell growth analysis, MTS assay, Flow cytometry analysis, Chemoinvasion assay
For cell growth analysis, T11hT parental cell line, T1, T10C and T10E clones, or T11hT, control pool and sh-TBP-1 pool, were plated in 100 mm dishes in presence of 10% FBS at the cell density of 1×105 cells/plate. Cells were cultured for 24, 48 and 72 hrs, collected, and counted in a Burker chamber. For growth in the absence of serum, after 6 hrs from plating, medium was removed and replaced with medium without serum. As above, cells have been grown for 24, 48 and 72 hrs, collected and counted. Each point is the result of triplicate samples.
Cell viability was evaluated using the MTS [3-(4,5-dimethylthiazhol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt] (Cell Titer 96AQueous assay G358 purchased from Promega) colorimetric assay. Briefly, T11hT parental cell line or cells derived from the T1 clone were plated at different cell densities as indicated in 96 well plates (2.5×103/well, 103/well, 3×102 /well) either in DMEM or in DMEM+10%FBS. After six hrs from plating, 1∶5 MTS solution was added to each well and the cells were incubated for 30′ at 37°C. Plates were read on a Microplate Reader (BIO-TEK Instruments, Model Elx800) at 492 nm. Survival was expressed as the percentage of viable cells in treated samples relative to non-treated control cells. All the experiments were repeated in quintuplicate.
For flow cytometry analysis, T11hT and T1 cells were counted and seeded at 2×105cells/35 mm plate. At 24 hours after plating the medium was replaced and the cells treated with DMEM containing 10% Fetal bovin serum or DMEM without serum. At the indicated time points, cells were collected, centrifuged, washed twice with PBS 1× and then fixed with ice-cold 70% ethanol. Fixed cells were incubated with staining buffer solution (50 µg/ml PI and 50 µg/ml RNase A in PBS pH 7.4) for 20 minute at room temperature in a dark box. Stained cells were analysed in a fluorescence-activated cytometer (DakoCytomation). Data on DNA cell-content were acquired on 20,000 events at a rate of 150±50 events/second and the percentages of cells in the SubG1, G0–G1, S and G2–M phases were quantified with Summit v4.1 software.
Chemoinvasion assays were performed in Boyden chambers using 8 µm pore size PVPF polycarbonate filters coated with 50 µg/ml of Matrigel. 1×105 cells were plated in the upper chamber in serum-free medium. 100 ng/ml EGF or serum free medium was added in the lower chamber. Cells were allowed to migrate for 4 h at 37°C, 5% CO2. To examine basal migration, serum free medium was added to both upper and lower chamber, and migration was allowed for 12 h at 37°C, 5% CO2, in the absence of chemoattractants.
The cells on the lower surface of the filter were then fixed in ethanol, stained with hematoxylin, and counted at 200× magnification (10 random fields/filter).
Western Blotting, Immunoprecipitations, Insulin treatments, MG132 treatment
Western Blots were performed as described [12]. Antibodies to Akt (used in 1∶1000 dilution), Phospho-Akt Ser473 (used in 1∶1000 dilution), Phospho-GSK-3β/pSer21/9 (used in 1∶1000 dilution), Caspase-3 (1∶1000) and PARP-1 (1∶1000) were purchased from Cell Signalling Technologies, Boston, MA, USA. Antibodies to MDM2 (used in 1∶500 dilution) was purchased from Calbiochem, to Rpt-1 (PSMC2) (used in 1∶6000 dilution), Rpt6 (PSMC5) (used in 1∶6000 dilution) and C8 (used in 1∶6000 dilution) were purchased from BioMol. Anti-Xpress antibody (used in 1∶1000 dilution) was purchased from Invitrogen. Secondary antibodies for Western Blot analysis (goat anti-rabbit IgG-HRP 1∶3000 dilution) were purchased from Santa Cruz Biotechnology, CA, USA. Proteins were visualized with an enhanced chemiluminescence detection system (Amersham ECL ™) and images were taken with ChemiDoc XRS System (Bio-Rad Laboratories) and analysed with the QuantityONE software.
For insulin treatment, U2OS cells were transfected by Lipofectamine with 0.2 and 0.5 µg of the pcDNATBP-1 plasmid. At 24 hrs after transfection, cells were starved for 4 hrs and then treated with 10 ng/ml insulin for 10′.
To analyze TBP-1 levels following insulin treatments, either U2OS cells or T11hT cells were starved for 4 hrs and then treated with 10 ng/ml insulin for the times indicated.
For immunoprecipitation in U2OS cells, 1.0×106 cells were seeded in 100 mm dishes and transfected with the plasmids indicated in the figure legend. Cells were harvested 24 hours after transfection and cell lysates were prepared as described [12]:
800 µg of whole cell extract were incubated overnight at 4°C with anti-TBP1 (BioMol) or anti-MDM2 C18 (Santa Cruz). Controls of immunoprecipitations were perceived with mouse anti-GFP (Roche) or rabbit anti-Flag (Sigma). Immunocomplexes were collected by incubation with 30 µl of protein A-agarose (Roche Applied Science) at 4°C for 4 hrs. The beads were washed with Co-Ip buffer (50 mM tris-HCL pH 7.5; 150 mM NaCl; 5 mM EDTA; 0,5% Np40), resuspended in 2× loading buffer (Sigma) and loaded on a SDS-8% polyacrylamide gel.
Treatment with proteasome inhibitor was performed as follows: U2OS cells were treated either with DMSO or 10 µM MG132 for five hours. Cells were harvested and total extracts prepared for subsequent analysis as described.
Constructs
pSUPERIORshTBP-1 has been obtained from pSUPERIOR.retro.neo (Oligoengine) by cloning into BglII-HindII sites a duplex oligonucleotide obtained by MWG-Biotech that could give rise to a short hairpin RNA specifically designed to silence TBP-1 expression.
Oligoseq:
5′GATCCCCAACAAGACCCTGCCGTACCTTCAAGAGAGGTACGGCAGGGTCTTGTTTTTTTA3′
pCA-Akt plasmid was kindly provided by Prof. G. Condorelli. The MDM21–441 and MDM2Δ150–230 expression plasmids were previously described [19].
Plasmids MDM2S166A and MDM2S166A/S186A mutant were generated by Quick Change Site Direct Mutagenesis Kit (Stratagene, La Jolla, CA, USA) and amplified using the following primers:
S166A (F)
5′GGAGAGCAATTGCTGAGACAGAAG 3′
,
S166A (R)
5′CTTCTGTCTCAGCAATTGCTCTCC 3′
S166A/S186A (F)
5′ CGCCACAAAGCTGATAGTATTTCCC 3′
S166A/S186A (R)
5′GGGAAATACTATCAGCTTTGTGGCG3′
PCR was performed with a 2720 Thermo Cycler Applied Biosystem.
We thank Prof. Eric Gilson for kindly providing h-TERT immortalized firbroblasts, Prof. Nicola Zambrano for kindly providing HEK 293-LinX packaging cells, Prof. G. Condorelli for generously providing pCA-Akt plasmid, and Dr. Hua Lu for generously providing the MDM21–441 and MDM2Δ150–230 plasmids used in this study.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by grants awarded to GLM and PR from PRIN (Programmi di ricerca di Rilevante Interesse Nazionale) and AIRC (Associazione Italiana Ricerca sul Cancro). VdF received funding from MIUR-FIRB (Ministero Istruzione Università e Ricerca-Fondo per gli Investimenti della Ricerca di Base) (RBIN04J4J7). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 21998744PONE-D-11-0950910.1371/journal.pone.0026003Research ArticleBiologyComputational BiologyPopulation GeneticsGenetic PolymorphismEvolutionary BiologyPopulation GeneticsGenetic PolymorphismGeneticsPopulation GeneticsGenetic PolymorphismPopulation BiologyPopulation GeneticsGenetic PolymorphismMedicineDiagnostic MedicinePathologyGeneral PathologyBiomarkersGastroenterology and hepatologyLiver diseasesInfectious hepatitisHepatitis BGastrointestinal CancersInfectious diseasesViral diseasesHepatitisHepatitis BOncologyCancers and NeoplasmsGastrointestinal TumorsHepatocellular CarcinomaSurgeryGastrointestinal SurgerySurgical OncologyTransplant SurgeryGenetic Variations in Plasma Circulating DNA of HBV-Related Hepatocellular Carcinoma Patients Predict Recurrence after Liver Transplantation Circulating DNA Genotypes Predict HCC RecurrenceHu Jie
1
Wang Zheng
1
Fan Jia
1
2
3
Dai Zhi
1
He Yi-Feng
1
Qiu Shuang-Jian
1
Huang Xiao-Wu
1
Sun Jian
1
Xiao Yong-Sheng
1
Song Kang
1
Shi Ying-Hong
1
Sun Qi-Man
1
Yang Xin-Rong
1
Shi Guo-Ming
1
Yu Lei
1
Yang Guo-Huan
1
Ding Zhen-Bin
1
Gao Qiang
1
Tang Zhao-You
1
2
Zhou Jian
1
2
3
*
1
Liver Cancer Institute, Zhong Shan Hospital, Fudan University, Key Laboratory for Carcinogenesis and Cancer Invasion, the Chinese Ministry of Education, Shanghai Key Laboratory for Organ Transplantation, Shanghai, People's Republic of China
2
Institute of Biomedical Sciences, Fudan University, Shanghai, People's Republic of China
3
Shanghai Key Laboratory of Organ Transplantation, Zhongshan Hospital, Fudan University, Shanghi, People's Republic of China
Hoheisel Jörg D. EditorDeutsches Krebsforschungszentrum, Germany* E-mail: [email protected] and designed the experiments: JZ ZW JH. Performed the experiments: JH ZD YFH GHY ZBD GMS LY. Analyzed the data: ZW JH XRY QG ZYT. Contributed reagents/materials/analysis tools: JF SJQ XWH JS YSX KS YHS QMS. Wrote the paper: JH ZW JZ JF.
2011 5 10 2011 6 10 e2600330 5 2011 15 9 2011 Hu et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
Recurrence prediction of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) patients undergoing liver transplantation (LT) present a great challenge because of a lack of biomarkers. Genetic variations play an important role in tumor development and metastasis.
Methods
Oligonucleotide microarrays were used to evaluate the genetic characteristics of tumor DNA in 30 HBV-related HCC patients who were underwent LT. Recurrence-related single-nucleotide polymorphism were selected, and their prognostic value was assessed and validated in two independent cohorts of HCC patients (N = 102 and N = 77), using pretransplant plasma circulating DNA. Prognostic significance was assessed by Kaplan-Meier survival estimates and log-rank tests. Multivariate analyses were performed to evaluate prognosis-related factors.
Results
rs894151 and rs12438080 were significantly associated with recurrence (P = .003 and P = .004, respectively). Multivariate analyses demonstrated that the co-index of the 2 SNPs was an independent prognostic factor for recurrence (P = .040). Similar results were obtained in the third cohort (N = 77). Furthermore, for HCC patients (all the 3 cohorts) exceeding Milan criteria, the co-index was a prognostic factor for recurrence and survival (P<.001 and P = .002, respectively).
Conclusions
Our study demonstrated first that genetic variations of rs894151 and rs12438080 in pretransplant plasma circulating DNA are promising prognostic markers for tumor recurrence in HCC patients undergoing LT and identify a subgroup of patients who, despite having HCC exceeding Milan criteria, have a low risk of post-transplant recurrence.
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Introduction
Hepatocellular carcinoma (HCC) is a malignant tumor responsible for approximately 600,000–700,000 deaths worldwide and is becoming more prevalent not only in Southeast Asia and Africa, but also in western countries [1], [2]. Because of high infection rates with hepatitis B virus (HBV), 55% of world's HCC cases occur in China [3]. Despite improvements in surveillance and clinical treatment strategies, the prognosis of HCC remains dismal [4]. Tumor resection is the first treatment choice for early-stage HCC. However, recurrence and metastasis after curative resection remain the main factors that decrease survival [5]. Moreover, for patients with multifocal HCC or advanced cirrhosis, resection is not always feasible. Liver transplantation (LT) removes both tumors and underlying liver cirrhosis; therefore, it plays an important role in HCC management. However, the scarcity of liver donors has limited its clinical application. Thus, risk estimation of post-transplant tumor recurrence and metastasis is an essential element in selecting HCC patients for LT.
Hepatocarcinogenesis is characterized by accumulation of genetic alterations including chromosomal rearrangements, activation of oncogenes, and inactivation of tumor suppressor genes [6]. Our previous study demonstrated that genes favoring HCC metastasis progression are initiated relatively early in primary tumors [7]. Genomic information may provide better insights into HCC behavior and patient-directed therapy [8]–[11]. These findings compelled us to further investigate genetic variations associated with HCC recurrence and metastasis, which could be valuable for prediction of prognosis and selection of subsequent treatment. Single-nucleotide polymorphisms (SNPs) may represent the most common genetic variations in the human genome. As genetic markers, SNPs have several advantages over microsatellite sequence repeats, including abundance (one in every 750–1000 bp), stability, and suitability for high-throughput analysis [10], [12]. Microarray-based high-density SNP analysis makes a reproducible and rapid determination of genome-wide alterations possible. Some SNPs exhibit different allele frequencies between tumor and normal tissues [12]. Therefore, studies on SNPs genotyped from tumor DNA may help clarify the biological importance of genetic variations in the tumor genome. However, tumor tissue can only be obtained after surgery or from invasive biopsy.
Detection of biomarkers from peripheral blood obtained before surgery is more convenient and more applicable for prognosis prediction than from post-surgery tissue samples. Tumor-derived DNA has been detected in the plasma or serum of cancer patients [13]–[15]. A recently published study demonstrated that circulating DNA from tumor patients can be used to assess tumor dynamics [16], [17]. Thus, circulating DNA may be a good target to study, instead of DNA from tumor tissues, for its accessibility, simple manipulation, and available prognostic information before surgery [18]. Here, we detected genetic variations associated with prognosis in pretransplant plasma circulating DNA and evaluated the predictive value for post-transplant recurrence and metastasis in HBV-related HCC patients undergoing LT.
Materials and Methods
Ethics statement
The study protocol was approved by The Research Ethics Committee of Zhongshan Hospital, Fudan University. Informed written consent was obtained according to the Declaration of Helsinki.
Patients and specimens
The LT program of Zhongshan Hospital started in 2001. At the beginning, the indication of LT in our hospital was quite expanded (without macrovascular invasion, lymph node invasion and extrahepatic metastasis). Through the year 2007, we started to use Shanghai criteria for selecting HCC patients for LT (HCC patients with a solitary lesion < or = 9 cm in diameter, no more than three lesions with the largest < or = 5 cm, a total tumor diameter < or = 9 cm without macrovascular invasion, lymph node invasion and extrahepatic metastasis [19]). The inclusion criteria for this study were as follows: 1) HBV background and distinctive HCC diagnosis by pathology; 2) full follow-up and clinicopathological data; and 3) well-preserved formalin-fixed, paraffin-embedded (FFPE) tissues or plasma samples extracted before LT. 4) without macrovascular invasion, lymph node invasion and extrahepatic metastasis. Our previous study and other literature reported that mTOR-based immumosuppression (Rapamycin) may improve the overall survival of HCC patients after LT [20], [21]. Thus, the present study excluded patients treated with mTOR-based immumosuppression and only included those patients using FK-506 (tacrolimus). Two hundred and nine patients were included in this study. All patients were received deceased donor liver transplantation. After LT, patients were followed with regular surveillance for recurrence or metastasis by chest radiograph, abdominal ultrasonogram, computed tomographic (CT) and AFP level measurement every 2 months in the first year, and at least every 3–4 months thereafter. A diagnosis of recurrence was based on typical imaging appearanceon and/or an elevated AFP level. Among the 209 patients included in the study, 91 developed HCC recurrence and 86 patients died (68 patients died of HCC recurrence and 18 died of transplantation-related complications). We use Lamivudine (100 mg/day) or Entecavir (0.5 mg/day) in combination with hepatitis B immunoglobulin (HBIG) to prevent HBV reactivation after LT. All patients were given 2400 IU HBIG intravenously in LT, followed by posttransplant 400 IU HBIG i.m. tid. until serum HBsAg became negative, and the titer of HBsAb is more than 150 IU/l. Thereafter, lamivudine or entecavir continued to use in the lifetime, and HBIG dosage was adjusted to keep the titer of HBsAb >150 IU/l.
We designed our study referring to REMARK guidelines for reporting prognostic biomarkers in oncology [22]. The study design is shown in Figure 1. The clinicopathological characteristics of three cohorts of HCC patients are summarized in Table S1. Peripheral blood and FFPE tumor tissues were collected for DNA extraction (Supplemental Text S1).
10.1371/journal.pone.0026003.g001Figure 1 Study design.
Discovery Set consisted of 30 patients randomly selected from recurrence patients and non-recurrence patients. Training Set consisted of 102 consecutive patients from January 2004 to December 2006, while Validation Set comprised 77 consecutive patients from January 2007 to June 2008. Thirty tumor DNA samples randomly selected from the recurrence and non-recurrence groups were hybridized on SNP microarrays. Signatures associated with tumor recurrence were selected and validated in circulating DNA using two other independent patient cohorts.
Microarray hybridization and genotyping
Tumor DNA was extracted from 30 FFPE tumor tissues (Discovery Set). Hybridization was performed on Affymetrix GeneChip Human Mapping SNP6.0 using the Human mapping SNP6.0 assay kit (Affymetrix Inc., Santa Clara, CA, USA). Plasma circulating DNA was genotyped using the MassARRAY system (Sequenom Inc., San Diego, CA, USA) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (Supplemental Text S1).
Statistical analysis
The Cochran-Armitage trend test was used to assess alleles associated with recurrence after LT. Unsupervised hierarchical clustering analysis was performed using Cluster software (version 3.0) and TreeView software (version 1.0.13). The Spearman rank test and Fisher's exact test were used to evaluate clinicopathological correlations. We used time to recurrence as the primary endpoint. Overall survival and tumor-related death were used as the secondary endpoint. Time to recurrence (TTR), Overall survival (OS) and tumor-related death were analyzed using the Kaplan–Meier method and the log-rank test. OS was defined as the interval between LT and death or the last observation. The data were censored at the last follow-up for living patients. TTR was measured from the date of LT until the detection of recurrent tumor or the last follow-up assessment. The data were censored for patients without tumor recurrence. Tumor-related death was defined as time from operation to HCC-related death. Patients alive at the end of follow-up were censored. Multivariate analysis using Cox proportional hazards model was used to evaluate prognosis-related factors. Data were analyzed using the statistical package SPSS 16.0 (SPSS Inc., Chicago, IL, USA). A statistical significance was set at P<.05. Receiver operating characteristic (ROC) curve analysis was used to determine the predictive value for tumor recurrence. All microarray data were registered into NCBI's Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/projects/geo/). (Accession number GSE29667).
Results
Microarrays
Call rates <65% were often associated with highly degraded DNA from FFPE samples [23]. After strict quality control before hybridization, all 30 samples passed the call rate threshold of 65% (average, 74.4%; range, 72.1–78.1%) and were subjected to further analysis. Before analysis, we removed SNPs that had call rates <90%. After this filtration, 230,802 SNPs were included for further analysis. We used the Cochran-Armitage trend test to assess alleles associated with recurrence or metastasis after LT. We identified 1272 SNPs with P<.01, and among these, 30 SNPs demonstrated P<.001 (Figure S1C). Hierarchical clustering was performed based on the 1272 SNPs (Figure S2A) and the top 30 SNPs (Figure S2B). The top 30 SNPs, rather than the 1272 SNPs, could separate recurrence (high-risk group) from non-recurrence patients (low-risk group). The low-risk group had longer OS and TTR than the high-risk group (Figure S2C and 2D).
Genotyping of plasma circulating DNA
To study the concordance between plasma circulating DNA and FFPE tumor DNA, we genotyped the top 30 SNPs in plasma circulating DNA from Discovery Set using the MassARRAY system. rs8059833 failed in PCR amplification and rs860056 was excluded because of low call rate (<90%) and finally we successfully genotyped 28 SNPs in plasma circulating DNA. Our results revealed that plasma circulating DNA and FFPE tumor DNA have a high concordance (98.2%). Plasma circulating DNA extracted from 102 patients in Training Set was genotyped for the 28 SNPs. As shown in Table S2, we calculated the allele frequencies between the recurrence and non-recurrence groups using Haploview software (vesion 4.1). We found that the minor alleles at rs894151 and rs12438080 were significantly associated with recurrence and metastasis in HCC patients after LT (P = .033 and P = .001, respectively. Table S2).
Clinicopathological characteristics and correlation with rs894151 and rs12438080
The two-year and four-year survival rates for Training Set (N = 102) were 58.6%, 48.5%, and the two-year and four-year recurrence rates were 36.3%, 45.1%, respectively. The genotype frequencies at rs894151 were 67.6% (69/102) for AA, 29.4% (30/102) for AG, and 2.9% (3/102) for GG. The genotype frequencies at rs12438080 were 47.1% (48/102) for AA, 47.1% (48/102) for AC, and 5.9% (6/102) for CC. Because of the low frequency of the minor allele homozygote at rs894151 and rs12438080 (3/102 and 6/102, respectively), we combined the heterozygote and minor allele homozygote patients as one group. We divided patients into two groups by rs894151 (AA and AG/GG). Similarly, patients were divided into two groups by rs12438080 (AA and AC/CC). We found that patients with AG/GG at rs894151 have larger tumor sizes, increased tumor number and higher probability of microvessel invasion (P = .027, P = .012 and P = .038, respectively), while younger age and worse tumor differentiation were associated with AC/CC at rs12438080 (P = .010 and P = .020, respectively; Table S3).
Prognostic significance of rs894151 and rs12438080
Univariate analysis revealed that patients with AG/GG at rs894151 or AC/CC at rs12438080 were significantly associated with a decreased OS (P = .011and P = .038, respectively; Figure 2G and 2H) and TTR (P = .003 and P = .004, respectively; Figure 2D and 2E). Patients with more minor alleles showed shorter TTR and OS (Figure S3, Table S4 and S5). Other unfavorable predictors for OS were pre-LT treatment, tumor size>5 cm, multiple tumor nodes, and microvessel invasion (P = .050, P<.001, P = .003, and P<.001, respectively). Tumor size, tumor number, tumor encapsulation, and microvessel invasion also had prognostic significance for TTR (P<.001, P = .015, P = .011, and P<.001, respectively; Table 1). Multivariate analyses demonstrated that rs12438080 was an independent prognostic factor for TTR (P = .042) rather than OS, while rs894151 was not an independent prognostic factor for TTR or OS (P = .382 and P = .935, respectively; Table 1).
10.1371/journal.pone.0026003.g002Figure 2 Prognostic significance assessed using Kaplan–Meier survival estimates and log-rank tests stratified by rs894151 (D, G), rs12438080 (E, H), and the co-index of the two factors (F, I).
A, B, and C show the frequency distributions of genotypes in the non-recurrence and recurrence groups with the p-value calculated by chi-square test. *B represents a minor allele. Patients were divided into two groups by the co-index: patients with AA at both rs894151/rs12438080 and patients with minor allele(s).
10.1371/journal.pone.0026003.t001Table 1 Univariate and Multivariate analyses of factors associated with survival and recurrence in Training Set.
OS TTR
Univariate Multivariate Univariate Multivariate
Factors HR (95% CI)
P
HR (95% CI)
P
HR (95% CI)
P
HR (95% CI)
P
Gender (male versus female) 0.669(0.241–1.855) .440 NA 1.185(0.287–4.891) .814 NA
Age (≥50 years versus <50 years) 0.870(0.507–1.491) .612 NA 0.757(0.423–1.354) .348 NA
Child-Pugh (B/C versus A) 1.291(0.725–2.299) .385 NA 0.791(0.440–1.423) .434 NA
Differentiation (III/IV versus I/II) 1.435(0.836–2.463) .190 NA 1.571(0.879–2.808) .127 NA
AFP (≥200 ng/ml versus <200 ng/ml) 0.914(0.529–1.578) .746 NA 1.086 (0.608–1.941) .780 NA
Encapsulation (none versus complete) 1.331(0.774–2.286) .301 NA 2.183(1.199–3.976) .011 1.527(0.827–2.819) .176
Pre-LT treatment (yes versus no) 1.722(1.001–2.962) .050 1.438(0.820–2.524) .205 1.431(0.793–2.580) .234 NA
Tumor size (>5 cm versus ≤5 cm) 3.369(1.821–6.234) <.001 2.078(1.025–4.214) .042 4.236(2.138–8.394) <.001 2.998(1.334–6.733) .008
Tumor number (multiple versus single) 2.439(1.352–4.401) .003 1.566(0.800–3.065) .190 2.150(1.159–3.988) .015 1.131(0.538–2.379) .746
Microvessel invasion (yes versus no) 3.495(1.862–6.562) <.001 2.219(1.088–4.525) .028 4.831(2.384–9.787) <.001 2.843(1.341–6.027) .006
rs894151 (AG/GG versus AA) 2.062(1.182–3.596) .011 1.026(0.549–1.920) .935 2.483(1.374–4.486) .003 1.333(0.700–2.538) .382
rs12438080 (AC/CC versus AA) 1.805(1.034–3.150) .038 1.425(1.798–2.546) .231 2.560(1.360–4.819) .004 1.951(1.025–3.716) .042
rs894151/rs12438080 (AB/BB
§ versus AA) 3.357(1.577–7.146) .002 2.032(0.878–4.704) .098 5.137(2.022–13.050) .001 2.791(1.048–7.436) .040
Abbreviations: OS, overall survival; TTR, time to recurrence; HR, hazard ratio; CI, confidence interval; LT, liver transplantation; AFP, alpha-fetoprotein; NA, not adopted.
The prognostic significance was assessed using Kaplan-Meier survival estimates and log-rank tests. In multivariate analysis variables were adopted for their prognostic significance by univariate analysis with enter-stepwise selection (p<.05) and the co-index of rs894151/rs12438080 was analyzed when both SNPs were excluded. §AB/BB represents patients with minor allele(s) at rs894151 and/or rs12438080. Pre-LT treatment, Tumor size, Tumor number, Microvessel invasion and rs894151/rs12438080 were adopted for Cox analysis for OS. While, Encapsulation, Tumor size, Tumor number, Microvessel invasion and rs894151/rs12438080 were adopted for Cox analysis for TTR.
Chi-square tests revealed no correlation between rs894151 and rs12438080 (P = .469). To increase predicting power, we combined the two SNPs as a co-index. We divided patients into two groups using the combined index of rs894151 and rs12438080 (group I: patients with genotype AA at both rs894151 and rs12438080; group II: patients with allele G at rs894151 and/or C at rs12438080). Multivariate analyses demonstrated that the co-index (rs894151/rs12438080), tumor size, and microvessel invasion were independent prognostic factors for TTR (P = .040, P = .008, and P = .006, respectively), while tumor size and microvessel invasion were independent prognostic factors for OS (P = .042 and P = .028, respectively; Table 1).
Validation
We validated the prognostic value of the co-index of rs894151 and rs12438080 in another independent cohort of 77 HBV-related HCC patients who undergoing LT (Validation Set) with results similar to those in Training Set. Patients with genotype AA at both rs894151 and rs12438080 had a longer TTR than patients with allele G at rs894151 and/or C at rs12438080. In multivariate Cox proportional hazards analyses, the co-index was still an independent predictor of TTR (P = .001; Table S6).
ROC analysis
ROC curve analysis were performed (N = 209) to evaluate the predictive power for recurrence. It showed that the predictive power of the co-index [area under the curve (AUC) = 0.788] was significantly higher than that of rs894151 (AUC = 0.683, P = 0.033), rs12438080 (AUC = 0.679, P = 0.027), tumor number (AUC = 0.614, P<0.001) and encapsulation (AUC = 0.583, P<0.001). Tumor size (AUC = 0.699) and microvessel invasion (AUC = 0.717) also had lower AUC when compared with the co-index, but the differences were not reach statistical significance (P = 0.066 and P = 0.136 respectively, Figure 3 and Table S7).
10.1371/journal.pone.0026003.g003Figure 3 The predictive ability of the co-index of rs894151 and rs12438080 compared with single markers and other clinical prognostic parameters by receiver operating characteristic (ROC) curves (A).
The areas under the curve (AUCs) with 95% CI are shown in B (*p<.05, compared with the co-index). The details for AUC and 95% CI are also shown in Table S7.
Milan criteria and the co-index of rs894151 and rs12438080
Among the 209 patients of the three cohorts, 94 met and 115 exceeded Milan criteria [24]. The Clinicopathological characteristics of patients exceeding Milan criteria were summarized in table S8. The three-year recurrent rates for patients within Milan criteria and exceeding Milan criteria were 12.3% and 67.3%. The survival rates were 83.8% and 40.6% respectively. We stratified the patients as within or beyond Milan criteria to evaluate the prognostic value of the co-index (rs894151/rs12438080). The log-rank test indicated that the co-index can be a novel predictor of recurrence regardless of Milan criteria (Figure 4A and 4D; Table 2). The co-index of the two SNPs (rs894151/rs12438080) in pretransplant plasma circulating DNA identified a subgroup of HCC patients with a low risk of post-transplant recurrence, despite having HCC beyond Milan criteria (Figure 4E). We also performed analysis of the co-index in patients within or exceeding UCSF criteria (Supplemental Text S1). The log-rank test indicated that, in patients exceeding UCSF criteria, the co-index of two SNPs was also associated with TTR and OS (Table S9). The results were similar to those in patients within UCSF (Table S10).
10.1371/journal.pone.0026003.g004Figure 4 The relationship of the co-index (rs894151/rs12438080) in patients within Milan criteria (upper panel) and exceeding Milan criteria (lower panel).
Time to recurrence of the co-index (rs894151/rs12438080) in patients within Milan criteria (A) and exceeding Milan criteria (D). The prognostic significance was assessed using Kaplan-Meier survival estimates and log-rank tests. B, E, The recurrences in patients with AA at both SNPs were 0/44 (within Milan criteria) and 8/26 (exceeding Milan criteria). In patients with a minor allele, the recurrences were 16/50 (within Milan criteria) and 67/89 (exceeding Milan criteria). C, F, Frequency distributions of genotypes in non-recurrence and recurrence patients. The p-value was calculated by chi-square test.
10.1371/journal.pone.0026003.t002Table 2 Kaplan-Meier survival estimates and log-rank tests of factors associated with TTR and OS in patients exceeding Milan criteria.
Factors TTR OS
HR (95% CI) P
HR (95% CI)
P
Pre-LT treatment (yes versus no) 1.016 (0.646–1.598) .946 1.083 (0.675–1.737) .741
Child-Pugh (B/C versus A) 1.178 (0.774–1.864) .485 0.175(0.726–1.900) .512
AFP (≥200 ng/ml versus <200 ng/ml) 1.134 (0.721–1.784) .587 1.070(0.668–1.717) .777
Differentiation (III/IV versus I/II) 1.245 (0.789–1.963) .346 1.454(0.906–2.333) .121
Encapsulation (none versus complete) 1.336 (0.844–2.115) .216 0.817(0.510–1.311) .403
Microvessel invasion (yes versus no) 2.975 (1.786–4.954) <.001 2.916(1.679–5.064) <.001
rs894151/rs12438080 (AB/BB
§ versus AA) 4.238 (2.021–8.886) <.001 3.244(1.546–6.805) .002
§ AB/BB represents patients with minor allele at rs894151 and/or rs12438080. Abbreviations: TTR, time to recurrence; CI, confidence interval; LT, liver transplantation; AFP, alpha-fetoprotein.
Discussion
As a radical treatment for HCC, LT has many advantages over resection such as simultaneously curing HCC and the underlying cirrhosis in a single surgery. However, tumor recurrence and metastasis after LT remain the main obstacles for long-term survival. To achieve a low recurrence rate and reasonable distribution of limited donors, meticulous evaluation of the prognosis in recipients is crucial [25]. However, conventional prognostic factors for HCC patients are limited to clinicopathological parameters. Including Milan criteria, most LT indications for HCC patients focus on the tumor size, number of tumor nodules, and the absence of macroscopic vascular invasion or lymph nodes [24], [26], [27]. Efforts to identify more accurate prediction algorithms have revealed that tumor size and number are imperfect surrogates for predicting the metastatic potential of HCC [28]. Increasingly, more attention is being given to biological tumor markers for insights into HCC behavior. Studies on epigenetics [29]–[31], gene expression [32]–[35], and proteomics [36], [37] are beginning to yield potentially useful information. Schwartz et al [28] reported that analysis of allelic imbalance (AI) of nine microsatellites may extend Milan criteria without increasing tumor recurrence after LT. Wu et al [38] found Histone Deacetylase 3 could serve as a biomarker for tumor recurrence following LT in HBV-Associated HCC. Our previous study demonstrated that overexpression of Capn4 in HCC tissues was associated with tumor invasion and metastasis in HCC patients after LT [39]. In these studies, all the samples used were obtained from tumor tissues that were only available after surgery. Therefore, prognosis biomarker studies in preoperative plasma or serum are urgently needed.
A small amount of circulating DNA can be detected in the plasma of healthy individuals. The levels of circulating DNA are elevated in cancer patients and are associated with poor prognosis [18], [40], [41]. Many studies suggested that the elevated circulating DNA of cancer patients was from apoptotic and necrotic tumor cells [18], [42]. Our previous study showed that circulating DNA extracted from the plasma of HCC patients displayed neoplastic characteristics [43]. Diehl et al [17] explored a new technology called BEAMing (beads, emulsion, amplification, and magnetics) to detect colorectal cancer-related genetic variations in circulating DNA and found that the genetic alterations could be used to monitor tumor dynamics in colorectal cancer patients undergoing surgery or chemotherapy. In this study, we tried to screen genetic variations in pretransplant plasma circulating DNA to identify promising biomarkers that are associated with tumor recurrence after LT. First, we used plasma circulating DNA for microarray hybridization, but the concentration and quantity did not meet the QC required for microarrays. Whole-genome amplification (WGA) offers new possibilities for genetic studies where limited DNA samples have been collected. We succeeded in harvesting sufficient DNA though WGA. However, the amplified plasma circulating DNA generated poor-quality array data, yielding a result similar to that in a previous report [44]. Therefore, we used FFPE tumor DNA for chip hybridization, then validated candidate SNPs in plasma circulating DNA using MALDI-TOF mass spectrometry. High concordance (98.2%) between FFPE tumor DNA and plasma circulating DNA was confirmed by our result.
We identified two novel SNPs (rs894151 and rs12438080) located in 8q22 and 15q26 from plasma circulating DNA that were associated with HCC recurrence after LT and validated using another independent cohort of patients. The TTR was negatively associated with the number of minor alleles at rs894151 and rs12438080 (G at rs894151 and C at rs12438080). However, HCC is a polygenic, complex disease caused by the interaction of many genetic and environmental factors [45]. Variations in any one gene in the polygenic pathway may have a small effect on tumor progression. Therefore, we used the co-index—a combination of the two SNPs (rs894151 and rs12438080)—to increase the predictive power of SNPs. Multivariate analyses demonstrated that the co-index was an independent prognostic factor for recurrence. ROC analysis also showed that the predictive power of the co-index was more robust than that of any single SNP. To our knowledge, the present study is the first one to evaluate the prognostic value of genetic variations in pretransplant plasma circulating DNA in HCC patients undergoing LT.
The co-index of rs894151and rs12438080 was an independent prognostic factor for TTR (P = .040) but not for OS (P = .098), which may be attributed to the complexity of underlying factors for post-transplant survival. Besides HCC recurrence, other long-term problems such as immunosuppression-related and technique-related complications, as well as organ rejection, are also important prognostic factors which may cause mortality. In order to decrease the effect of these factors on survival, we used the endpoint of tumor-related death for further analysis. We found that the co-index of rs894151 and rs12438080 was an independent prognostic factor for tumor-related death (Table S11).
Previous studies reported several recurrence-associated factors for HCC patients. Nucleotide analogs may reduce the risk of HBV-related HCC development and prevent recurrence after resection [46], [47]. Active replication of HBV may initiate malignant transformation through a direct carcinogenic mechanism by increasing the probability of viral DNA insertion in or near proto-oncogenes, tumor-suppressor genes [48], [49]. However, new tumors developing from noncirrhosis liver graft need relatively long period. The main aim of administration of nucleoside analogues was to prevent function loss of liver graft, which was caused by HBV reactivation. Further studies that include a larger number of patients and a longer follow-up period are necessary to assess the benefits of nucleoside analogues for preventing long-term HCC recurrence after LT. Des-gamma-carboxyprothrombin (DCP), also known as the protein induced by vitamin K absence or antagonist II (PIVKA-II), is reported to be an effective tumor marker for HCC. This tumor marker is currently confirmed in Japan, Korea and Indonesia, while many studies have been performed worldwide. Fujiki et al [50] reported superiority of DCP and AFP over preoperative tumor size or number for predicting recurrence after living donor liver transplantation. Unfortunately, DCP detection is unavailable yet in our hospital. This restricted our ability to get the pre-LT DCP data of the studied patients. The prognostic value of the two SNPs in combination with DCP will be the next phase of our study.
Milan criteria have been accepted worldwide and the outcomes of HCC patients within Milan criteria were similar to that of patients undergoing LT without HCC. However, the dichotomous yes/no nature of Milan criteria has been challenged for being too strict [27], [51], [52], some patients with tumors exceeding Milan criteria are also potentially curable by LT [26]–[28], [51], [52]. In the present study, for those patients exceeding Milan criteria, we found the co-index of rs894151 and rs12438080 also acted as a prognostic factor for recurrence (P<.001; Figure 4D and Table 2). The co-index may be used as a tool to identify patients with AA at both rs894151 and rs12438080 who, despite having HCC beyond Milan criteria, have an acceptable outcome profile (Figure 4E). Therefore, incorporation of tumor genetic variations into selection algorithms for LT candidates is a promising way to extend Milan criteria and allow more HCC patients to benefit from LT.
SNP rs894151 is intergenic between the pleckstrin homology domain-containing family F member 2 (PLEKHF2) and chromosome 8 open reading frame 37 (C8orf37) in chromosome 8q22. PLEKHF2 encodes an endoplasmic reticulum (ER)-associated protein. Overexpression of PLEKHF2 enhances tumor necrosis factor (TNF)-α-induced cellular apoptosis through an ER-mitochondrial apoptotic pathway [53]. In 15q26, rs12438080 lies on the longevity assurance homolog 3 (LASS3) gene, which is evolutionarily conserved from yeast to mammals [54] and encodes a ceramide synthase. 8q22 was reported to be associated with elevated expression of the metastasis gene metadherin (MTDH) with poor clinical outcomes in breast cancer [55]. Amplification of 15q26 in gastric cancer cell lines was associated with an up-regulated negative regulator of cell-cell adhesion [56]. Genome copy-number alteration and loss of heterozygosity in 15q26 were found in pediatric malignant astrocytomas [57] and breast cancer [58]. We genotyped healthy individuals and HBV infected patients to evaluate the distribution of genotypes on the two loci. We found the co-index of the two loci in either healthy group or HBV group showed significant difference from that in HCC group (Supplemental Text S1). Whereas, there was no difference between healthy group and HBV group. This result suggested that the two variations may result from acquired somatic mutations accumulating in the tumor genome.
Patients with AG/GG at rs894151 have larger tumor sizes, increased tumor number and higher probability of microvessel invasion, while worse tumor differentiation was associated with AC/CC at rs12438080. The genetic variations of the two loci seem to represent the different features of the progression of HCC. Minor allele in the two loci may result in more aggressive tumor biologic behavior via interactions with neighboring tumor associated genes. Through this biologic change, HCC patients may get poorer prognosis after LT. However, the precise mechanism by which rs894151 and rs12438080 are associated with HCC recurrence and metastasis remains to be clarified and further investigated.
Like most studies focusing on organ transplantation, the sample size of this study is not very large, which made us to adopt a less stringent statistical cut-off for microarray analysis. And the sub-group analyses also came across this problem. This limitation predispose to a risk of false positive associations. Therefore, our result need to be further validated using a sufficiently large independent cohort from different transplantation centers. On the other hand, since the primary etiology of HCC in China is HBV infection, we do not have enough patients to be included in the study at that time. So, our study focused on HBV-related HCC. The prognostic value of the variation of the two loci shall be further evaluated for its clinical value across heterogeneous HCC patients, such as HCV-related or alcohol-related HCC, and Nonalcoholic steatohepatitis-related HCC.
In conclusion, the present study suggests that HBV-related HCC patients with allele G at rs894151 and/or C at rs12438080 in pretransplant plasma circulating DNA may bear an increased risk for HCC recurrence after LT. In contrast, patients with AA at both rs894151 and rs12438080 may experience a low recurrence risk, even in HCC patients exceeding Milan criteria. Genetic variations of rs894151 and rs12438080 in pretransplant plasma circulating DNA from HCC patients may predict recurrence after LT.
Supporting Information
Figure S1 Pre-array quality control and distribution of genome-wide p-values. Pre-array quality control using Mapping PCR test, PCR products were visualized on 2% agarose gels. Sty and Nsp represent different restriction enzyme digestions. Samples with fragments >750 bp indicate good quality for microarray hybridization. A, samples passing PCR-based QC tests. B, sample 11203 failed the QC test. C, genome-wide p-values of the Cochran-Armitage trend test. After filtration, 230,802 SNPs were included for analysis. The chromosomal distribution of P-values from the trend test is shown. The blue horizontal line represents a threshold of 10−3 for suggestive significance: 30 SNPs were above the horizontal line. The inset shows the quantile-quantile (Q-Q) plots of the observed p-values for association.
(TIF)
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Figure S2 Hierarchical clustering of selected SNPs. Each SNP value was assigned based on the number of minor alleles. A, Hierarchical clustering of 1272 SNPs with P<.01. Each row represents an individual SNP and each column represents an individual tumor sample. B, Hierarchical clustering of 30 SNPs with P<.001. Each row represents an individual tumor sample and each column represents an individual SNP. Patients can be separated into two groups (high-risk or low-risk groups) using 30 top SNPs. The high-risk group had a shorter overall survival time (C) and time to recurrence (D) than the low-risk group (P<.001).
(TIF)
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Figure S3 Kaplan-Meier analysis for recurrence (A) and survival (B) of patients harboring different numbers of risk alleles. Patients possessing more risk alleles had a higher recurrent risk and shorter survival time. The prognostic significance was assessed using Kaplan-Meier survival estimates and log-rank tests.
(TIF)
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Table S1 Clinicopathological characteristics of three cohorts of HCC patients.
(DOC)
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Table S2 Differences in allele frequencies between recurrence and non-recurrence groups in Training Set 2 (N = 102).
(DOC)
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Table S3 Correlation between rs894151, rs1248080, and clinicopathological characteristics in Training Set (N = 102).
(DOC)
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Table S4 Pairwise comparisons of TTR between patients with 0, 1, 2, and 3 risk alleles.
(DOC)
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Table S5 Pairwise comparisons of OS between patients with 0, 1, 2, and 3 risk alleles.
(DOC)
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Table S6 Univariate and multivariate analyses of factors associated with TTR in Validation Set (N = 77).
(DOC)
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Table S7 AUC of the co-index and other factors.
(DOC)
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Table S8 Clinicopathological characteristics of HCC patients beyond Milan criteria.
(DOC)
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Table S9 Kaplan-Meier survival estimates and log-rank tests of factors associated with TTR and OS in patients exceeding UCSF criteria.
(DOC)
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Table S10 Kaplan-Meier survival estimates and log-rank tests of factors associated with TTR and OS in patients within UCSF criteria.
(DOC)
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Table S11 Univariate and Multivariate analyses of factors associated with tumor-related death in Training Set.
(DOC)
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Text S1 Supporting information for methods and analysis.
(DOC)
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Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was funded by grants from National Natural Science Foundation of China (No. 30972949 and No. 30700815, http://www.nsfc.gov.cn/), Shanghai Key-Tech Research and Development Program (No. 09411951700, www.stcsm.gov.cn), Program for Shanghai Excellent Subject Leaders (No. 10XD1401200, www.stcsm.gov.cn), Leading Academic Discipline Program, 211 Project for Fudan University (the 3rd phase, No. 211XK21, http://www.fudan.edu.cn/) and Research Fund for the Doctoral Program of Higher Education of China (No. 20090071110022, http://www.moe.edu.cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Mol CancerMolecular Cancer1476-4598BioMed Central 1476-4598-10-1152192974510.1186/1476-4598-10-115ResearchDifferential modulatory effects of GSK-3β and HDM2 on sorafenib-induced AIF nuclear translocation (programmed necrosis) in melanoma Liu Qingjun [email protected] James W [email protected] David J [email protected] Division of Hematology-Oncology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA2 Division of Urology, Beijing Friendship Hospital, Capital Medical University, Beijing, China2011 19 9 2011 10 115 115 5 8 2011 19 9 2011 Copyright ©2011 Liu et al; licensee BioMed Central Ltd.2011Liu et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
GSK-3β phosphorylates numerous substrates that govern cell survival. It phosphorylates p53, for example, and induces its nuclear export, HDM2-dependent ubiquitination, and proteasomal degradation. GSK-3β can either enhance or inhibit programmed cell death, depending on the nature of the pro-apoptotic stimulus. We previously showed that the multikinase inhibitor sorafenib activated GSK-3β and that this activation attenuated the cytotoxic effects of the drug in various BRAF-mutant melanoma cell lines. In this report, we describe the results of studies exploring the effects of GSK-3β on the cytotoxicity and antitumor activity of sorafenib combined with the HDM2 antagonist MI-319.
Results
MI-319 alone increased p53 levels and p53-dependent gene expression in melanoma cells but did not induce programmed cell death. Its cytotoxicity, however, was augmented in some melanoma cell lines by the addition of sorafenib. In responsive cell lines, the MI-319/sorafenib combination induced the disappearance of p53 from the nucleus, the down modulation of Bcl-2 and Bcl-xL, the translocation of p53 to the mitochondria and that of AIF to the nuclei. These events were all GSK-3β-dependent in that they were blocked with a GSK-3β shRNA and facilitated in otherwise unresponsive melanoma cell lines by the introduction of a constitutively active form of the kinase (GSK-3β-S9A). These modulatory effects of GSK-3β on the activities of the sorafenib/MI-319 combination were the exact reverse of its effects on the activities of sorafenib alone, which induced the down modulation of Bcl-2 and Bcl-xL and the nuclear translocation of AIF only in cells in which GSK-3β activity was either down modulated or constitutively low. In A375 xenografts, the antitumor effects of sorafenib and MI-319 were additive and associated with the down modulation of Bcl-2 and Bcl-xL, the nuclear translocation of AIF, and increased suppression of tumor angiogenesis.
Conclusions
Our data demonstrate a complex partnership between GSK-3β and HDM2 in the regulation of p53 function in the nucleus and mitochondria. The data suggest that the ability of sorafenib to activate GSK-3β and alter the intracellular distribution of p53 may be exploitable as an adjunct to agents that prevent the HDM2-dependent degradation of p53 in the treatment of melanoma.
SorafenibMI-319HDM2p53GSK-3βApoptosis-Inducing Factor (AIF)apoptosisBcl-2
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Background
Glycogen synthase kinase-3β (GSK-3β) is a constitutively active kinase regulated primarily by an inhibitory phosphorylation at Ser9 [1] and activated by endoplasmic reticular (ER) and other forms of cellular stress [2,3]. The enzyme has a variable modulatory effect on the response to apoptotic stimuli in that it can either enhance or suppress apoptosis depending on the nature of the stimulus [4]. GSK-3β activation, for example, generally inhibits apoptosis triggered by the engagement of death receptors [4,5] but enhances the apoptotic response to death signals originating in the mitochondria [4,6]. GSK-3β activates NF- κB [7] and phosphorylates hexokinase II, facilitating its association with VDAC [8] in the outer mitochondrial membrane, both of which would be expected to promote cell survival. On the other hand, it phosphorylates c-myc, β-catenin, and numerous other survival-associated proteins leading to their degradation in the proteasome [9,10], thereby facilitating programmed cell death.
Among the downstream targets of GSK-3β are the tumor suppressor p53 and its negative regulator, the E3 ligase HDM2 [2,3,11]. The interaction between these two proteins is governed largely by the extent to which they are phosphorylated by upstream kinases. The phosphorylation of p53 on any of several serines in its N-terminal region, for example, prevents its interaction with HDM2 and enhances its stability in response to stress such as DNA damage or hypoxia [11-15]. N-terminal phophorylation also enhances the acetylation of p53 by the acetyl transferases p300/CBP and PCAF, which facilitates sequence-specific DNA binding by p53 as well as p53-dependent transcription [16]. JNK, p38, ATM and ATR are among the kinases that phosphorylate p53 in this region and promote its activity [11]. The C-terminal phosphorylation of p53 by GSK-3β at Ser315 and Ser376, on the other hand, directs the export of p53 from the nucleus and its subsequent degradation in the proteasome [2,17,18]. GSK-3β also phosphorylates HDM2, enhancing its ability to bind and ubiquitinate p53 [8,19]. It is likely that these destabilizing effects on p53 contribute to the prosurvival agenda of GSK-3β in some circumstances.
p53 mediates cell cycle arrest, senescence, and/or programmed cell death in response to DNA damage, hypoxia, and other cellular stresses [20,21]. Although many of these effects of p53 are attributable to its ability to promote gene expression, several are due to the expression of non-coding RNAs or to transcriptional repression. Although p53 resides primarily in the nucleus, there is a substantial cytosolic pool of p53 that in response to an apoptotic stimulus, translocates to the mitochondria, binds to Bax and Bak directly, and induces programmed cell death in a manner similar to that mediated by certain BH3-only members of the Bcl-2 family (i.e. Bim, tBid, and Puma)[22-28]. This particular function of p53 can trigger the release of cytochrome c from the mitochondria, the activation of caspases, and death through a classical apoptotic mechanism. It can also induce a caspase-independent form of death mediated by the translocation of Apoptosis-Inducing Factor (AIF) from the mitochondria to the nuclei. Once in the nucleus, AIF associates with histone H2AX and recruits nucleases such as CypA or EndoG, resulting in the cleavage of DNA into high molecular weight fragments (i.e. programmed necrosis, necroptosis)[29-31]. Both of these mechanisms of programmed cell death are independent of p53-dependent gene expression.
Recently, several small molecule antagonists of HDM2 have been developed which interfere with the interaction between p53 and HDM2, resulting in enhanced p53 stability. Most of these small molecule inhibitors (e.g. the Nutlins, MI-319, and TDP665759) target HDM2 [32-36] whereas others (e.g. RITA) bind to p53 itself [9,37,38]. Both classes of drug increase p53 levels and p53-dependent gene expression without damaging the genome. In the absence of HDM2 blockade, GSK-3β activation (in response to ER stress, for example) leads to the nuclear export of p53 and its subsequent degradation in the proteasome [2,3]. In the setting of HDM2 blockade, however, the p53 exported from the nucleus in response to GSK-3β activation remains available for translocation to the mitochondria in response to apoptotic signaling. Its pro-apoptotic function in the mitochondria is further enhanced by its physical association with GSK-3β [39]. The ability of HDM2 inhibitors to prevent the degradation of p53 that usually follows its nuclear export and the ability of GSK-3β to facilitate the redistribution and mitochondrial function of p53 suggest that combining an HDM2 antagonist with an agent that activates GSK-3β might be a particularly useful antitumor strategy.
We previously demonstrated a high degree of variability in the extent of GSK-3β-Ser9 phosphorylation among BRAFV600E (+) melanoma cell lines and showed that GSK-3β activity in these cells was increased in response to the multikinase inhibitor sorafenib [40], presumably through an ER stress-dependent mechanism. This GSK-3β activation blocked the down modulation of Bcl-2 and Bcl-xL and the nuclear translocation of AIF otherwise induced by sorafenib and limited the toxicity of the drug. In this report, we show that in the presence of the HDM2 antagonist MI-319, sorafenib induces the disappearance of p53 from the nucleus and its translocation to the mitochondria in melanoma cells. Both of these effects are GSK-3β-dependent. Although MI-319 alone is minimally toxic in melanoma cells as a single agent, it amplifies the toxicity of sorafenib. The cell death elicited by the combination of sorafenib and MI-319 can be inhibited by pifithrin-μ, an agent known to selectively block p53 function in the mitochondria without affecting p53-dependent gene expression [41]. We further show that, in contrast to the suppressive effect of GSK-3β on the down modulation of Bcl-2 and Bcl-xL and the nuclear translocation of AIF induced by sorafenib alone, the ability of the sorafenib/MI-319 combination to induce these effects requires the participation of GSK-3β.
The nuclear accumulation of p53 induced by MI-319 alone appears to be well-tolerated by melanoma cells both in vitro and in vivo. The multikinase inhibitor sorafenib has been extensively evaluated in melanoma patients both as a single agent and in combination with chemotherapy with disappointing results [42,43]. Our data suggest that the ability of sorafenib to activate GSK-3β and alter the intracellular redistribution of p53 may be exploitable as an adjunct to HDM2 blockade in the treatment of melanoma.
Results
Effects of sorafenib on MI-319-induced cytotoxicity and p53-dependent gene expression
To assess the effect of sorafenib on MI-319-induced cytotoxicity, A375 and SKMEL5 melanoma cells were exposed to various concentrations of MI-319 and sorafenib for 20 hr, stained with PI, and then analyzed for viability by flow cytometry. The interaction between the two drugs was evaluated in two studies. In the first, A375 and SKMEL5 cells were exposed to increasing concentrations of MI-319 in the presence (black bars ■) or absence (white bars □) of 10 μM sorafenib (Figure 1A) and in the second, the cells were exposed to increasing concentrations of sorafenib in the presence (black bars ■) or absence (white bars □) of 10 μM MI-319 (Figure 1B). As shown in Figure 1A, MI-319 had negligible single agent toxicity for A375 cells and only modest toxicity for SKMEL5 cells, even at the highest concentration tested (20 μM). However, in the presence of 10 μM sorafenib, MI-319 induced a concentration-dependent increase in PI staining in A375 cells (p < 0.0003, < 0.0023, and < 0.0023 for the drug combination vs. 20 μM MI-319 alone, 10 μM sorafenib alone, and 20 μM sorafenib alone, respectively). SKMEL5 cells were much more sensitive than A375 cells to single agent sorafenib but were unaffected by single agent MI-319. Furthermore, the toxicity of sorafenib in these cells was not appreciably augmented by the addition of MI-319. As shown in Figure 1B, the toxicity of single agent sorafenib was concentration-dependent for both cell lines and in the case of A375 cells, augmented by 10 μM MI-319. MI-319 had no such enhancing effect on the toxicity of sorafenib in SKMEL5 cells.
Figure 1 (A and B). Induction of apoptosis in A375 and SKMEL5 cell lines treated with (A) variable concentrations of MI-319 with (black bars) or without (white bars □) 10 μM sorafenib or with (B) variable concentrations of sorafenib with (black bars) or without (white bars □) 20 μM MI-319. Data are presented as the percentage of total cells staining positive for propidium iodide. (C and D). p53 and p21waf levels in A375 and SKMEL5 cell lines treated with (C) variable concentrations of MI-319 with or without 10 μM sorafenib or with (D) variable concentrations of sorafenib with or without 20 μM MI-319. (E). p53-luciferase reporter activity in transfected A375 and SKMEL5 cells treated with 10 μM sorafenib (S) and/or 20 μM MI-319 (M) for 6 hours. Data are presented as fold increase relative to the activity in the untreated control cells.
To assess the effects of sorafenib on MI-319-induced p53 accumulation and p53-dependent gene expression, A375 and SKMEL5 cells were exposed to increasing concentrations of MI-319 in the presence or absence of sorafenib. As shown in Figure 1C, MI-319 increased p53 levels in A375 and SKMEL5 melanoma cells in a concentration-dependent manner. The expression of the cdk inhibitor p21waf was also induced by the drug. The addition of sorafenib (10 μM) partially inhibited the increase in p53 levels induced by MI-319 and almost completely suppressed the expression of p21waf. Similar data were obtained in a related experiment in which a fixed concentration of MI-319 (20 μM) was added to varying concentrations of sorafenib. As shown in Figure 1D, 10 μM sorafenib completely inhibited the expression of p21waf induced by MI-319 in both cell lines. To verify that sorafenib was active as a raf kinase inhibitor, the lysates were also probed for pERK. Since p21waf levels can be regulated by ubiquitination and degradation [44,45], we considered the possibility that the observed effects of MI-319 and sorafenib on p21waf levels were due to changes in protein stability rather than p53-dependent gene expression. To more directly assess the ability of sorafenib to antagonize MI-319-induced p53-dependent gene expression, we examined the effects of both drugs on the activity of a p53-luciferase reporter. As shown in Figure 1E, p53 reporter activity was induced by MI-319 (p < 0.0041 and < 0.0048 for A375 and SKMEL5, respectively) and this induction was prevented with sorafenib (p < 0.0060 and < 0.0010 for A375 and SKMEL5, respectively; MI-319 alone vs. the MI-319/sorafenib combination).
To assess the contribution of p53 to the cytotoxic effects induced by sorafenib and MI-319, A375 cells were stably transfected with a tetracycline-inducible p53 shRNA. The transfectants were then treated with doxycycline and evaluated for their susceptibility to MI-319/sorafenib-induced programmed cell death as determined by flow cytometry. As shown in Figure 2A, exposure to doxycycline blocked the induction of p53 and p21waf by MI-319, confirming our hypothesis that the increase in p21waf levels induced by exposure to MI-319 was p53-dependent and not just due to protein stabilization [44,45]. Doxycycline markedly reduced the toxicity of the MI-319/sorafenib combination (p < 0.0271, Figure 2B), indicating that the toxicity of the MI-319/sorafenib combination was at least partly p53-dependent.
Figure 2 The effect of p53 downmodulation on p21waf expression and programmed cell death induced in A375 cells by the combination of 10 μM sorafenib (S) and 20 μM MI-319 (M). A375 cells expressing a tetracycline-regulable p53 shRNA were treated with 3 μM doxycycline overnight and then with sorafenib and MI-319 for 6 hr for the western blots shown in (A) or 24 hours for the cell death assays shown in (B). Cell death data are presented as the percentage of total cells staining positive for propidium iodide.
Effects of sorafenib on the intracellular distribution of p53: Role of GSK-3β
To determine if the dissociation between p53 levels and p53-dependent gene expression observed in cells exposed to sorafenib might be due to a change in the intracellular distribution of p53, A375 and SKMEL5 cells were exposed to MI-319 and sorafenib, lysed, and the lysates fractionated as described in Methods into nuclear and mitochondrial fractions. Cox-4 and c-myc were used as markers to assess the purity of the mitochondrial and nuclear fractions, respectively. As shown in Figure 3A, exposure to MI-319 markedly increased the amount of p53 present in the nucleus of both SKMEL5 and A375 cells. The addition of sorafenib, however, prevented this increase in nuclear p53 and induced the accumulation of p53 in the mitochondria in A375, but not SKMEL5 cells.
Figure 3 The role of GSK-3β in the redistribution of p53 induced by sorafenib in the setting of HDM2 blockade. (A). Nuclear and mitochondrial p53 levels in A375 and SKMEL5 cells treated with 10 μM sorafenib (S) and/or 20 μM MI-319 (M). In these studies, c-myc and COX4 were used as markers for the nuclear and mitochondrial subcellular fractions, respectively. (B). (Upper Panel). Nuclear and mitochondrial p53 and AIF levels in A375 cells expressing a tetracycline-regulable GSK-3β shRNA treated with 10 μM sorafenib (S) and/or 20 μM MI-319 (M) with or without doxycycline pretreatment. The downmodulation of GSK-3β in the doxycycline-treated cells was validated by western blot. (Lower Panel). Nuclear and mitochondrial p53 and AIF levels in SKMEL5 and SKMEL5-GSK-3β S9A cells treated with 10 μM sorafenib and/or 20 μM MI-319. (C). Effect of GSK-3β on the pro-apoptotic effects of single agent sorafenib and the sorafenib/MI-319 combination as determined by flow cytometry. Cell death data are presented as the percentage of total cells staining positive for propidium iodide.
We previously demonstrated that sorafenib activates GSK-3β [40], a kinase that phosphorylates p53 at two sites within its nuclear export sequence and regulates its intracellular distribution [2,3]. The constitutive and sorafenib-enhanced activities of GSK-3β were previously shown to be greater in A375 than in SKMEL5 cells [40]. To assess the role played by GSK-3β in the redistribution of p53 induced by sorafenib in the setting of HDM2 blockade, we stably transfected A375 melanoma cells with a tetracycline-inducible GSK-3β shRNA and SKMEL5 cells with a constitutively active GSK-3β (GSK-3β S9A) and examined the response of the transfectants to MI-319 and sorafenib. As shown in Figure 3B, treatment with MI-319 markedly increased the nuclear pool of p53 in all of the transfectants regardless of their GSK-3β status. In A375 cells, the addition of sorafenib largely abolished this nuclear accumulation of p53 and induced its translocation to the mitochondria. Suppression of GSK-3β by adding doxycycline prevented the redistribution of p53 induced by sorafenib. The results obtained with SKMEL5 were comparable to those generated with the GSK-3β-downmodulated A375 cells and consistent with the previous observation that SKMEL5 cells have lower GSK-3β activity than A375 cells [40]. To further implicate GSK-3β activity as a critical determinant of how sorafenib affects the intracellular distribution of p53, we examined the effects of sorafenib and MI-319 in SKMEL5 cells infected with an adenovirus expressing a constitutively active form of GSK-3β (GSK-3βS9A). The expression of the GSK-3βS9A construct was verified in these studies by western blot with an antibody to hemaglutinin (HA). As shown in Figure 3B (lower panel), exposure to MI-319 increased the nuclear pool of p53 in SKMEL5-GSK-3βS9A cells and the addition of sorafenib induced its disappearance from the nucleus and translocation to the mitochondria, similar to what was observed in melanoma cell lines with high constitutive GSK-3β activity such as A375. As mentioned above, sorafenib had no effect on the intracellular distribution of p53 in uninfected SKMEL5 cells. These results indicate that GSK-3β activity determines that effect of sorafenib on the intracellular distribution of p53.
We previously showed that the GSK-3β activation induced by sorafenib exposure was prosurvival in melanoma cells in that either the pharmacologic inhibition or downmodulation of the kinase enhanced sorafenib toxicity [40]. To determine if the activation of GSK-3β had a similar protective role in cells exposed to both sorafenib and MI-319, A375 cells stably transfected with a tetracycline-regulable GSK-3β shRNA were treated with 3 μM doxycycline overnight or left untreated and then exposed to sorafenib and MI-319. The cells were then stained with PI and analyzed for viability by flow cytometry. As shown in Figure 3C, the downmodulation of GSK-3β enhanced the toxicity of single agent sorafenib (as previously reported) but reduced the toxicity of the sorafenib/MI-319 combination (p < 0.0062 for sorafenib/MI-319 with or without doxycycline). These data suggest that the toxicity of this drug combination is due to both the increase in p53 levels induced by MI-319 and its mitochondrial translocation, the latter of which is dependent on the activation of GSK-3β.
Regulation of sorafenib-induced AIF nuclear translocation by p53 and GSK-3β
We previously demonstrated that sorafenib induced the mitochondrial release and nuclear translocation of AIF in melanoma cells sensitive to the drug (e.g. A2058) and that AIF translocation was responsible for the cytotoxic effects of sorafenib in these cells [46]. AIF translocation could not be induced in the more resistant cell line A375. To better define the roles of GSK-3β and p53 in sorafenib-induced AIF nuclear translocation, nuclear and mitochondrial fractions were prepared from various drug-treated melanoma cells and analyzed by western blot for AIF. As shown in Figure 3B (upper panel), the sorafenib/MI-319 combination (but not sorafenib alone) was able to induce AIF nuclear translocation in A375 cells stably transfected with a tetracycline-regulable GSK-3β shRNA in the absence of doxycycline. This pattern of AIF translocation, however, was completely reversed in the presence of doxycycline (i.e. with GSK-3β down modulated). In the absence of GSK-3β, sorafenib alone (but not the sorafenib/MI-319 combination) induced AIF nuclear translocation. Data obtained with SKMEL5 were similar to those obtained with the GSK-3β-down modulated A375 cells in that sorafenib as a single agent induced AIF nuclear translocation in a setting in which the sorafenib/MI-319 combination appeared unable to do so. The results obtained with the SKMEL5-GSK-3βS9A cells were identical to those obtained with A375 in that the drug combination, but not sorafenib alone, induced the nuclear translocation of AIF. These data are consistent with the ability of GSK-3β activation to reduce the toxicity of single agent sorafenib but to enhance that of the sorafenib/MI-319 combination.
Role of mitochondrial p53 in MI-319/sorafenib toxicity
To assess the contribution of mitochondrial p53 to the cytotoxicity induced by the sorafenib/MI-319 combination, cells were pretreated with pifithrin-μ (10 uM), an agent that blocks the pro-apoptotic effects of p53 in the mitochondria without affecting its transcriptional activity [41]. As shown in Figure 4A, pifithrin-μ pretreatment reduced the toxicity of the sorafenib/MI-319 combination by approximately half (p < 0.0267) in A375 cells, implicating the mitochondria as the dominant site of action of p53 in cells treated with this drug combination.
Figure 4 Inhibition of the mitochondrial function of p53 with pifithrin-μ diminishes the cytolytic activity of sorafenib/MI-319. (A). Effect of pifithrin-μ(10 μM) on the apoptotic effects of 10 μM sorafenib (S) and 20 μM MI-319 (M) in A375 cells. Cell death data are presented as the percentage of total cells staining positive for propidium iodide. (B). Effect of pifithrin-μ on the nuclear translocation of AIF induced by the sorafenib/MI-319 combination (but not sorafenib alone). c-myc and COX4 were used as markers for the nuclear and mitochondrial subcellular fractions used in these assays. (C). GSK-3β activity determines whether pifithrin-μ inhibits the cell death induced in melanoma cells by sorafenib and MI-319. (Left Panel). A375 cells expressing the tetracycline-regulable GSK-3β shRNA with and without doxycycline pretreatment were treated with sorafenib and MI-319 in the presence or absence of pifithrin-μ. Cell death data are presented as the percentage of total cells staining positive for propidium iodide. (Right Panel). SKMEL5 and SKMEL5-GSK-3βS9A cells were treated with sorafenib and MI-319 in the presence or absence of pifithrin-μ. Cell death data are presented as the percentage of total cells staining positive for propidium iodide. (D). Effect of MI-319 and sorafenib on PARP cleavage in A375 cells.
To determine if the mitochondrial translocation of p53 was responsible for the nuclear translocation of AIF induced by sorafenib/MI-319, A375 cells were exposed to various combinations of sorafenib and MI-319 in the presence or absence of pifithrin-μ. The cells were then fractionated into nuclear and mitochondrial subsets and analyzed for AIF by western blot. As shown in Figure 4B, single agent sorafenib again failed to induce the nuclear translocation of AIF in A375 cells. The translocation was, however, readily achieved with the sorafenib/MI-319 combination but could be blocked with pifithrin-μ, suggesting that it was mediated by mitochondrial p53. Since the mitochondrial translocation of p53 accounts for much of the toxicity induced by the sorafenib/MI-319 combination and depends on sorafenib-induced GSK-3β activation, we suspected that the suppressive effect of pifithrin-μ on drug-induced cytotoxicity might be similarly GSK-3β dependent. To test this hypothesis, the experiments shown in Figure 4A were repeated in additional melanoma cell lines with variable GSK-3β activity. As shown in Figure 4C, pifithrin-μ reduced the toxicity of the sorafenib/MI-319 combination by approximately half (p < 0.001) in A375 cells stably transfected with a tetracycline-inducible GSK-3β shRNA in the absence of doxycycline, similar to its effects on the parent A375 cell line shown in Figure 4A. Suppression of GSK-3β by the addition of doxycycline, however, nullified this protective effect (Figure 4C). Pifithrin-μ also failed to protect SKMEL5 cells from the proapoptotic effects of sorafenib/MI-319 unless the constitutively low GSK-3 activity of these cells was enhanced by the forced expression of GSK-3βS9A (p < 0.0212). Collectively, these data establish a causal link between the activation of GSK-3β, the mitochondrial translocation of p53, and the toxicity of the sorafenib/MI319 combination.
We previously showed that single agent sorafenib induced the release of cytochrome c but not AIF from the mitochondria of A375 cells. Sorafenib-induced caspase activation (PARP cleavage) was delayed in these cells (evident at only 24 hours) and did not appear to contribute to the lethality of the drug as the cells were not protected by the pancaspase inhibitor ZVAD [46]. The combination of sorafenib with MI-319, on the other hand, readily induced the translocation of AIF within 6 hours, at which point PARP was still undetectable (Figure 4D), suggesting that the early toxicity of this drug combination was caspase-independent.
Effects of GSK-3β activation and HDM2 blockade on sorafenib-induced Bcl-2 and Bcl-xL down modulation
As with Bim, tBid, and Puma, the ability of p53 to bind to and activate Bak and Bax in the mitochondria is limited by the relative abundance of anti-apoptotic Bcl-2 family members [22-28]. We previously showed that single agent sorafenib down modulated Bcl-2 and Bcl-XL in cells with low constitutive GSK-3β activity (e.g. SKMEL5), but not in cells with high GSK-3β activity (e.g. A375, SKMEL5-GSK-3βS9A)[40]. To determine how GSK-3β might affect the ability of the sorafenib/MI-319 combination to down modulate these anti-apoptotic BCL-2 family members, A375-GSK-3β-shRNA cells were exposed to MI-319 and/or sorafenib and then evaluated for Bcl-2 and Bcl-XL expression by western blot. As predicted from our earlier studies with unmodified A375 cells [40], single agent sorafenib failed to reduce Bcl-2 and Bcl-xL levels in these A375 transfectants in the absence of doxycycline or in SKMEL5-GSK-3βS9A cells (Figure 5). However, the drug down modulated these proteins in SKMEL5 cells and A375 cells in which GSK-3β expression was suppressed by doxycyline. Exactly the opposite results were obtained from cells treated with the sorafenib/MI-319 combination. The combination, for example, induced the down modulation of Bcl-2 and Bcl-XL in A375-GSK-3β-shRNA cells in the absence of doxycycline and in SKMEL5-GSK-3βS9A cells, but not in SKMEL5 or A375 cells in which GSK-3β expression was down modulated. These results are in agreement with the data shown in Figure 3B, which demonstrate a similar dichotomous effect of GSK-3β as an enhancer or inhibitor of AIF nuclear translocation depending on the status of HDM2. The data shown in Figure 5 suggest that the mitochondrial translocation of p53 and the pifithrin-μ-suppressible component of the toxicity of the sorafenib/MI-319 combination are both augmented by the GSK-3β-dependent down modulation of Bcl-2 and Bcl-xL. The data also demonstrate a hitherto unknown ability of HDM2 activity to determine how GSK-3β activation (e.g. by sorafenib) affects Bcl-2 and Bcl-xL expression.
Figure 5 GSK-3β activity and p53 levels (HDM2 blockade) determine whether sorafenib exposure downmodulates Bcl-2 and Bcl-xL in melanoma cells. (Left Panel). A375 cells stably transfected with a tetracycline-inducible GSK-3β shRNA were first treated with doxycycline (or not) and then exposed to sorafenib and/or MI-319. Cell lysates were then probed for Bcl-2 and Bcl-xL. (Right Panel). SKMEL5 and SKMEL5-GSK-3βS9A cells were treated with sorafenib and MI-319 and the cell lysates then probed for Bcl-2 and Bcl-xL.
Effects of MI-319 and sorafenib on A375 xenografts
To determine if the antitumor effects of the sorafenib/MI-319 combination on A375 melanoma cells in vitro could be reproduced in vivo, A375 melanoma xenografts were established in nude/beige mice and the mice then treated with sorafenib (80 mg/kg) and MI-319 (200 mg/kg) individually and in combination. As shown in Figure 6A, the tumor growth curve from mice treated with MI-319 was nearly identical to that of the control group. Treatment with single agent sorafenib had a modest growth-retarding effect (p < 0.007 for sorafenib vs. control). Treatment with the drug combination, on the other hand, resulted in a marked decrease in tumor growth (p < 0.001 for the drug combination vs. each of the other treatment groups).
Figure 6 Antitumor effects of sorafenib and MI-319 on A375 melanoma xenografts. (A). Growth curves of A375 xenografts from mice treated with control vehicle (C), sorafenib (S), MI-319 (M), or both drugs (MS). Data is presented as tumor volume relative to tumor volume when treatment started. (B). Western blot analyses of whole tumor lysates prepared from tumors excised on day 21.
To assess the effects of drug treatment on Bcl-2 and Bcl-xL levels, tumors from the different treatment groups were excised on day 21 and analyzed by western blot. As shown in Figure 6B, Bcl-xL levels appeared to be increased by treatment with either single agent MI-319 or sorafenib. The protein was undetectable, however, in the tumors excised from mice treated with the drug combination. A similar pattern was noted for Bcl-2 except that the baseline levels were lower. Of note, erk phosphorylation was not diminished in the tumors from mice receiving either single agent sorafenib or the sorafenib/MI-319 combination, indicating that the antitumor effect of these agents was not the result of raf inhibition.
To assess the mechanism by which the sorafenib/MI-319 combination impaired tumor growth, tumor tissue sections were examined by H&E staining for necrosis, IHC for proliferation (Ki-67) and microvessel density (CD31), and by TUNEL assay. Routine H&E staining revealed a marked increase in the extent of necrosis in tumors from mice treated with either single agent sorafenib or the drug combination Ki-67 staining and TUNEL assays limited to areas of tumor that were not overtly necrotic revealed no differences among the treatment groups (data not shown). Analysis of microvessel density using antibodies to CD31 showed a statistically significant decrease in vascularity in tumors treated with sorafenib alone relative to controls (p < .024) and an even greater decrease in vascularity in tumors treated with the MI-319/sorafenib combination relative to sorafenib alone (p < .006) (Figures 7A and 7B).
Figure 7 (A) Effect of MI-319 and sorafenib on microvessel density in A375 xenografts. Tumors were stained with CD31. A. A representative tumor section from each treatment group is shown. (B) Endothelial cells were quantitated using Image Pro 6.0 software. Each bar graph is representative of 6 tumors. (C). Immunofluorescence microscopic evaluation of p53 (red) in tumor cells from A375 xenografts. Drug treatment was as indicated in the figure. COX4 (green) was used as a mitochondrial marker in this study. (D). Immunofluorescence microscopic evaluation of AIF (red) in tumor cells from A375 xenografts. Drug treatment was as indicated in the figure. (E). Immunofluorescence microscopic evaluation of AIF in tumor cells from A375 xenografts. All colors are shown here (i.e. blue = Hoescht (Bisbenzimide H33342) stain, green = COX4, AIF = red). Drug treatment was as indicated in the figure.
Histologic sections were also subjected to wide field immunofluorescence microscopy to assess the intracellular distribution of p53. As shown in Figure 7C, p53 (red color) was scarcely detectable in the tumor cells from control or sorafenib-treated mice. Treatment with single agent MI-319 resulted in a marked increase in p53, which appeared to be confined primarily to the nuclei. In the tumor cells from mice treated with both MI-319 and sorafenib, however, a substantial amount of p53 was present outside the nucleus (yellow color) in apparent association with COX4 (green color), consistent with mitochondrial translocation.
Immunofluorescence microscopy was also used to assess the effect of treatment on the intracellular distribution of AIF. As shown in Figure 7D (red filter only), AIF was excluded from the nuclei of tumors excised from control mice and those treated with either sorafenib or MI-319. However, AIF was clearly present in the nuclei of tumors from mice that received both drugs. Figure 7E shows the same tissue sections with all colors displayed. As shown, the control and single agent-treated tumors have a prominent yellow to red cytosolic signal that is presumably due to the proximity of AIF (red) to COX4 (green) in the mitochondria. The nuclei (Hoescht stain) appear dark blue in each of the tumor sections except those from the mice treated with the drug combination, in which case the blue color is replaced by violet, indicating AIF nuclear translocation. These data suggest that the antitumor activity of the sorafenib/MI-319 combination may be due to a direct apoptotic effect mediated by the p53-dependent mitochondrial translocation of AIF as well as an additive antiangiogenic effect.
Discussion
Although the HDM2 antagonist MI-319 failed to induce an increase in PI-staining, AIF nuclear translocation, or any other manifestation of programmed cell death in melanoma cells when used as a single agent, it was markedly toxic in some (e.g. A375), but not all, melanoma cell lines when used in conjunction with the multikinase inhibitor sorafenib. The cytotoxic effect of the MI-319/sorafenib drug combination in responsive melanoma cells appears to depend on p53 acting in the mitochondria, an effect determined primarily by the GSK-3β activity of the cell line. Our data indicate that GSK-3β activity is not only required for the drug combination to induce the mitochondrial translocation of p53 but also the down modulation of Bcl-2 and Bcl-xL and the nuclear translocation of AIF. The critical role played by GSK-3β in these events contrasts with the largely inhibitory function of the kinase on these parameters when sorafenib is used as a single agent. In the absence of HDM2 blockade, for example, exposure to sorafenib induced the down modulation of Bcl-2 and Bcl-xL and the nuclear translocation of AIF only in cells with low GSK-3β activity.
An alternative way of presenting these data would be point out that sorafenib is able to down modulate Bcl-2 and Bcl-xL and induce AIF nuclear translocation in cells with low GSK-3β activity only when HDM2 is functional and that HDM2 blockade inhibits these effects. HDM2 blockade, on the other hand, is essential for sorafenib-induced Bcl-2 and Bcl-xL down modulation and AIF nuclear translocation in cells with high constitutive GSK-3β activity.
The basis for these context-dependent effects of HDM2 and GSK-3β on the cytotoxicity of sorafenib is unknown but several potential mechanisms may be operative. The cleavage of AIF from the inner mitochondrial membrane prior to its release, for example, is mediated by calpain and this proteolytic event is enhanced by oxidative modification of AIF by ROS [47]. The generation of ROS is regulated by the transcription factor Nrf2, whose activity is in turn enhanced by an association with p21waf [48]. In cells with low GSK-3β activity, p53 remains largely nuclear and it is therefore conceivable that in these circumstances the increase in p21waf induced by MI-319 limits ROS production and the processing and subsequent release of AIF from the mitochondria.
In cells with high GSK-3β activity, HDM2 blockade enhances the ability of sorafenib to induce AIF nuclear translocation and to down modulate Bcl-2 and Bcl-xL. There are several mechanisms by which p53 and GSK-3β could collaborate to achieve this effect. For example, GSK-3β is known to phosphorylate CREB, β-catenin, c-myc, and other transcriptional factors that regulate Bcl-2 and Bcl-xL expression [1,4,49,50]. Once phosphorylated by GSK-3β, these transcriptional factors become substrates for p53-regulated E3 ligases such as β-TrCP or FBW7 [51-53] and are polyubiquitinated and degraded in the proteasome. It is therefore possible that GSK-3β and a p53-inducible E3 ligase work in tandem to destabilize these transcription factors, resulting in the reduced expression of Bcl-2 and Bcl-xL.
Using drug doses that have been previously reported for other xenograft models (see Materials and Methods), MI-319 as a single agent appears to be completely ineffective at constraining the growth of A375 xenografts and sorafenib has only a modest effect. The two drugs together, however, markedly delay tumor growth. The growth suppression induced by the drug combination is associated with many of the biochemical changes observed in vitro in A375 cells including the down modulation of Bcl-2 and Bcl-xL, the mitochondrial translocation of p53, and the nuclear translocation of AIF. In addition, the vascularity of xenografts from mice treated with MI-319 and sorafenib was decreased relative to that of mice treated with sorafenib alone, which was in turn decreased relative to controls. Since the reduced vascularity of the sorafenib group was not associated with a demonstrable retardation in tumor growth, it is unclear whether enhanced suppression of angiogenesis resulting from the addition of MI-319 accounts for the superior anti-tumor activity of the combination.
Conclusions
The multikinase inhibitor sorafenib has been extensively evaluated in melanoma patients both as a single agent and in combination with chemotherapy with disappointing results. Our data suggest that the ability of sorafenib to activate GSK-3β and alter the intracellular redistribution of p53 may be exploitable as an adjunct to HDM2 blockade in the treatment of melanoma. Our data suggest that the high p53 levels inducible in melanoma cells with an HDM2 antagonist may not result in programmed cell death in vitro or appreciable tumor regression in vivo unless the drug is administered in conjunction with a second agent that can facilitate these GSK-3β-dependent cytotoxic effects. The ability of HDM2 inhibitors to prevent the degradation of p53 that usually follows its nuclear export and the ability of GSK-3β to facilitate the redistribution and mitochondrial function of p53 suggest that combining an HDM2 antagonist with an agent that activates GSK-3β might be a particularly useful antitumor strategy.
Materials and Methods
Cell lines and reagents
The human melanoma cell lines A375 and SKMEL5 were obtained from ATCC and maintained in RPMI-1640 medium containing 10% fetal bovine serum (USA Scientific), 2 mM glutamine and 50 μg/ml gentamycin at 37°C in 5 percent CO2. The SKMEL5 cells are heterozygous for the constitutively active BRAFV600E mutation [54] while the A375 line is homozygous as determined by sequence analysis. Sorafenib was provided by Bayer Pharmaceuticals, New Haven, CT. The MI-319 was provided by Ascenta Therapeutics (Malvern, PA) and Sanofi-Aventis (Paris, France).
Western blots
Cells were treated as described in Results and then lysed in Lysis Solution (Cell Signaling) supplemented with sodium fluoride (10 μM, Fisher Scientific, Hampton, NH) and phenylmethylsulfonyl fluoride (100 μg/ml, Sigma-Aldrich, St Louis, MO). Lysates were fractionated in 8-16% gradient SDS-polyacrylamide gels as indicated and the separated proteins were transferred to nitrocellulose. The blots were probed for the proteins of interest with specific antibodies followed by a second antibody-horse radish peroxidase conjugate and then incubated with SuperSignal chemiluminescence substrate (Pierce, Rochford, IL). The blots were then exposed to Kodak X-Omat Blue XB-1 film. The p21, phospho-p53 (Thr55), Bcl-xL and AIF antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); the phospho-erk, c-myc, Bcl-2, p53, phospho-p53 (Ser315), Bax, HA-tag, PARP and GSK-3β antibodies were purchased from Cell Signaling (Beverly, MA). The Bak antibody was from Calbiochem (San Diego, CA). The vinculin antibody was obtained from Sigma (St. Louis, MO); the COX4 antibody was obtained from ABCAM (Cambridge, MA).
Cell death assays
In each of these assays, the adherent cells were detached from the underlying plastic by treatment with trypsin/EDTA in PBS for 5 minutes and then combined with the floating, nonadherent cells. Propidium iodide (PI, 5 ng/ml, Sigma) was added to the cell pool and after 20 minutes at room temperature, the cells were analyzed by flow cytometry with a BD Biosciences FACScan. The percentage of cells staining with propidium iodide (PI) was recorded and each experiment reported was carried out at least 3 times. Data were reported as the mean +/- standard error for each experimental condition. In each of these assays, the percentage of cells staining with PI was taken to represent the extent of cell death induced by the experimental condition being tested.
p53 reporter assay
A p53 reporter vector containing the p53 response element coupled to firefly luciferase was purchased from Stratagene (Agilent Technologies, Santa Clara, CA). Briefly, tumor cell lines were transfected with the p53 luciferase and a CMV renilla luciferase vector (a gift of Stephen Balk, BIDMC) using Superfect (Qiagen) following the manufacturer's protocol. Twenty-four hours later, the cells were treated with sorafenib (10 μM) and MI-319 (20 μM) for 6 hours. The lysates were assayed using the Dual-Luciferase Reporter Assay System from Promega Corporation (Madison, WI). The data are presented as the ratio of firefly to renilla luciferase activity normalized to untreated controls.
Subcellular fractionations
Mitochondria-enriched and cytosolic fractions were isolated from Dounce-homogenized cells using the ApoAlert Cell Fractionation Kit (BD Clontech). The quality of the mitochondria-enriched fractions was validated by Western blot using an antibody for the mitochondrial protein cytochrome c oxidase subunit IV (COX4) [55]. Cytosolic fractions were obtained during the isolation of the mitochondria. Nuclei were isolated according to a standard protocol [56], lysed, and analyzed by western blot.
Design and construction of genetically modified melanoma cell lines
The generation of SKMEL5 cells expressing a constitutively active GSK-3β was previously described [40]. To generate the p53 and GSK-3β shRNA transfectants, the shRNA sequence selector and shRNA hairpin oligonucleotide sequence designer software provided by BD Clontech was used to select optimal sequences. Three shRNAs were generated for each gene to be silenced. To produce tetracycline-regulable shRNAs, the oligonucleotides selected were cloned into the pSingle-tTS-shRNA vector (BD Clontech). This vector is a tet-on vector. The three shRNA constructs were transfected as a group into A375 cells and stable transfectants obtained by selection in G418. Clones were screened individually for inducible expression of the shRNA (i.e. the downmodulation of the gene of interest as determined by Western blot) and 2-3 representative clones were selected for each shRNA based on the degree to which tetracycline exposure suppressed the expression of the gene of interest.
Immunoprecipitation experiments
Immunoprecipitations were carried out using a Protein A Immunoprecipitation Kit purchased from Roche Diagnostics (Mannheim, Germany). Briefly, treated cells were lysed and subjected to Dounce homogenization, followed by a pre-clearing step with protein A-sepharose. The cleared lysates were incubated with 10 μg primary antibody for 3 hours at 4°C, followed by an overnight incubation with protein A-sepharose. After washing with increasingly stringent buffers, the immunoprecipitated proteins were subjected to western blot analysis as described above.
Xenograft model
All animal studies were conducted according to Animal Investigation Committee (AIC)-approved protocol of Beth Israel Deaconess Medical Center. Six to eight week old athymic nude/beige female mice (Charles River Labs) were implanted subcutaneously with 1.0 × 107 A375 melanoma cells. When the tumors reached 7-8 mm in diameter, the mice were divided into 4 treatment groups of 6 mice each and treated daily for 21 days by gavage with sorafenib (80 mg/kg), MI-319 (200 mg/kg), sorafenib + MI-319, or saline (control). The doses of sorafenib [57,58] and MI-319 [59,60] were as previously reported. Tumors were measured bidimensionally daily. Tumor tissue from the sacrificed mice was frozen in liquid N2 for western blot analysis as described in Results or fixed in formalin for paraffin embedding.
Immunohistochemistry and immunofluorescence microscopy
The paraffin-embedded tumor tissue was sectioned at 5 microns using a Leica RM 2125 rotary microtome. The sections were dewaxed at 60°C, serially immersed in solutions of decreasing alcohol concentration, and then boiled in 10 mM sodium citrate, pH 6.2, for 30 minutes to unmask antigens. The tissue was then incubated in 3% hydrogen peroxide for 5 minutes, blocked with 1% BSA and 5% goat serum, and incubated overnight at 4°C with an antibody to Ki-67 (Dako, Carpinteria, CA). The Ki-67 epitope was detected using a biotinylated anti-mouse Ig antibody and an avidin-horseradish peroxidase conjugate (Vector Laboratories, Burlingame, CA). Similarly, sections were stained for endothelial cells with an antibody to CD 31 (ABCAM), followed by a biotinylated anti-rabbit Ig antibody (Vector Laboratories, Burlingame, CA). Slides were then counterstained with hematoxylin, dehydrated, and mounted.
The sections were assayed for apoptosis using the TUNEL method (Millipore, Billerica, MA) in accordance with an established protocol [61]. The tissue was hydrated and treated sequentially with proteinase K and hydrogen peroxide, and then blocked as described above for the Ki-67 staining. The sections were then exposed to a solution containing mixed nucleotides, some of which were digoxygenin-labeled, and terminal deoxynucleotidyl transferase (TdT). The slides were developed with an anti-digoxigenin antibody-peroxidase conjugate and DAB substrate. Tissue staining was quantitated using IMAGE Pro 6.0 software (MediaCybernetics, Inc, Bethesda, MD).
Immunofluorescence microscopy was utilized to assess the translocation of p53 and AIF to the nuclei and mitochondria, respectively. For p53, the above protocol for IHC was followed using a COX 4 antibody conjugated to Alexa 488 (green) and a p53 antibody conjugated to Alexa 555 (red) (both from Cell Signaling, Danvers, MA). For AIF staining, a primary AIF rabbit antibody was used (Santa Cruz) followed by a secondary antibody to rabbit IgG conjugated to Alexa 555, along with the COX 4 antibody-Alexa 488 conjugate. Finally, nuclei were stained with Bisbenzimide H33342 (Alexis Biochemicals, San Diego, CA). Immunofluoresence microscopy was carried out with a Nikon TE-2000E microscope at 100X magnification and a Hamamatsu Orca ER camera. The data was acquired with Nikon's NIS-Elements and analyzed with ImageJ software.
Statistical analysis
In vitro data depicted as bar graphs represent mean values from at least 3 separate experiments +/- standard error. For most of the studies shown, the significance of an apparent difference in mean values for any parameter (e.g. the percent of cells staining with propidium iodide) was validated by a Student's unpaired t test and the difference considered significant if p < 0.05. For the xenograft studies, the growth curves of the different treatment groups were statistically compared using one-way ANOVA.
List of abbreviations
AIF: apoptosis inducing factor; GSK: glycogen synthase kinase; ER: endoplasmic reticular; HDM2: human double mutant 2; HA: hemaglutinin.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
QL carried out many of the xenograft experiments, immnuohistochemistry, wide field fluorescence and western blots. JM conceived of the study, and participated in its design and coordination and helped to draft the manuscript. DP also conceived of the study, and participated in its design and coordination and helped to draft the manuscript. In addition, DP performed all in vitro experiments including the generation of tet-regulable shRNA cell lines and their implementation, cell death assays, western blots, reporter assays and cellular fractionations. All authors read and approved the final manuscript.
Acknowledgements
This work was supported by the NCI SPORE in Skin Cancer 2P50CA93683 and the Egan Memorial Laboratory for Melanoma Translational Research.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22022384PONE-D-11-1458610.1371/journal.pone.0025224Research ArticleBiologyDevelopmental BiologyMorphogenesisCell MigrationMedicineComplementary and Alternative MedicineDermatologySkin NeoplasmsMalignant Skin NeoplasmsMelanomasOncologyCancer TreatmentComplementary and Alternative MedicineCancers and NeoplasmsSkin TumorsMalignant MelanomaBasic Cancer ResearchGreen Tea Catechins Reduce Invasive Potential of Human Melanoma Cells by Targeting COX-2, PGE2 Receptors and Epithelial-to-Mesenchymal Transition Green Tea Catechins Inhibit Melanoma Cell InvasionSingh Tripti
1
Katiyar Santosh K.
1
2
3
4
*
1
Department of Dermatology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America
2
Nutrition Obesity Research Center, University of Alabama at Birmingham, Birmingham, Alabama, United States of America
3
Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama, United States of America
4
Birmingham Veterans Affairs Medical Center, Birmingham, Alabama, United States of America
Batra SK EditorUniversity of Nebraska Medical Center, United States of America* E-mail: [email protected] and designed the experiments: TS SKK. Performed the experiments: TS. Analyzed the data: TS SKK. Contributed reagents/materials/analysis tools: SKK. Wrote the paper: SKK TS.
2011 13 10 2011 6 10 e2522427 7 2011 29 8 2011 Singh, Katiyar.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Melanoma is the most serious type of skin disease and a leading cause of death from skin disease due to its highly metastatic ability. To develop more effective chemopreventive agents for the prevention of melanoma, we have determined the effect of green tea catechins on the invasive potential of human melanoma cells and the molecular mechanisms underlying these effects using A375 (BRAF-mutated) and Hs294t (Non-BRAF-mutated) melanoma cell lines as an in vitro model. Employing cell invasion assays, we found that the inhibitory effects of green tea catechins on the cell migration were in the order of (-)-epigallocatechin-3-gallate (EGCG)>(-)-epigallocatechin>(-)-epicatechin-3-gallate>(-)-gallocatechin>(-)-epicatechin. Treatment of A375 and Hs294t cells with EGCG resulted in a dose-dependent inhibition of cell migration or invasion of these cells, which was associated with a reduction in the levels of cyclooxygenase (COX)-2, prostaglandin (PG) E2 and PGE2 receptors (EP2 and EP4). Treatment of cells with celecoxib, a COX-2 inhibitor, also inhibited melanoma cell migration. EGCG inhibits 12-O-tetradecanoylphorbol-13-acetate-, an inducer of COX-2, and PGE2-induced cell migration of cells. EGCG decreased EP2 agonist (butaprost)- and EP4 agonist (Cay10580)-induced cell migration ability. Moreover, EGCG inhibited the activation of NF-κB/p65, an upstream regulator of COX-2, in A375 melanoma cells, and treatment of cells with caffeic acid phenethyl ester, an inhibitor of NF-κB, also inhibited cell migration. Inhibition of melanoma cell migration by EGCG was associated with transition of mesenchymal stage to epithelial stage, which resulted in an increase in the levels of epithelial biomarkers (E-cadherin, cytokeratin and desmoglein 2) and a reduction in the levels of mesenchymal biomarkers (vimentin, fibronectin and N-cadherin) in A375 melanoma cells. Together, these results indicate that EGCG, a major green tea catechin, has the ability to inhibit melanoma cell invasion/migration, an essential step of metastasis, by targeting the endogenous expression of COX-2, PGE2 receptors and epithelial-to-mesenchymal transition.
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Introduction
The melanoma remains the leading cause of death from skin diseases due to its propensity to metastasis. The statistical analysis from American Cancer Society indicated that in 2008, there were 8,420 melanoma-associated deaths in the U.S. and the number of new cases of invasive melanoma was estimated at 62,480 [1]. The incidence of melanoma has increased in the past few decades in the United States and is increasing rapidly in children [1]–[3]. If recognized and treated early, melanoma is curable, but as the disease progresses its propensity to metastasize make it difficult to treat. Chronic exposure to solar ultraviolet (UV) radiation has been implicated in melanoma and non-melanoma skin cancers [4], [5]. Exposure of the skin to UV radiation induces an increase in the expression levels of cyclooxygenase-2 (COX-2), a rate-limiting enzyme that catalyzes the conversion of arachidonic acid to prostaglandins (PGs). The enhanced generation of PGs, specifically PGE2, plays a central role in orchestrating the multiple events involved in cancer invasion and metastasis. PGE2 is an important metabolite which has been implicated in these risks more than other PG metabolites, and has been shown to exert its effects through G protein-coupled receptors, EP1, EP2, EP3 and EP4, and has been implicated in angiogenesis, decreased host immunity and enhanced invasion and metastasis [6], [7]. Although, melanoma is less common than other skin cancers, it causes the majority (75%) of skin cancer-related deaths. Once diagnosed with metastatic melanoma, most patients will die of their disease within 2 years [1], [8]. Since, melanoma is a highly malignant cancer with a potent capacity to metastasize distantly, an approach that reduces its metastatic ability may facilitate the development of an effective strategy for its treatment and/or prevention.
Catechins isolated from the leaves of green tea (Camellia sinensis) have a number of beneficial health effects including anti-carcinogenic activity, which has been demonstrated in various tumor models [9], [10]. In previous studies, we and others have shown that oral administration of an aqueous extract of green tea or green tea catechins, which are commonly called as polyphenols (a mixture of catechins), in drinking water inhibits UV radiation-induced non-melanoma skin cancer in mice in terms of tumor incidence, tumor multiplicity and tumor growth/size [11], [12]. Multiple mechanisms or molecular targets have been reported by which green tea polyphenols protect the skin from skin tumors. These mechanisms include the DNA repair [13], [14], stimulation of immune system [14], [15], anti-inflammatory effects [16] and anti-oxidant activity [17] of green tea polyphenols in vitro and in vivo models. However, the beneficial effects of green tea polyphenols on melanoma are not well studied and less understood. As green tea is commonly consumed as a beverage world-wide, we assessed the effect of its polyphenolic components on the invasive potential of melanoma cells using melanoma cell lines as an in vitro model. Beverage grade green tea leaves contains 5 major catechins or epicatechin derivatives: (-)-epicatechin, (-)-gallocatechin, (-)-epicatechin gallate, (-)-epigallaocatechin and (-)-epigallocatechin-3-gallate (EGCG) [18].
In the present study, first we assessed the chemotherapeutic potential of various catechins on the migration capacity of human melanoma cells, as the migration of cancer cells is a major event in the metastatic cascade. For this purpose, two highly metastasis-specific melanoma cancer cell lines were selected: one is A375 which is BRAF mutated and activating mutations of the protooncogene BRAF have been observed in approximately 50% of malignant melanomas. Second cell line is Hs294t, which is also highly metastatic but not BRAF mutated. These two cell lines were used as an in vitro model for this study. In preliminary screening experiments, we identified that EGCG is a major active component of green tea polyphenols which significantly blocks the migration/invasion of melanoma cells compared to other catechins or epicatechin derivatives. We further characterized the role of COX-2 and its metabolite PGE2 on the migration of human melanoma cancer cells and ascertained whether EGCG has any suppressive effects on the COX-2-mediated migration of these cells. Epithelial-to-mesenchymal transition (EMT), the process whereby epithelial cells transform into mesenchymal cells, has recently been shown to be relevant for cancer growth and cancer metastasis. During EMT, cells lose expression of proteins that promote cell-cell contact such as E-cadherin and acquire mesenchymal markers such as vimentin, fibronectin and N-cadherin, which promote cell invasion and metastasis [19]. The EMT has also been associated with higher levels of inflammation and inflammatory mediators, and therefore we have also checked whether inhibition of COX-2 expression and PGE2 production by EGCG in melanoma cells is associated with reversal of EMT and that leads to inhibitory effect on melanoma cell migration. Here, we present evidence that EGCG inhibits the invasive potential of melanoma cells through transition of mesenchymal state to epithelial state in melanoma cells and that EGCG do so through a process that involves the reduction of COX-2 expression and lowering the levels of PGE2 and PGE2 receptors in melanoma cells.
Materials and Methods
Source of green tea catechins
Various purified tea catechins used in this study were obtained from Dr. Y. Hara of Mitsui Norin Company (Tokyo, Japan). These catechins or polyphenols are stable for at least two years when refrigerated at 4°C.
Antibodies, chemicals and reagents
Celecoxib, PGE2, 12-O-tetradecanoylphorbol-13-acetate (TPA) and EP2 agonist were purchased from Sigma Chemical Co. (St. Louis, MO). Boyden Chambers and polycarbonate membranes (8 µm pore size) for cell invasion assays were obtained from Neuroprobe, Inc. (Gaithersburg, MD). The antibodies specific to N-cadherin, keratin-18 and fibronectin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), while antibodies for EP1, EP2, EP3, EP4, vimentin, E-cadherin, NF-κB, IKKα and IκBα and their secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA). Desmoglein-2 was obtained from Abcam (Cambridge, MA). Antibodies specific for COX-2, EP4 agonist and an enzyme immunoassay kit for PGE2 analysis were obtained from Cayman Chemicals (Ann Arbor, MI).
Cell lines and cell culture conditions
The normal human epidermal melanocytes (HEMa-LP, Catalogue #C-024-5C) were commercially obtained from Invitrogen (Carlsbad, CA), and were cultured in HMGS-2 medium supplemented with human melanocyte growth supplement provided by the supplier. The human melanoma cells lines, A375 and Hs294t, were purchased from the American Type Culture Collection (Manassas, VA). The cell lines were cultured as monolayers in RPMI 1640 culture medium supplemented with 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT), 100 µg/mL penicillin, and 100 µg/mL streptomycin and maintained in an incubator with 5% CO2 at 37°C. The EGCG was dissolved in a small amount of acetone, which was added to the complete cell culture medium [maximum concentration of acetone, 0.1% (v/v) in media] prior to addition to sub-confluent cells (60–70% confluent). Cells treated with acetone only served as a vehicle control. To determine the effect of EGCG on TPA- or PGE2-mediated effects, EGCG was added in cell culture medium at least 30 minutes before the treatment of the cells with TPA, PGE2, PGE2 receptor or PGE2 receptor agonists.
Cell invasion assay
The invasion capacity of melanoma cells was determined in vitro using Boyden Chambers (Gaithersburg, MD) in which the two chambers were separated with matrigel coated Millipore membranes (6.5 mm diameter filters, 8 µM pore size), as detailed previously [20]. Briefly, melanoma cells (1.5×104 cells/100 µL serum-reduced medium) were placed in the upper chamber of Boyden chambers, test agents were added alone, or in combination, to the upper chamber (200 µL), and the lower chamber contained the medium alone (150 µL). Chambers were assembled and kept in an incubator for 24 h. After incubation, cells from the upper surface of Millipore membranes were removed with gentle swabbing and the migrant cells on the lower surface of membranes were fixed and stained with crystal violet. Membranes were then washed with distilled water and mounted onto glass slides. The membranes were examined microscopically and cellular migration was determined by counting the number of stained cells on each membrane in at least 4–5 randomly selected fields using an Olympus BX41 microscope. Representative photomicrographs were obtained using a Qcolor5 digital camera system fitted to an Olympus BX41 microscope. Resultant data are presented as a mean of migrating cells ± SD/microscopic field of three independent experiments.
Scratch assay or wound healing assay
Scratch or wound healing assay was performed to detect melanoma cell migration, as detailed previously [20]. Briefly, melanoma cells were grown to full confluency in six-well plates and incubated overnight in starvation medium. Cell monolayers were wounded with a sterile 100 µL pipette tip, washed with starvation medium to remove detached cells from the plates. Cells were left either untreated or treated with indicated doses of tea catechins in full medium and kept in a CO2 incubator for 48 h. After 48 h, medium was replaced with phosphate-buffered saline (PBS) buffer, the wound gap was observed and cells were photographed using an Olympus BX41 microscope fitted with digital camera.
Quantitation of prostaglandin E2 using PGE2 immunoassay kit
The levels of PGE2 in cell homogenates were measured using the Cayman PGE2 Enzyme Immunoassay Kit (Ann Arbor, MI) following the manufacturer's protocol. Briefly, at indicated time point, cells were harvested and homogenized in 100 mM phosphate buffer, pH 7.4 containing 1 mM ethylenediamine tetraacetic acid and 10 µM indomethacin using a homogenizer. Homogenates were centrifuged and the supernatants were collected for the analysis of PGE2 concentrations following the manufacturer's protocol.
Western blot analysis
Cell lysates were prepared to analyze the expression levels of different proteins, as described previously [21]. Briefly, following treatment of melanoma cells for the indicated time periods with or without EGCG or any other agent, the cells were harvested, washed with cold PBS and lysed with ice-cold lysis buffer supplemented with protease inhibitors. Equal amounts of proteins were resolved on 10% Tris-Glycine gels and transferred onto a nitrocellulose membrane. After blocking the non-specific binding sites, the membrane was incubated with the primary antibody at 4°C overnight. The membrane was then incubated with the appropriate peroxidase-conjugated secondary antibody and the immunoreactive bands were visualized using the enhanced chemiluminescence reagents. Equal protein loading was verified on the membrane after stripping it and re-probed with anti-β actin antibody.
Assay for NF-κB/p65 activity
Quantitative analysis of NF-κB/p65 activity was performed using the NF-κB TransAM Activity Assay Kit (Active Motif, Carlsbad, CA) following the manufacturer's protocol. Briefly, the nuclear extracts of cells were prepared using the Nuclear Extraction Kit (Active Motif, Carlsbad, CA) following the manufacturer's instructions. Absorbance was recorded at 450 nm using absorbance at 650 nm as the reference. The results are expressed as the percentage of the optical density of the non-EGCG-treated control group.
Statistical analysis
For migration assays, the control and EGCG-, TPA- PGE2- or EP2- and EP4-agonists treatment groups or combined-treatment groups separately were compared using one-way analysis of variance (ANOVA) followed by post hoc Dunn's test using GraphPad Prism version 4.00 for Windows, GraphPad Software, San Diego, California, USA, www.graphpad.com. All quantitative data for cell migration are shown as mean ± SD/microscopic field. In each case P<0.05 was considered statistically significant.
Results
Effect of green tea catechins on human melanoma cell migration/invasion
In the present study, first we have assessed and compared the effect of various green tea catechins on the invasive potential of human melanoma cells in vitro using Boyden chamber. The molecular structures of five major tea catechins, EC, GC, ECG, EGC and EGCG are shown in Figure 1A. Using in vitro cell invasion assay, we found that treatment of A375 and Hs294t cells with equimolar concentration (25 µM) of catechins for 24 h resulted in inhibition of migration/invasion of these cells. The relative inhibitory effect of catechins on melanoma cell migration or invasion was in the order of: EGCG>EGC>ECG>GC>EC, as shown in Figure 1B. The experiment was repeated three times and resultant data of cell migration from each treatment group has been summarized in Figure 1C. These results also suggest that the cell migration ability of BRAF-mutated A375 cells was higher than non-BRAF-mutated Hs294t cells; however, the difference was not statistically significant. This screening preliminary experiment revealed that EGCG has greater inhibitory effect on melanoma cell migration compared to other tea catechins; therefore, EGCG was selected for further studies of cell invasion behavior of human melanoma cells and the molecular mechanisms underlying these effects.
10.1371/journal.pone.0025224.g001Figure 1 Comparative effects of green tea catechins on the cell invasion capacity of melanoma cells.
(A) Chemical structures of major green tea catechins. EC, (-)-epicatechin; GC, (-)-gallocatechin; ECG, (-)-epicatechin-3-gallate; EGC, (-)-epigallocatechin, and EGCG, (-)-epigallocatechin-3-gallate. (B) Effect of equimolar concentration (25 µM) of various green tea catechins on the migration of human melanoma cells (A375 and Hs294t) after treatment for 24 h. (C) The migrating cells were counted and the results expressed as the mean number of migratory cells ± SD/microscopic field. Data collected from three independent experiments. Significant inhibition by EGC and EGCG versus non-tea catechins-treated controls, ¶
P<0.01; *
P<0.001.
EGCG inhibits human melanoma cell invasion
Next, we determined dose-dependent effect of EGCG on the cell migration potential of A375 and Hs294t human melanoma cells using Boyden chamber cell migration assays. First, screening experiments were performed to determine the effects of lower concentrations of EGCG (µg/mL), which should not reduce the cell viability or induce apoptosis in these cells. As shown in Figure 2A, relative to untreated control cells, treatment of cells with EGCG at concentrations of 0, 10, 20 and 40 µg/mL reduced the invasive potential of A375 and Hs294t cells in a concentration-dependent manner. The density of the migrating cells on the membrane after staining with crystal violet is shown in Figure 2A, and the numbers of migrating cells/microscopic field are summarized in Figure 2B. The melanoma cell invasion potential was inhibited by 13% to 65% (P<0.01–0.001) in A375 cells and by 7% to 70% (P<0.001) in Hs294t cells in a concentration-dependent manner after treatment with EGCG for 24 h. The density of cell migration was higher after 48 h, and the inhibitory effect of EGCG on melanoma cells migration was also comparatively higher than 24 h (data not shown). To verify that the inhibition of cancer cell migration by EGCG was a direct effect on migration ability, and that was not due to a reduction in cell viability, a trypan blue assay was performed using cells that were treated identically to those used in the migration assays. Treatment of A375 and Hs294t cells with various concentrations of EGCG (0, 10, 20 and 40 µg/mL) for 24 h had no significant inhibitory effect on cell viability or cell death (data not shown).
10.1371/journal.pone.0025224.g002Figure 2 EGCG inhibits melanoma cell invasion/migration.
(A) Treatment of human melanoma cells with EGCG for 24 h inhibits migration of A375 and Hs294t cells in a concentration-dependent manner. (B) The migrating cells were counted and the results expressed as the mean number of migratory cells ± SD/microscopic field. Significant inhibition by EGCG versus non-EGCG-treated controls, ¶
P<0.01; *
P<0.001. (C) Scratch or wound healing assay was performed to assess the effect of EGCG on the migration of A375 and Hs294t melanoma cells. Incubation of A375 or Hs294t cells with EGCG (10 and 20 µg/mL) for 48 h inhibits migration of cells compared to non-EGCG-treated control cells. Assay was repeated three times and representative pictures are shown. Space between dotted lines in each panel shows the space without or negligent number of migrating cells.
We have further confirmed the inhibitory effect of EGCG on melanoma cell migration by employing scratch or wound healing assay, as described in Material and Methods. As shown in Figure 2C, relative to untreated control cells, treatment of cells with EGCG (10 and 20 µg/mL) reduced the migration capacity of A375 and Hs294t cells in a concentration-dependent manner after the treatment of cells for 48 h. The part of gap or wounding space between cell layers after making a wound was occupied by the migrating Hs294t cells which were not treated with EGCG. However, the healing of the wound or the empty space of the cells was not occupied by the migrating cells treated with EGCG and this effect was dose-dependent. The gap or wounding space between the cell layers is highlighted by broken white lines, as shown in Figure 2C. Similar inhibitory effects of EGCG on cell migration using scratch assay was also found with A375 cells (Figure 2C, lower panels). These observations suggest that EGCG has the ability to inhibit the migration ability of melanoma cells.
EGCG reduces endogenous COX-2 expression in melanoma cells
To examine whether the inhibitory effect of EGCG on the migration of the melanoma cells is associated with the reduction of endogenous expression of COX-2, we determined the levels of COX-2 in cell lysates of the various treatment groups using western blot analysis. Western blot analysis revealed that the treatment of A375 and Hs294t cells with EGCG reduced the levels of COX-2 expression in a concentration-dependent manner as compared to the expression in untreated controls (Figure 3A).
10.1371/journal.pone.0025224.g003Figure 3 EGCG inhibits invasion of melanoma cells by reducing endogenous COX-2 expression.
(A) Treatment of A375 and Hs294t cells with EGCG for 24 h resulted in down-regulation of COX-2 protein expression. (B) Treatment of A375 and Hs294t cells with celecoxib, an inhibitor of COX-2, for 24 h inhibits melanoma cell migration in a dose-dependent manner. The data are expressed as the mean number of migratory cells± SD/microscopic field. Significant difference versus non-celecoxib-treated control cells, *
P<0.001; †
P<0.05. (C) Treatment of A375 cells with EGCG (20 and 40 µg/mL) inhibits TPA (a COX-2 stimulator)-enhanced cell migration capacity. The data on cell migration capacity are summarized in terms of mean number of migrating cells/microscopic field ± SD, n = 3. Significant inhibition versus TPA treatment alone, *
P<0.001. (D) EGCG down regulates TPA-induced COX-2 expression in A375 cells. The levels of COX-2 were determined in cell lysates using western blot analysis.
Celecoxib, a selective COX-2 inhibitor, inhibits melanoma cell migration
This experiment was performed to verify whether the inhibitory effect of EGCG on melanoma cell migration is mediated through its inhibitory effect on COX-2 expression. For this purpose, equal numbers of A375 and Hs294t cells were subjected to the cell invasion assay after treatment with various concentrations of celecoxib (0, 5, 10, 20 µM), a well known inhibitor of COX-2, for 24 h. As shown in Figure 3B, treatment of the A375 and Hs294t cells with celecoxib resulted in a significant reduction in the cell migration capacity of melanoma cells in a concentration-dependent manner as compared with non-celecoxib-treated controls (P<0.05–0.001). These data suggested that the inhibition of COX-2 expression by the treatment of cells with EGCG is associated with the inhibition of melanoma cell migration.
EGCG inhibits TPA (an inducer of COX-2)-induced cell migration
TPA is a well known skin tumor promoter and has been shown to stimulate the expression of COX-2 in the skin cells [22]; therefore, the melanoma cells were treated with TPA for the stimulation of COX-2 expression in vitro, and thereafter determined the effect of TPA on the migration of melanoma cells. As shown in Figure 3C, treatment of A375 cells with TPA (40 ng/mL) for 24 h resulted in a significantly enhanced cell migration (P<0.001) compared to non-TPA-treated control cells. To determine whether EGCG inhibits TPA-induced cell migration in human melanoma cells, A375 cells were treated with TPA (40 ng/mL) with and without the treatment of EGCG for 24 h. The treatment of A375 cells with EGCG (20 and 40 µg/mL) resulted in a dose-dependent inhibition of TPA-induced cell migration. A summary of the cell migration data for the various treatment groups is provided in Figure 3C. Treatment of EGCG at the doses of 20 µg/mL and 40 µg/mL inhibited TPA-induced cell migration by 95% (P<0.001) and >100% (P<0.001) respectively. To verify whether this inhibition of cell migration by EGCG is mediated through the inhibition of TPA-induced COX-2 expression, cell lysates were prepared and subjected to western blot analysis to estimate the levels of COX-2 expression. Western blot analysis data revealed that treatment of A375 cells with TPA for 24 h resulted in higher expression of COX-2 as compared to the expression in cells that were not treated with TPA (Figure 3D). Pretreatment of A375 cells with EGCG (20 and 40 µg/mL) for 24 h resulted in inhibition of TPA-induced COX-2 expression (Figure 3D). These data suggest that inhibition of TPA-induced cell migration by EGCG is mediated through the downregulation of COX-2 expression.
Inhibition of melanoma cell migration by EGCG is mediated through its inhibitory effects on PGE2 production
PGE2 is one of the metabolites of COX-2, and most of the biological activities of COX-2 are mediated through its metabolites, therefore, we examined whether treatment of melanoma cells with EGCG reduced the levels of PGE2 production. For this purpose, Hs294t melanoma cells were treated with EGCG for 24 h. Cells were harvested and the levels of PGE2 were determined using PGE2 immunoassay kit. As shown in Figure 4A, treatment of cells with EGCG resulted in a dose-dependent reduction in the levels of PGE2 in these cells. Next, we examined the effect of PGE2 on the Hs294t cell migration using in vitro cell invasion assay. Cell invasion data revealed that treatment of PGE2 significantly enhanced (P<0.01, P<0.001) the cell migration potential in a dose-dependent manner (Figure 4B). Further, the effect of EGCG on PGE2-induced cell migration was evaluated. For this purpose, Hs294t cells were treated with PGE2 (10 µM) with and without the treatment with EGCG for 24 h and cell migration determined. We found that the treatment of melanoma cells with PGE2 resulted in a significant increase of cell migration (P<0.05) compared to the cells which were not treated with PGE2 (Figure 4C and 4D). Treatment of Hs294t cells with EGCG (20 or 40 µg/mL) for 24 h resulted in a dose-dependent inhibition of PGE2 (10 µM)-induced melanoma cell migration, as shown in Figure 4C and data are summarized in Figure 4D. Similar inhibitory effect of EGCG was observed on PGE2-induced cell migration in A375 melanoma cells (data not shown).
10.1371/journal.pone.0025224.g004Figure 4 EGCG inhibits PGE2-induced melanoma cell migration.
(A) Dose-dependent effect of EGCG on the levels of PGE2 in melanoma Hs294t cells. The levels of PGE2 are expressed in terms of pg/mg protein± SD, n = 3 independent experiments. Significant inhibition by EGCG versus non-EGCG-treated controls, *
P<0.001. (B) Treatment of Hs294t cells with PGE2 enhances cell migration in a concentration-dependent manner. Significant difference versus control, ¶
P<0.05, *
P<0.001. (C) Treatment of cells with EGCG (20 and 40 µg/mL) inhibits PGE2-enhanced cell migration capacity of Hs294t cells. Representative photomicrographs of cell migration were presented from three independent experiments. (D) The data on cell migration are summarized as a mean number of migratory cells ± SD/microscopic field. Significant inhibition versus PGE2 alone: *
P<0.001. Cell invasion assays under each experiment were repeated three times and in each case the migrating cells were counted and the results are expressed as a mean number of migratory cells ±SD/microscopic field.
EGCG decreases the levels of PGE2 receptors in melanoma cells
COX-2 metabolite PGE2 has been shown to manifest its biological activity via four known G-protein-coupled receptors (i.e., EP1-EP4) [7], [23]. Therefore, we determined the effect of EGCG on the basal levels of PGE2 receptors in melanoma cells. Western blot analysis revealed that treatment of A375 cells with EGCG (0, 10, 20 and 40 µg/mL) for 24 h resulted in a dose-dependent reduction in the levels of EP2 and EP4 (Figure 5A). The inhibitory effect of EGCG was also observed on EP1 and EP3 but was less prominent than the effect on EP2 and EP4 (data not shown).
10.1371/journal.pone.0025224.g005Figure 5 EGCG inhibits PGE2 receptors-induced human melanoma cell migration.
(A) Treatment of A375 melanoma cells with EGCG for 24 h decreases the expression levels of PGE2 receptors EP2 and EP4 in a concentration-dependent manner. The cells were harvested 24 h after the treatment and cell lysates were prepared and subjected to western blot analysis, as detailed in Materials and Methods. (B) Treatment of A375 cells with EP2 agonist (butaprost) for 24 h enhances melanoma cell migration (left panels). The data on cell migration are summarized and expressed as a mean number of migratory cells ± SD/microscopic field, n = 3 (right panel). (C) Treatment of A375 cells with EGCG inhibits EP2 agonist (1.0 µM)-induced melanoma cell migration (left panels). The data on cell migration are summarized as a mean number of migratory cells ± SD/microscopic field, n = 3 (right panel). (D) Treatment of A375 cells with EP4 agonist (Cay10580) for 24 h enhances cell migration (left panels). The data on cell migration in each group are expressed as a mean number of migratory cells ± SD/microscopic field, n = 3 (right panel). (E) Treatment of A375 cells with EGCG inhibits EP4 agonist (1.0 µM)-induced cell migration (left panels). The data on cell migration are summarized as a mean number of migratory cells ± SD/microscopic field, n = 3 (right panel). Representative photomicrographs of cell migration were presented from three independent experiments in each case. Significant inhibition versus non-EGCG-treated controls, ¶
P<0.01; *
P<0.001; Significant increase versus non-EP2 agonist-treated or non-EP4 agonist-treated controls, †
P<0.001.
An EP2 agonist and EP4 agonist promote the migration of melanoma cells while EGCG inhibits EP2 and EP4 agonists-stimulated cell migration
To determine whether PGE2 receptor EP2 and EP4 has a role in melanoma cell migration, and whether EGCG inhibits their effects on cell migration, we further conducted cell invasion experiments with A375 melanoma cells. In vitro cell invasion experiments revealed that treatment of A375 cells with EP2 agonist (butaprost) significantly enhanced the migration ability of melanoma cells, as shown in Figure 5B. A summary of number of migrating cells/microscopic field is also shown (Figure 5B, right panel). Pretreatment of A375 cells with EGCG inhibits EP2 agonist (1 µM)-induced melanoma cell migration by 36% and 58% (P<0.001) at the dose of 20 and 40 µg/mL, respectively (Figure 5C right panel). Similarly, the effect of EP4 agonist was determined on the migration of A375 melanoma cells. As shown in Figure 5D, EP4 agonist treatment of A375 cells for 24 h significantly enhanced (P<0.001) the cell migration in a dose-dependent manner. Treatment of cells with EGCG (20 and 40 µg/mL) significantly inhibited (41–66%, P<0.01 and P<0.001) EP4 agonist-induced cell migration (Figure 5E, left and right panels). These data suggest that the expressions of PGE2 receptors in melanoma cells have roles in cell migration, and that EGCG inhibits the migration of melanoma cells, at least in part, by decreasing the levels of PGE2 receptors.
EGCG decreases the nuclear level and activity of NF-κB/p65 in melanoma cells: NF-κB acts as a mediator of melanoma cell invasion
NF-κB is an upstream regulator of COX-2, therefore we assessed whether EGCG also affects the levels and activation of NF-κB in melanoma cells. To examine this effect, A375 cells were treated with various concentrations of EGCG (0, 10, 20 and 40 µg/mL) for 24 h, and thereafter cells were harvested and cell lysates prepared for western blot analysis. Western blot analysis revealed that treatment of cells with EGCG decreased the translocation of NF-κB/p65 in to the nucleus in a dose-dependent manner (Figure 6A). The activity of NF-κB also was significantly reduced (20–65%, P<0.001) after the treatment of cells with EGCG in a concentration-dependent manner (Figure 6B). The results also indicated that treatment of EGCG resulted in the downregulation of IKKα and degradation of IκBα (Figure 6A), which leads to the inactivation of NF-κB and its translocation to the nucleus. Further, to assess whether NF-κB has a role in melanoma cell migration, A375 melanoma cells were treated with caffeic acid phenethyl ester (0, 5.0 and 10.0 µg/mL), a potent inhibitor of NF-κB, and cell migration was studied. As shown in Figures 6C and 6D, treatment of A375 cells with caffeic acid phenethyl ester resulted in a significant reduction of cell migration (26% and 57%; P<0.05, and P<0.001) compared to non-cafeic acid phenethyl ester-treated control cells, and these results are similar to that observed on treatment of the cells with EGCG (Figure 2).
10.1371/journal.pone.0025224.g006Figure 6 Effect of EGCG on NF-κB activation in melanoma cells.
(A) Treatment of A375 cells with EGCG decreases the basal level of NF-κB/p65 and IKKα while inhibiting the degradation of IκBα. After 24 h treatment with various concentrations of EGCG the cells were harvested and cytosolic and nuclear fractions were prepared and subjected to the analysis of NF-κB, IKKα and IκBα using western blot analysis. Representative blot is shown from three independent experiments with identical observations. (B) The activity of NF-κB/p65 in the nuclear fraction of the cells after treatment with and without EGCG for 24 h was measured using NF-κB/p65-specific activity assay kit, n = 3. Activity of NF-κB is expressed in terms of percent of control (non-EGCG-treated) group. Significant decrease versus control: *
P<0.001. (C) Treatment of A375 cells with caffeic acid phenethyl ester (CAPE), an inhibitor of NF-κB, inhibits melanoma cell migration. Representative photomicrographs are shown from three separate experiments. (D) Data on cell migration capacity are summarized as the mean number of migratory cells ± SD/microscopic field, n = 3. Significant inhibition versus non-CAPE-treated cells: ¶
P<0.05; *
P<0.001.
EGCG reverses epithelial-to-mesenchymal transition in melanoma cells
Activation of NF-κB has been implicated in inflammation-induced tumor growth and progression, and has been identified as an important regulator of EMT in several cancer cell types [24]–[27]. As the inhibition of melanoma cell migration by EGCG is associated with the inactivation of NF-κB, we sought to examine whether EGCG targets EMT biomarkers or whether EGCG transforms mesenchymal biomarkers to epithelial biomarkers in melanoma cells and that is responsible for its inhibitory effect on cell migration. To examine this effect, A375 cells were treated with EGCG for 24 h, and cell lysates were prepared for the analyses of various epithelial and mesenchymal biomarkers using western blot analysis. As shown in Figure 7, western blot analyses revealed that EGCG increased the levels of the epithelial biomarkers, such as E-cadherin, keratin-18 and desmoglein 2 dose-dependently in melanoma cells compared to untreated controls. In contrast, the levels of mesenchymal biomarkers, such as vimentin, fibronectin and N-cadherin were reduced in melanoma cells after treatment with EGCG in a dose-dependent manner.
10.1371/journal.pone.0025224.g007Figure 7 Treatment of melanoma cells with EGCG results in mesenchymal-to-epithelial transition.
Treatment of A375 cells with EGCG for 24 h enhances the levels of epithelial biomarkers in the cells, such as, the levels of E-cadherin, keratin-18 and desmoglein 2. Simultaneously the levels of mesenchymal biomarkers in melanoma cells, such as, vimentin, fibronectin and N-cadherin were decreased dose-dependently. Western blot analysis was performed as detailed under Materials and Methods. Representative blots are shown from three independent experiments with similar results.
Discussion
Melanoma remains the leading cause of death from skin disease, in large part, due to its propensity to metastasize. Although melanoma is less common than non-melanoma skin cancers, which includes squamous cell and basal cell carcinoma, however, it causes approximately 75% skin cancer-related deaths. World Health Organization report indicated 48,000 melanoma-related deaths worldwide per year [28]. Most patients suffering from malignant melanoma ultimately die of their disease within two years [29], [30]. The development of new treatment options and novel strategies are required which can prevent the invasiveness of melanoma cells and that can inhibit the metastatic ability of melanoma cells. Majority of cancers over-express COX-2, an enzyme responsible for the biosynthesis of PGs metabolites. Enhanced production of PGs, and particularly PGE2, has been linked with tumor progression, invasion and metastasis [5], [7]. Because of its important role in tumor invasion and metastasis, COX-2 is considered as a promising target for cancer therapy [7], [31]. Therefore, the search of novel and non-toxic inhibitors of COX-2 as well as the inhibitors of PGE2 may provide a better option for the treatment of malignant melanoma and that may prove a better strategy for its prevention or treatment.
Green tea polyphenols/catechins have been shown to have anti-carcinogenic activities in various tumor models, including skin cancers [4], [10], [14]. In the present study, we have found that EGCG has significantly greater anti-invasive activity in melanoma cells compared to EC, GC, ECG and EGC. The significant findings of the present study are that the treatment of melanoma cells with EGCG for 24 h inhibits cell migration in a dose-dependent manner, and that is associated with the inhibition of COX-2 expression and PGE2 production. Based on our experimental observations, cells will go under apoptosis or cell death if melanoma cells are treated with higher concentrations of EGCG or for more than 24 h. Under these conditions, cell migration will decrease, and this reduction in cell migration could be due to cell death and not due to changes in migrating behavior of cells. In this study, cell death or apoptosis is not a reason of EGCG-caused inhibition of melanoma cell migration. The melanoma cells over-express COX-2, and the reduction in the levels of COX-2 by EGCG may be responsible for the inhibition of cell migration of melanoma cells. This notion is supported by the evidence that treatment of the melanoma cells with a potent COX-2 inhibitor (celecoxib) resulted in a reduction of cell migration. Studies have shown that TPA promotes COX-2 expression and subsequently enhances cell migration [32], and we have found that TPA-induced cell migration was blocked by the treatment of cells with EGCG. These observations further suggest that inhibition of melanoma cell migration by EGCG is mediated through the inhibition of COX-2 expression. PGE2 exerts its biologic functions through four G protein-coupled receptors, EP1, EP2, EP3 and EP4 [7], [23], [33], that can stimulate cell survival signals as well as invasive potential of cancer cells [34]–[36]. PGE2 has been shown to promote lung cancer and melanoma cell migration, and that this effect of PGE2 is associated with the activation of PGE2 receptors [32], [37]. Based on these investigations, we determined the involvement of the PGE2 receptors in EGCG-mediated inhibition of melanoma cell migration. It was found that the levels of EP2 and EP4 were decreased when melanoma cells were treated with EGCG. These data suggest that inhibition of the EP2 and EP4 levels by EGCG may contribute to the inhibition of melanoma cell migration. The inhibitory effect of EGCG on melanoma cell migration through the inhibitory effect on EP2 or EP4 was further verified by treating the cells with an EP2 agonist (butaprost) and an EP4 agonist (cay10580) with and without the treatment of cells with EGCG. Our data revealed that the treatment of A375 cells with the EP2 agonist and EP4 agonist resulted in enhanced cell migration, and that EP2 agonist- and EP4 agonist-induced cell migration was significantly inhibited by the treatment of cells with EGCG using identical in vitro conditions. These observations support the evidence that inhibition of PGE2 receptors by green tea catechin EGCG may have contributed to the blocking of melanoma cell migration. These findings also demonstrate the feasibility of using EGCG as an alternative to COX-2 inhibitors, which show toxicity in some patients, given the fact that COX-2 remains an attractive target for cancer therapy. As EGCG acts by decreasing the expression of both COX-2 and EP receptors, this could be more effective because EGCG targets both ligand (PGE2) and receptor (EP). EGCG has also been shown to inhibit mammary cancer cell migration through the inhibition of nitric oxide and nitric oxide-mediated mechanisms [38]. Other phytochemicals also have been assessed for their inhibitory effect on cancer cell invasion and migration. Punathil and Katiyar [39] have reported that treatment of non-small cell lung cancer cells with proanthocyanidins from grape seeds resulted in inhibition of cell migration following the inhibition of nitric oxide and guanylate cyclase pathways.
As NF-κB is an upstream regulator of COX-2, we further checked the effect of EGCG on the levels of NF-κB/p65 in melanoma cells using western blot analysis. EGCG inhibits the activation of NF-κB/p65 in a dose-dependent manner. Caffeic acid phenethyl ester, an inhibitor of NF-κB, inhibits melanoma cell migration. These observations further support the hypothesis that the inhibitory effect of EGCG on melanoma cell migration is mediated, at least in part, through the downregulation of COX-2, PGE2 and PGE2 receptors. However, it remains to be examined whether down-regulation of other NF-κB target genes also contribute to the inhibition of invasive potential of melanoma cells. NF-κB regulates a wide spectrum of biological processes, including inflammation, cell proliferation and apoptosis. Additionally, NF-κB has a role in tissue invasion, cancer cell migration and metastasis. Importantly, NF-κB has been identified as an important regulator of EMT in several cancer cell types [24]–[27]. EMT has been shown to play a major role in invasion and metastasis of epithelial tumors. EMT can render tumor cells migratory and invasive following its effect on all stages, which includes invasion, intravasation and extravasation [19]. During EMT, cells can change from an epithelial to a mesenchymal state. They lose their characteristic epithelial traits and instead acquire properties of mesenchymal cells. This process is primarily coordinated by the disappearance or loss of epithelial biomarkers such as E-cadherin and certain cytokeratins with the concomitant appearance of mesenchymal markers such as vimentin, fibronection and N-cadherin, etc. In the present study, we found that treatment of melanoma cells with EGCG resulted in suppression of mesenchymal biomarkers, such as vimentin, fibronectin and N-cadherin while restored the levels of epithelial biomarkers such as, E-cadherin, keratin and desmoglein 2, in melanoma cells which suggest that EGCG has the ability to transform mesenchymal characteristics to epithelial characteristics in melanoma cancer cells and this transition may also be one of the possible mechanisms through which EGCG reduce the invasiveness of melanoma cells and that lead to inhibition of migration of melanoma cells in our system.
Together, the results from this study have identified for the first time that EGCG, a major component of green tea catechins or polyphenols, inhibit the invasive potential of melanoma cells and that involves: (i) the inhibitory effect of EGCG on endogenous COX-2 expression and successive down-regulation of PGE2 and PGE2 receptors, (ii) the inhibitory effect of EGCG on the activation of NF-κB/p65, which is the upstream regulator of COX-2, and (iii) the mesenchymal-to-epithelial transition. Further mechanism-based in vivo studies are required which can establish the importance of EGCG and its development as a pharmacologically safe non-toxic agent for the treatment of malignant melanoma by using either alone or in combination with other phytochemicals or anti-metastatic drugs.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by funds from the 5RO1 AT002536 by National Center for Complementary and Alternative Medicine, NCCAM/NIH (SKK) (http//www.cancer.gov), and Veterans Administration Merit Review Award (SKK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22046282PONE-D-11-0810810.1371/journal.pone.0026420Research ArticleBiologyMolecular Cell BiologySignal TransductionSignaling CascadesStress Signaling CascadeCellular Stress ResponsesNeuroscienceMedicineNeurologyNeurodegenerative DiseasesSilencing GADD153/CHOP Gene Expression Protects against Alzheimer's Disease-Like Pathology Induced by 27-Hydroxycholesterol in Rabbit Hippocampus Gadd153 Mediates 27-OHC-Induced AD PathologyPrasanthi Jaya R. P. Larson Tyler Schommer Jared Ghribi Othman
*
Department of Pharmacology, Physiology and Therapeutics, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota, United States of America
Ferreira Sergio T. EditorFederal University of Rio de Janeiro, Brazil* E-mail: [email protected] and designed the experiments: JP OG. Performed the experiments: JP TL JS. Analyzed the data: JP OG. Contributed reagents/materials/analysis tools: JP TL JS OG. Wrote the paper: JP OG.
2011 14 10 2011 18
10 2011 6 10 e264209 5 2011 26 9 2011 Prasanthi et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Endoplasmic reticulum (ER) stress is suggested to play a key role in the pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD). Sustained ER stress leads to activation of the growth arrest and leucine zipper transcription factor, DNA damage inducible gene 153 (gadd153; also called CHOP). Activated gadd153 can generate oxidative damage and reactive oxygen species (ROS), increase β-amyloid (Aβ) levels, disturb iron homeostasis and induce inflammation as well as cell death, which are all pathological hallmarks of AD. Epidemiological and laboratory studies suggest that cholesterol dyshomeostasis contributes to the pathogenesis of AD. We have previously shown that the cholesterol oxidized metabolite 27-hydroxycholesterol (27-OHC) triggers AD-like pathology in organotypic slices. However, the extent to which gadd153 mediates 27-OHC effects has not been determined. We silenced gadd153 gene with siRNA and determined the effects of 27-OHC on AD hallmarks in organotypic slices from adult rabbit hippocampus. siRNA to gadd153 reduced 27-OHC-induced Aβ production by mechanisms involving reduction in levels of β-amyloid precursor protein (APP) and β-secretase (BACE1), the enzyme that initiates cleavage of APP to yield Aβ peptides. Additionally, 27-OHC-induced tau phosphorylation, ROS generation, TNF-α activation, and iron and apoptosis-regulatory protein levels alteration were also markedly reduced by siRNA to gadd153. These data suggest that ER stress-mediated gadd153 activation plays a central role in the triggering of AD pathological hallmarks that result from incubation of hippocampal slices with 27-OHC. Our results add important insights into cellular mechanisms that underlie the potential contribution of cholesterol metabolism in AD pathology, and suggest that preventing gadd153 activation protects against AD related to cholesterol oxidized products.
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Introduction
Alzheimer's disease (AD), the most common neurodegenerative disorder, is histopathologically characterized by the accumulation of β-amyloid (Aβ) peptide and the hyperphosphorylation of tau protein. In addition to increased levels of Aβ and phosphorylated tau, oxidative stress, inflammation, and cell death contribute to the neurodegenerative features of AD [1]. Accumulation of Aβ peptides in soluble and insoluble forms is suggested to be the trigger of the neurodegenerative processes that lead to the development of AD. Currently, there is no consensus as to what are the factors or cellular mechanisms that lead to Aβ accumulation in the brain. We have shown that cholesterol-enriched diets increase Aβ production and oxidative damage involving the activation of the growth and arrest DNA damage protein gadd153 [2]. Gadd153 (also called CHOP) is activated by endoplasmic reticulum (ER) stress and can cause cellular damage by mechanisms that include induction of oxidative stress, generation of reactive oxygen species (ROS), triggering of apoptosis, and disturbing iron homeostasis [3]–[5]. The ability of gadd153 to increase ROS generation can lead to increased production of β-secretase (BACE1), the rate limiting enzyme that cleaves β-APP to yield Aβ, thus leading to increased Aβ levels [6], [7]. ER stress-induced gadd153 activation may thus triggers AD-like pathology by generating oxidative damage and increasing Aβ production. Silencing gadd153 gene expression would therefore represent a potential strategy to reduce ROS generation, BACE1 activation, and Aβ accumulation and ultimately protect against AD.
We have recently shown that the cholesterol oxidized metabolite (oxysterol) 27-hydroxycholesterol (27-OHC) causes AD-like pathology in human neuroblastoma cells and in organotypic slices from adult rabbit hippocampus [8]–[10]. In this study, we incubated organotypic slices from adult rabbit hippocampus with 27-OHC, in the presence or absence of siRNA to gadd153, and determined the effects on levels of Aβ, phosphorylated tau, ROS, oxidative and ER stress, iron homeostasis and apoptosis-regulatory proteins, which are all relevant to AD pathology. The organotypic slice system has many advantages in that connectivity between neurons, interneurons and glia is maintained. In addition, rabbits have a phylogeny closer to humans than rodents [11], and their Aβ sequence, unlike that of rodents, is similar to the Aβ sequence of the human [12]. We found that siRNA to gadd153 dramatically reduces the generation of a wide range of events that are relevant to AD.
Materials and Methods
Preparation of organotypic slices and treatments
Organotypic slices were prepared as we have previously shown [8], [10]. In brief, hippocampi from adult male rabbits (n = 4; 1.5–2 year old) were dissected and sectioned with a MacIlwain chopper (300 µm thick). Each hippocampus yields about 60 sections (120 sections per rabbit). Five sections were plated on membrane inserts (Millipore, Bedford, MD), with a total of 12 inserts per hippocampus (24 inserts per rabbit). Inserts were placed in 35 mm culture dishes containing 1.1 ml growth media (Neurobasal A with 20% horse serum, 0.5 mM l-glutamine, 100 U/ml penicillin, and 0.05 µM/ml streptomycin). Slices were exposed to a humidified incubator atmosphere (4.5% CO2 and 35°C). Media was changed at day 1, and at day 4 slices were switched to a defined medium consisting of Neurobasal A, 2% B27 supplement and 0.5 mM l-glutamine. At day 8, slices were divided to 4 groups (twenty four dishes of five slice each per group) as the following: Control slices, 27-OHC-treated slices, gadd153 siRNA-treated slices, and gadd153 siRNA+27-OHC-treated slices. All animal procedures were carried out in accordance with the U.S. Public Health Service Policy on the Humane Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the University of North Dakota (protocol number 1101-1C).
siRNA to gadd153/CHOP and non silencing control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The following human gadd153 gene sequences (5′→3′ orientation) were used (A): Sense GAAGGCUUGGAGUAGACAAtt, Antisense UUGUCUACUCCAAGCCUUCtt; (B): Sense GGAAAGGUCUCAGCUUGUAtt, Antisense UACAAGCUGAGACCUUUCCtt; (C): Sense GUCUCAGCUUGUAUAUAGAtt, Antisense UCUAUAUACAAGCUGAGACtt. The transfection of siRNA was performed in the slices with siRNA transfection reagent (Santa Cruz Biotechnology) and siRNA transfection medium (Santa Cruz Biotechnology) according to the manufacturer's recommendation on day 8. The siRNAs (final concentration, 200 nM) were mixed with 100 µl of siRNA transfection medium. This mixture was gently added to a solution containing 20 µl of siRNA transfection reagent in 100 µl of siRNA transfection medium. The mixture solutions were incubated for 45 minutes at room temperature and 800 µl of siRNA transfection medium was added to the solution. Half of the final solution was added to the bottom of the inserts and remaining half onto the top of the slices with total of 1 ml in each dish. Transfected slices were incubated at 37°C for 48 hours without changing medium and then, treated with either 25 µM of 27-OHC (Medical Isotopes, Pelham, NH) or vehicle (0.1% ethanol). After 72 hours of treatment, samples were collected for ELISA, Western blot, real-time RT-PCR, reactive oxygen species, hydrogen peroxide, isoprostane, glutathione levels and confocal microscopy studies.
Quantification of secreted Aβ and TNF-α levels by ELISA
Aβ levels were quantified in the media of treated organotypic slices by ELISA using a kit from Invitrogen (Carlsbad, CA) as per the manufacturer's protocol. Briefly, following treatments, the culture medium was collected, supplemented with protease and phosphatase inhibitor cocktail, and centrifuged at 16,000× g for 5 min at 4°C. 100 µl of supernatant was used for Aβ40 and Aβ42 quantification. The quantity of Aβ in each sample was measured in duplicate and expressed as mean ± standard error for the samples. Aβ40 and Aβ42 levels are expressed in pg/ml.
TNF-α released in media was quantified using a colorimetric sandwich human TNF-α ELISA (Invitrogen, Camarillo, CA) according to the manufacturer's protocol. Treatments were performed in triplicate, and the quantity of TNF-α in each sample was measured in duplicate and expressed as mean ± standard error for the samples. TNF-α levels are expressed in pg/ml.
Western blot analysis
Organotypic slices were homogenized with the tissue protein extraction reagent (T-PER, Thermo Scientific, Rockford, IL) supplemented with protease and phosphatase inhibitors. Protein concentrations were determined with the BCA protein assay reagent by standard protocol. Proteins (10 µg) were separated in 10% and 12.5% SDS-PAGE gels, transferred to a polyvinylidene difluoride membrane (Millipore) and incubated with antibodies to APP (1∶100, Chemicon International, Temecula, CA), BACE1 (1∶100, Chemicon International), phosphorylated tau (PHF-1 and CP13, 1∶500, gift from Dr. Peter Davis, Albert Einstein College of Medicine), total tau (1∶200, Calbiochem, La Jolla, CA), to the apoptosis-regulatory proteins Bax (1∶100, Santa Cruz Biotechnology), caspase 3 (1∶100, Santa Cruz Biotechnology) and Bcl-2 (1∶100, Santa Cruz Biotechnology), to the ER- glucose regulated proteins grp 78 (1∶100, Assay Designs Stressgen, Ann Arbor, MI) and grp 94 (1∶100, Affinity Bioreagents, Golden, CO), to the ER-stress marker gadd153 (1∶100, Abcam, Cambridge, MA), to the iron regulatory proteins transferrin receptor (TfR, 1∶100, Abcam), ferritin light chain (FLC, 1∶100, Santa Cruz Biotechnology), ferritin heavy chain (FHC, 1∶100, Santa Cruz Biotechnology), and iron regulatory proteins-1 and 2 (IRP-1, 1∶500, Millipore; IRP-2, 1∶100, Chemicon International), and to the inflammation marker Tumor necrosis factor (TNF-α, 1∶100, Abcam). β-actin (1∶5000) was used as a gel loading control. The blots were developed with enhanced chemiluminiscence (Immun-star HRP chemiluminiscent kit, Biorad, Herculus, CA). The results were quantified by densitometry and represented as total integrated densitometric values.
Real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR)
Total RNA was isolated and extracted from organotypic slices using the 5 prime PerfectPure RNA tissue kit (5 Prime, Inc., Gaithersburg, MD) as we have previously described [8]. 1 µg of purified RNA was converted to cDNA by using qScript CDNA SuperMix (Quanta Biosciences, Gaithersburg, MD). The cDNA was amplified using PerfeCTa SYBR Green FastMix for iQ (2X, Quanta Biosciences) in Bio-Rad iCycler iQ Multicolor Real Time PCR Detection System (BioRad, Hercules, CA). Primers for gene of interest and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed with Beacon Designer 4.0. The primer sequences (forward; reverse, both in the 5′ to 3′ direction) are: Gadd153 (TGCTTCTCTGGCTTGGCTGAC; CTGGTTCTCCCTTGGTCTTCC), TNF-α (CGCTTCGCCGTCTCCTACC; GGCAAGGTCCAGGTACTCAGG), and GAPDH (AGGTCATCCACGACCACTTC; GTGAGTTTCCCGTTCAGCTC). The amplification process time was 15 min at 95°C followed by 80 cycles of 30 s at 95°C, 1 min at 55°C and 30 s at 72°C. Values were expressed in cycle threshold time (Ct) and were normalized to the Ct times for the housekeeping gene GAPDH. Primer specificity was confirmed by a single peak in a dissociation curve and/or by a single band following gel electrophoresis of the primer products.
RNA band shift assay
The IRP-IRE interactions were performed using LightShift Chemiluminescent RNA Electrophoretic mobility shift assay kit (REMSA, Pierce Biotechnology, Rockford, IL). Briefly, rabbit ferritin light chain iron regulatory element (IRE) probe UCUUGCUUCAACAGUGUUUGAACGGAAC was biotin labeled (Sigma, St. Louis, MO) at 3′. 10 µg of cytoplasmic protein of organotypic slices following treatments was incubated with 2 nM (final concentration) of biotin labeled IRE probe in binding buffer provided in the kit following manufacturer's protocol. 5 µl of IRP-IRE complexes formed were resolved on 5% DNA retardation gel (Biorad) and transferred on to nylon membrane (Roche Diagnostics, Indianapolis, IN). After the membrane was cross linked with UV-light, the IRP-IRE complexes were visualized with enhanced chemiluminiscence (Immun-star HRP chemiluminiscent kit, Biorad, Herculus, CA) according to the manufacturer's protocol. Electrophoretic mobility shift experiments were performed at least three times and one representative experiment was shown.
Reactive Oxygen Species (ROS) Assay
ROS generation was determined using 2′–7′-dichlorofluorescein-diacetate (DCFH-DA) and fluorometric detection of H2O2 as we have previously described [13]. For DCFH-DA measurements, 25 µg of homogenate from control and treated organotypic slices were diluted in PBS and incubated with 5.0 µM DCFH-DA (Sigma, St. Louis, MO) in the dark for 15 minutes at 37°C. Fluorescence was measured every 15 minutes for 1 hr with excitation and emission wavelengths of 488 nm and 525 nm respectively, using a Spectramax Gemini-EM (Molecular Probes, Sunnyvale, CA). Values are expressed as percent increase in fluorescence compared to controls. H2O2 was measured using the horseradish peroxidase (HRP)-linked fluorometric assay (Amplex Ultra Red; Invitrogen, Carlsbad, CA) following the manufacturer's recommendation and as we described previously [13]. Resorufin fluorescence was followed by a Spectramax Gemini-EM (Molecular Probes, Sunnyvale, CA) with excitation 530–560 nm and emission at 590 nm.
Oxidative stress measurements
Isoprostanes and glutathione depletion assays were used to measure oxidative stress levels. The F2-isoprostane 8-Iso-prostaglandin F2α (8-iso-PGF2α) is produced by lipid peroxidation and is a marker of oxidative stress. 8-iso-PGF2α levels were quantified in the control and treated organotypic slices using the isoprostane oxidative stress assay kit B (Biomol International, Plymouth Meeting, PA) as we have shown previously [13]. The color developed in the standards and samples was read on a SpectraMax Plus microplate reader (Molecular Devices, Sunnyvale, CA) at 405 nm. The measured optical density was used to calculate the concentration of 8-iso-PGF2α. Glutathione (gamma-glutamyl-cysteinyl-glycine; GSH) plays an important role in antioxidant defense in animal cells. Increased levels of oxidative stress lead to the accumulation of oxidized glutathione (GSSG) and a subsequent decrease in the ratio of reduced glutathione (GSH) to GSSG. A luminescent based GSH-Glo Assay (Promega Corporation, Madison, WI) was used for quantification of glutathione according to the manufacturer's recommendation and as we have previously reported [13].
Confocal microscopy
Alterations in total intracellular iron distribution were determined using the fluorescent indicator Phen-green SK, the fluorescence of which is quenched by iron. Organotypic slices were incubated with phen green SK (20 µM of the diacetate) [14] for 1 hour at 37°C. The slices were mounted with vectasheild containing DAPI (Vector laboratories, Inc., Burlingame, CA). Phen-green SK staining was visualized with a Zeiss LSM 510 META confocal system coupled to a Zeiss Axiophot 200 inverted epifluorescence microscope. Imaging was performed with a 40× oil immersion objective.
Statistical Analysis
Data was analyzed for statistical significance using one-way analysis of variance (ANOVA) followed by Newman-Keuls Multiple Comparison Test with GraphPad Prism software 4.01. All values in each group were expressed as mean value ± SEM. All group comparisons were considered significant at p<0.05.
Results
Silencing gadd153 with siRNA reversed 27-OHC-induced increase in gadd153 levels
Western blot and densitometric analysis (Fig. 1a,b) show that treatment with 27-OHC induced a substantial increase in gadd153 levels in comparison to control levels in hippocampal slices. Treatment with siRNA to gadd153 completely suppressed basal gadd153 levels and markedly reduced the 27-OHC-induced increase in gadd153 levels. Real-time RT-PCR demonstrated that 27-OHC also increases gadd153 mRNA and that treatment with siRNA to gadd153 reduces the basal as well as the 27-OHC-induced increase in gadd153 mRNA (Fig. 1c). Treatment of slices with siRNA to gadd153 reduced both the basal distribution of gadd153 and the large increase induced by 27-OHC treatment. Western blot and real-time RT-PCR demonstrate the efficiency of gadd153 silencing.
10.1371/journal.pone.0026420.g001Figure 1 siRNA to gadd153 reduces 27-OHC-induced alteration in ER protein levels in organotypic slices from rabbit hippocampus.
27-OHC significantly increased protein levels (a,b) as well as mRNA (c) of gadd153 and treatment with siRNA to gadd153 markedly reduced the 27-OHC-induced increase in expression levels of gadd153. Treatment with siRNA also reduced the increase in levels of the ER chaperones grp 78 and and grp 94 induced by 27-OHC (d–f). *p<0.05, **p<0.01 and ***p<0.001 versus controls; #p<0.05 and ###p<0.001 versus 27-OHC.
Our results also show that the 27-OHC increased the levels of the ER-resident chaperone proteins grp 78 and grp 94 in organotypic slices (Fig. 1d–f). Treatment of slices with siRNA to gadd153 didn't affect levels of grp 78 or grp 94 in slices untreated with 27-OHC but significantly reduced levels of these chaperones in slices treated with 27-OHC. Together, with increased gadd153, the increase in grp 78 and grp 94 levels demonstrates that 27-OHC targets the ER and causes stress in this organelle.
siRNA to gadd153 reduced 27-OHC-induced increase in Aβ levels
Treatment with 27-OHC led to an increase in secreted levels of both Aβ40 and Aβ42 in media of the organotypic slices as determined with ELISA assay (Fig. 2a,b). While levels of Aβ40 increased from 24.05±0.79 in media of control slices to 31.31±1.11 pg/ml in media of 27-OHC-treated slices, levels of Aβ42 were 4.49±0.59 pg/ml in media of control slices and 7.26±0.55 pg/ml in media of 27-OHC treated organotypic samples. In media of slices treated with siRNA to gadd153 alone, levels of Aβ40 and Aβ42 were decreased by 45% and 52% respectively compared to control levels. siRNA to gadd153 also reduced levels of Aβ40 and Aβ42 by 36% and 54% in slices treated with 27-OHC compared to slices treated with 27-OHC alone (Fig. 2a,b). The decrease in Aβ levels with siRNA to gadd153 suggests that gadd153 regulates the production of Aβ from APP by BACE1 enzyme. To confirm the specificity of siRNA, we performed gadd153 silencing using the three siRNA probes individually, rather than a mixture of the three. Each of the three probes markedly reduced gadd153 as well as BACE1 levels (Supporting Figure S1).
10.1371/journal.pone.0026420.g002Figure 2 siRNA to gadd153 reduces 27-OHC-induced increase in Aβ production and tau phosphorylation.
27-OHC significantly increased levels of both secreted Aβ40 and Aβ42, and treatment with siRNA, alone or in presence of 27-OHC, significantly reduced Aβ40 and Aβ42 levels (a,b). siRNA to gadd153 also reduced 27-OHC-induced increase in APP and BACE1 levels (c–e). Note that siRNA to gadd153 markedly reduced basal levels of BACE1, suggesting that gadd153 regulates BACE1 transcription (c,e). Treatment with 27-OHC also increases phosphorylation of tau as detected with PHF-1 and CP13 antibodies (f–h). The increase in phosphorylated tau is reduced by treatment with siRNA to gadd153. *p<0.05, **p<0.01 and ***p<0.001 versus controls; #p<0.05, ##p<0.01 and ###p<0.001 versus 27-OHC.
Our results show that the increase in the levels of Aβ40 and Aβ42 in the 27-OHC-treated slices was accompanied with an increase in the levels of parent protein APP as well as of BACE1, the enzyme that initiates the cleavage of APP to yield Aβ (Fig. 2c–e). Treatment with siRNA to gadd153 alone, while didn't affect APP levels, reduced BACE1 levels. Treatment with siRNA to gadd153 markedly reduced 27-OHC-induced increase in APP and BACE1 to control levels. These results suggest that gadd153 regulates Aβ production by primarily controlling BACE1 levels.
siRNA to gadd153 reversed 27-OHC-induced phosphorylation of tau
The phosphorylation of tau protein was determined with PHF-1 and CP13, antibodies that detects tau phosphorylated at Ser396/404 and Ser202 respectively. Our results show that 27-OHC-induced about a two-fold increase in phosphoylated tau as detected by both PHF-1 and CP13 antibodies (Fig. 2 f–h). siRNA to gadd153 did not affect basal levels of phosphorylated tau but significantly reduced the increase in tau phosphorylation induced by 27-OHC. These results suggest that siRNA to gadd153 reduces tau phosphorylation in conditions where there is an abnormal increase in the phosphorylation of tau but not in the basal state.
siRNA to gadd153 reduces 27-OHC-induced alteration in apoptosis-regulatory proteins
A growing body of evidence suggests an active role for the ER in the regulation of apoptosis by mechanisms that involve regulation of apoptosis-regulatory protein levels [15]. We determined the effect of siRNA to gadd153 on levels of Bax, caspase 3 and Bcl-2. Our data shows that 27-OHC significantly increased level of the pro-apoptotic proteins Bax and the effector of apoptosis caspase 3 (Fig. 3). While siRNA to gadd153 didn't affect basal levels of Bax and caspase 3, it significantly reduced the increase in levels of these proteins induced by 27-OHC. On the other hand, levels of the anti-apoptotic protein Bcl-2 were markedly reduced by 27-OHC treatment and incubation of slices with siRNA to gadd153 in presence or absence of 27-OHC markedly increased levels of Bcl-2 beyond control levels. These latter results suggest that gadd153 may regulate the basal expression levels of Bcl-2 (Fig. 3).
10.1371/journal.pone.0026420.g003Figure 3 siRNA to gadd153 gene reverses 27-OHC-induced apoptosis.
Treatment of slices with 27-OHC while increases levels of the pro-apoptotic Bax and of the active caspase 3, this treatment reduces levels of the anti-apoptotic Bcl-2 (a–d). siRNA to gadd153 reduces the increase in Bax as well as caspase 3 levels and markedly increases levels of Bcl-2 in presence or absence of 27-OHC. *p<0.05 and ***p<0.001 versus controls; #p<0.05, ##p<0.01 and ###p<0.001 versus 27-OHC.
siRNA to gadd153 protects from 27-OHC-induced oxidative stress
ER stress is known to induce oxidative stress and oxidative stress can also cause ER stress [16]. We determined the extent to which 27-OHC-induced ER stress is associated with ROS generation, isoprostane formation and glutathione depletion, which are all markers for oxidative stress. Our results show that the 27-OHC increased levels of ROS, H2O2 and 8-iso-PGF2α, and reduced levels of glutathione in rabbit hippocampus organotypic slices (Fig. 4). While treatment with siRNA to gadd153 alone didn't alter the levels of ROS, H2O2 and 8-iso-PGF2α, it significantly reduced 27-OHC-induced increase in levels of these oxidative stress markers. Interestingly, as it was the case with BACE1 and Bcl-2, treatment with siRNA to gadd153 alone markedly increased glutathione levels in comparison to levels in control untreated slices. Additionally, siRNA to gadd153 reversed the depletion of glutathione induced by 27-OHC (Fig. 4). Altogether, these results show that silencing the expression of gadd153 protects against the oxidative damage that can be triggered by 27-OHC.
10.1371/journal.pone.0026420.g004Figure 4 siRNA to gadd153 gene reduces 27-OHC-induced oxidative stress.
Silencing gadd153 gene expression reduces 27-OHC-induced increase in reactive oxygen species (ROS) generation, isoprostane formation and glutathione depletion. ROS generation was measured with increased fluorescence with the DCFH-DA assay (a), H2O2 levels were measured using Amplex red (b), isoprostane level were detected with formation of 8-iso-PGF2α (c) and glutathione depletion was assessed with reduction in glutathione (GSH): reduced glutathione (GSSG) ratio (d). *p<0.05, **p<0.01 and ***p<0.001 versus controls; #p<0.05, ##p<0.01 and ###p<0.001 versus 27-OHC.
27-OHC altered iron metabolism and increased TNF-α levels, effects reversed by siRNA to gadd153
Dyshomeostasis of iron metabolism has been suggested to mediate the neurodegenerative processes that characterize AD [17]–[21]. We determined levels of transferrin receptor (TfR) which regulates iron uptake and ferritin light and heavy chains that regulate iron storage. While treatment with 27-OHC reduced levels and expression of TfR, expression levels of ferritin light chain and ferritin heavy chain were significantly increased in slices treated with 27-OHC compared to untreated slices (Fig. 5a–d). Treatment with siRNA to gadd153, alone or in presence of 27-OHC, increased expression levels of TfR. siRNA to gadd153 significantly increased the 27-OHC-induced decrease in expression levels of TfR (Fig. 5a,b). On the other hand, 27-OHC increased expression levels of both ferritin light and heavy chain (Fig. 5a,c,d). Treatment with siRNA to gadd153 didn't affect basal levels of these proteins but reduced the increase in their expression levels induced by 27-OHC. The decrease in TfR and increase in ferritin levels resulting from 27-OHC treatment was associated with cellular accumulation of iron, as evidenced by the intense staining of phen-green SK, a sensitive fluorescence probe for free iron (Fig. 5e). The 27-OHC-induced iron accumulation was reduced with siRNA to gadd153. We also determined protein levels of the iron-regulatory proteins IRP-1 and IRP-2 that regulate levels of ferritin post-transcriptionally. Levels of IRP-1 and IRP-2 were significantly decreased with 27-OHC treatment compared to controls (Fig. 6a–c). Treatment with siRNA to gadd153 alone didn't alter levels of these proteins but reversed the decrease induced by 27-OHC.
10.1371/journal.pone.0026420.g005Figure 5 siRNA to gadd153 reduces alterations in iron homeostasis caused by 27-OHC.
Treatment with 27-OHC reduces transferrin receptor (TfR) levels and increases levels of ferritin light chain (FLC) as well as ferritin heavy chain (FHC) (a–d). Staining with Phen-green SK, a probe which the fluorescence of which is quenched by iron, shows an accumulation of iron in slices treated with 27-OHC compared to control slices and treatment with siRNA to gadd153 reduces the intensity of Phen-green SK staining (e). *p<0.05 and **p<0.01 versus controls; #p<0.05 versus 27-OHC.
10.1371/journal.pone.0026420.g006Figure 6 Reduction in iron-regulatory protein levels by 27-OHC is prevented by siRNA to gadd153.
While treatment with 27-OHC reduces levels of IRP-1 and IRP-2, siRNA to gadd153 reverses the effects of 27-OHC on IRP-1 and IRP-2 levels (a–c). REMSA assay shows a clear shift in bands resulting from binding of IRP to IRE in control (line 4) as well as in gadd153 siRNA alone- treated slices (line 6). No shift in band was observed in the slices treated with 27-OHC alone (line 5) demonstrating that 27-OHC inhibits binding of IRP to IRE. Treatment with siRNA to gadd153 in presence of 27-OHC causes a band shift indicating that siRNA to gadd153 partially, but significantly, restores the binding of IRP to IRE (line 7). Lanes 1–3 – positive controls; lane 1 - biotin labeled IRE probe, lane 2 - biotin labeled IRE probe+cytosolic liver extract, lane 3 - biotin labeled IRE probe+cytosolic liver extract+200 fold molar excess of unlabeled IRE RNA. *p<0.05 versus controls; #p<0.05 versus 27-OHC.
REMSA assay (Fig. 6d) shows a clear shift in bands in control as well as in gadd153 siRNA alone-treated slices indicating the binding of IRP to IRE. In the contrary, no shift in band was observed in the slices treated with 27-OHC alone, demonstrating that 27-OHC inhibits binding of IRP to IRE. A significant band shift was observed in slices treated with both siRNA to gadd153 and 27-OHC showing that siRNA to gadd153 partially, but significantly, restores the binding of IRP to IRE.
As activation of TNF-α has been shown to induce the expression of ferritin in a variety of cell lines [22], [23] and thus can dysregulate iron homeostasis, we determined levels of TNF-α, which is also a prominent marker of inflammation. Levels of TNF-α in tissue (a,b) and in media (d) were significantly increased with 27-OHC treatment compared to control levels (Fig. 7). Treatment with siRNA to gadd153, while didn't alter basal levels of TNF-α, it reduced the 27-OHC-induced increase in TNF-α levels. Real-time RT-PCR analysis also showed that siRNA to gadd153 reduced 27-OHC-induced increase in TNF-α expression (Fig. 7c).
10.1371/journal.pone.0026420.g007Figure 7 siRNA to gadd153 reduces 27-OHC-induced increase in TNF-α levels.
Western blot (a), densitometric analysis (b), and real-time RT-PCR (c) showing an increase in TNF-α expression levels with 27-OHC and a significant decrease in expression levels of TNF-α in slices treated with siRNA to gadd153. Levels of TNF-α released in media are also increased with 27-OHC treatment and these levels are reduced by siRNA to gadd153 (d). Values expressed are mean value ± SEM from three different experiments. *p<0.05 and **p<0.01 versus controls; #p<0.05 and ##p<0.01 versus 27-OHC.
Discussion
In the present study we show that treatment of organotypic slices with the oxysterol 27-OHC causes ER stress as evidenced with increased levels of the ER-specific proteins gadd153, grp 78 and grp 94. Stress in the ER is associated with increased Aβ production, phosphorylated tau levels, apoptosis, oxidative stress, and iron dyshomeostasis, which are all pathological hallmarks of AD. Remarkably, silencing gadd153 gene expression substantially reduced the generation of the AD hallmarks. Our data strongly suggest that increased levels of gadd153 plays an important role in the 27-OHC-induced effects.
Disturbances in ER functions leads to activation of ER stress response that involves various pathways and proteins (see for review [24]). Gadd153 is a member of the C/EBP family of bZIP transcription factors, and is expressed at low levels in normal conditions and is highly expressed in response to sustained stress in the ER [3], [4], [25]. Various transcription factors that activate the unfolded protein response activate gadd153 gene transcription. Overexpression of gadd153 has been shown to trigger apoptosis and to contribute to cell death by downregulating the anti-apoptotic protein Bcl-2 [4], [26]. Gadd153 deficiency protects against apoptosis in mice [27], [28] and cultured macrophages [29]–[31]. The mechanisms by which gadd153 induces apoptosis are not well known but downregulation of the anti-apoptotic protein Bcl-2 may play an important role [15]. We demonstrate here that silencing gadd153 gene expression reversed the 27-OHC-induced reduction in levels of Bcl-2 and increased levels of the apoptotic proteins Bax and caspase 3. Interestingly, siRNA to gadd153 increases the basal levels of Bcl-2, suggesting that gadd153 regulate the transcription of Bcl-2 gene. Previous studies have also shown that gadd153 sensitizes cells to ER stress by downregulating Bcl-2 [3] and upregulating Bax [4], [26]. It may also be possible that 27-OHC-induced accumulation of Aβ may trigger apoptosis by mechanisms independent of gadd153 as accumulation of Aβ is known to increase cell death by increasing Bax and decreasing Bcl-2 levels [32]–[34]. siRNA to gadd153 may reduce apoptosis indirectly by reducing BACE1 and APP, which are responsible for Aβ production. Whether 27-OHC causes apoptosis through increased levels of Aβ and/or directly by increasing levels of gadd153 is yet to be elucidated.
In addition to promoting apoptosis, gadd153 can increase ROS production [3], [16], [27], [35], leading to oxidative damage. Oxidative damage is considered as an early events in AD [36]. As both ER stress and oxidative damage are tightly linked and increased levels of ROS also triggers ER stress [16], the extent to which ER stress precedes oxidative damage or vice versa is not clear. Increased levels of ROS are known to enhance the activity of BACE1, the enzyme that initiates cleavage of β-APP to yield Aβ, thereby causing Aβ overproduction [6], [7]. Interestingly, we demonstrate that siRNA to gadd153 markedly reduces basal levels of BACE1 enzyme. These results suggest that gadd153 regulates transcription activity of BACE1. Further studies are needed to determine the cellular mechanisms by which gadd153 regulate BACE1 expression. There is evidence that BACE1 expression is transcriptionally regulated by NF-κB [37] and that gadd153 activated by ER stress regulates NF-κB signaling [38]. On the other hand, the ER has been shown to regulate Aβ production by controlling the protein folding for APP and delaying the access for BACE1 to process APP [39]. Alterations in the ER function subsequently to stress would lead to alterations in the processing and regulation of Aβ levels. Further studies are needed to determine the cellular mechanisms by which gadd153 regulate BACE1 expression in our system.
The increase in the levels of isoprostanes and depletion in the glutathione system foster oxidative stress. Decreased levels of glutathione (GSH) are a marker for increased free radical levels in AD [40]. Free radicals increase production of APP, Aβ as well as ROS [41], [42]. Our results show that 27-OHC-induced increase in ROS, isoprostanes and glutathione depletion are greatly reduced by siRNA to gadd153. Of seminal relevance, siRNA to gadd153 alone increases levels of glutathione to levels higher than basal levels suggesting, as is the case for Bcl-2 and BACE1, that gadd153 regulates glutathione activity. It has been suggested that gadd153 may interfere with glutathione synthesis by regulating γ-glutamyl cysteine synthase enzyme [3]. Effects of gadd153 on γ-glutamy cysteine synthase enzyme and glutathione synthesis are still to be investigated.
ER stress is associated with a number of diseases including neurodegenerative diseases, obesity, and atherosclerosis, [43]–[46]. Increased levels of gadd153 have been observed in PS-1 transgenic mice for AD [47]. In addition to increasing levels of gadd153, 27-OHC increases levels of ER molecular chaperone grp 78 and grp 94. Grp 78 has been shown to bind to β-APP in the ER and to reduce Aβ secretion in human embryonic kidney 293 cells [48]. Grp 78 has also been demonstrated to stimulate Aβ42 clearance in rat mixed glial cell cultures [49]. These results suggest that grp 78 facilitates the correct folding of APP in the ER and that alterations in grp 78 levels may cause the accumulation of extracellular Aβ42 [49]. Regarding grp 94, the expression of this protein has been found to increase in the parietal cortex of AD patients [50]. We have found increased levels of both grp 78 and grp 94. While the significance of increased levels of these proteins to 27-OHC-induced apoptosis, oxidative stress and Aβ accumulation is still to be elucidated, alterations in these levels is a direct indication of triggering of ER stress by 27-OHC. Gadd153 gene silencing reduced the increase in levels of these proteins.
Our results show that 27-OHC also increased phosphorylation of tau protein, another important hallmark of AD. The exact mechanism by which gadd153 regulates tau phosphorylation is yet to be determined. siRNA to gadd153 may reduce levels and activities of enzymes responsible for phosphorylation of tau. It may also be possible that reduction in Aβ by siRNA to gadd153 reduces phosphorylation of tau as tau phosphorylation is considered a downstream event to Aβ accumulation [51], [52]. Another important pathological hallmark observed in AD is disturbances in iron homeostasis. Iron is an essential element and participate in various toxic reactions and generate free radicals by Fenton reaction [2], [53], [54]. The iron homeostasis in the cells is maintained by interactions of iron regulatory proteins (IRP), ferritin, transferrin and transferrin receptor proteins. Ferritin is the iron storage protein and regulates the quantity of iron in the cell [55]. Transferrin receptor (TfR) is a transmembrane protein that transports iron into cells and its density is regulated by intracellular iron levels [56]–[58]. Transferrin (Tf), with the support of TfR, mobilizes iron [59], [60]. IRPs can sense iron levels in the cell and regulate the expression of proteins that are involved in iron metabolism [61]–[66]. Disruption of iron metabolism has been suggested to contribute to the pathogenesis of AD and other neurodegenerative diseases [67]. The iron accumulation we show with 27-OHC was associated with an increase in ferritin and a decrease in TfR as well as IRP-2 levels. An increase in level of ferritin was observed in AD compared to normal [68], varying depending on the brain regions [18]. Low levels of TfR have been observed in specific regions in the brain of AD in a study by Kalaria and colleagues [56]. The increase in the levels of iron with 27-OHC may have resulted from decreased expression of TfR, both at protein and mRNA levels, as TfR expression is strictly regulated by the intracellular iron levels and increased iron levels can lead to degradation of TfR mRNA [69]. Accumulation of ferritin and dysregulation of iron metabolism were observed in IRP-2 mutated mice [70] and alterations in the IRP-2 localization were reported in AD [71]. IRP-IRE binding regulates the iron levels in the cells. The high levels of iron in slices treated with 27-OHC is confirmed by REMSA assay that shows no band shift. A band shift would normally indicate that IRP did not bind to IRE probe. If the cells are overloaded with iron IRPs does not bind to IRE and the synthesis of ferritin and degradation of TfR increase. The increase in the levels of iron induced by 27-OHC can cause or exacerbate oxidative damage [72], [73] and ER stress [74]. Remarkably, siRNA to gadd153 substantially reduced disturbances in iron dyshomeostasis induced by 27-OHC. Activation of TNF-α may also potentially dysregulate iron homeostasis. Alteration in ferritin expression may result from TNF-α activation and may lead to the release of iron from ferritin stores, thereby increasing free iron levels. The increased levels of TNF-α with 27-OHC may also be a cause for the alterations in iron metabolism and neuronal death, as TNF-α can cause neuronal damage [75], [76]. Inflammation and TNF are known to contribute to the progression of AD. Interestingly, silencing gadd153 in the slices with siRNA has reduced the increased levels of TNF-α by 27-OHC, thus potentially reducing inflammation, oxidative stress and iron dyshomeostasis.
In summary, we demonstrate that the oxysterol 27-OHC induces AD-like pathology in organotypic slices from rabbit hippocampus. Therapeutic strategies for management of AD and other neurological disorders characterized by disturbed cholesterol and oxysterol metabolism have been suggested [77]–[79]. We also show that ER stress is an important event induced by the oxysterol 27-OHC. Interestingly, inhibition of downstream events to ER stress by silencing gene expression of the growth arrest and DNA-damage-inducible protein gadd153 markedly protects against 27-OHC-induced AD-like pathology. Several studies showing increased levels of 27-OHC in AD brains suggest that increased turn-over of cholesterol to 27-OHC may contribute to AD pathology. Our study is of seminal relevance to AD studies in that it adds new information on the role of gadd153 in underlying Aβ production, phosphorylated tau accumulation, oxidative stress generation and iron dyshomeostasis, which all are pathological hallmarks of AD. Preventing ER stress and silencing gadd153 expression may represent a strategy to prevent or reduce AD pathology.
Supporting Information
Figure S1 Western blot analyses showing that each of the three siRNA to gadd153 individually reduces gadd153 as well as BACE1 levels, thus confirming the specificity of the silencing of gadd153. *p<0.05, **p<0.01 and ***p<0.001 versus controls. ###p<0.001 versus 27-OHC.
(TIF)
Click here for additional data file.
The authors thank Dr. Byron Grove and Sarah Rolling (Department of Anatomy and Cell Biology, School of Medicine & Health Sciences, University of North Dakota) for their technical assistance in confocal microscopy.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by a grant from the National Institutes of Health (NIH) (RO1ES014826). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Mult Scler IntMult Scler IntMSIMultiple Sclerosis International2090-26542090-2662Hindawi Publishing Corporation 2209663110.1155/2011/246412Review ArticleConsensus Guidelines for CSF and Blood Biobanking for CNS Biomarker Studies Teunissen Charlotte E.
1
*Tumani Hayrettin
2
Bennett Jeffrey L.
3
Berven Frode S.
4
Brundin Lou
5
Comabella Manuel
6
Franciotta Diego
7
Federiksen Jette L.
8
Fleming John O.
9
Furlan Roberto
10
Hintzen Rogier Q.
11
Hughes Steve G.
12
Jimenez Connie R.
13
Johnson Michael H.
14
Killestein Joep
15
Krasulova Eva
16
Kuhle Jens
17
Magnone Maria-Chiara
18
Petzold Axel
19
Rajda Cecilia
20
Rejdak Konrad
21, 22
Schmidt Hollie K.
23
van Pesch Vincent
24
Waubant Emmanuelle
25
Wolf Christian
26
Deisenhammer Florian
27
Giovannoni Gavin
28
Hemmer Bernhard
29
1Department of Clinical Chemistry, Neurological Laboratory and Biobank, VU University Medical Center, FdG, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands2University of Ulm, Ulm 89081, Germany3Departments of Neurology & Ophthalmology, University of Colorado Denver, Aurora, USA4Proteomics Unit (PROBE), Department of Biomedicine, University of Bergen, Bergen 5020, Norway5Division Neurology, Department Clin Neuroscience, Karolinska University Hospital, Stockholm, Sweden6Neurology, Centre d'Esclerosi Múltiple de Catalunya, CEM-Cat, Unitat de Neuroimmunologia Clínica, Hospital Universitari Vall d'Hebron, Barcelona, Spain7Laboratory of Neuroimmunology, IRCCS, “C. Mondino Neurological Institute”, Pavia, Italy8Neurology, Glostrup Hospital, University of Copenhagen, Glostrup 2600, Denmark9Department of Neurology, University of Wisconsin, Madison 53705, WI, USA10Clinical Neuroimmunology Unit, Institute of Experimental Neurology, Division of Neuroscience, San Raffaele Scientific Institute, 20132 Milan, Italy11Neurology, Erasmus MC, Rotterdam, The Netherlands12Clinical Developement Department, Isis Pharmaceuticals, Carlsbad, California 92024, USA13OncoProteomics Laboratory, Department of Medical Oncology, VU University Medical Center, 1007 MB Amsterdam, The Netherlands14Department of Neurology, Leeds Teaching Hospitals NHS Trust, Leeds LS1 3EX, UK15Department of Neurology and Center of Clinical Neuroscience, 1st Faculty of Medicine and General University Hospital, Charles University, 128 21 Prague, Czech Republic16Department of Neurology, VU University Medical Center, 1007 MB Amsterdam, The Netherlands17Neurology and Clinical Neuroimmunology, University Hospital, University of Basel, Basel 4031, Switzerland18F. Hoffmann- La Roche Pharma, Basel, Switzerland 19Department of Neuroimmunology, UCL Institute of Neurology, Queen Square, wc1n 3bg London, UK20Department of Neurology, University of Szeged, Szeged, Hungary21Department of Neurology, Medical University of Lublin, Lublin, Poland22Department of Experimental Pharmacology, Medical Research Center, Warsaw, MA 02451, Poland23Accelerated Cure Project for Multiple Sclerosis, Waltham, MA, USA24Neurology Department UCL, Université Catholique de Louvain, Brussels CA94117, Belgium25Clinical Development, Lycalis sprl, 1180 Brussels, Belgium26Clinical Development, UCB Pharma S.A., 6020 Braine l'Alleud, Belgium27Department of Clinical Neurology, Innsbruck Medical University, Innsbruck E1 2AT, Austria 28Queen Mary University of London, Neuroscience & Trauma Centre, Blizard Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, London, UK29Deptartment of Neurology, Klinikum rechts der Isar, Technische Universität, Munich 81245, Germany*Charlotte E. Teunissen: [email protected] Editor: Helmut Butzkueven
2011 18 7 2011 2011 24641225 11 2010 5 4 2011 Copyright © 2011 Charlotte E. Teunissen et al.2011This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.There is a long history of research into body fluid biomarkers in neurodegenerative and neuroinflammatory diseases. However, only a few biomarkers in cerebrospinal fluid (CSF) are being used in clinical practice. Anti-aquaporin-4 antibodies in serum are currently useful for the diagnosis of neuromyelitis optica (NMO), but we could expect novel CSF biomarkers that help define prognosis and response to treatment for this disease. One of the most critical factors in biomarker research is the inadequate powering of studies performed by single centers. Collaboration between investigators is needed to establish large biobanks of well-defined samples. A key issue in collaboration is to establish standardized protocols for biobanking to ensure that the statistical power gained by increasing the numbers of CSF samples is not compromised by pre-analytical factors. Here, consensus guidelines for CSF collection and biobanking are presented, based on the guidelines that have been published by the BioMS-eu network for CSF biomarker research. We focussed on CSF collection procedures, pre-analytical factors and high quality clinical and paraclinical information. Importantly, the biobanking protocols are applicable for CSF biobanks for research targeting any neurological disease.
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1. Introduction: The Need for Collaborative Biobanking and Biomarker Studies
NMO can be diagnosed based on a blood-derived biomarker, that is antibodies against aquaporin-4, a channel protein present on astrocytes, extensively discussed in other contributions in this special issue. The presence of antibodies against aquaporin-4 has been proven as one of the most successful results of biomarker studies, and is supportive for the idea that central nervous system (CNS) abnormalities are reflected in changes in body fluids. It also proofs the autoimmune component of this disorder and of pathologies that are related to the NMO spectrum disorders, such as longitudinally extensive transverse myelitis.
Determination of serum anti-aquaporin-4 antibody levels is a mainstay in the diagnosis of NMO, but the discovery of such disease-specific antibodies is relatively recent [1], and, therefore, further studies in body fluids are warranted. One case report suggested that NMO-immunoglobulin G (IgG), the NMO-associated antibodies that are reactive to cerebellar tissue [1], can be absent in serum, but present in CSF [2]. However, another study on a relative large cohort of patients showed that testing CSF does not increase diagnostic sensitivity [3]. Another recently identified candidate biomarker for NMO is glial fibrillary acid protein (GFAP). Takano and colleagues observed that the analysis of CSF glial fibrillary acid protein is useful in the differential diagnosis between NMO and multiple sclerosis or acute demyelinating encephalomyelitis, and that its CSF levels at disease onset correlated with expanded disability score scale (EDSS) in NMO [4]. However, studies on larger cohorts are needed before drawing definite conclusions. Taken together, no biomarkers are available yet for prognosis or therapy response in NMO and in NMO-related disorders. Therefore, biomarker studies on CSF are ongoing.
One important flaw in several previously performed biomarker studies in CNS diseases has been the lack of large cohorts to sufficiently power the study. This is especially an issue for such a rare disease as NMO, where a single center will not be able to collect a large cohort within a reasonable time frame. The need for collaboration was the reason for biomarker researchers in Multiple Sclerosis to start a network (BioMS-eu, http://www.bioms.eu/). The aim of this collaboration is to obtain well-proven, high-quality biomarkers, which will be achieved by sharing patient samples, standardization, and improvement of procedures important in the research area. One of the most urgent prerequisites for collaboration was felt to be standardization of biobanking protocols. Therefore, a consensus-meeting was organised and the result was collection and biobanking guidelines, which the network developed and published in 2009 [5]. There are currently major efforts worldwide to professionalize biobanks and the collection and biobanking guidelines established by consensus among 26 groups participating in BioMS-eu (http://www.bioms.eu/) is a major achievement in the CNS biomarker field [5]. One year after publication of the guidelines, over 90% of the BioMS-eu laboratories had already adapted their procedures in agreement with the guidelines. A great use of the guidelines is the applicability for any neurological disease, including NMO, and that it provides guidelines for setting up a novel biobank. Furthermore, it will greatly facilitate biomarker studies in the CNS biomarker research area. In the concensus discussions, we have sought a balance between practicality and scientific rationale, and the background of each decision is provided. Before the consensus, it was clear that large differences were present between collection protocols, highlighting the need to address these differences (Figure 1 and Table 1). In the current paper, we include only the items and their rationale from the original paper that are relevant for biobanking for NMO. Other modifications from the original protocol is an adaptation of item 1 (samples should be pooled if multiple collection tubes are used for one patient), and the inclusion of an item addressing transportation (item 20) and information of some more physiological confounders (item 25).
We would like to stress that researchers adhere to these protocols for optimal collaboration in the field of CSF biomarker research. We suggest using Tables 2 and 3 as a checklist for CSF biomarker research and recommend that future studies of CSF biomarker take these issues into account. In discovery-based biomarker research, all these items should be considered carefully before initiating a study. Some procedures may not be possible in everyday clinical practice (e.g., processing within one hour), but less stringent requirements will suffice for specific research questions. Therefore, careful documentation of these issues is crucial to facilitate retrieval of appropriate samples dictated by specific study aims. As indicated before, the procedures for withdrawal and storage of CSF (Table 2) are broadly applicable for any neurological disease.
Besides methodological issues, ethical approval is a crucial prerequisite for collaboration between international or national centers. The signed informed consent should include a statement that exchange of samples between (international) centers is allowed. Furthermore, to bring a biomarker to clinical practice, one may need patents and the involvement of industrial partners could be needed, who have the infrastructure for large-scale production, quality control procedures, and to reach as many laboratories as possible. For large-scale validation studies, patient samples are of course also needed, and it will be wise to consider this possibility at the start of biobank formation and, if ethical laws permit and the option is perceived to be important, indicate the possibility for industrial cooperation in the patient information and consent.
Lastly, researchers should be willing to share their samples and information for the benefit of the whole, that is, obtaining reliable biomarkers that can be used for patient care and cure.
2. Guidelines for CSF Biobanking for Biomarker Research, Rationale, and Details
2.1. Procedure of CSF Collection (Table 2)
(A) Collection Procedures:
Item 1 Volume of Withdrawal of at Least 12 mL
The CSF volume taken can influence the concentration of biomarkers. Most molecules and cell numbers have a rostrocaudal concentration gradient [6, 7]. If a small volume is taken, the CSF will reflect the composition of the lumbar dural sac, whereas large volumes may reflect the rostral spinal or even ventricular CSF. Therefore, if biomarker concentrations in a sample from a puncture of 2 mL are compared to that in a puncture of 15 mL, this can lead to erroneous results. Also collecting different portions of the CSF for biobanking (e.g., initial and final volumes of the puncture) may introduce errors. Thus, a standard volume of CSF should be collected during lumbar puncture, the first 2 mL can be used for basic CSF analysis (item 26), and the remainder of the sample should be pooled before spinning and aliquoting. At least, the procedure must be recorded. The volume of collected CSF does not correlate with the risk of postlumbar puncture headache [8, 9].
Item 2 Location of Puncture: Vertebral Body L3–L5
Usually, diagnostic CSF is obtained by lumbar puncture. Because of the increasing gradient in protein concentration from ventricular to lumbar CSF [10], the site of CSF withdrawal must be recorded. When CSF is taken from other locations such as the cervical cisterns or from the lateral ventricles (e.g., ventricular drainage), this should be documented.
Item 3 Removal of Bloody CSF Samples
A traumatic tap causing blood contamination of CSF occurs in about 14–20% of standard lumbar punctures [11]. For biomarkers that have high serum concentrations, such as coagulation factors, blood contamination can lead to false positive results. In addition, blood proteins lead to suppressed matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-TOF/MS) proteomics patterns in CSF. This suppression by blood proteins is, however, highly reduced after removal of the blood cells by centrifugation prior to initial freezing [12, 13]. Recording of erythrocyte count is essential to select CSF samples appropriate for these measurements. CSF samples with an erythrocyte count above 500/μL should not be used for biomarker studies.
Item 4 Use of Atraumatic Needle (Sprotte or Whitacre Needle)
There is no evidence that the type of lumbar puncture needle influences biomarker concentrations. However, atraumatic needles are best tolerated by patients, and are associated with a lower risk for postlumbar puncture headache, that is about 12% for a needle size of 20–22 G compared to about 70% for a needle size of 16–19 G [14, 15].
Item 5 Use of Polypropylene Collection Tubes
There are several reports showing that the type of collection tube influences biomarker outcomes, for example, total tau proteins and amyloid β peptides [16]. Therefore, standardization is important. We propose to use polypropylene tubes, with their low protein binding potential, for collecting CSF. No additives should be used. Glass tubes should be avoided, due to safety reasons for personnel. When multiple tubes are used, the total volume should be mixed after centrifugation to avoid gradient effects.
Item 6 Time of the Day of Withdrawal
For biomarkers that are influenced by circadian rhythm, time of withdrawal is important [17]. Since it is often difficult to accomplish standardization of withdrawal time in everyday clinical practice, documentation is necessary to select the appropriate samples to minimize the effect of this variable.
Items 7 and 8 Serum, Plasma, and DNA Linked to the CSF Sample
It is important to collect matched serum and/or plasma samples for evaluation of CSF biomarkers because the concentration of the marker in blood often influences that in CSF [18]. Further, serum/plasma pairs are essential to study the intrathecal origin of a biomarker and its CNS specificity. Furthermore, the presence of CNS markers in serum/plasma may aid in disease monitoring. Vacuum tubes that use EDTA (in dried format) are preferred over those that use citrate (in solution) because if tubes containing a standard volume of citrate are filled incompletely, the final biomarker concentration is diluted unequally compared to other samples. Depending on the type of biomarkers and methods of study, we recommend collecting both serum and plasma [19]; for some methods, plasma is preferred over serum and vice versa. Serum/plasma samples should not be haemolysed. We advise to perform a blood draw using vacuum systems, since tourniquet use is related to additional confounding factors in the preanalytic phase include tourniquet time and posture [20]. Furthermore, instructions of the supplier should be followed, such as mixing.
Lastly, DNA collection expands the possibilities for studying the phenotypes and genotypes within individuals. A protocol for storage and handling of DNA can be found in the supplementary files (E-Appendix 1).
(B) Processing for Storage:
Item 9 Storage at Room Temperature Until Spinning and Aliquoting
For CSF, there are no data available yet that support a preference for leaving the samples at room temperature or at 4°C until processing. For serum/plasma preprocessing temperature is more crucial. To avoid platelet activation [21], serum/plasma samples should be kept at room temperature before centrifugation. Therefore, processing at room temperature for both serum/plasma and CSF, including during and after spinning, is suitable for most studies. Relatively few systematic studies have been performed on this issue. We would recommend exploratory studies to define the effect of temperature on specific biomarkers.
Item 10 Standardized Spinning Conditions
We propose to adhere to a standardized spinning protocol of 400 g for 10 minutes at room temperature when fragile cells need to be preserved for RNA of cell isolation, and otherwise at 2,000 g. For serum/plasma, we propose to spin at 2,000 g for 10 min at room temperature. Standardization of spinning temperature and speed may be important for some biomarkers, although no studies have addressed these specific preanalytical variables for CSF. For plasma and serum, temperature of processing is known to be critical for specific biomarkers [22]. After centrifugation, the supernatant must be aliquoted and stored immediately. If this is not done, the processing time should be documented.
Item 11 Standardization of Time-Delay between Withdrawal, Spinning, and Freezing
Studies of the effects of preanalytical variables by MALDI-TOF/MS proteomics (proteins/peptides <20 kD) have shown that the time between sampling and storage is more crucial for specific serum proteins or peptides than for CSF, [12, 13, 23]. For CSF, it was observed that processing within two hours does not lead to artefactual results [12, 13]. For serum, it was observed that small differences in processing time (~10–30 min) can result in changes in the protein profile [19]. Some biomarkers, such as antibodies or specific cytokines, are not very sensitive to sampling and storage conditions [24]. For practical reasons, and in view of the standard of 30–60 min clotting time for serum, we recommend a time delay of 1.5 hours (±30 min) for both matrices. When CSF cells are to be preserved, processing as soon as possible is to be advised as cell numbers decrease quickly. However, in most of the centers, processing of the body fluid samples within one hour is not common practice. Therefore, documentation of time of withdrawal and storage is required in order to select uniform samples. For newly discovered biomarkers, these preanalytical variables should be evaluated.
Item 12 Use of Small Polypropylene Tubes for Aliquoting
Due to the same rationale as for CSF withdrawal (item 5), we recommend that polypropylene tubes should be used for aliquoting and storage. Furthermore, vials with screw caps should be used for a secure sealing. The proposed tube size is 0.25, 0.5, and 1 mL.
Item 13 Aliquoting
Freeze/thaw cycles can influence biomarker concentrations [25]. For example, one-time freezing of CSF samples can lead to a highly significant loss of amyloid β(1-42) which is decreased a further 20% after three more thawing cycles [26, 27]. By contrast, no effects on CSF proteome profiles obtained by MALDI-TOF/MS have been observed after up to four freeze/thaw cycles [13].
In principle, repeated freeze/thawing of samples should be avoided, as data addressing this topic are available for only a few biomarkers and the response to freeze/thaw cycles of new biomarkers is not known. Thus, splitting the pooled sample in multiple small aliquots is optimal, and possible freeze/thaw cycles should be recorded.
Item 14 Volumes of Aliquots of 0.2, 0.5, and 1 mL
Small aliquot volumes are optimal to avoid freeze/thawing and to avoid waste of CSF. Tubes should be filled up to 75% to prevent freeze-drying within the tube, which will affect the concentration of biomarkers, although it may only be a problem if the seal of the cryogenic tubes are not airtight. This issue has not been formally studied and is not referred to in related standard operating procedures [28].
Item 15 Coding and Use of Freezing-Proof Labels
Unique codes are necessary to track samples and pair with clinical data. Ideally barcodes should be used to facilitate searching, to aid in blinding the analysis, and to protect the privacy of patients. It is important to have center-unique codes, to track data retrospectively. Labels must be water and frost (−80°C) resistant.
(C) Storage and Administration of Samples (Table 2, Lower Part):
Item 16 Freezing Temperature of −80°C
Proteins may not be stable at −20°C for years. In one study, the effect of storing CSF at −20°C and −80°C on cystatin C, an abundant CSF protein, was investigated. Cleavage of this protein occurred in all samples stored at −20°C but not in samples stored at −80°C [29]. Apart from the cystatin C truncation, changes in the low molecular weight polypeptide profile due to CSF sample storage at −20°C for three months appeared to be minimal [12, 13]. Oligoclonal bands in CSF may be recovered after several years of storage at −20°C indicating a high stability of immunoglobulins. Nevertheless, self-defrosting freezers must not be used. No data are available showing the benefit of storage of CSF or serum in liquid nitrogen. As this is expensive and not practical for CSF biobanking, there is no basis yet to recommend storage in liquid nitrogen.
Taken together, we recommend that samples are stored at −80°C to ensure long-term stability of biomarkers.
Item 17 Location of Samples
To enable easy tracking and fast relocation of samples, storage information should include freezer location, freezer identification, and sample location within freezer.
Items 18 and 19: Surveillance of Freezers and Splitting of Samples
Freezers should be alarm controlled and a sample rescue plan established and documented. All freezers must be registered in a freezer log file. Ideally, daily temperature logs should be available for all freezers. Aliquots of samples should be distributed among different freezers, although not absolutely needed if good surveillance is in place. An empty, an empty back-up freezer should be available.
Item 20: Transport Conditions and Thawing before Use
Transport of frozen samples should always be performed on dry-ice, and the volume should be sufficient for transport for minimal 3 days. Preferably, transports are initiated on Monday for the samples to arrive within the same week. Once the samples have arrived and are ready for experiments, excessive thawing temperatures (such as 37°C) are to be avoided to prevent protein degradation. Furthermore, inadequate agitation can cause salt and protein gradients to form in thawed samples.
(D) Patient Information Requirement in Database:
Items 21-22 Basic Demographics, such as Age and Gender
Information on the age at sampling is needed to allow comparability to age-matched reference values, since many proteins show age-dependent changes, for example, albumin or IgG [30]. Ideally, date of birth and date of sampling are recorded. Gender has to be provided due to variability of markers influenced by hormones.
Item 23 Ethnicity
Reference ranges of biomarkers can be influenced by the genetic status [31]. For example, a recent study observed a higher IgG index in African Americans than in Caucasians, unrelated to socioeconomic status [32]. Criteria for race and ethnicity are available via the website of the National Institutes of Health [33].
Item 24 Treatment at Sampling and Year before Sampling
It is well known that commonly used drugs for treatment of MS, including immunomodulatory agents and use of methylprednisolone for treatment or prevention of relapses, have an influence on expression of biomarkers [34, 35]. Other treatments could likewise influence biomarker results in NMO patients. Therefore, type and duration of treatment should be documented in detail, preferably beginning at least one year before CSF collection.
Item 25 Fasting, Infections and Pregnancies
Other relevant physiological variables that can influence CSF and blood analyte levels should be recorded including fasting versus non fasting, pregnancy, and underlying nonneurological conditions such as infections [20].
Item 26 Basic CSF Analysis (Protein, Cell Counts, Erythrocytes, Etc…)
To enable stratification of patients according to their CSF findings and to evaluate suitability of samples for further analysis, results of basic CSF analysis should be recorded. Primarily, the CSF profile serves for exclusion of other diseases. In addition, quantitative changes of immunological markers are likely to occur depending on disease stage, relapse activity, and medication. Inflammatory processes may influence the blood-CSF barrier function and thereby biomarker concentrations [18].
The presence of Oligoclonal IgG bands (OGB) in NMO is quite distinct from that in MS in that OGBs in MS are persistent, while they are transient in NMO [36, 37]. For example, OGBs were detected in 399 of 411 MS patients (97%) and never disappeared. In NMO, OGBs were detected in three of 11 patients (27%) and always disappeared. The sensitivity of oligoclonal IgG bands is strongly dependent on the method used. We strongly recommend isoelectric focusing followed by immunoblotting and staining for IgG [38, 39]. Preferably, the methods of all routine diagnostic procedures, including oligoclonal banding, should be documented.
Item 27 Data in the CSF Database in English
The mask on the database screen could be in the local language, but the underlying files will need to be in English. It is strongly suggested to use a commercially available program, if not a common database for networks like BioMS-eu. The database should also adhere to standardized international units.
3. Concluding Remarks
The lists provided in Table 2 can be used as an easy checklist for CSF biobanking for any CNS disease, applicable during setup of the procedures and also as a checklist for recording sample characteristics. It is expected that these standardizations will pave the way for large biomarker studies and fruitful collaborations. In the original paper, we present guidelines for outcome measures to be included for MS biomarker studies [5]. For NMO, standardisation of outcome measures is still needed. Ultimately, these endeavors are to arrive at validated biomarker assays for diagnosis, prognosis, and treatment of CNS diseases and a potential to elucidate relevant disease mechanisms.
Acknowledgments
Other participants of the BioMS-eu meeting in London, March 2007, are acknowledged for their contribution to the discussions resulting in this guideline: H. Abderrahim, S. Al-Izki, D. Baker, R. Banks, Y. Ben-Schloma, A. Berthele, A. Bertolotto, R. Farrell, J. Furby, R. Gani, F. Gilli, S. Gnanapavan, B. Gomez, B. Greco, E. Hauben, E. Havrdova, T. Hayton, E. Iacobaeus, A. Jaber, N. Jafari, S. Jackson, R. Kapoor, G. Keir, A. Kok, A. Konieczny, A. Kroksveen, V. Lampasona, R. Lindberg, A. Lockhart, C. Luk, C. Maggiore, M. Mauritzio, A. Millonig, H. Parkes, T. Plitz, D. Sadovnik, A. Sala, R. Sachse, K. Schmierer, K. Smith, S. Suessmith, P. Thompson, H. L. Weiner, B. Wilson, D. Wright, J. Zajicek, and P. Zaratin.
Figure 1 Results of inventory of collection procedures among 14 European centers with CSF biobanks for MS research in 2006. (a) Other body fluids that are collected simultaneously with CSF. Filled area indicates “yes”; open areas: not collected. (b) Storage temperature of CSF and serum. Open area: −80; closed area: −20; grey area: not collected. (c) Average volume of CSF that is collected per patient per CSF withdrawal. Bars indicate the average and ranges of volume per center. (d) Time-delay between CSF withdrawal, spinning and storage into the freezer. Bars indicate the average and ranges of time per center.
Table 1 Results of inventory on collection protocols among 14 MS Biomarker Research Centers.
Procedure of CSF withdrawal Previous status among European CSF centers
Type of needle: 71% atraumatic, 21% traumatic, 8% both
Time of the day of withdrawal (important for markers that are sensitive for circadian rhythm) 71% no specific day/time of withdrawal, 29% in the afternoon only
Temperature until storage 57% room temperature, 43% at 4°C
Type of tube: 50% Sarstedt, 29% Eppendorf, 21% other
aliquoting: Range from 0.2 mL to 2 mL
(1) surveillance of freezers Present at 93% of the centers
(2) several freezers to split the samples (backup) Present at 14% of the centers
Table 2 Guidelines for procedure of CSF withdrawal.
Item no. Procedure Ideal situation
(A) Collection procedures
(1) Preferred volume At least 12 mL. First 1-2 mL for basic CSF assessment (item 26). Last 10 mL for biobanking. Record volume taken and fraction used for biobanking, if applicable.
(2) Location Vertebral body L3–L5
(3) If bloody Do not process further.
Criteria for bloody: more than 500 red blood cells/μL.
Record number of blood cells in diagnostic samples.
(4) Type of needle Atraumatic
(5) Type of collection tube Polypropylene tubes, screw cap, volume >10 mL.
(6) Time of day of withdrawal and storage Preferably standardized within each center allowing for intercenter differences in local logistics.
Record date and time of collection.
(7) Other body fluids that should be collected simultaneously Serum
(8) Other body fluids that should be collected simultaneously Plasma: EDTA (preferred over citrate).
(B) Processing for storage
(9) Storage temperature until freezing Room temperature before, during, and after spinning.
(10) Spinning conditions Serum: 2,000 g, 10 min at room temperature.
CSF: 400 g, 10 min at room temperature/2,000 g if no cells are to be preserved.
(11) Time delay between withdrawal and spinning and freezing Optimal for CSF: 1-2 hours
Optimal for serum: 30–60 min.
Thus doing “both body fluids simultaneously”: ideally within one hour.
After spinning, samples must be aliquoted and frozen immediately for storage at −80ºC.
(12) Type of tube for aliquoting Small polypropylene tubes (1 to 2 mL) with screw caps. Record manufacturer.
(13) Aliquoting A minimum of two aliquots is recommended. The advised research sample volume of 10 mL should be enough for >10 aliquots.
(14) Volume of aliquots Minimum 0.1 mL. Depending on total volume of tube: 0.2, 0.5, and 1 mL. Preferably, the tubes are filled up to 75%.
(15) Coding Unique codes. Freezing-proof labels. Ideally barcodes to facilitate searching, to aid in blinding the analysis and to protect the privacy of patients.
(C) Storage conditions and administration
(16) Freezing temperature −80ºC
(17) Additional items on sample collection protocols that must be recorded Location of samples
(18) Additional items on sample collection protocols that must be recorded Surveillance of freezers
(19) Additional items on sample collection protocols that must be recorded Splitting of samples over two or more freezers
(20) Transport conditions Always on dry-ice, sufficient volume of dry-ice for minimal 3 days of transport.
Initiated on Mondays.
Avoid high temperatures for thawing and mix thoroughly.
Table 3 Guidelines for patient information requirement in databases of MS patients.
Item no. (D) Patient information requirement in databases
(a) Basic demographics
(21) (1) date of birth (age if date of birth is not available)
(22) (2) Gender
(23) (3) Ethnicity
(24) (4) Use of drugs, at sampling and year before sampling.
(25) (5) Actual nonneuronal infections, fasting or nonfasting, pregnancy.
(26) (6) Basic CSF analysis (CSF cell count, differential cytology, erythrocyte count, oligoclonal IgG bands (which is at least two bands by definition), albumin ratio, total protein (if albumin is not measured), and IgG index)
(7) Record the methods of routine analysis
(27) (8) The data in the CSF database should be in English and use Standardized International Units
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22028792PONE-D-11-1274310.1371/journal.pone.0025826Research ArticleBiologyMolecular cell biologySignal transductionSignaling CascadesAkt Signaling CascadeSignaling in cellular processesAntiapoptotic signalingG-protein signalingGTPase signalingRas signalingMechanisms of Signal TransductionMembrane Receptor SignalingMedicineOncologyCancer TreatmentAntiangiogenesis TherapyPlexin-B1 Activates NF-κB and IL-8 to Promote a Pro-Angiogenic Response in Endothelial Cells Plexin-B1 Activates NF-κB and IL-8Yang Ying-Hua
1
Zhou Hua
1
Binmadi Nada O.
1
Proia Patrizia
1
2
Basile John R.
1
3
*
1
Department of Oncology and Diagnostic Sciences, University of Maryland Dental School, Baltimore, Maryland, United States of America
2
Department of Sports Science (DISMOT), University of Palermo, Palermo, Italy
3
Marlene and Stuart Greenebaum Cancer Center, Baltimore, Maryland, United States of America
Bozza Patricia T. EditorFundação Oswaldo Cruz, Brazil* E-mail: [email protected] and designed the experiments: JRB Y-HY. Performed the experiments: Y-HY HZ NOB PP. Analyzed the data: JRB Y-HY. Contributed reagents/materials/analysis tools: Y-HY HZ NOB PP. Wrote the paper: JRB Y-HY.
2011 18 10 2011 6 10 e258266 7 2011 11 9 2011 Yang et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
The semaphorins and their receptors, the plexins, are proteins related to c-Met and the scatter factors that have been implicated in an expanding signal transduction network involving co-receptors, RhoA and Ras activation and deactivation, and phosphorylation events. Our previous work has demonstrated that Semaphorin 4D (Sema4D) acts through its receptor, Plexin-B1, on endothelial cells to promote angiogenesis in a RhoA and Akt-dependent manner. Since NF-κB has been linked to promotion of angiogenesis and can be activated by Akt in some contexts, we wanted to examine NF-κB in Sema4D treated cells to determine if there was biological significance for the pro-angiogenic phenotype observed in endothelium.
Methods/Principal Findings
Using RNA interference techniques, gel shifts and NF-κB reporter assays, we demonstrated NF-κB translocation to the nucleus in Sema4D treated endothelial cells occurring downstream of Plexin-B1. This response was necessary for endothelial cell migration and capillary tube formation and protected endothelial cells against apoptosis as well, but had no effect on cell proliferation. We dissected Plexin-B1 signaling with chimeric receptor constructs and discovered that the ability to activate NF-κB was dependent upon Plexin-B1 acting through Rho and Akt, but did not involve its role as a Ras inhibitor. Indeed, inhibition of Rho by C3 toxin and Akt by LY294002 blocked Sema4D-mediated endothelial cell migration and tubulogenesis. We also observed that Sema4D treatment of endothelial cells induced production of the NF-κB downstream target IL-8, a response necessary for angiogenesis. Finally, we could show through co-immunofluorescence for p65 and CD31 that Sema4D produced by tumor xenografts in nude mice activated NF-κB in vessels of the tumor stroma.
Conclusion/Significance
These findings provide evidence that Sema4D/Plexin-B1-mediated NF-κB activation and IL-8 production is critical in the generation a pro-angiogenic phenotype in endothelial cells and suggests a new therapeutic target for the anti-angiogenic treatment of some cancers.
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Introduction
The semaphorins are a family of secreted, transmembrane and glycosylphosphatidylinositol-linked proteins characterized by large cysteine-rich semaphorin domains that were originally identified based on their ability to provide both attractive and repulsive axon guidance cues during neural development [1]. They are now known to be expressed in tissues outside of the nervous system where they are involved in many motility responses including regulation of cell-cell contacts and branching morphogenesis in epithelium [2], promotion of angiogenesis [3], [4], and growth and metastasis of tumors [5], [6]. The main functional receptors for semaphorins are a family of single pass transmembrane proteins known as plexins [7], [8]. The intracellular portion of the plexins contain a GTPase-activating protein (GAP)-like motif that downregulates activity of the G protein R-Ras, interrupted by a region capable of binding small Rho family GTPases [9], [10]. In the case of Plexin-B1 for example, this Rho GTPase binding domain (RBD) can associate with Rnd1, Rac1, and RhoD, the binding of which influences plexin functioning [10]. The small GTPases act as molecular switches that cycle between an active GTP-bound and inactive GDP-bound form to regulate microtubule dynamics, cell shape and cell mobility [11]. Therefore, it is likely that the binding of semaphorins to plexins initiates a signaling cascade that impinges upon the cytoskeleton [12]. In addition to the small GTPases, kinase activity plays a vital role in plexin signaling. The plexins themselves are devoid of any intrinsic kinase activity, so this function is provided by kinases activated by the signaling complex, depending upon the class of plexins and the context.
The nuclear factor (NF)-κB family of transcription factors plays an important role in the ability of a cell to adapt to environmental changes and figures prominently in many biological processes. NF-κB dimers positively or negatively regulate expression of target genes in response to bacterial products, cytokines, viral infection, growth factors and other stressful stimuli [13]. In the inactive form, NF-κB is bound to the inhibitor of κB (I-κB) family of proteins that sequester NF-κB dimers in the cytoplasm. Upon stimulation, the I-κB kinase (IKK) complex phosphorylates the I-κB proteins on two conserved N-terminal serine residues, which target them for E2- and E3-ligase-mediated polyubiquitination and subsequent 26S proteasomal degradation [14]. This process releases and activates NF-κB, freeing it up to move to the nucleus where it undergoes a series of posttranslational modifications and binds to specific DNA sequences in target genes (designated as “κB elements”) that regulate the transcription of over 500 genes involved in inflammation, the immune response, cell growth control and the regulation of cell survival [14]. Importantly, dysregulated NF-κB activity has been associated with tumor promotion, suppression of apoptosis, tumor-induced angiogenesis and metastasis [15].
Our lab and others have shown that Semaphorin 4D (Sema4D) is pro-angiogenic when acting through its receptor Plexin-B1 on endothelial cells [3], [4] and may be produced by malignancies for the purposes of promoting blood vessel growth into the tumor [16]. There is a PDZ binding motif at the C-terminus of Plexin-B1 that associates with PDZ-Rho guanine nucleotide exchange factor (GEF) and leukemia-associated RhoGEF (LARG), proteins that activate Rho [17], [18]. Sema4D binding to Plexin-B1 also promotes Rnd1-dependent activation of receptor GAP activity and R-Ras inhibition [9], [10]. Finally, there is evidence that Plexin-B1 competes with p21-activated kinase (PAK) for Rac binding, sequestering the active form of Rac and inhibiting Rac-dependent processes [19]. We previously have demonstrated that pro-angiogenic Plexin-B1 signaling is dependent upon its ability to activate Rho, specifically by signaling through the downstream effector Rho kinase (ROK) and activating Akt, Src and Pyk2 [20].
Since NF-κB has been linked to promotion of angiogenesis in some malignancies [21] and IKK can be phosphorylated by Akt, leading to activation of NF-κB [22], we wanted to examine NF-κB in response to Plexin-B1 stimulation and determine if there was any biological significance for the pro-angiogenic phenotype observed in endothelial cells. Here we show that NF-κB and its pro-angiogenic effector IL-8 are activated downstream of Plexin-B1. In endothelial cells, this response occurred following treatment with Sema4D and was necessary for chemotaxis, capillary tube formation and resistance to apoptosis. We could elicit NF-κB activation in endothelial cells when treated by conditioned media from head and neck squamous cell carcinoma (HNSCC) cell lines expressing Sema4D and demonstrate activation of NF-κB in vivo in a tumor xenograft model. Taken together, these results demonstrate further versatility in plexin signaling that may hint at new diagnostic and therapeutic approaches in the anti-angiogenic treatment of some cancers.
Results
Sema4D activates NF-κB through Plexin-B1
Though Sema4D is also known to bind to lower-affinity receptors such as CD72 (on cells of hematopoietic origin, which include endothelial cells) [23] and Plexin-B2 (which has only been observed in neurons) [24], we previously have demonstrated that promotion of a pro-angiogenic phenotype in endothelial cells is due to ligation by Sema4D of its high-affinity receptor, Plexin-B1 [4], which in turn activates Akt [25]. The pro-inflammatory and pro-cell survival protein NF-κB is a known functional target of Akt [22], [26] and has been linked to angiogenesis. Therefore, we wanted to look for Plexin-B1-mediated NF-κB activation in Sema4D treated endothelial cells. NF-κB is sequestered in the cytoplasm by I-κB, which needs to be phosphorylated and degraded to allow NF-κB to translocate to the nucleus, so rapid phosphorylation and loss of I-κB signal in an immunoblot (and then recovery, as I-κB is induced by NF-κB) is a good marker for activation of NF-κB. To determine the role of Plexin-B1 in the activation of NF-κB, we infected HUVEC with control lentivirus or lentivirus coding for Plexin-B1 shRNA and confirmed protein knockdown in an immunoblot (Figure 1A). We then treated these cells with 400 ng/ml Sema4D and observed I-κB phosphorylation (Figure 1B, upper panel, left) and degradation (Figure 1B, middle panel, left), but loss of this response in cells expressing reduced levels of Plexin-B1 (Figure 1B, right panels). We treated control cells and those infected with Plexin-B1 shRNA-expressing lentivirus with soluble Sema4D, lysed them and incubated the nuclear fraction with labeled oligonucleotides containing κB elements to look for binding in an electrophoretic mobility shift assay. NF-κB binding was detected by a gel shift in Sema4D treated cells but not in cells infected with Plexin-B1 shRNA (Figure 1C). Specificity of this assay was demonstrated by a supershift upon incubation with a p65 antibody and a loss of signal when incubated with a 100-fold excess of unlabelled oligonucleotide (Figure 1C). We also detected the NF-κB subunit p65 migrating to the nucleus in an immunofluorescence analysis of Sema4D treated HUVEC (Figure 1D) and in an immunoblot of nuclear and cytoplasmic fractions from these cells (Figure 1E). We then looked for NF-κB activation in a reporter assay in HUVEC expressing an NF-κB responsive luciferase construct, following treatment with Sema4D. We observed a concomitant increase in fluorescence with increasing concentrations of Sema4D (Figure 1F). Taken together, these results demonstrate that Sema4D induces NF-κB translocation to the nucleus and transcriptional activity in endothelial cells in a Plexin-B1-dependent manner.
10.1371/journal.pone.0025826.g001Figure 1 Sema4D activates NF-κB downstream of Plexin-B1.
A. Immunoblot analysis for Plexin-B1 on lysates from endothelial cells infected with empty vector control lentivirus (control) or virus coding for Plexin-B1 shRNA (PB1shRNA). GAPDH was used as a loading control (lower panel). B. Immunoblot for phospho- and total I-κB (upper and middle panels, respectively) in endothelial cells infected with empty vector control lentivirus (control, left column) or virus coding for Plexin-B1 shRNA (PB1shRNA, right column) treated with Sema4D for the times indicated. GAPDH was used as a loading control (lower panels). C. EMSA performed on control infected HUVEC or cells infected with lentivirus coding for Plexin-B1 shRNA, in the presence of Sema4D with and without competition from unlabeled oligos (competition) or anti-p65 antibody (p65 antibody). “ns” denotes a non-specific band; “NF-κB” indicates a shift in oligo migration; “supershift” indicates the higher band resulting from antibody binding. D. Immunofluorescence for the presence of the p65 subunit of NF-κB in nuclei of HUVEC, controls (top row) or treated with Sema4D (bottom row). p65 is shown in green (center column). DAPI was used to stain cell nuclei (left column). The merged image is shown in the right column. E. Immunoblot for the presence of p65 in cytoplasmic and nuclear fractions of HUVEC treated with Sema4D. GAPDH was used as a loading control (lower panel). F. HUVEC were infected with lentiviruses coding for an NF-κB reporter construct and fluorescence measured in arbitrary units (AU) in increasing concentrations of Sema4D, as indicated. Error bars represent the standard deviation from three experiments.
Sema4D induces a pro-angiogenic phenotype and apoptotic protection in endothelial cells through NF-κB but has no effect on proliferation or VEGF-mediated angiogenesis
To determine the biological significance of the activation of NF-κB we observed in endothelial cells, we performed a Boyden chamber migration assay on HUVEC, as an in vitro measure of angiogenesis, with and without the NF-κB inhibitor BAY11-7085 and an I-κB mutant protein resistant to phosphorylation and degradation (the “super-repressor”), using Sema4D as the chemoattractant. HUVEC migrated towards Sema4D, as we and others have observed before [3], [4], except when co-treated with BAY11-7085 (Figure 2A), and where cells were infected with lentiviruses coding for the I-κB super-repressor (Figure 2B, results quantified for both migration assays in the bar graph, lower panels). HUVEC continued to migrate towards FBS in both instances, indicating the specificity of NF-κB activation in Sema4D-mediated chemotaxis. We also looked for the ability of these cells growing on reconstituted basement membrane material to form tube-like capillary structures in tissue culture under similar conditions, which is indicative of a pro-angiogenic response. HUVEC formed capillary tubes when growing on reconstituted basement membrane material in the presence of Sema4D except when co-treated with BAY11-7085 (Figure 2C) or when expressing the I-κB super-repressor (Figure 2D, results quantified for both tubulogenesis assays in the bar graph, lower panels). NF-κB is also known to promote cell survival and proliferation in different cell lines. In order to determine the effects of Sema4D-mediated activation of NF-κB on endothelial cell survival, we treated HUVEC with soluble Sema4D and looked for resistance to apoptosis under conditions of serum starvation, with or without the NF-κB inhibitor BAY 11-7085. We noted the presence of cleaved, and hence activated, caspase 3 in serum starved cells, a response which decreased in increasing concentrations of Sema4D but returned when the cells were co-treated with BAY 11-7085, indicating that this effect was dependent upon Sema4D-induced NF-κB activity (Figure 2E). To evaluate if endothelial cell proliferation is enhanced by Sema4D, we measured HUVEC proliferation by [3H] thymidine incorporation assay, using vascular endothelial growth factor (VEGF) treatment as a positive control. As expected, VEGF treated HUVEC were stimulated to proliferate, incorporating [3H] at three times the rate of controls (Figure 2F). Interestingly, no concentrations of Sema4D seemed to influence HUVEC proliferation (Figure 2F).
10.1371/journal.pone.0025826.g002Figure 2 Sema4D induces migration, capillary tube formation and apoptotic protection in endothelial cells in an NF-κB-dependent manner, but has no effect on endothelial cell proliferation or VEGF-mediated angiogenesis.
A. Migration assays on HUVEC towards BSA, FBS or Sema4D in the presence of control media or media containing BAY11-7085 (BAY). B. Migration assays on HUVEC, control infected (C), or infected with the I-κB super-repressor (SR) towards BSA, FBS or Sema4D. C. HUVEC treated with vehicle control (neg ctrl), or BAY11-7085 (BAY), with or without Sema4D were grown on reconstituted basement membrane material in serum free media and examined for formation of capillary tubes. Representative photographs are shown. D. HUVEC mock electroporated (mock), or electroporated with the I-κB super-repressor (SR) were grown on reconstituted basement membrane material in serum free media with or without Sema4D and examined for formation of capillary tubes. Representative photographs are shown. E. Immunoblot performed for the cleaved fragment of caspase 3 (top panel) in HUVEC growing under conditions of low serum concentration, treated with the indicated concentrations of Sema4D with or without the NF-κB inhibitory compound BAY11-7085 (BAY). GAPDH was used as a loading control (lower panel). F. An [3H] thymidine incorporation assay was performed on HUVEC growing in the presence of Sema4D or VEGF for the purposes of measuring proliferation. Counts per minute (cpm) are represented on the Y-axis. Error bars represent the standard deviation from four independent experiments. G. Migration assay on HUVEC, control infected (control), or infected with lentiviruses expressing Plexin-B1 shRNA (Plexin-B1 shRNA) towards BSA, Sema4D or VEGF. For all migration assays, the results are quantified by the bar graphs in the bottom panels as the pixel intensity of scanned migration assay membranes, which are shown on the top. Error bars represent the standard deviation from six wells (*, p<0.05; n.s., not significant). H. HUVEC, control infected (control), or infected with lentiviruses expressing Plexin-B1 shRNA (Plexin-B1 shRNA) were grown on reconstituted basement membrane material in serum free media with BSA, Sema4D or VEGF and examined for formation of capillary tubes. Representative photographs are shown. Quantification of the results of all tubulogenesis assays are shown in the bar graphs in the lower panels, with the Y-axis representing tube formation as measured by summing the length of capillary tubular structures observed in 10 microscopic fields, relative to the negative control wells (error bars represent the standard deviation from three independent experiments; *, p<0.05; n.s., not significant).
Because we noted endothelial cell proliferation in the presence of VEGF but not Sema4D, we suspected that these two pro-angiogenic factors elicited different signaling pathways and possibly functioned independently of each other. To further establish their independence and evaluate the role of Sema4D and Plexin-B1 in the broader context of the angiogenic process, we performed in vitro angiogenesis assays on endothelial cells, with and without silenced Plexin-B1, in the presence of Sema4D and VEGF, and compared the responses. Sema4D and VEGF induced robust endothelial cell migration compared to control populations (Fig. 2G). Sema4D-mediated cell migration was greatly reduced in cells infected with lentiviruses expressing Plexin-B1 shRNA, while migration toward VEGF remained unaffected (Figure 2G, results quantified in the bar graph, lower panel). We then confirmed these results in a tubulogenesis assay, growing endothelial cells on reconstituted basement membrane extract under the same conditions. Once again, HUVEC formed capillary tube-like structures when growing in the presence of Sema4D except when infected with Plexin-B1 shRNA-expressing lentivirus, while cells growing in VEGF formed these structures regardless of their Plexin-B1 status (Figure 2H, results quantified in the bar graph, lower panel).
Taken together, these findings indicate that NF-κB activation is necessary for the Sema4D-mediated pro-angiogenic phenotype in endothelial cells. This response is probably due to endothelial cell chemotaxis and differentiation into capillary structures and not due to an effect on cell proliferation, unlike what was observed for VEGF. Furthermore, insensitivity of endothelial cells to Plexin-B1 status in the presence of VEGF suggests that Sema4D and VEGF work through independent, parallel pathways to achieve their respective pro-angiogenic responses.
RhoA and Akt are necessary for Plexin-B1-mediated activation of NF-κB and promotion of endothelial cell migration and tube formation by Sema4D
To dissect the pathways involved in Plexin-B1-mediated NF-κB activation, and to rule out involvement of CD72 or Plexin-B2 in this process, we looked for phosphorylation and degradation of I-κB in cells expressing plasmids coding for the chimeric receptors TrkA fused to full length Plexin-B1, Plexin-B1 lacking the C-terminal PDZ binding domain necessary to activate Rho, and a chimera containing Plexin-B1 with mutations at the arginine residues required for R-RasGAP activity [4], [17]. We have previously shown that we could isolate and study Plexin-B1 specific signaling in cells expressing these constructs when treated with NGF [4], [17]. We also co-treated with the Rho inhibitor C3 toxin or the PI3K inhibitor LY294002, where indicated, in order to clarify further the role of Ras, Rho and Akt in the NF-κB response. Phosphorylated I-κB was observed in control treated cells transfected with the full-length chimera, which was then rapidly degraded as expected (Figure 3A). However, this response was absent in cells expressing the full-length chimera but incubated with C3 or LY294002 and in all cells expressing the ΔPDZ chimera incapable of activating Rho (Figure 3A). The R-RasGAP mutant was still able to elicit phosphorylation and degradation of I-κB except when co-treated with C3 or LY294002, indicating that this effect was independent of R-RasGAP activity (Figure 3A). To further confirm the role of Rho and Akt in this process, we looked for activation of NF-κB transcription in a reporter assay in HUVEC transfected with an NF-κB responsive luciferase construct, treated with Sema4D with and without C3 or LY294002. Fluorescence was greatly increased in Sema4D treated HUVEC except when co-treated with C3 or LY294002 (Figure 3B). We then evaluated the ability of HUVEC to exhibit chemotaxis or form capillary tube structures in Sema4D, with or without Rho and Akt inhibition. HUVEC migrated towards Sema4D, a response that was blocked by C3 and LY294002 (Figure 3C, results quantified in the bar graph, lower panel), and could form tubes on reconstituted basement membrane material in the presence of Sema4D except when co-incubated with C3 or LY294002 (Figure 3D, results quantified in the bar graph, right panel). Taken together, these results indicate that Plexin-B1 activates NF-κB in a Rho and Akt-dependent manner, and that this pathway needs to remain intact for Sema4D-mediated promotion of angiogenesis.
10.1371/journal.pone.0025826.g003Figure 3 RhoA and Akt are necessary for Plexin-B1-mediated activation of NF-κB and promotion of endothelial cell migration and tube formation.
A. Immunoblot for phospho-I-κB (top panel) and total I-κB (middle panel) in cells expressing chimeric receptors coding for the extracellular portion of Trk-A fused to the wild-type intracellular segment of Plexin-B1 (wtPB1), Plexin-B1 lacking the PDZ binding motif (ΔPDZ) or Plexin-B1 mutated in the RasGAP domain (RasGAP mut), treated with NGF with and without the Rho inhibitor C3 toxin (C3) and the PI3K inhibitor LY294002 (LY), for the times indicated. GAPDH was used as a loading control (lower panel). B. NF-κB reporter assay performed on control treated HUVEC or HUVEC treated with Sema4D, with or without C3 toxin or LY294002 (LY), with fluorescence expressed as arbitrary units (AU). Error bars represent the standard deviation from three experiments. C. Migration assays on HUVEC towards BSA or Sema4D in the presence of control media, or media containing C3 or LY294002 (LY). Quantification of migration is shown in the bar graph in the lower panel. Error bars represent the standard deviation from six wells (*, p<0.05). D. HUVEC were plated on reconstituted basement membrane material in serum free media and treated with vehicle control (control) or Sema4D, with or without co-treatment with C3 toxin (C3) or LY294002 compound (LY), and examined for formation of capillary tubes. Representative photographs are shown. Quantification of the results observed in the tubulogenesis assay are shown in the right panel (error bars represent the standard deviation from three independent experiments; *, p<0.05).
Sema4D treatment of HUVEC activates the NF-κB downstream target IL-8, which is necessary for the promotion of angiogenesis
We performed an ELISA on media conditioned by Sema4D treated HUVEC, looking for the presence of IL-8, a known pro-angiogenic downstream target of NF-κB [27]. HUVEC treated with increasing concentrations of soluble Sema4D produced increasing amounts of IL-8, unless when co-treated with the NF-κB inhibitory compound BAY11-7085 (Figure 4A). To determine the biological significance of NF-κB-mediated IL-8 production, we performed a migration assay on HUVEC toward Sema4D, with and without the presence of IL-8 blocking antibody. As expected, Sema4D attracted HUVEC, but this response was attenuated by increasing concentrations of IL-8 blocking antibody (Figure 4B, results quantified in the bar graph, lower panel). Sema4D-induced tube formation in HUVEC growing on reconstituted basement membrane material was also reduced by co-administration of IL-8 blocking antibody (Figure 4C, results quantified in the bar graph, lower panel). These results show that NF-κB-dependent IL-8 production is necessary for Sema4D to promote a pro-angiogenic response in endothelial cells.
10.1371/journal.pone.0025826.g004Figure 4 Sema4D treatment of HUVEC induces NF-κB-dependent production of IL-8, which is necessary to promote angiogenesis.
A. ELISA for production of IL-8 in HUVEC treated with the indicated concentrations of Sema4D or 400 ng/ml Sema4D with BAY11-7085 (BAY). Results are expressed as averages in pg/ml. Error bars represent the standard deviation for three independent experiments. B. Cell migration assay for HUVEC migrating towards BSA or Sema4D in the presence of IgG control (IgG) or the indicated concentrations of anti-IL-8 blocking antibody. The results are quantified in the bar graph below as the pixel intensity of the scanned migration assay membrane. Error bars represent the standard deviation from six wells (*, p<0.05). C. HUVEC were plated on reconstituted basement membrane material in control media (BSA) or media containing Sema4D, with IgG control (IgG) or the indicated concentrations of IL-8 blocking antibody and examined for formation of capillary tubes. Representative photographs are shown. Quantification of the results observed in the tubulogenesis assay are shown in the bar graph below (error bars represent the standard deviation from three independent experiments; *, p<0.05).
NF-κB is activated in endothelium by Sema4D-expressing tumor cells
We have previously shown that many different solid tumors produce Sema4D for the purposes of inducing angiogenesis [16]. Therefore, we wanted to determine if Sema4D production by tumor cells could activate NF-κB in endothelial cells. We infected the HNSCC cell line HN13 with control lentivirus or lentivirus coding for Sema4D shRNA and confirmed protein knockdown in an immunoblot (Figure 5A). We then treated HUVEC with media conditioned by these cell lines and looked for phospho- and total I-κB levels in an immunoblot. A phospho-I-κB response and I-κB degradation was noted in HUVEC treated with media conditioned by control infected HN13 but not in HUVEC treated with media conditioned by Sema4D shRNA infected cells (Figure 5B). To further determine biological significance in vitro, we looked for the presence of the p65 NF-κB subunit in the nuclei of endothelial cells lining blood vessels from the tumor stroma of HN13 xenografts implanted into the flanks of nude mice, either control infected or infected with Sema4D shRNA-expressing lentivirus. We observed the presence of p65 in the nuclei of endothelial cells of blood vessels associated with tumors comprised of control infected cells, but less so from the vessels associated with tumors made up of Sema4D shRNA infected cells (Figure 5C). These results are quantified in Figure 5D. Taken together, these results strongly suggest that Sema4D production by tumors activates NF-κB signaling in endothelial cells via Plexin-B1 in order to promote tumor-induced angiogenesis.
10.1371/journal.pone.0025826.g005Figure 5 Sema4D produced by tumor cells activates NF-κB in endothelial cells.
A. Immunoblot analysis for Sema4D on lysates from HN13 cells, infected with empty vector control lentivirus (control) or virus coding for Sema4D shRNA (S4DshRNA). GAPDH was used as a loading control (lower panel). B. Immunoblot for phospo- (upper panel) and total (middle panel) I-κB in HUVEC growing in media conditioned by control infected HN13 cells (control, left column) or cells infected with Sema4D shRNA expressing lentivirus (S4DshRNA, right column) for the times indicated. GAPDH was used as a loading control (lower panel). C. Immunofluorescence on tumor xenografts composed of control infected HN13 cells or cells infected with Sema4D shRNA, for CD31 (endothelial cells, red) and p65 (green). The white arrows indicate endothelial cell nuclei. D. The results of the xenograft immunofluorescence expressed as the percentage of p65 positive endothelial cell nuclei counted in 10 high power fields (*, p<0.05).
Discussion
Most cancers arise from an altered pre-malignant progenitor cell that accumulates genetic damage over time, a factor that contributes to the ability of tumor cells to proliferate inappropriately, avoid natural defenses and acquire resistance to chemotherapy. For this reason, patients often are poor candidates for treatment with traditional cytoreductive therapies but might benefit from the development of anti-angiogenic agents, which instead target the normal, genetically stable endothelial cells that line the vessels that feed the tumor. Indeed, growth and metastasis of solid tumors requires induction of angiogenesis, the creation and remodeling of new blood vessels from a pre-existing vascular network, to ensure the delivery of oxygen, nutrients and growth factors to rapidly dividing transformed cells.
VEGF is an endothelial cell specific growth factor that plays a unique role in the regulation of vascular permeability and physiological and pathological angiogenesis, mainly by acting through its receptor VEGF-receptor 2 (VEGF-R2) on endothelial cells. Many different solid tumors have been shown to produce VEGF, and it has become a tempting target for neutralizing antibodies in the treatment of advanced neoplasms. We have demonstrated that Sema4D is over expressed by many different aggressive carcinomas in a manner analogous to VEGF, and that its activity on Plexin-B1-expressing endothelial cells promotes angiogenesis in vitro and in vivo
[4]. However, the mechanisms of Plexin-B1 signaling continue to be investigated. Our group and others have found that Plexin-B1 activates downstream kinases such as Akt and triggers a G-protein response in Sema4D treated cells [4], [17], [18]. Though the role of NF-κB activation by Akt remains controversial [28], here we show that stimulation of Plexin-B1 signaling induces a Rho and Akt-dependent activation of NF-κB. This effect is independent of the RasGAP activity of Plexin-B1, as a RasGAP mutant receptor construct was still able to induce phosphorylation and degradation of I-κB. It is also a purely Plexin-B1-mediated response and likely does not involve the low-affinity Sema4D receptors CD72 or Plexin-B2 or any components of VEGF signaling, as RNA interference directed at Plexin-B1 blocked Sema4D-mediated NF-κB activation and promotion of a proangiogenic response while having no effect on HUVEC responses to VEGF. Furthermore, stimulation of Plexin-B1 specific signaling through NGF-mediated activation of TrkA/Plexin-B1 chimeric receptors promoted I-κB phosphorylation and degradation.
NF-κB is known to be a crucial mediator of inflammation-induced tumor growth and progression, as well as a strong promoter of oncogenesis and tumor cell survival [29] but its effects in endothelial cells on the promotion of angiogenesis is not well studied. There is conflicting evidence in the literature demonstrating that NF-κB activation can be both pro- and anti-angiogenic, mainly by influencing endothelial cell apoptosis [30], [31]. In our system, we show that NF-κB activation by Sema4D results in promotion of endothelial cell survival, migration and tube formation, but unlike VEGF fails to induce significant cell proliferation. These results led us to speculate that Sema4D might elicit a parallel but slightly more limited angiogenic repertoire in endothelial cells when compared to VEGF. However, we also observed robust Sema4D-dependent stimulation of IL-8. This is a significant finding, as IL-8 is secreted by a variety of cells, including endothelial cells, and plays important roles in inflammation and tumor-induced angiogenesis [21]. Many reports have identified NF-κB as the main transcription factor for stimulating IL-8 promoter activity in response to various stimuli [32]. Our study confirmed that the NF-κB inhibitor BAY 11-7085 abrogated the release of IL-8 induced by Sema4D. Stimulation of the NF-κB/IL-8 axis by Sema4D was crucial, since its blockade attenuated the pro-angiogenic response. We also looked for evidence that tumor cells making Sema4D could induce NF-κB activation in endothelium. We showed in vitro that media conditioned by HNSCC cells could induce I-κB phosphorylation and degradation in HUVEC in a Sema4D-dependent manner. In vivo we observed p65 translocation in the nucleus of endothelial cells in the tumor stroma when tumors were expressing Sema4D but much less so in vessels associated with tumors where Sema4D had been silenced by shRNA. Taken together these findings suggest that NF-κB induction in endothelial cells is crucial for endothelial cell survival and chemotaxis and that it is induced by tumors producing Sema4D for the purpose of promotion of angiogenesis.
There is at least one report of plexins being involved in activation of NF-κB that also appears to promote cell survival. Catalano et al. demonstrated that binding of Semaphorin 6D (Sema6D) to its receptor, Plexin-A1, triggered NF-κB transcriptional activity that supported a pro-survival program in malignant pleural mesothelioma cells [33]. Interestingly, they discovered that unlike for the system studied here, Sema6D-induced NF-κB transcriptional activity was dependent upon Plexin-A1-mediated phosphorylation of VEGF-R2, which associated with Plexin-A1 in a signaling complex [33]. These findings raise the question as to whether or not a co-receptor tyrosine kinase or other signaling protein might be involved in Plexin-B1-mediated activation of NF-κB, particularly since Plexin-B1 itself cannot act as a tyrosine kinase. It has been reported that the tyrosine kinase receptor c-Met cooperates with Plexin-B1 to elicit a Sema4D-mediated signal [2], [3]. Such a possibility is currently being investigated.
In summary, we have provided evidence that Sema4D/Plexin-B1-mediated NF-κB activation and IL-8 production is critical in the generation a pro-angiogenic phenotype in endothelial cells, where it promotes migration and survival. However, further studies likely will be necessary to investigate the mechanisms of signaling. Sema4D production by tumors may represent an alternative angiogenesis pathway and therefore represent another treatment target in addition to anti-VEGF therapy for particularly aggressive cancers.
Materials and Methods
Ethics statement
All animal studies were approved by the University of Maryland Office of Animal Welfare, Institutional Animal Care and Use Committee (protocol #: 07-03-01) in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
Cell culture
Human umbilical vein endothelial cells (HUVEC, ATCC, Manassas, VA) were cultured in Endothelial Cell Medium-2 (EGM-2, Lonza). 293T (ATCC) cells and HN13 cells [34] were cultured in DMEM (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (unless otherwise indicated) and 100 units/ml penicillin/streptomycin/amphotericin B (Sigma).
Purification of soluble Sema4D
Sema4D was produced and purified as described previously [4]. Briefly, the extracellular portion of Sema4D was subjected to PCR and the resulting product cloned into the plasmid pSecTag2B (Invitrogen, Carlsbad, CA). This construct was transfected into 293T cells growing in serum free media. Media containing soluble Sema4D was collected 65 hours post-transfection and purified with TALON metal affinity resin (Clontech Laboratories, Palo Alto, CA) according to manufacturer's instructions. Concentration and purity of the TALON eluates was determined by SDS PAGE analysis followed by silver staining (Amersham Life Science, Piscataway, NJ) and the Bio-Rad protein assay (Bio-Rad, Hercules, CA). In all cases, media collected from cells transfected with the empty pSecTag2B vector were used as control.
Immunoblot analysis
Cells were lysed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP 40) supplemented with protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 1 µl/ml aprotinin and leupeptin, Sigma) and phosphatase inhibitors (2 mM NaF and 0.5 mM sodium orthovanadate, Sigma) for 15 minutes at 4°C. After centrifugation, protein concentrations were measured using the Bio-Rad protein assay (Bio-Rad). 100 µg of protein from each sample was subjected to SDS-polyacrylamide gel electrophoresis and transferred onto a PVDF membrane (Immobilon P, Millipore Corp., Billerica, MA). The membranes were then incubated with the appropriate antibodies. The antibodies used were as follows: Plexin-B1 (Santa Cruz A8); GAPDH (Sigma); phospho-I-κB (Santa Cruz, S.C.-101713); total I-κB (Santa Cruz, S.C.-371); p65 (Neomarkers, Fremont, CA); cleaved caspase 3 (Cell Signaling, Beverly, MA); Sema4D (BD Transduction Labs, BD Biosciences, Palo Alto, CA). Proteins were detected using the ECL chemiluminescence system (Pierce, Rockford, IL). Where indicated, cells were treated with the indicated concentrations of soluble Sema4D, C3 toxin (List Biological Laboratories, Campbell, California), LY294002 (Sigma), LPS (Sigma), or BAY11-7085 (Sigma).
Nuclear extraction and electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared using NE-PER Nuclear and Cytoplasmic extraction kit (Thermo Scientific, Waltham, MA) according to the manufacturer's instructions. EMSAs were performed with the LightShift chemiluminescent EMSA kit (Thermo Scientific). DNA-binding probes for NF-κB (forward: 5′-GATCGAGGGGACTTTCCCTAGC-3′; reverse: 5′-GCTAGGGAAAGTCCCCTCGATC-3′) were annealed and biotin labeled according to the Biotin 3′ End DNA labeling kit (Thermo Scientific). 20 fM labeled oligonucleotides were added to a 20 µl reaction mix consisting of 6 µg of nuclear extracts, DNA binding buffer, Poly (dI-dC), 1% NP-40, and MgCl2 in concentrations based on manufacturer's recommendations. For competition assays, unlabeled oligonucleotides were allowed to bind the nuclear extracts (30 min at room temperature) before the addition of labeled probes. Supershift experiments were performed by incubating 1 µg of the anti NF-κB/p65 antibody (Neomarkers) with nuclear extract proteins (6 µg for 30 min at room temperature). The reactions were incubated for additional 20 min with biotin labeled probe. The binding complexes were separated on 6% native DNA polyacrylamide gel, transferred to a positively charged nylon membrane (Thermo Scientific) and then detected using a Chemiluminescent Nucleic acid detection Module (Thermo Scientific) according to the manufacturer's instructions and as previously reported [35].
Immunofluorescence for p65
HUVEC were grown on sterile glass coverslips in 35-mm six-well plates and treated with 400 ng/ml Sema4D for 60 min. The cells were washed in PBS, fixed in 3% paraformaldehyde for 15 min, and permeabilized in 0.5% Triton X-100 for 5 min. The cells were then incubated with anti-p65 antibody (Neomarkers) diluted 1/200 in PBS with 0.5% BSA at 4°C overnight. After three more washings with PBS, coverslips were placed in a humidity chamber for 1 h and covered with FITC-conjugated anti-rabbit secondary antibody (Sigma, 1∶200 dilution in PBS with 0.5% fetal bovine serum). Coverslips were inverted and mounted onto glass slides with Vectashield containing 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA) and viewed using a immunofluorescence microscope.
NF-κB reporter assay
HUVEC were seeded in 96-well plates at a density of 5×103 cells/well. The following day, the cells were infected with lentiviruses expressing the NF-κB reporter and renilla control (SA Biosciences, Frederick, MD) according to the manufacturer's instructions. After 24 h, the cells were changed to DMEM containing 1% FBS and treated with varying concentrations of Sema4D and the indicated inhibitors (3 µg/ml C3 toxin (List Biological Laboratories) or 50 µM LY294002 (Sigma)) for 18 h, as shown. Firefly and renilla luciferase activities were measured using a Dual-Glo luciferase reporter assay system (Promega, Fitchburg WI), and the ratio of firefly luciferase/renilla luciferase luminescence was calculated as previously described [36]. Assays were performed in triplicate and average and standard deviation calculated.
Short hairpin (sh) RNA and lentivirus infections
The shRNA sequences for human Sema4D and Plexin-B1 were obtained from Cold Spring Harbor Laboratory's RNAi library (RNAi Codex, http://katahdin.cshl.org:9331/homepage/portal/scripts/main2.pl) [37], [38]. The sequences used as PCR templates for Sema4D shRNA have been previously reported [16]. The sequence used for Plexin-B1 shRNA was 5′- TGC TGT TGA CAG TGA GCG CGC CCA GTA TGT GGC CAA GAA CTA GTG AAG CCA CAG ATG TAG TTC TTG GCC ACA TAC TGG GCA TGC CTA CTG CCT CGG A -3′. Oligos were synthesized (Invitrogen) and cloned into pWPI GW, a Gateway compatible CSCG based lentiviral destination vector. Viral stocks were prepared and infections performed as previously reported [16].
Migration assays
Media containing 10% FBS (positive control), serum free media containing 0.1% BSA (negative control), or serum free media containing 400 ng/ml of purified Sema4D or 50 ng/ml VEGF, where indicated, were placed in the bottom well of a Boyden chamber to serve as chemoattractants. 50,000 serum starved HUVEC cells infected with control lentivirus, lentivirus coding for Plexin-B1 shRNA, electroporated with empty vector or electroporated with the I-κB super-repressor plasmid [39] were added to the top chamber along with the indicated inhibitors (3 µg/ml C3 toxin, 50 µM LY294002, or 10 µM BAY11-7085) or blocking antibodies (IgG control or 2 µg/ml or 10 µg/ml anti-IL-8 antibody (Lifespan Biosciences)) where indicated. The two chambers were separated by a PVPF membrane (Osmonics, GE Water Technologies, Trevose, PA, 8 µm pore size) coated with 10 µg/ml fibronectin (GIBCO, Carlsbad, CA). The migration assay was then performed as described [20]. Briefly, after 7 h, the chamber was disassembled and the membrane stained with Diff-Quick Stain (Diff-Quick, Dade Behring, Deerfield, Illinois), placed on a glass slide and scanned. Densitometric quantitation was performed with NIH image software and cell migration expressed as pixel intensity. Each experiment was performed six times and average and standard deviation calculated.
Tubulogenesis assays
HUVEC cells infected with control lentivirus, lentivirus coding for Plexin-B1 shRNA, electroporated with empty vector or electroporated with the I-κB super-repressor plasmid [39], were grown in 35 mm plates coated with 150 µl of Cultrex basement membrane extract (Trevigen, Gaithersburg, MD) and incubated overnight in serum free DMEM or serum free media containing 400 ng/ml of Sema4D or 50 ng/ml VEGF, with or without inhibitors or blocking antibody (10 µM BAY11-7085, 3 µg/ml C3 toxin, 50 µM LY294002, or 2 µg/ml or 10 µg/ml anti-IL-8 antibody, where indicated). Cells were then fixed in 0.5% glutaraldehyde and photographed. Media containing 0.1% BSA served as the negative control. Quantification of results was determined using NIH Image, measuring and summing the length of all tubular structures observed in 10 random fields for three independent experiments.
[3H] Thymidine incorporation assay
[3H] thymidine incorporation assay was performed as described elsewhere [40]. Briefly, HUVEC were seeded in 24-well culture plates at 5×104 cells/well and growth-arrested in serum-free medium overnight. Cells were left as controls or incubated with media containing 50 ng/ml VEGF or various concentrations of Sema4D (100–400 ng/ml) for 24 h. 0.5 µCi of [3H] thymidine was added to each well, and the cells were incubated for a further 4 h. The final incorporation of [3H] thymidine into cells was measured with a liquid scintillation counter (LS-6500; Beckman Instruments, Inc., Fullerton, CA) and results of four independent experiments expressed as average counts-per-minute (cpm) relative to untreated controls.
Trk/Plexin-B1 fusion proteins
Trk-A/Plexin-B1 fusion proteins were made as previously described [4]. Briefly, the intracellular portion Plexin-B1, with or without the PDZ binding motif, was cut out of the plasmid pCEFL EGFP Plexin-B1 with NheI/NotI and cloned in frame with the N-terminal extracellular and transmembrane portion of the NGF receptor Trk-A in the vector pCEFL-myc. A Trk-A/Plexin-B1 mutant lacking key residues involved in RasGAP activity was generated as previously described [4] using the QuikChange II XL Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). Mutations were confirmed by sequencing.
IL-8 ELISA
Confluent HUVECs were serum starved for 4 h, then cultured in serum free medium with or without 100 ng/ml, 200 ng/ml or 400 ng/ml Sema4D or 400 ng/ml Sema4D with 10 µM BAY 11-7085, where indicated, for 12 h. The culture supernatant was collected and used to analyze IL-8 by ELISA (Cytokine Core Facility, University of Maryland School of Medicine). Results are expressed as the average and standard deviation for three independent experiments.
Tumor xenografts
2×106 HN13 cells infected ex vivo with control lentiviruses and virus coding for Sema4D shRNA were resuspended in 250 µl of serum-free DMEM with an equal volume of Cultrex basement membrane extract (Trevigen) and injected subcutaneously into nude mice. After tumor growth had been recorded, animals were sacrificed and tumors were removed and processed for co-immunofluorescence, as described [41]. Briefly, OCT-embedded 8 µm thick frozen tissue sections were cut onto silanated glass slides, air-dried, and stored at −80°C. They were then thawed, hydrated, fixed and washed in PBS. The following antibodies were used: anti-PECAM (BD Pharmingen; 1∶100 dilution); Anti-p65 (Neomarkers, 1∶100 dilution); Fluorescein anti-rabbit secondary and Texas-red anti-mouse secondary (Vector Laboratories; 1∶200 dilution).
The authors would like to thank Dr. Daniel Martin and Dr. Silvio Gutkind of the National Institute of Dental and Craniofacial Research for advice on generation of shRNA lentiviruses and for providing the I-κB super-repressor construct.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by the National Cancer Institute (R01 CA133162 to JRB) and a scholarship to NOB from the King Abdulaziz University in Jeddah, Saudi Arabia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22046297PONE-D-11-0921410.1371/journal.pone.0026503Research ArticleBiologyMolecular Cell BiologyCellular TypesNeuronsSignal TransductionSignaling in Selected DisciplinesNeurological SignalingNeuroscienceNeurophysiologyCentral Nervous SystemMotor SystemsCellular NeuroscienceSensory Coding by Cerebellar Mossy Fibres through Inhibition-Driven Phase Resetting and Synchronisation Metronome Mossy FibresHoltzman Tahl
1
*
Jörntell Henrik
2
1
Behavioural and Clinical Neuroscience Institute and Department of Experimental Psychology, University of Cambridge, Cambridge, United Kingdom
2
Section for Neurophysiology and Neuronano Research Center, Lund, Sweden
Sugihara Izumi EditorTokyo Medical and Dental University, Japan* E-mail: [email protected] and designed the experiments: TH. Performed the experiments: TH. Analyzed the data: TH HJ. Contributed reagents/materials/analysis tools: TH HJ. Wrote the paper: TH.
2011 26 10 2011 6 10 e2650324 5 2011 28 9 2011 Holtzman, Jörntell.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Temporal coding of spike-times using oscillatory mechanisms allied to spike-time dependent plasticity could represent a powerful mechanism for neuronal communication. However, it is unclear how temporal coding is constructed at the single neuronal level. Here we investigate a novel class of highly regular, metronome-like neurones in the rat brainstem which form a major source of cerebellar afferents. Stimulation of sensory inputs evoked brief periods of inhibition that interrupted the regular firing of these cells leading to phase-shifted spike-time advancements and delays. Alongside phase-shifting, metronome cells also behaved as band-pass filters during rhythmic sensory stimulation, with maximal spike-stimulus synchronisation at frequencies close to the idiosyncratic firing frequency of each neurone. Phase-shifting and band-pass filtering serve to temporally align ensembles of metronome cells, leading to sustained volleys of near-coincident spike-times, thereby transmitting synchronised sensory information to downstream targets in the cerebellar cortex.
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Introduction
Oscillatory neuronal activity is considered fundamental for enabling co-ordinated activity during normal brain functioning [1]–[3] whilst disturbances of oscillatory activity are associated with a variety of brain disorders including epilepsy, Parkinson's disease and schizophrenia [4], [5]. In general, oscillogenesis is considered to arise from the concerted interplay of excitation and inhibition within a local network [for review see 6], [7], although intrinsic oscillatory behaviour can also operate at the single neurone level. Electrical coupling and intrinsic membrane currents may interact to produce prominent oscillatory activity, such as that seen in cells of the inferior olive [8], [9], prompting the suggestion that synchronous oscillations provide a temporal reference for control of motor performance. Passive and active membrane conductances can bias cells to oscillatory activity at preferred, resonant frequencies [reviewed by 10]. For example, cerebellar granule cells and Golgi cells show a low frequency resonance [11]–[13] suggesting that they may be tuned to respond to oscillatory afferent signals in a narrow frequency range.
At the cellular level, representation of information using oscillatory schemes gives rise to phase-of-firing coding, where neurones fire at particular times during an on-going oscillation thereby implementing a temporal code [see 14]. Sub-threshold oscillations, and thus spike-time reliability, can be phase shifted by excitatory inputs [15], [16] and by inhibitory inputs [17]–[19] suggesting that oscillatory information coding can adapt dynamically. Downstream neurones must be able to read-out such codes and might employ a variety of mechanisms including spike counts and spike-time dependent plasticity [see 20]–[23]. In this regard, cortical neurones are well-suited to detect correlated oscillatory activity [24] as are the much smaller, electrotonically compact cerebellar granule cells [25], [26].
In this study we examine a novel class of neurones located in the lateral reticular nucleus of the brainstem, whose axons form a major supply of afferents to the cerebellar cortex [27]. These units fire with remarkable regularity at idiosyncratic frequencies ranging from ∼7–22 Hz. Sensory evoked inhibition serves to phase reset their regular spike firing enabling spike-time locking to particular sensory stimulation frequencies. Using phase-response curves, joint-peristimulus time histograms and simulations we show that phase resetting can generate synchronised volleys of near-coincident firing capable of representing a temporal code of sensory input frequency that is well-suited to influence downstream neurones such as Golgi cells and granule cells in the cerebellar cortex.
Materials and Methods
Experiments were performed, in vivo, on 50 adult Wistar rats weighing 300–450 g. All procedures were conducted so as to minimise suffering and were approved by the local ethical review panel of the University of Cambridge and by UK Home Office regulations (Project number 80/2234). The methods for general preparation have been described previously [28]. Under urethane general anaesthesia (1–1.5 g/kg i.p.) supplemented with 0.1 ml Hypnorm (i.p.) rats were fixed in a stereotaxic frame and the cerebellum was exposed. Single unit recordings were made from units located in lobules Crus Ic/II a/b and in some experiments recordings were also obtained from the lateral reticular nucleus (LRN) in which the foramen magnum was opened, exposing the brainstem landmark obex. For cerebellar penetrations electrode angles were ∼45 degrees from vertical so as to be perpendicular to the cerebellar folial surface; depth from surface rarely exceeded 700 µm and crossing of Purkinje cell layers was carefully determined in each electrode track. LRN recordings were targeted using stereotaxic co-ordinates [29] – electrode angle 30 degrees from vertical, interaural −4.2 mm AP, interaural −0.3 to −0.5 DV and midline +1.9 mm, following the histologically verified approach of [30]. Signals from the microelectrodes were amplified (gain ×1000–10000), filtered (band-pass 0.3–10 kHz for spikes and 0.1–300 Hz for local field potentials [LFP]) and digitised at 25 kHz (spikes) and 5 kHz (LFP). Some recordings were made using platinum/tungsten electrodes coated with quartz glass – 80 µm shaft diameter – impedance 2–3 MΩ Thomas Recording - Giessen, Germany) arranged in a 4×4 array or concentric 6+1 array (Eckhorn & Thomas, 1993) whilst on other occasions we used glass micropipettes pulled from filament glass broken to give tip impedances of 6–15 MΩ when filled with 0.5 M NaCl.
During LRN recording experiments, a stainless steel stimulation electrode (100 kΩ, Microprobes, MD 20879, USA) was inserted into the cerebellar cortex/white matter. Biphasic stimulus pulses (0.2 ms, 2 Hz, 100–400 µA) were used to elicit antidromically activated spikes in LRN units with all-or-none characteristics and constant latency (typically ∼1.5 ms). Since all of our units were spontaneously active, antidromicity was confirmed by cancellation of evoked spikes by collision with spontaneously occurring spikes (Figure 1B), confirming the identity of these LRN units as cerebellar-projecting mossy fibres.
10.1371/journal.pone.0026503.g001Figure 1 Spontaneous firing properties of metronome cells.
A shows a 5 second excerpt of spontaneous spiking activity from an example metronome cell recorded in the brainstem, alongside the superimposed spike waveforms. B plots the ISIH for this neurone which had an idiosyncratic frequency ∼13 Hz (bin size 1 ms). The inset shows 10 traces of raw data where spontaneous spikes were used to trigger delayed antidromic stimulation in the cerebellum. In the upper traces, antidromically evoked spikes occur at fixed latency (1.3 ms). When stimuli are triggered at less than this latency, no antidromic spikes are evoked (lower traces indicated by * - stimulus delay 1 ms) due to collision with spontaneous orthodromically conducted spikes. Similar data for an example cerebellar metronome unit (slower idiosyncratic frequency ∼8 Hz) are plotted in C and D. Group data for mean firing frequency and CV2 calculated from spontaneous activity of brainstem and cerebellar units is plotted in E (note that whilst all units were spontaneously active, records of spontaneous activity were obtained from a subset of our sample). No significant differences were found between these groups comparing either parameter. F plots CV2 for a variety of neurones in the cerebellum and the neocortex, indicating that metronome cells are particularly regular.
Mixed low-threshold somatosensory afferents were stimulated using percutaneous pin electrodes inserted in to the foot pads and vibrissal skin at rates generally <0.66 Hz. During some experiments we used trains of stimuli at frequencies ranging from 6–30 Hz, with trains lasting for ∼1000 ms or 500 ms (frequencies >20 Hz). Spike trains were discriminated using a custom written spike shape analysis and cluster-cutting package (LabSpike, Dr. Gary Bhumbra, Dept. of Physiology, Development and Neuroscience, Downing Street, Cambridge, UK; available from http://www.pdn.cam.ac.uk/staff/dyball/labspike.html) and the time series were used to construct peri-stimulus time-histograms (PSTHs) and interspike interval histograms (ISIHs). Phase response curves for somatosensory input were calculated for some metronome cells, using stimuli delivered at random relative to the spontaneous firing. Prior to each stimulus, a period of spontaneous firing (typically 5–6 spikes) was used to estimate the time of the expected spike if no stimulus had occurred, thus perturbation phase was calculated using the time of the expected spike and its predecessor, taking into account peripheral conduction delays (∼10 ms; estimated from the PSTH).
To assess spike-time accuracy during stimulus trains we calculated a spike-stimulus synchronisation index (SI), by treating the time interval between stimulus pulses with a periodic function, thus: where hi = number of spikes occurring in the ith phase bin of n (typically 36 bins, corresponding to 10 degrees per bin).
ISI distributions were expressed as probability density distributions using a kernel density estimation algorithm [31] allowing grouping of datasets and normalisation for idiosyncratic frequencies of individual cells. Spike triggered averages of LFP (STA-LFP) were constructed by taking epochs of LFP, typically 100 ms either side of each spike. Confidence limits were calculated using Monte Carlo simulations (spike-times and LFP segments shuffled, minimum 100 simulations). Coherence between spikes and LFP was calculated using spike-field coherence (SFC) as detailed by [32] and Monte Carlo simulations were used to generate confidence bounds (1000 simulations). For metronome cell ensemble recordings we used the joint-peristimulus time-histogram (JSPTH) technique to assess time-resolved stimulus-evoked correlations between cell pairs [33]. JPSTHs were constructed using 2 ms time-bins and smoothed using a gaussian where σ = 2 ms.
A simple model of granule cell synaptic integration, similar to that used in our earlier work [25], [26], was used to calculate the summation of mossy fiber-EPSPs. The only purpose of this model was to analyze the pattern of membrane potential fluctuations, which could be obtained as a result of varying the number and temporal density of synaptic inputs. In granule cells in vivo, the spike output is essentially a linear function of the membrane potential [25], so these membrane potential fluctuations should be closely correlated to spike output, although this remains to be confirmed in actual recordings from granule cells receiving inputs from this type of LRN cell. In this paper, we set the model to operate at a membrane potential of −59 mV to prevent spiking. The model assumes that different mossy fiber synapses have different average EPSP amplitudes [25]. As the model was used for simulations within a narrow membrane potential range, it was simplified to not include any active membrane conductances or Golgi cell inhibition. Time-courses and amplitudes of each mossy fiber-EPSP (at −59 mV) and the paired-pulse depression ratio when an individual mossy fiber input is activated at high rates were based on data for the mossy fiber-EPSPs in vivo
[25]. The following parameters were used for the EPSPs: EPSP peak amplitudes at 800 MΩ (membrane resistance for granule cells in vivo) (synapse 1–4): 5.2; 4.0; 3.2; 2.5 mV; time-to-peak: 0.95 ms; half-time decay: 5.5 ms; paired-pulse depression, max. 63% (at 1 ms interval); paired-pulse depression time constant: 8 ms. The spike responses, recorded at a temporal resolution of 0.1 ms, of single metronome cells were fed to each of the four mossy fiber inputs.
Results
Metronome-like activity in LRN cells
We made recordings from brainstem units (n = 41, 15 animals) located in the lateral reticular nucleus (LRN) and also from mossy fibre terminals in the cerebellar cortex (n = 70, 35 animals). All of these units were spontaneously active (i.e. in absence of overt sensory stimulation) and their firing patterns were distinctive in their regularity. The raw spike train and interspike interval histogram (ISIH) shown in Figure 1A and 1B show data from an example LRN unit with a mean firing rate of ∼14 Hz. We only included units recorded in the LRN which were positively identified as cerebellar-projecting using antidromic collision testing (see Methods). Example collision data are shown inset in Figure 1B. Across the population of cells tested, collision latencies ranged from 1.1–1.9 ms, consistent with fast-conducting fibres (c.f. ∼5 ms climbing fibre conduction latency from the inferior olive [34]). In other experiments, we recorded units with similar firing patterns in Crus I/II of the cerebellar cortex. An example unit is illustrated in Figure 1C and 1D. The action potential waveform of this unit (and all others like it recorded in the cerebellum – see also Figure S1C and S1D) comprised an early fast- and later variable slow-component typical of mossy fibre terminals [25], [35]–[37]; the fast component represents the axonal action potential and the slow, variable amplitude component (negative after-wave; NAW) reflecting elements of the synaptic action potential and post-synaptic response. Such units were commonly encountered alongside spikes belonging to Golgi cells and granule cells [see 28], [38].
Comparing LRN units with cerebellar units, their mean firing rates and the regularity of their firing patterns [measured using CV2; 39] are plotted in Figure 1E, for a selection of units from each population. Across our sample, individual units fired at a range of idiosyncratic mean frequencies ranging from ∼7–22 Hz; the distinct regularity of these firing patterns generated CV2 values which were generally <0.13 - moreover, our brainstem neurones showed highly similar characteristics when compared to those units recorded in the cerebellum (Figure 1E). A one way multivariate analysis of variance test did not find evidence of a significant difference between these two groups (p<0.1494) consistent with these samples being drawn from the same underlying population. Metronome cells appear to be the most regular firing of cell types in the cerebellum (perfect regularity generates CV2 = 0) compared to other cell types such as Purkinje cells, Golgi cells and granule cells [28], [40], [41], and in comparison to neocortical cells [39] (Figure 1F).
In summary, the similarity of the cerebellar metronome firing patterns to those in the brainstem alongside their dissimilarity to other cerebellar cortical neurones in the granular layer considered along with action potential waveforms characteristic of mossy fibres (see also insets in Figure S1 – see also identical response patterns between cerebellar and brainstem unit), make it likely that our sample of cerebellar units represents the synaptic terminals of metronomic LRN neurones, none-the-less in the absence of definitive proof, our classification remains putative.
The highly regular firing patterns of metronome cells could arise via intrinsic oscillatory currents, network activity or regular afferent inputs – these scenarios are not mutually exclusive. We used spike-triggered averaging (STA) of local field potentials (LFP) recorded in the LRN to address this question. LFP-STAs are useful for assessing the relationship between spikes (supra-threshold) and peri-spike membrane activity (sub-threshold) since the LFP represents the average of both supra- and sub-threshold events from a volume of several 100 µm [for review see 42]–[45].
Example STAs calculated for a pair of simultaneously recorded LRN units (different electrodes) are drawn in Figure 2A and 2B. Peri-spike periodic voltage oscillations dominate the STA; the period of these oscillations closely matches idiosyncratic firing frequency of each cell, 16 Hz and 12 Hz, respectively. All LRN STA's showed peri-spike periodic voltage oscillations (n = 6). We used a Monte Carlo shift predictor (spike times randomised, STA recomputed; 100 shuffles - superimposed broken grey lines) to calculate 95% confidence interval for the STA, thus values exceeding these bounds (grey lines) are considered significant (p<0.05). This neurone pair was separated by approximately 300 µm – we also computed the STA using spikes from one cell and the LFP signal from its counterpart, giving rise to a ‘flat’ STA (Figure 2C). Similar results were obtained for other cross-referenced STAs using simultaneous recordings (data not shown). STA oscillations were typically 40–80 µV peak to peak, comparable in size to STA oscillations reported elsewhere in the brain [46]–[49]. These results suggest that peri-spike oscillations are generated focally (i.e. spatially restricted), rather than representing distributed network activity such as that seen during hippocampal theta oscillations [50].
10.1371/journal.pone.0026503.g002Figure 2 Spike-LFP coherence of metronome cell firing.
A and B show spike-triggered averages of LFP for two example metronome cells (recorded simultaneously on separate electrodes). In each case, spikes referenced to their own LFP signal revealed peri-spike periodic voltage fluctuations (solid lines). Monte Carlo based confidence limits (95%) are superimposed on each plot (broken grey lines). C shows the STA using spikes from the cell in A referenced to LFP of the cell in B using the same format – although recorded simultaneously and separated by ∼300 µm no relationship between spikes and LFP was apparent. D and E plot spike-field coherence (solid lines) for the same cell pair. Monte Carlo based confidence limits (95%) are superimposed on each plot (broken lines) along with probability density estimates for the spiking frequency of each cell (grey curves). Grouped data for SFC values (mean ±2 S.E.M.), normalised for idiosyncratic firing frequency are plotted in F.
We also calculated spike-field coherence (SFC) to assess phase synchronization between the LFP and spike times as a function of frequency [32]. SFC is a unitless measure between zero and one – values of one suggest a constant phase synchronisation of spikes with the LFP. Figure 2D and 2E show SFC spectra for each STA (solid lines) with maximal peak values at 0.66 and 0.37, respectively. Confidence limits were generated using Monte Carlo simulations (95th centile of 1000 shift predictors – broken lines), thus SFC values above these limits are considered to be significantly above levels expected by chance synchronisations between spikes and LFP frequency components (p<0.05). Dominant peaks in the SFC spectra were closely linked to idiosyncratic firing frequency, c.f. superimposed spike frequency distributions (grey curves) in each example. Grouped data are plotted in Figure 2F (normalised for the idiosyncratic firing rate of each cell) which shows a 95% confidence interval for SFC values with respect to normalised idiosyncratic firing frequency; maximal phase synchronisation occurs at frequencies closely corresponding to the cell's idiosyncratic firing frequency. We conclude that peri-spike voltage oscillations and spike output are closely synchronised.
The localised origin, spike-time dependence and the spike field coherence for peri-spike periodic STAs suggests they represent membrane voltage fluctuations from the individual neurones under study. These findings are consistent with the regular firing patterns of metronome cells being underpinned by intrinsic oscillatory currents [44].
Sensory evoked silent periods phase reset metronome cells
Somatosensory activation interrupted the regular firing of metronome cells by causing a brief silent period (duration 40–50 ms) following which the spontaneous spiking resumed, thus sensory evoked silent periods served to reset the spike-times of the regular spiking metronome cells (Figure 3A). Responses of this type could be evoked from widespread areas of the skin, including the face and limbs, and consistent with previous studies of LRN neurones [51], [52], metronome cells had widely convergent, often bilateral receptive fields (see Text S1 and Figure S1). In the example PSTH shown in Figure 3A, stimuli were delivered at random relative to the firing of the cell. Reorganising the raster based on the expected spike times (see Methods), reveals the phase response properties of this neurone (Figure 3B). Intuitively, many of the observed spikes following the perturbations are delayed (i.e. evoked silent periods extend the interspike interval = phase procession; trials 1∼30) whilst others show little or no deviation (trials 31∼50). However, the perturbation caused many spikes to occur earlier than expected (trials 51∼71: phase precession). A spike-time phase response curve (PRC) for this cell is shown in Figure 3C. The shape of the PRC is related to spike-generation mechanisms and thus offers a precise characterisation of the effect a perturbation has on an on-going oscillation and the timing of subsequent spikes [53]. The PRC shown in Figure 3C indicates that perturbations of the regular firing of this metronome cell occurring soon after spontaneously generated spikes (phase 0–0.3) tended to advance the subsequent spike-time by 5–10 ms (triangles <0). Spike-time delays up to 60 ms were observed for perturbations occurring later in the firing cycle. Given the idiosyncratic firing frequency of this cell was 12–13 Hz these changes in spike-time range from ∼10% advancement to ∼75% delay.
10.1371/journal.pone.0026503.g003Figure 3 Somatosensory stimulation briefly silences and phase-resets metronome cell spike timing.
A shows a PSTH (bin size 5 ms) and raster from an example metronome cell following sensory stimulation (limb stimulus, 0.6 Hz). Note the evoked silent period shortly after each stimulus (onset latency ∼10 ms, duration 40–50 ms) which stops the spontaneous spiking of the cell, thereby ‘resetting’ the subsequent spike-timings, evident as peaks in the PSTH. B shows the same raster as in A reordered by the expected spike-time (see Methods) for each trial (red triangles). The first observed spike-times after each stimulus are indicated by blue triangles – note the spike-time delays (lower half of panel) and spike-time advancements (upper half of panel). C plots the phase-response curve for this same cell, summarising the raster shown in B. The phase response curve in D show grouped data, normalised for idiosyncratic firing frequency; solid lines represent a 95% confidence interval for the median in each of 10 phase bins. The phase response curves in E and F extend the analysis to the subsequent interspike intervals (cf. green and pink triangles in C).
The PRC for group data from 18 cells tested in the same way (40 perturbations each) is shown in Figure 3D – data are normalised for idiosyncratic firing frequencies. Each triangle represents a perturbed spike-time from an individual cell (cf. blue triangles in Figure 3B). Data are grouped into 10 phase bins with 95% confidence intervals calculated for the mean of each bin (solid lines). The PRC indicates that for a typical metronome cell, phase advancement of 5–10% is likely to occur for perturbations that arrive within the first third of a typical firing cycle (i.e. observed spike-times are 5–10% earlier than expected). Little or no change in spike-time was apparent for perturbations delivered across 30–40% of a ‘typical’ firing cycle, whereas for all later-arriving perturbations subsequent spike-times were delayed by up to 50%.
We extended the analysis to the 2nd and 3rd spike-times in the series (green and pink triangles, respectively in Figure 3B). We found that the adjacent interspike interval (2nd spike) tended to show a modest spike-time advancement of 5–10% (Figure 3E, cf. Figure 3B), irrespective of the phase of the initial perturbation. The spike-times of the 3rd (and subsequent spikes – not shown) did not show this effect. This finding indicates that the influence of the evoked silent periods may extend to 1st and 2nd spike-times after the perturbation, but not beyond.
The spike-time advancement arising through presumed inhibitory input (i.e. evoked silent periods) suggests that inhibition-activated depolarising mechanisms, such as Ih
[54], might be brought into play following sensory input. Metronome cells can thus reflect the precise timing of sensory events by the phase response of their spike-time resetting, on a trial-by-trial basis.
Spike-time alignment of metronome cell firing to rhythmic sensory input patterns
The regular firing pattern of metronome cells at idiosyncratic frequencies ranging from ∼7–22 Hz prompted us to examine the effects of rhythmic somatosensory stimulation on spike-timing. We used trains of stimuli ranging from 6–30 Hz; a bandwidth that encompasses rhythmic movements such as licking, grooming, locomotion and also the distribution of idiosyncratic firing rates of metronome cells (Figure 1E).
Metronome cells showed a pronounced ability to stimulus-lock their spike times across a broad range of sensory stimulation frequencies. Data from an example cell are shown in Figure 4. Ascending stimulation frequencies (2 Hz steps) cause pronounced shifts in the overall activity of this cell (cf. Figure 3C), as shown by the close relationship between spike-times and individual stimulus pulses (black triangles – Figure 4A). During sensory stimulation, spike firing shifted toward the stimulation frequency; this cell was able to lock onto input frequencies <10 Hz (cf. ISI probability distributions, Figure 4B), whilst for input frequencies 10–16 Hz, the firing was split into at least two distributions each tending toward the stimulation frequency or half of this value, respectively. With increasing stimulation frequency, spike firing became increasingly locked to half of the stimulation frequency, reaching a ceiling at >24 Hz, where the stimulus-locking relationship broke down. In order to assess spike-time alignment precision at different input frequencies, we calculated a spike-stimulus synchronisation index (SI – see Methods; Figure 4C). This provided a unitless measure between 0 and 1, thus if all spikes occur at the same phase relative to each stimulus pulse, SI = 1. In this particular example, spike-stimulus synchronisation exceeded 0.5 for frequencies in the range 8–20 Hz, thus at least half of the spikes had a consistent relationship to the stimulation frequency; this particular cell had an idiosyncratic frequency of ∼14 Hz. Given that stimulation could last for ∼1000 ms (see Methods), these findings indicate that metronome cells can show sustained spike-time alignment to a broad range of sensory input frequencies.
10.1371/journal.pone.0026503.g004Figure 4 Rhythmic somatosensory stimulation entrains metronome cell spiking in a band-pass like manner.
The PSTHs in A show the responses of an example metronome cell during a variety of rhythmic stimulation patterns (stimulus frequency indicated beside each PSTH, stimulus pulse times represented by solid triangles under each PSTH; bin-size 10 ms). Simple visual inspection of the PSTHs is misleading as it obscures trial-by-trial variability, thus we computed probability density estimates for the interspike interval frequencies (B) before and during the stimulus trains (white and black distributions, respectively). To aid visual inspection, in each plot in B stimulation frequency is indicated by the open triangles whereas the grey triangles represent half of this value – note the tendency for spiking frequencies to centre around these values at different stimulation frequencies. Spike-time accuracy with respect to each stimulus pulse in the train was calculated using a spike-stimulus synchronisation index (see Methods).
In order to group data across cells, we normalised for the idiosyncratic frequencies of our cells. Data for 12 cells tested with the range of input frequencies are plotted in Figure 5A, where the ISI frequency distributions during stimulation are represented as colour-coded probability density. Grouping the data in this way suggests that stimulus-locking (either at the stimulation frequency or half of this value) will occur for inputs in the range of 0.5 to <2-times the cell's idiosyncratic frequency, indicated by the diagonal trends in the colour plot (cf. Figure 4B). Grouped data for the spike-stimulus synchronisation index are plotted alongside (Figure 5B). Analogous to tuning curves and best-frequencies for auditory hair cells, maximal spike-time accuracy for individual cells occurs with input frequencies close to the idiosyncratic frequency.
10.1371/journal.pone.0026503.g005Figure 5 Population entrainment in response to rhythmic somatosensory stimulation.
A plots grouped data for 12 metronome cells tested with the gamut of rhythmic stimulation frequencies – data were grouped as probability density estimates and normalised for idiosyncratic firing rates, thus stimulation frequencies are expressed relative to these values for each cell. The diagonal trends in the probability density distributions indicate the tendency for the metronome cells to alias stimulation frequencies in the range of 0.5 - <2 times their idiosyncratic frequencies. B plots the spike-stimulus synchronisation index derived from the same grouped data, indicating that maximal spike-time accuracy with respect to individual stimulation pulses occurred for stimulation frequencies close to the idiosyncratic firing frequency. Data are plotted as mean ±2 S.E.M.
It is not straight-forward to interpret the PRC analysis alongside these findings, although it is noteworthy that on average, cells tend to decelerate (i.e. phase delay) and similarly they may also show modest acceleration (i.e. 5–10% phase advance) for input frequencies <1.5-times the idiosyncratic frequency, cf. white dotted line in Figure 5A. These findings suggest that across the population of metronome cells subsets of cells are capable of stimulus-locking to a broad range of input frequencies, consistent with individual cells performing a ‘band-pass’ operation similar to that seen in cortical and trigeminal neurones [55], [56].
Sensory evoked silent periods drive correlated firing in metronome cells
Phase resetting of spike-times, and stimulus-locking during rhythmic sensory activation will have important consequences for the downstream target neurones; the cerebellar granule cells. Anatomical convergence and synchronisation of multiple metronome cells could transform relatively slow spike-trains (∼7–20 Hz across the metronome cell population) into high frequency, near-coincident, volleys of excitatory post-synaptic potentials – an essential requirement for spiking in granule cells [25], [26].
To assess time-resolved stimulus-induced correlations between simultaneously recorded metronome cells, we used the joint peri-stimulus time histogram technique [JPSTH – 33]. Our analysis suggests that the evoked silent periods following sensory stimulation can synchronise metronome cells, for the duration of the sensory stimulation. We analysed data from 11 LRN neurones (12 possible pairs recorded simultaneously) tested with a range of stimulation frequencies (see Figure 4). Assessment of the normalised JPSTHs (i.e. normal JPSTH minus shift-predictor) showed no residual correlations (data not shown) indicating that any correlations between metronome cells were underpinned by stimulus-induced spike-time changes, rather than arising through mutual connections [33]. Since we found no evidence for correlations other than those driven exclusively by stimulation, henceforth we only consider the bin-by-bin cross-product of the two PSTHs (‘predictor’, alternatively the PST-product) – this represents the null hypothesis that spiking probabilities are related to the stimulus, although each neurone fires independently of its counterpart. We first examined near-coincident firing arising from single stimuli – example data for a metronome cell pair are drawn in Figure 6A (idiosyncratic frequencies ∼12 Hz and 14 Hz, respectively). The main diagonal of the JPSTH gives rise to the ‘PST coincidence histogram’ (Figure 6B) which displays the observed rate at which both neurones fire simultaneously (to within the accuracy of the bin-width of the histogram – 2 ms). Near-coincident activity is most likely at ∼50 ms latency (i.e. the first spike following the evoked silent periods in each neurone). Subsequent near-coincident firing occurs due to stochastic interaction of the cells' idiosyncratic frequencies, producing the interference pattern visible in the correlation delay matrix (paradiagonal to JPSTH diagonal) shown in Figure 6C, which highlights the lead-lag times for near-coincident spike-timings. The arbitrarily chosen lead-lag range of −25 to +25 ms corresponds to a minimum instantaneous frequency of 40 Hz – if these metronome cells projected to the same granule cell, the principal coincidences for this cell pair would generate EPSPs at rates >200 Hz (taken as the instantaneous frequency of the spikes generated by the cell pair). Grouped data for 11 metronome cell pairs tested with single stimuli are shown in the same format in Figure 6D – thus, the population response of metronome cells (of differing idiosyncratic frequencies) following a single stimulus is a well synchronised, but brief lived increase in near-coincident firing at ∼50 ms (see expanded time-base in lower panel).
10.1371/journal.pone.0026503.g006Figure 6 Near-coincident firing in metronome cells induced by single somatosensory stimuli.
A shows the JPSTH for a pair of simultaneously recorded metronome cells following a single sensory stimulus (limb); the matrix represents the PST-product, from which the diagonal is used to give rise to the PST coincidence histogram shown in B. A further extension of this is the correlation delays matrix shown in C, which highlights epochs of near-coincident firing for the cell pair, along with lead-lag times between counterpart spikes corresponding to a minimum of 40 Hz. Note that near-coincident firing is most probable ∼50 ms after the stimulus onset (i.e. latency of the first phase-reset spike-time) with subsequent stochastic interactions between the cells as they resume their idiosyncratic firing patterns (see Figure 3). The correlation delay matrix shown in D plots grouped data for 11 cell pairs tested with single sensory stimuli. E plots correlation delay matrices for the same cell pair illustrated in A–C, during a variety of rhythmic stimulation patterns – stimulation frequency is indicated besides each panel, and individual pulse times are represented by vertical white lines. Lead-lag times in each panel are 0±25 ms (arbitrarily chosen), corresponding to a minimum instantaneous frequency of 40 Hz. Note the tendency for epochs of near-coincident firing to persist for the duration of the stimulation trains (1000 ms), and the apparent ‘preference’ of this cell pair for input frequencies <16 Hz.
The stimulus-locking properties of metronome cells suggest that spike-timing can be altered for prolonged periods during rhythmic somatosensory stimulation (see Figures 4 and 5). This may create ideal conditions for stimulus-induced synchronisation of multiple metronome cells across the population. Using the same format as Figure 6D, the correlation delay matrices shown in Figure 6E for the same cell pair show that stimulus-locking gives rise to repeated epochs of near-coincident firing closely tied to the stimulation frequency. Moreover, this behaviour persists for the duration of each stimulation train (1000 ms), although with the cessation of stimulus-locking at the termination of the stimulus train, the cell pair rapidly de-correlated and resumed their idiosyncratic firing (cf. Figure 6C). As stimulus locking degrades with increasing stimulation frequencies, especially >20 Hz (correlation delay matrices not shown) so too does the likelihood of synchronisation, thus near-coincident firing appears most prominent for this cell pair for stimulation frequencies <16 Hz (cf. idiosyncratic frequencies ∼12 Hz and ∼14 Hz).
Considered across the population, correlation delay matrices for grouped data are plotted in Figure 7 showing that the population of metronome cells, each with idiosyncratic frequencies, are capable of temporally encoding a variety of stimulation frequencies into sustained ‘packetised’ near-coincident firing. The stimulus-locking preferences for input frequencies <20 Hz considered alongside the decrement in synchronisation likelihood suggests that the population of metronome cells may behave as a series of band-pass filters (≤20 Hz; cf. individual cells behaving as band-pass filters – Figure 4) able to broadcast temporally aligned near-coincident activity to the cerebellum, indexed by the underlying frequency of sensory events.
10.1371/journal.pone.0026503.g007Figure 7 Near-coincident firing in metronomes cells is paced by rhythmic sensory stimulation.
Following the same format as Figure 6E, correlation delay matrices are shown for grouped data derived from 11 metronome cell pairs tested with the range of stimulation frequencies. Note the tendency for epochs of near-coincident firing closely indexed by the stimulation frequency and the decline of near-coincident firing for input frequencies ≥20 Hz.
Synaptic integration of metronome cell spike-timing in granule cells
Our analysis suggests that common inhibitory drive can temporally-align metronome cell spike-timing, leading to a synchronised population broadcast of mossy fibre activity to the cerebellum. Prior modelling work has highlighted precise timing of mossy fibre inputs across granule cell dendrites as a requirement to reach spike-threshold [25], [26]. We examined how metronome mossy fibre signals might summate in a simulated granule cell (see Methods), employing real spike-times recorded from a range of metronome cells (n = 10 cells, covering the range of firing frequencies, only ipsilateral hindlimb responsive cells were arbitrarily selected for consistency and to normalise for peripheral conduction delays). In the first of two scenarios, we assumed that the simulated granule cell received convergent mossy fibre inputs with similar frequencies on each of 4 dendrites, in contrast to the alternative scenario where convergent input frequencies were dissimilar, instead covering a broad range (note that spiking and inhibition were not included in the simulation). As expected for the similar input scenario, a single stimulus reveals a resonant EPSP pattern, reflecting the auto-correlation of similar frequencies, particularly close to the idiosyncratic frequency of the mossy fibres (∼16 Hz; Figure 8A). In keeping with the stimulus-locking of individual metronome cells, no patterned EPSP summation occurred for stimulation frequencies of 20 Hz and above (only 20 Hz case shown; cf. 24 Hz and above in Figure 4 and inputs >20 Hz in Figures 6 and 7). In our alternative scenario using dissimilar frequencies, a diverse mixture of metronome cells with firing frequencies ranging from 8 to 16 Hz converging onto a single granule cell produced more complex results. In this situation, single stimuli failed to evoke EPSP summation (Figure 8B). Although our JPSTH analysis suggests that single stimuli evoke mass synchronisation across the population (onset latency ∼50 ms; see Figure 6D), it is not guaranteed that all granule cells receiving convergent metronome mossy fibre inputs would experience this, as precise synchronisation depends on the idiosyncratic firing frequencies of the particular mossy fibres reaching each granule cell. During rhythmic stimulation, modest EPSP patterning is evident at 6 Hz and 12 Hz, with highly coincident patterned EPSP summation visible during 18 Hz stimulation, with a similar failure of summation at the highest frequency. The pronounced resonance at 18 Hz is sharply in contrast to the relative lack of resonance at frequencies bordering this (16 Hz and 20 Hz). Although these models are speculative, they suggest that frequency-specific EPSP patterning may occur in individual granule cells and that these properties may emerge as a function particular pattern of idiosyncratic frequency convergence of metronomic mossy fibres.
10.1371/journal.pone.0026503.g008Figure 8 Simulation of EPSP summation arising from near-coincident firing of metronome cells converging in granule cells.
The cartoons in A & B illustrate the input characteristics used for each simulation of averaged EPSP summation in a simulated granule cell, i.e. similar or dissimilar convergence of metronome mossy fibres. Spike times from a real metronome cell with 16 Hz idiosyncratic firing frequency were fed to each dendrite in A, giving rise to resonant patterning of EPSPs at a variety of rhythmic stimulation frequencies (stimulus pulse times indicated by triangles). In B, spike times from 4 individual metronome cells, each with differing idiosyncratic firing frequency, were fed into the model. In contrast to A, single stimuli did not bring about EPSP summatioan although rhythmic stimulation, particularly at 18 Hz gave rise to resonant EPSP patterning.
Discussion
Our experiments reveal an unusual spike-time coding strategy driven by inhibition imposed upon highly regular firing in metronomic neurones which in turn form a major mossy fibre input to the cerebellum.
Phase resetting of spike-time by sensory evoked inhibition can produce either spike-time advancements and delays indexed by the arrival time of the inhibition relative to preceding spontaneous spike. This type of biphasic phase response is known as a type II PRC [57] in contrast to type I PRCs where only spike-time delays are seen. Many PRC studies in vitro rely on depolarising current or sinusoidal current injection to evoke firing [54], [58], [59]. Our study has the advantage of exploiting rhythmic spontaneous firing and uses an intact sensory pathway to drive inhibition-mediated phase resetting. Although we do not address the mechanisms behind this inhibition, comparison with GABAergic resetting of intrinsic oscillations and spike-timing mechanisms has been reported in subthalamic neurones [18], also in Type A cells of the globus pallidus (Fig. 4 of [19]) and hippocampal pyramidal cells [17] and indeed GABAergic inhibition has a prominent role in the generation and maintenance of oscillatory activity [7], [60], [61]. Our spike-time advancements (extending over two ISIs; approximately 200 ms) are consistent with GABAergic activation of hyperpolarisation-activated cation currents, such as Ih [54]. Computationally, a pool of oscillators at differing frequencies can efficiently represent the passage of time [62] and although inhibitions appear brief-lived (40–50 ms), the ionic mechanisms underlying regular metronome cell firing create a de facto spike-time ‘memory’ of inhibition arrival times, extending over many hundreds of milliseconds (Figures 3 and S1 of [63]). Neurones considered under integrate-and-fire models (which fire solely on the basis of synaptic events) do not show this property, it being instead a property of resonate-and-fire models. Such models rely on sub-threshold oscillations imparting a membrane resonance to the neurone – inputs timed according to the eigenfrequency, or its factors, may bring the cell to spiking threshold. Inhibitory inputs can also produce post-inhibition spiking, via anodal break excitation (Figures 4 and 8 of [64]) suggesting a close correlate between metronome cells and the resonate-and-fire model. Resonance is generally considered in terms of sub-threshold oscillations, although our metronome cells were spontaneously active raising the possibility that spikes and associated after-potentials might serve as an oscillatory ‘internal’ reset mechanism as well as an output signal [see 63]. As highlighted in Figure 2, metronome cells appear to behave as independent neuronal oscillators since their spikes are intimately associated with locally generated periodic voltage oscillations intimately linked to the cell's idiosyncratic frequency. Since we used spikes and LFP from the same electrode it is possible that slow components of the spikes (afterpotentials) contribute to these LFP oscillations as suggested by Buszaki [45]. However, sub-threshold soma-dendritic membrane voltage oscillations also make a direct contribution to local LFP [44] and we interpret the peri-spike voltage oscillations as reflecting these processes. An additional concern surrounds our use of anaesthetic which may have induced regular firing in metronome cells. However, other cerebellar cell types fire with much greater irregularity under the same conditions (see Figure 1F). Our data suggest that metronome cells possess intrinsic pacemaker-like properties which may be underpinned by persistent sodium currents and calcium activated potassium currents leading to membrane resonance [for review see 10] and auto-rhythmic firing [65], [66]. A further limitation of our experiments concerns the use of electrical micro-stimulation of sensory afferents capable of producing highly synchronous afferent volleys. In contrast, natural sensory stimuli may well evoke lower levels of metronome cell population synchrony although rhythmic behaviours such as whisking, sniffing, etc. are likely to produce synchronous afferent input (discussed later). It is also noteworthy that cerebellar Golgi cells recorded in the same preparation show qualitatively similar responses to electrical or air-puff stimulation of the same sensory areas (Figure 8 of [28]).
Ensemble recordings revealed that single sensory evoked inhibitions can briefly synchronise ensembles of metronome cells (Figure 6C and 6D). Comparable behaviour has been observed in Type A globus pallidus cells sharing inhibitory input [19]. Similarly, olfactory bulb mitral cells can form synchronous ensembles when receiving common inhibitory ‘noise’ [67] suggesting a close correlate between our metronome cell synchrony arising via common inhibitory inputs. Across the population, Type II phase-resetting of spike-times may promote temporal alignment between cells; neurones with Type II PRCs receiving common noisy input synchronise more readily than those with Type I PRC [68]. Thus far, we have only considered the response of metronome cells following a single, randomly delivered, sensory evoked inhibition. The large receptive fields of metronome cells suggest they may gather sensory information from many sources including the face and limbs (Figure S1). Vibrissae movements in rats exhibit a range of frequencies from 2–20 Hz with predominant frequencies around 2 Hz, 5–9 Hz and ∼16 Hz [69] similar to licking [4–7.5 Hz 70], rapid sniffing during odour discrimination [see 71] and stereotyped grooming behaviour [2–7 Hz 72]. Moreover, in cats, cerebellar-projecting LRN cells can receive locomotor and/or rhythmic central respiratory inputs [73]. These sources of rhythmic inputs would be expected to generate periodic/rhythmic barrages of inhibition in metronome cells. Rhythmic stimulation in our experiments created a forced-periodic synchronisation that persisted for the duration of the stimulus (1000 ms), bearing a close correlate with periodic stimulation in the resonate-and-fire model [64]. Metronome spike-timing can reliably represent the sensory stimulation frequency and its factors, with maximal spike-time accuracy occurring for frequencies close to the cell's idiosyncratic frequency. Similar spike-time alignment via phase-resetting of intrinsic oscillations and synchronisation of multiple hippocampal principal cells can be induced by rhythmic stimulation of inhibitory inputs at 5 Hz [17], although in that study other frequencies were not examined. Such behaviour suggests that metronome cells can behave in a manner similar to band-pass filters [c.f. 55], [56] and that as a population they broadcast a ‘packetised’ code of sensory input frequency [74], established through brief-lived epochs of synchronisation, to their downstream targets in the cerebellum. However, the reliability of such a code at the single neurone level may be low since stimulus-spike locking can be <1∶1 (see Figure 4 and 5). None-the-less, these limitations might be compensated for at the population level by synchronised ensembles of cells and through the possible anatomical convergence of metronome cell projections upon their downstream targets.
Functionally, our data complement the view that that one of the major roles for inhibition is to determine precise spike-timing [18], [75]. The most numerous targets for metronome cells are cerebellar granule cells, although Golgi cells may also be targeted. Prior modelling work has highlighted precise timing of inputs across granule cell dendrites as a requirement to reach spike-threshold [25], [26]; we examined these issues using the same granule cell model. Although we did not specifically examine spiking (nor was inhibition included) our simulations suggest that metronomic mossy fibres, if converging on individual granule cells, may impart their resonant properties predisposing granule cells to respond favourably at particular input frequencies and thereby creating preferred times for spike generation. In this regard, the spiking output of granule cells is expected to linearly reflect their membrane potential level [25], [76], although the exact timing of granule cell spikes is subject to stochasticity [77]. Alternatively, synchronised metronomic mossy fibre input may be equally well suited to induce NMDA-receptor mediated plasticity [c.f. theta burst stimulation used by 78]. However, metronome cells are not the only source of mossy fibres and we cannot address the question of whether all LRN cells are metronomic; in contrast, other mossy fibre types tend to fire at much higher frequencies (>100 Hz) during signalling [25], [35], [37], [79], [80]. Indeed, a subset of metronome cells do show brief-lived high frequency excitations (see Figure S1C, S1D, S1F and S1H). In agreement with our own observations, other studies have described metronome-like mossy fibre terminals in the anterior lobe of decerebrate cats [81]. These mossy fibres discharged regularly at ∼20 Hz and showed spike-time resetting responses initiated by sensory evoked silent periods, identical to our own responses. Taken together, these observations suggest that metronome cells are at least common to both rats and cats and that they may represent a generalised coding scheme of cerebellar mossy fibres.
If metronome cells converge on individual granule cells our data suggest that the tight synchronisation of their activity across multiple dendrites could generate an EPSP profile similar to the higher frequency firing mossy fibre types. Prior investigations have not described granule cell EPSP patterns predicted by our simulations [25], [26], [80], [82] although it is noteworthy that none of these studies employed rhythmic stimulation – our simulations suggest that this is a key requirement to reveal frequency-patterned EPSP summation, in particular, no EPSP patterning is seen when convergent mossy fibre inputs of dissimilar frequency respond to a single stimulus (Figure 8B) so the possibility remains that frequency-patterned EPSP summation is yet to be observed experimentally. Nonetheless, granule cells in vitro show membrane resonance around 4–10 Hz and Golgi cells show pacemaker oscillations at similar frequencies [11], [12], [66], whilst Golgi cells in vivo may participate in 5–30 Hz oscillatory activity via gap-junction coupling to other Golgi cells [13]. LFP oscillations (7–25 Hz) in the granular layer have been reported in awake monkeys, rabbits and rats during periods of quiet attentiveness or learning [13], [83]–[85] although the origin of these signals remains unclear their frequency range is a very close correlate with metronome cell firing frequencies (∼7–22 Hz). The resonant properties of Golgi cells and granule cells predisposes them to ‘tune-in’ to oscillatory inputs, thus metronome cells may be one of the principal orchestrators of oscillatory sensorimotor information processing at the input stage of the cerebellum.
Supporting Information
Figure S1
Characterisation of somatosensory responses in metronome cells. The PSTHs in A and B show typical spike-time resetting responses beginning with silent periods only, for an example brainstem unit and cerebellar unit, respectively. Action potential waveforms are inset – note the dual component action potential of the cerebellar units. Similar data are presented in C and D illustrating responses beginning with short-lasting excitations (SLE; highest bins in each PSTH) for example brainstem and cerebellar units, respectively. The vibrisssal-evoked SLE response shown in D consisted of two discrete excitations – see expanded time-base (inset). The bar charts summarise the response likelihoods from each of six routinely tested skin areas (E and F) alongside data for onset latencies and durations of resetting responses overall (G) and SLE excitations only (H). Note that two bars are presented for vibrissal responses (cf. D). PSTH bin-size 10 ms. Error bars represent mean ±2 S.E.M.
(TIF)
Click here for additional data file.
Text S1 Widespread, bilateral receptive fields of metronome cells.
(DOC)
Click here for additional data file.
The authors express their gratitude to Dr. Steve Edgley and Prof. Ole Paulsen, (Dept of Physiology, Development and Neuroscience, Cambridge University) and to Mr. Patrick Dylan Rich (Janelia Farm Research Campus) for insightful discussions and helpful comments on the manuscript and to Dr. Wei Xu (Dept of Physiology, Development and Neuroscience, Cambridge University) for practical assistance during some experiments.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was funded by the Medical Research Council, United Kingdom and the Isaac Newton Trust, Cambridge, United Kingdom. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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BMC Complement Altern MedBMC Complementary and Alternative Medicine1472-6882BioMed Central 1472-6882-11-762193343310.1186/1472-6882-11-76Research ArticleIn vitro antioxidant and anticancer activity of young Zingiber officinale against human breast carcinoma cell lines Rahman Shahedur [email protected] Faizus [email protected] Asif [email protected] Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia-7003, Bangladesh2 Department of Biotechnology and Genetic Engineering, University of Development Alternative, Dhaka, Bangladesh2011 20 9 2011 11 76 76 7 8 2011 20 9 2011 Copyright ©2011 Rahman et al; licensee BioMed Central Ltd.2011Rahman et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
Ginger is one of the most important spice crops and traditionally has been used as medicinal plant in Bangladesh. The present work is aimed to find out antioxidant and anticancer activities of two Bangladeshi ginger varieties (Fulbaria and Syedpuri) at young age grown under ambient (400 μmol/mol) and elevated (800 μmol/mol) CO2 concentrations against two human breast cancer cell lines (MCF-7 and MDA-MB-231).
Methods
The effects of ginger on MCF-7 and MDA-MB-231 cell lines were determined using TBA (thiobarbituric acid) and MTT [3-(4,5-dimethylthiazolyl)-2,5-diphenyl-tetrazolium bromide] assays. Reversed-phase HPLC was used to assay flavonoids composition among Fulbaria and Syedpuri ginger varieties grown under increasing CO2 concentration from 400 to 800 μmol/mol.
Results
Antioxidant activities in both varieties found increased significantly (P ≤ 0.05) with increasing CO2 concentration from 400 to 800 μmol/mol. High antioxidant activities were observed in the rhizomes of Syedpuri grown under elevated CO2 concentration. The results showed that enriched ginger extract (rhizomes) exhibited the highest anticancer activity on MCF-7 cancer cells with IC50 values of 34.8 and 25.7 μg/ml for Fulbaria and Syedpuri respectively. IC50 values for MDA-MB-231 exhibition were 32.53 and 30.20 μg/ml for rhizomes extract of Fulbaria and Syedpuri accordingly.
Conclusions
Fulbaria and Syedpuri possess antioxidant and anticancer properties especially when grown under elevated CO2 concentration. The use of ginger grown under elevated CO2 concentration may have potential in the treatment and prevention of cancer.
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Background
Cancer is a multi-step disease incorporating physical, environmental, metabolic, chemical and genetic factors, which play a direct and/or indirect role in the induction and deterioration of cancers. Diet containing antioxidant rich fruits and vegetables significantly reduces the risk of many cancer diseases suggesting that antioxidants could be effective agents for the inhibition of cancer spread. These agents are present in the diet as a group of compounds with low toxicity, safe and generally accepted [1]. The Isolated polyphenols from different plants have been considered as indicator in a number of cancer cell lines at different evolutionary stages of cancer. Anticancer activities of Flavonoids were described in various studies [2]. Some tests showed antitumor properties of quercetin including the inhibition of cancer cell proliferation and migration [3]. The isolated polyphenols from strawberry including kaempferol, quercetin, anthocyanins, coumaric acid and ellagic acid were shown to inhibit the growth of human cancer cell lines originated from breast (MCF-7), oral (KB, CAL-27), colon (HT-29, HCT-116), and prostate (LNCaP, DU-145) [4]. Similar results have also been reported in other studies with wine extracts, isolated polyphenols (resveratrol, quercetin, catechin, and epicatechin) and green tea polyphenols (epigallocatechin, epicatechin) [5,6]. Arts et al. reported of catechin's ability to control postmenopausal cancer in woman [7]. They found that catechin intake may prevent rectal cancer. Epicatechin and gallocatechin-3-gallate induce reduction in experimental lung tumour metastasis (77% and 46%). Epigallocatechin-3-gallate is an effective antiangiogenesis agent, which inhibits tumour cell invasion and proliferation [8]. It also inhibits the growth of the NBT-II bladder tumour cells and breast cancer cell lines [9]. Manthey et al. reported that citrus flavonoids inhibited the growth of HL-60 leukaemia cells [10]. Kaempferol belongs to the flavonoids group. Luo et al. showed kaempferol inhibited the growth of ovarian cancer cell lines (91%) and A2780/CP70 (94%) by concentration of 20 μM and 40 μM respectively [11]. Inhibition of breast cancer cell lines (MCF-7 and MDA-MB-231) by quercetin was reported by Gibellini et al. [12]. In recent years, researches about anticarcinogenic potential of quercetin have exhibited its promise as an anticancer agent. Likewise, in vitro and in vivo studies showed that quercetin was able to inhibit viability of leukemic cells, colon and ovarian carcinoma cells, and especially human breast cancer cells.
The Zingiberaceae family is well-known in Southeast Asia and many of its species are being used as traditional medicine, which is found to be effective in the treatment of several diseases. Zingiber officinale is generally used as a culinary spice in Bangladesh and as well as for the treatment of oral diseases, leucorrhoea, stomach pain, stomach discomfort, diuretic, inflammation and dysentery. Shukla et al. reported cancer preventive properties of ginger and showed that this ability is related to flavonoid and polyphenolic components of fresh ginger extract especially quercetin [13]. Kuokkanen et al. showed that the concentration of total phenolics was significantly increased in the birch leaves produced in the CO2-enriched air, as has also been observed in the experiments of Ibrahim et al. [14,15]. Emerging management strategies are using eco-physiological factors to elevate phytochemical concentrations in food crops. Some eco-physiological conditions that are thought to have significant impact on the enhancement of health-promoting phytochemicals in a number of plants include environmental conditions, cultural and management practices [16]. In addition, there is an increasing interest in using appropriate strategies of management practices to improve the quality of food crops by enhancing their nutritive and health-promoting properties. The results of previous studies indicated that the synthesis of phenolics and flavonoids in ginger can be increased and affected by using CO2 enrichment and following that, the antioxidant activity in young ginger extracts could also be improved [17]. Information about anticancer and antioxidant activities of enriched ginger by elevated CO2 concentration is scarce. On the other hand, the impacts of cultural conditions and CO2 concentration on biopharmaceutical production in herbs have not been widely investigated and it is needed to be understood, especially when the objective is the optimization of the herb chemistry. In this study, we aimed to explore antioxidant potential and anticancer activities of two Bangladeshi ginger varieties (Zingiber officinale) at young age and grown under different CO2 concentration.
Methods
Plant material
Two varieties of Zingiber officinale Roscoe (Fulbaria and Syedpuri) rhizomes were germinated for two and half weeks and then transferred to polyethylene bags which were filled with soilless mixture of burnt rice husk and coco peat in a ratio 1:1. After two and half weeks, those plants were transferred to CO2 growth chamber with two different CO2 concentrations (400 μmol/mol, ambient; 800 μmol/mol, elevated CO2 concentration). Pure carbon dioxide (99.6% purity) was supplied from high pressure carbon dioxide cylinder and injected through a pressure regulator into the growth chamber. Irradiance, relative humidity and air temperature of chamber were controlled using integrated control, monitoring and data management system software. Plants were harvested at 15 weeks and aerial parts and rhizomes separated and freeze dried and kept in -90°C for future analysis.
Extract preparation
Aerial parts and rhizomes were dried (freeze dry) to constant weights. Aerial parts and rhizomes (1 g) were powdered and extracted using methanol (50 ml), with continuous swirl for 1 h at room temperature using an orbital shaker. Extracts were filtered under suction, evaporated and crude extract stored at -25°C. These crude extracts were used in this study [18].
Determination of antioxidant activity
TBA assay
The method of Ottolenghi (1959) was used to determine the TBA (thiobarbituric acid) values of the samples [19]. The formation of malonaldehyde is the basis for the well-known TBA method used for evaluating the extent of lipid peroxidation. At low pH and high temperature (100°C), malonaldehyde binds TBA to form a red complex that can be measured at 532 nm. The increase amount of the red pigment formed correlates with the oxidative rancidity of the lipid. 2 ml of 20% trichloroacetic acid (CCI3COOH) and 2 ml TBA aqueous solution were added to 1 ml of sample solution and incubated. The mixture was then placed in a boiling water bath for 10 min. After cooling, it was centrifuged at 3,000 rpm for 20 min and the absorbance of the supernatant was measured at 532 nm. Antioxidant activity was determined based on the absorbance.
Cell culture and treatment
Human breast cancer cell lines (MCF-7 and MDA-MB-231) were obtained from the American Tissue Culture Collection (ATCC) (Rockville, MD) and were cultured in 100 μl of Roswell Park Memorial Institute medium (RPMI) 1640 media supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. MCF-7 and MDA-MB-231 cells were incubated overnight at 37°C in 5% CO2 for cells attachment [20].
Both non invasive MCF-7 and highly invasive MDA-MB231 cancer cells were used in this study to verify the effectiveness of ginger extract against them.
Determination of anticancer activity
MTT assay
The assay detects the reduction of MTT [3-(4, 5-dimethylthiazolyl)-2, 5-diphenyl-tetrazolium bromide] by mitochondrial dehydrogenase to blue formazan product, which reflects the normal functioning of mitochondria and hence the cell viability. The experiment was conducted as described by Mosmann (1983) [21]. Briefly, the cancer cells were seeded in 96-well plates at a density of 1 × 104 cells/well in 100 μl RPMI. After twenty-four hours of seeding, the medium was removed and then the cells were incubated for 3 days with RPMI with the absence and/or the presence of various concentration of ginger extracts. Ginger extract was added at various concentrations ranging from 4.6, 9.3, 18.7, 37.5, 75, 150 and 300 μg/ml. After incubation, 20 μl of MTT reagent was added into each well. These plates were incubated again for 4 h in CO2 incubator at 37°C. The resulting MTT-products were determined by measuring the absorbance at 570 nm using ELISA reader [22]. The cell viability was determined using the formula:
Viability %=(optical density of sample∕optical density of control)×100
IC50 values were calculated as the concentrations that show 50% inhibition of proliferation on any tested cell line.
Same batch of ginger extracts were used for both TBA and MTT assay.
High performance liquid chromatography (HPLC)
Flavonoid extract preparation
Aliquots of aerial parts and rhizomes (0.25 g) were extracted with 60% aqueous methanol (20 ml). 6 M HCl (5 ml) was added to each extract to give a 25 ml solution of 1.2 M HCl in 50% aqueous methanol. Extracts were refluxed at 90°C for 2 h. Extract aliquots of 500 μl, taken both before and after hydrolysis, were filtered through a 0.45 μm filter [23].
Analysis of flavonoids composition
Reversed-phase HPLC was used to assay flavonoid compositions. The Agilent HPLC system used consisted of a model 1100 pump equipped with a multi-solvent delivery system and an L-7400 ultraviolet (UV) detector. The column was an Agilent C18 (5 μm, 4.0 mm internal diameter 250 mm). The mobile phase composed of: (A) 2% acetic acid (CH3COOH) and (B) 0.5% acetic acid-acetonitrile (CH3CN), (50:50 v/v), and gradient elution was performed as follows: 0 min, 95:5; 10 min, 90:10; 40 min, 60:40; 55 min, 45:55; 60 min, 20:80 and 65 min, 0:100. The mobile phase was filtered under vacuum through a 0.45 μm membrane filter before use. The flow rate was 1 ml/min and UV absorbance was measured at 280-365 nm. The operating temperature was maintained at room temperature [24]. Identification of the flavonoids was achieved by comparison with retention times of standards, UV spectra and calculation of UV absorbance ratios after coinjection of samples and standards [25].
Statistical analysis
The experimental results were expressed as mean ± standard deviation of three replicates. Where applicable, the data were subjected to one-way analysis of variance (ANOVA) and the differences among samples were determined by Duncan's Multiple Range Test using the SPSS v14 and MSTATC programs. P-value of ≤ 0.05 was regarded as significant.
Results and discussion
Antioxidant activity
The results obtained from the preliminary analysis of antioxidant activity are shown in Table 1. According to the data obtained significant differences were observed among treatments for antioxidant activities. From the result, the antioxidant activity of aerial parts was higher than rhizomes extracts in both varieties that were grown under ambient CO2 concentration. The results also had indicated that antioxidant activities increased significantly by elevated CO2 concentration. Antioxidant activity was enhanced in rhizomes by elevated CO2 concentration more than in aerial parts with highest value of TBA (77.98%) were obtained from Syedpuri rhizomes. The aerial parts extract of Fulbaria and Syedpuri in ambient and elevated CO2 condition exhibited strong potential of free radical scavenging activity. According to the results, TBA content of the Syedpuri aerial parts grown in ambient CO2 concentration reached to 70.59%, while at the same extract concentration, that of the rhizomes was 67.79%. In ambient CO2 concentration, differences between aerial parts and rhizomes in both varieties for TBA activity was not significant, while in elevated CO2 concentration significant differences was observed between different parts of each variety. Many researchers had shown that high total flavonoids content increases antioxidant activity and there was a linear correlation between flavonoids content and antioxidant activity [18,26].
Table 1 Antioxidant activity of Zingiber officinale extracts grown under different CO2 concentrations (measured by the TBA method)
CO2 (μmol/mol) Varieties Parts TBA
Fulbaria Aerial parts
Rhizomes 69.29 ± 2.32c,d
67.93 ± 1.81d
400
Syedpuri Aerial parts
Rhizomes 70.59 ± 1.89a,c,d
67.79 ± 0.64d
Fulbaria Aerial parts
Rhizomes 71.01 ± 2.52a,c
75.05 ± 1.63b,e
800
Syedpuri Aerial parts
Rhizomes 73.78 ± 1.21a,e
77.98 ± 1.20b
All analyses are the mean of triplicate measurements ± standard deviation. Means not sharing a common letter were significantly different at P < 0.05.
Results expressed in percent.
Anticancer activity
As shown in Table 2, parts (aerial parts and rhizomes) of two ginger varieties were found to express MCF-7 and MDA-MB-231 cancer cell inhibitory activity when tested at concentrations of 4.6-300 μg/ml. At a concentration of 37.5 μg/ml, though, most of the extracts exhibited strong anticancer activity towards MCF-7 and MDA-MB-231 cells, at this concentration, extract of Syedpuri rhizomes grown under elevated CO2 concentration exhibit lowest MCF-7 and MDA-MB-231 cell viability at 39.01% and 40.16% respectively. Moreover, MCF-7 and MDA-MB-231 treated with tamoxifen (positive control) showed 24.9% and 26.7% viability in same concentration (37.5 μg/ml). In contrast, for MCF-7 cell, the anticancer activity of aerial parts extract in ambient and elevated CO2 concentration was significantly stronger than that of the rhizomes extract especially in Syedpuri variety. In addition, for MDA-MB-231 cell, the anticancer activity of aerial parts extract in ambient CO2 concentration was significantly stronger than that of the rhizomes extracts, but, with increasing of CO2 concentration anticancer power increased significantly in rhizomes of both varieties. However, of all extracts investigated, Syedpuri rhizomes that were obtained from plants grown under elevated CO2 concentration exhibited the strongest anticancer activities towards cancer cells. The IC50 values for MCF-7 and MDA-MB-231 cells were 25.7 and 30.2 μg/ml respectively (Table 3). While IC50 value of rhizomes extract of Syedpuri grown in ambient CO2 for MCF-7 and MDA-MB-231 cells were 47 and 38.8 μg/ml accordingly. However, with the increase of CO2 concentration, IC50 value decreased significantly in both varieties. Furthermore, IC50 values of tamoxifen as a positive control for MCF-7 and MDA-MB-231 cells were 19.7 and 22.89 μg/ml respectively.
Table 2 Anticancer activities of Zingiber officinale extracts against MCF-7 and MDA-MB-231 cell lines (determined by the MTT assay at concentration 37.5 μg/ml)
CO2 (μmol/mol) Varieties Parts MCF-7 MDA-MB-231
Fulbaria Aerial parts
Rhizomes 59.65 ± 2.55b
57.56 ± 1.68b 63.31 ± 1.85e
69.41 ± 2.30b
400
Syedpuri Aerial parts
Rhizomes 50.65 ± 0.56e
56.98 ± 1.74b 58.12 ± 1.09a
66.61 ± 2.31b,e
Fulbaria Aerial parts
Rhizomes 40.37 ± 1.46c
48.97 ± 1.04e 48.16 ± 1.03c
44.35 ± 1.86d
800
Syedpuri Aerial parts
Rhizomes 44.93 ± 1.53a
39.01 ± 2.1c 43.02 ± 1.99d
40.16 ± 2.42f
Positive control Tamoxifen 24.9 ± 1.6 26.70 ± 2.11
All analyses are the mean of triplicate measurements ± standard deviation. Means not sharing a common letter were significantly different at P ≤ 0.05.
Results expressed in percent of cell viability.
Table 3 IC50 values of Zingiber officinale extracts against MCF-7 and MDA-MB-231 cancer cell lines (expressed in μg/ml)
CO2 (μmol/mol) Varieties Parts MCF-7 MDA-MB-231
Fulbaria Aerial parts
Rhizomes 51.39 ± 1.32b
52.01 ± 2.11b 56.12 ± 2.15e
62.81 ± 1.60b
400
Syedpuri Aerial parts
Rhizomes 36.80 ± 1.32a
47.00 ± 1.16e 46.87 ± 0.45a
38.80 ± 1.81c
Fulbaria Aerial parts
Rhizomes 29.83 ± 1.37c
34.80 ± 1.80a 34.60 ± 2.16d
32.53 ± 1.07d
800
Syedpuri Aerial parts
Rhizomes 27.21 ± 2.01d
25.70 ± 0.64f 32.85 ± 0.89d
30.20 ± 0.81f
All analyses are the mean of triplicate measurements ± standard deviation.
Means not sharing a common letter were significantly different at P ≤ 0.05.
HPLC analysis of flavonoids
The results obtained from the preliminary analysis of flavonoids are shown in Table 4. Increasing the CO2 concentration from 400 to 800 μmol/mol resulted in enhanced quercetin, catechin, kaempferol and fisetin levels in the aerial parts and rhizomes of both varieties. On the other hand, the contents of epicatechin and morin decreased in ginger parts with rising of CO2 concentration from ambient to 800 μmol/mol. Some study results indicated that increasing the CO2 concentration from 400 to 800 μmol/mol resulted in enhanced quercetin, catechin, kaempferol and fisetin levels in the aerial parts and rhizomes of Zingiber officinale varieties and following that, the antioxidant activity in young ginger extracts could also be improved [25]. Findings of this current study supported previous researcher's findings and showed that anticancer effect of ginger extracts increase with increasing CO2 concentration.
Table 4 The concentrations of some flavonoids compounds in two varieties of Zingiber officinale, Fulbaria and Syedpuri grown under various CO2 concentrations
Fulbaria Syedpuri
Flavonoid compounds 400 800 400 800
Aerial parts Rhizomes Aerial parts Rhizomes Aerial parts Rhizomes Aerial parts Rhizomes
Quercetin 0.961 ± 0.013a 0.894 ± 0.039a 1.22 ± 0.06e 1.137 ± 0.023e 1.19 ± 0.122b,e 0.985 ± 0.032a 1.33 ± 0.124b 1.26 ± 0.01b
Epicatechin 0.128 ± 0.028b 0.085 ± 0.007a,e 0.073 ± 0.009a 0.049 ± 0.018c 0.12 ± 0.004b 0.103 ± 0.0034d,e 0.096 ± 0.021a,e 0.038 ± 0.009c
Catechin 0.416 ± 0.024c 0.492 ± 0.020a,c 0.673 ± 0.044b,e 0.637 ± 0.044e 0.668 ± 0.079b,e 0.533 ± 0.034a 0.734 ± 0.014b 0.684 ± 0.05b,e
Kaempferol 0.041 ± 0.006d 0.052 ± 0.003c,d 0.117 ± 0.014a 0.147 ± 0.023e 0.051 ± 0.002dd 0.067 ± 0.005c 0.162 ± 0.011b,e 0.184 ± 0.019b
Fisetin 0.982 ± 0.022d 0.633 ± 0.033f 2.051 ± 0.27a 2.88 ± 0.19b 1.53 ± 0.121c 1.32 ± 0.13c 2.37 ± 0.397e 3.12 ± 0.185b
Morin 0.532 ± 0.057d 0.464 ± 0.014d 0.491 ± 0.052d 0.876 ± 0.046b 0.765 ± 0.024e 0.607 ± 0.006c 0.662 ± 0.029a 0.517 ± 0.025d
All analyses are the mean of triplicate measurements ± standard deviation.
Means not sharing a common letter were significantly different at P ≤ 0.05.
Results expressed in mg/g of dry plant material.
Flavonoids are among the best candidates for mediating the protective effect of diets which are found in fruits and vegetables with respect to colorectal cancer. Study shows relative activity being as quercetin > apigenin > fisetin> kaempferol. Quercetin belongs to the flavonoids group due to its powerful antioxidant activity. Previous studies showed that quercetin may help to prevent cancer, especially prostate cancer [27]. Scambia et al. reported quercetin inhibited human breast cancer cells (MCF-7 and MDA-MB231) significantly [28]. Du et al. explained mechanism of breast cancer inhibition by quercetin [29]. In ginger quercetin is abundant flavonoid compound [25,26,30]. Antioxidant activity of quercetin was believed to have cytoprotective role against oxidative stress. It seemed that quercetin not only protects cells from free radical damage through antioxidant effect, but also motivates apoptotic cell death via pro-oxidant activity and inhibits tumourigenesis. Hence, anticancer power maybe related to quercetin content in those varieties. In addition, flavonoid compounds could probably be responsible for the anticancer activity of Zingiber officinale. Further research is required to untangle the specific bioactive compounds responsible for the anticancer properties of the extracts of Zingiber officinale varieties.
Conclusions
Currently, about 50% of drugs used in clinical trials for anticancer activity were isolated from natural sources such as herbs and spices or related to them [31]. A number of active compounds such as flavonoids, diterpenoids, triterpenoids and alkaloids have been shown to possess anticancer activity. According to the report of the American National Cancer Institute (NCI), the criterion of anticancer activity for the crude extracts of herbs is an IC50<30 μg/ml [32]. Thus, according to the results from current study seems that enriched ginger varieties developed by elevated CO2 concentration could be employed in ethno-medicine in the treatment of cancerous diseases.
There are some limitations of this study. Relationship between flavonoids concentration and antioxidant activity were not determined. Moreover, only cytotoxicity was determined but apoptosis and cell cycle analysis were not performed.
Our results in this study indicate that some compounds in Bangladeshi ginger varieties at young age possess anticancer activities and may contribute in the therapeutic effect of this medicinal herb. However, there is a need of detailed scientific study on traditional medical practices to ensure that valuable therapeutic knowledge of some plants is preserved and also to provide scientific evidence for their efficacies.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SR and FS participated in the design, coordinating and carried out the study and also drafted the manuscript. AI performed the statistical analysis. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1472-6882/11/76/prepub
Acknowledgements
We thank staffs and kind support of the Department of Biotechnology and Genetic Engineering, Islamic University.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22066019PONE-D-11-1496910.1371/journal.pone.0026943Research ArticleBiologyImmunologyImmunologic TechniquesImmunohistochemical AnalysisMedicineClinical ImmunologyImmunologic TechniquesImmunohistochemical AnalysisComplementary and Alternative MedicineOncologyBasic Cancer ResearchMetastasisCancer Risk FactorsChemopreventionCancer TreatmentChemotherapy and Drug TreatmentComplementary and Alternative MedicineCancers and NeoplasmsGastrointestinal TumorsPancreatic CancerCancer PreventionBoswellic Acid Suppresses Growth and Metastasis of Human Pancreatic Tumors in an Orthotopic Nude Mouse Model through Modulation of Multiple Targets Effect of AKBA on Growth of Pancreatic CancerPark Byoungduck Prasad Sahdeo Yadav Vivek Sung Bokyung Aggarwal Bharat B.
*
Cytokine Research Laboratory, Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America
Goel Ajay EditorBaylor University Medical Center, United States of America* E-mail: [email protected] and designed the experiments: BDP BBA. Performed the experiments: BDP SP VY BS. Analyzed the data: BDP. Contributed reagents/materials/analysis tools: BDP SP VY BS. Wrote the paper: BDP BBA.
2011 31 10 2011 6 10 e269432 8 2011 6 10 2011 Park et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Pancreatic cancer (PaCa) is one of the most lethal cancers, with an estimated 5-year survival of <5% even when patients are given the best treatment available. In addition, these treatments are often toxic and expensive, thus new agents which are safe, affordable and effective are urgently needed. We describe here the results of our study with acetyl-11-keto-β-boswellic acid (AKBA), an agent obtained from an Ayurvedic medicine, gum resin of Boswellia serrata. Whether AKBA has an activity against human PaCa, was examined in in vitro models and in an orthotopic nude mouse model of PaCa. We found that AKBA inhibited the proliferation of four different PaCa cell lines (AsPC-1, PANC-28, and MIA PaCa-2 with K-Ras and p53 mutations, and BxPC-3 with wild-type K-Ras and p53 mutation). These effects correlated with an inhibition of constitutively active NF-κB and suppression of NF-κB regulating gene expression. AKBA also induced apoptosis, and sensitized the cells to apoptotic effects of gemcitabine. In the orthotopic nude mouse model of PaCa, p.o. administration of AKBA alone (100 mg/kg) significantly inhibited the tumor growth; this activity was enhanced by gemcitabine. In addition, AKBA inhibited the metastasis of the PaCa to spleen, liver, and lungs. This correlated with decreases in Ki-67, a biomarker of proliferation, and CD31, a biomarker of microvessel density, in the tumor tissue. AKBA produced significant decreases in the expression of NF-κB regulating genes in the tissues. Immunohistochemical analysis also showed AKBA downregulated the expression of COX-2, MMP-9, CXCR4, and VEGF in the tissues. Overall these results demonstrate that AKBA can suppress the growth and metastasis of human pancreatic tumors in an orthotopic nude mouse model that correlates with modulation of multiple targets.
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Introduction
The National Cancer Institute estimated that 18,770 men and 18,030 women in the United States would die of pancreatic cancer (PaCa) in 2010 [1]. Because of a lack of early detection methods and an absence of effective biomarkers, patients with PaCa are usually diagnosed at a late stage, by which time the 5-year survival rate is less than 5% [2]. This low survival rate emphasizes the increased need for effective chemopreventive strategies, early detection methods, and novel treatments. Only 12% of the patients have partial or complete responses to gemcitabine, the standard treatment for advanced PaCa [3], and this treatment is associated with multiple adverse events and drug resistance. Erlotinib, also approved by the U.S. Food and Drug Administration (FDA) to treat pancreatic cancer, does not show significant efficacy either. Thus novel agents that are safe, inexpensive and effective are needed for the treatment of this disease.
One potential source of novel agents is the large pharmacopeia of traditional medicines. One with promise in the treatment of PaCa is the AKBA (acetyl-11-keto-β-boswellic acid), obtained from the medicinal plant Boswellia serrata. It contains beta boswellic acid, a pentacyclic triterpene and the active component of the gum resin (also called frankincense in European pharmacopeia) secreted by the bark of the tree Boswellia serrata (Sallai guggul) [4].
AKBA has been used for centuries in traditional Ayurvedic medicine for a wide variety of inflammatory diseases, including inflammatory bowel disease [5] and rheumatoid arthritis [6]. AKBA has been shown to inhibit the growth of a wide variety of tumor cells including glioma [7], colon cancer [8], [9], [10], leukemia cells [11], [12], [13], [14], [15], human melanoma [16], hepatoma [8], and prostate cancer cells [17]. It has been also reported that AKBA has apoptotic effects through a wide variety of mechanisms. AKBA inhibits topoisomerase I and II without inhibiting DNA fragmentation [7], [13], and induces death receptor (DR)-5 but not DR-4 or Fas through increased expression levels of CAAT/enhancer binding protein homologous protein (CHOP), which led to the activation of caspase-8 in prostate cancer cells [18]. The anti-inflammatory effects of this agent were further demonstrated by studies that showed that LPS-induced TNF production is blocked by this drug [19]. Anti-proliferative and anti-inflammatory effects of AKBA are also mediated through the suppression of the NF-κB pathway [20] and STAT3 pathway [21]. More recently our laboratory showed that AKBA can downregulate the expression of CXCR4, a chemokine receptor that has been closely linked with invasion of various cancers [22]. All these studies suggest an anti-inflammatory and anticancer potential for AKBA.
Because several of these targets play a critical role in growth and metastasis of PaCa, we decided to measure the effects of AKBA on a panel of PaCa cell lines and determine whether AKBA alone or in combination with gemcitabine, the current standard of care, affects the growth and metastasis of human pancreatic tumors in nude mice. We found that AKBA inhibited the proliferation and enhanced the apoptosis of gemcitabine in four PaCa cell lines. Moreover, it suppressed the growth and metastasis of human pancreatic tumors in an orthotopic nude mouse model through modulation of multiple targets.
Results
AKBA inhibits the proliferation of pancreatic cancer cells in vitro
We first examined whether AKBA could inhibit the proliferation of human PaCa cell lines. We treated these cell lines with different doses of AKBA for various time periods and determined the inhibition of proliferation by examining mitochondrial activity using the MTT uptake method. As Fig. 1A shows, AKBA suppressed the proliferation of all four pancreatic cancer cell lines in a dose- and time-dependent manner. BxPC-3 was most sensitive as 25 µmol/L inhibited proliferation, whereas for the other cell lines, a 50 µmol/L dose was required to fully suppress proliferation.
10.1371/journal.pone.0026943.g001Figure 1 AKBA inhibits proliferation of PaCa cells and enhances the apoptosis of gemcitabinein vitro.
A, MTT assay results showed dose-dependent suppression of cell proliferation in all four pancreatic cancer cell lines tested. Data are representative of three independent experiments. B, PANC-28 (1×106) cells were treated with AKBA (10, 25, and 50 µmol/L) for 12 h; nuclear extracts were prepared and then assayed for NF-κB activation by EMSA. C, Western blot analysis showed that AKBA inhibited constitutive expression of NF-κB–regulated gene products that regulate antiapoptosis, proliferation, and metastasis in pancreatic cancer cells. The MIA PaCa-2 (1×106) cells were treated with AKBA (10, 25 and 50 µmol/L) for 24 h. Whole-cell lysates were prepared and assayed for NF-κB–regulated gene products by Western blotting. Data represent two independent experiments. D, AKBA potentiates the apoptotic effects of gemcitabine in pancreatic cancer cells in vitro. LIVE/DEAD assay results indicated that AKBA potentiates gemcitabine-induced cytotoxicity. Percentages are proportions of apoptotic pancreatic cancer cells. Data are representative of two independent experiments. E, The MIA PaCa-2 (1×106) cells were treated with AKBA (25 µmol/L) for 12 h and then gemcitabine (500 nmol/L) was added for 24 h. Whole-cell lysates were prepared and subjected to Western blotting. Data represent two independent experiments.
AKBA inhibits constitutive NF-κB activation and NF-κB-regulated proteins in pancreatic cancer cells
We next determined how AKBA inhibits proliferation. Because NF-κB has been linked with proliferation, we examined AKBA's ability to inhibit constitutive NF-κB activation in PANC-28 cell lines. Our results showed that AKBA inhibited constitutive NF-κB activation in PANC-28 (Fig. 1B). We next examined whether AKBA modulates the NF-κB-regulated gene products linked to survival, proliferation, invasion, and angiogenesis. As shown in Figure 1C, AKBA downregulated the expression of antiapoptotic (Bcl-2, Bcl-xL, and survivin), proliferative (COX-2, c-myc, and cyclin D1), and metastatic (MMP-9, CXCR4) proteins in a dose-dependent manner.
AKBA potentiates the apoptotic activity of gemcitabine in pancreatic cancer cells
To determine whether AKBA could enhance the apoptotic effects of gemcitabine in these cell lines, we performed the LIVE/DEAD assay, which measures esterase activity. At a dose at which AKBA (25 µmol/L) and gemcitabine (500 nmol/L) alone had minimally apoptotic effects, the two together were highly effective in inducing apoptosis (Fig. 1D). Again BxPC-3 was found to be most sensitive to the combination and MiaPaCa-2 was found to be least sensitive. AKBA also potentiated caspase-mediated cleavage of PARP induced by gemcitabine (Fig. 1E).
AKBA inhibits the growth of human pancreatic tumors in an orthotopic nude mice model
On basis of our in vitro results, we designed studies to determine the effects of AKBA alone and in combination with gemcitabine in human pancreatic tumors orthotopically implanted in nude mice (Fig. 2A). Luciferase-transfected PANC-28 cells were implanted in the tails of the pancreas in nude mice. On the basis of IVIS imaging, the mice were randomized into four groups 1 week later. The treatment was started 1 week after tumor cell implantation and continued per experimental protocol for 4 weeks. The animals were euthanized 6 weeks after tumor cell injection and 5 weeks from the start of treatment. The bioluminescence imaging results indicated a gradual increase in tumor volume in the control group as compared with the three treatment groups (Fig. 2B). The tumor volume in AKBA group was decreased and combination group was significantly lower than in the group treated with AKBA or gemcitabine alone as well as in the vehicle-treated control group (P<0.01). On the 35th day, we euthanized the mice and measured the tumor volume with Vernier calipers. The results were in concordance with those from bioluminescence imaging and showed that the combination reduced the tumor volume more than AKBA did (P<0.01; Fig. 2C and D).
10.1371/journal.pone.0026943.g002Figure 2 AKBA potentiates the effect of gemcitabine in blocking the growth of pancreatic cancer in nude mice.
A, schematic of experimental protocol described in
Materials and Methods.
Group I was given corn oil (100 µL, p.o., daily), group II was given AKBA (100 mg/kg, p.o., daily), group III was given gemcitabine (25 mg/kg, i.p., twice weekly), and group IV was given AKBA (100 mg/kg, p.o., daily) and gemcitabine (25 mg/kg, i.p., twice weekly; n = 5). B, bioluminescence IVIS images of orthotopically implanted pancreatic tumors in live, anesthetized mice and measurements of photons per second depicting tumor volume at various time points using live IVIS imaging at the indicated times (n = 5). Points, mean; bars, SEM. C, Necropsy photographs of mice. D, tumor volumes in mice measured on the last day of the experiment at autopsy with Vernier calipers and calculated using the formula V = 2/3πr3 (n = 5). Columns, mean; bars, SE.
AKBA suppresses distant organ metastasis in mice
We evaluated whether AKBA alone or in combination with gemcitabine can suppress distant organ metastasis in mice. After sacrificing the mice, we found PaCa in the spleen, liver and lungs of vehicle-treated mice; AKBA alone suppressed metastasis to those organs. The combination of AKBA and gemcitabine inhibited metastasis significantly more in these mice and in fact completely suppressed metastasis to the lung (Fig. 3).
10.1371/journal.pone.0026943.g003Figure 3 AKBA potentiates gemcitabine to inhibit distant organ metastasis in orthotopic PaCa nude mice.
AKBA and gemcitabine combined inhibited metastases to the spleen, liver, and lungs. Mice were killed, and their abdomen was opened surgically, and then the number of metastasis foci in each organ was counted. Columns, mean; bars, SE.
AKBA inhibits Ki-67 and CD31 expression
The Ki-67-positive index is used as a marker for cell proliferation, and the CD31 index is a marker for microvessel density. We found that AKBA and gemcitabine downregulated the expression of these biomarkers. AKBA and gemcitabine alone significantly downregulated the expression of Ki-67 in tumor tissues compared with the control group (P<0.05 versus control), and the combination of these two was significantly more effective (Fig. 4A and B). The results also showed that combination of AKBA and gemcitabine remarkably suppressed the expression of CD31 when compared with the control (Fig. 4C and D).
10.1371/journal.pone.0026943.g004Figure 4 AKBA enhances the effect of gemcitabine to inhibit tumor cell proliferation and angiogenesis in PaCa.
A, the results of an immunohistochemical analysis of proliferation marker Ki-67 indicated that PaCa cell proliferation was inhibited in mice treated with AKBA alone and in combination with gemcitabine. B, quantification of Ki-67 positive cells was described in
Materials and Methods
. Columns, mean; bars, SE. C, the results of an immunohistochemical analysis of CD31 for microvessel density in PaCa indicated that angiogenesis was inhibited by AKBA alone and in combination with gemcitabine. D, quantification of CD31 positive cells was described in
Materials and Methods
. Columns, mean; bars, SE.
AKBA inhibits constitutive NF-κB activation and NF-κB–regulated biomarkers in pancreatic tumors
We next investigated whether the effects of AKBA on tumor growth in mice were associated with the inhibition of NF-κB activation. Fig. 5A shows that AKBA alone suppressed the constitutive activation of NF-κB in the pancreatic tumor tissue, and this down-modulation was enhanced in tissue obtained from animals treated with both AKBA and gemcitabine together.
We also examined expression of various biomarkers in the tumor tissue by western blot. As shown in Fig. 5B, AKBA significantly downregulated the expression of proteins related to antiapoptosis (Bcl-xL), proliferation (cyclin D1, c-myc), metastasis (CXCR4) and invasion (MMP-9, ICAM-1). Especially, expression of Bcl-xL and MMP-9 was reduced more by the combination than just by AKBA.
10.1371/journal.pone.0026943.g005Figure 5 AKBA enhances the effect of gemcitabine against the expression of NF-κB and NF-κB–regulated gene products in pancreatic cancer tissues.
A, detection of NF-κB by DNA binding in orthotopic tumor tissue samples showed the inhibition of NF-κB by AKBA. B, Western blot analysis showed that AKBA alone or combination with gemcitabine inhibited the expression of NF-κB–dependent genes. C, immunohistochemical analysis of NF-κB–regulated genes (COX-2, MMP-9, CXCR4 and VEGF) in pancreatic tumor tissues from mice. Percentages indicate positive staining for the given biomarker. Samples from three animals in each group were analyzed, and representative data are shown.
Our immunohistochemical analysis also showed that COX-2, MMP-9, CXCR4 and VEGF expression was downregulated by AKBA, and the combination was even more effective (Fig. 5C).
AKBA can be detected in plasma and pancreatic tumor tissue
To determine whether AKBA is directly detectable in plasma and pancreatic tissue, serum and PaCa tissue levels of AKBA were examined by HPLC. Serum levels of AKBA were determined 4 h after oral administration of drug in mice. 454±23 ng/ml of AKBA was found in plasma and 273±13 ng/ml of AKBA was found in pancreatic tissue respectively (Fig. 6). These results indicate that there is a strong correlation between tumor regression and the amount of AKBA in the blood.
10.1371/journal.pone.0026943.g006Figure 6 HPLC chromatogram of AKBA in plasma (left) and PaCa tissue (right) in mice.
Discussion
In the present study, we investigated whether AKBA, a derivative of boswellic acid, could enhance the antitumor activity of gemcitabine against human PaCa. We found that AKBA was highly effective in suppressing the proliferation of various pancreatic cancer cell lines, and when it was combined with gemcitabine, this activity was further enhanced. This activity in vitro correlated with the suppression of constitutive NF-κB activation and downregulation of cell survival, proliferative, and metastatic proteins. When examined in an orthotopic model, again AKBA was highly effective in suppressing tumor growth. It was also quite effective in suppressing PaCa metastasis, again in correlation with the downregulation of various biomarkers. The effects of AKBA were more pronounced when it was combined with gemcitabine.
Ours is the first report to suggest that AKBA can suppress the proliferation and enhance the apoptotic effect of gemcitabine in PaCa cell lines. Gemcitabine alone had a minimal effect on apoptosis in these cell lines. While BxPC-3 cells have been shown to be unaffected by gemcitabine [23], we found that they were highly sensitive to AKBA. This difference may be because BxPC-3 harbors wild-type K-Ras, unlike the other cell lines, in which K-Ras is mutated. The mechanism by which AKBA potentiates the apoptotic effects of gemcitabine may involve suppression of NF-κB. Because NF-κB has been linked with chemoresistance [24], it is very likely that downregulation of NF-κB by AKBA sensitized the cells to gemcitabine. These results agree with a previous report in which MG132, sulfasalazine, and the IκBα super-repressor sensitized pancreatic cancer cells to gemcitabine [23]. NF-kB silencing has been shown to induce apoptosis and increase sensitivity to gemcitabine in a subset of pancreatic cancer cells [25]. Similarly genistein has been shown to sensitize prostate cancer cells to gemcitabine through the downregulation of NF-κB [26].
In addition to these in vitro results, we found that AKBA potentiated the antitumor effects of gemcitabine in an orthotopic model of PaCa. Tumor growth was inhibited by almost 50% by 100 mg/kg AKBA alone, and when given in combination with gemcitabine its inhibitory effects were even greater. How AKBA exhibited its effects in vivo was investigated in several ways. First, AKBA alone suppressed metastasis of PaCa to the spleen, liver, and lungs. Further, the combination of AKBA and gemcitabine inhibited metastasis significantly in these mice. Second, markers of proliferation (Ki-67) and microvessel density (CD31) were significantly decreased by AKBA. Third, analysis of NF-κB in pancreatic tumors showed that AKBA alone and in combination with gemcitabine inhibited constitutive activation of NF-κB. Fourth, immunohistochemical analysis also showed that AKBA downregulated the expression of proinflammatory biomarker COX-2, suppressed the expression of invasion biomarker MMP-9 and CXCR4, and inhibited the angiogenic biomarker VEGF in the PaCa tissues. All of these effects were further enhanced by gemcitabine. The synergistic effects of the two agents were confirmed by Western blot analysis. Finally, the important aspect of an agent's ability to suppress tumor is the bioavailability, which serves primarily a protective regulatory function constraining diffusion across capillaries in relation to lipid solubility and molecular weight.
For a successful chemotherapy, agent must be able to reach the blood. AKBA shows high lipid solubility, and we detected it in concentration of 273 ng/ml in PaCa tissues, which corresponded to concentration of 454 ng/ml in the plasma. Thus, these data demonstrate substantial levels of AKBA in PaCa tissue after oral administration and confirm the bioavailability of AKBA. These results are further correlated with the tumor volume regression and decrease in metastases to the spleen, liver, and lungs.
All these potential mechanisms of AKBA we examined, such as inhibition of proliferation, inflammation and metastasis, are related to NF-κB suppression. As we discussed above, because NF-κB has been linked with chemoresistance we would suggest that chemosensitization of PaCa to gemcitabine by AKBA is through an inhibitory effect on NF-κB. Our results are consistent with a recent report in which small hairpin RNA and pharmacological inhibitor of TGF-β-activated kinase (TAK)-1 that activates NF-κB was used to sensitize pancreatic cancer to gemcitabine [27]. Furthermore, it has been reported that NF-κB targeting agent such as curcumin strongly enhanced the antitumor efficacy of radiation [28]. So we propose that AKBA may also sensitize the tumors to radiation therapy.
AKBA has also been shown to bind and inhibit 5-LOX, elastase, and topoisomerase II. AKBA directly inhibits 5-LOX with an IC50 as low as 1.5 µM [29]. Further studies have revealed that the pentacyclic triterpene ring structure, hydrophilic group on C4 ring A, and 11-keto functions are all essential for 5-LOX inhibitory activity [30]. Unlike other pentacyclic triterpenes, AKBA has also been shown to inhibit leukocyte elastase with an IC50 of 15 µM [31]. Besides 5-LOX and elastase, AKBA was also found to bind to and inhibit topoisomerase (topo) I and II α [32]. The affinity constant (KD) of AKBA for topo I and II α were 70.6 nM and 7.6 nM, respectively. When compared with camptothecin, amascrine or etoposide, AKBA was found to be more potent. Although, we did not measure these proinflammatory and apoptotic biomarkers, it is possible that these also play an important role in the action of AKBA against PaCa.
Our finding that AKBA downregulated COX-2 expression in pancreatic cancer tissue is notable, as COX-2 is overexpressed in chronic pancreatitis [33] and in human pancreatic cancer tissue. COX-2 expression has been associated with a lower apoptotic index [34], increased cell proliferation [35], increased risk of metastasis [36], [37], and enhanced VEGF production, leading to angiogenesis in pancreatic tumors [38], [39]. Thus inhibition of COX-2 expression in the nude mouse implies that anti-inflammatory effect of AKBA contributes to its anti-tumor effects.
Overall, our results show for the first time that AKBA potentiates the antitumor activity of gemcitabine by modulating multiple biomarkers, leading to the inhibition of proliferation, angiogenesis, invasion, and metastasis. Several clinical trials have been done with AKBA indicating that it is safe agent to use in humans [40], [41]. Our work presented here further provides the basis for clinical trial in patients with pancreatic cancer, one of the most lethal cancers known to mankind.
Ethics Statement
Our experimental protocol was reviewed and approved by the International Animal Care and Use Committee at University of Texas MD Anderson Cancer Center. Our IACUC protocol number for pancreatic cancer is 10–05–11032.
Materials and Methods
Materials
Purified AKBA was supplied by Sabinsa Corp. A 50 mmol/L stock solution of AKBA was prepared and then stored at −20°C as small aliquots until needed. Polyclonal antibodies against cyclin D1, matrix metalloproteinase 9 (MMP-9), survivin, COX-2, c-Myc, Bcl-2, and Bcl-xL were obtained from Santa Cruz Biotechnology. A rabbit polyclonal antibody to CXCR4 was obtained from Abcam. Anti-XIAP antibody was obtained from BD Biosciences. Antiobodies against VEGF and Ki-67 were purchased from Thermo Fisher Scientific. The liquid DAB+ substrate chromogen system–horseradish peroxidase used for immunocytochemistry was obtained from Dako. Penicillin, streptomycin, RPMI 1640, and fetal bovine serum (FBS) were obtained from Invitrogen. Tris, glycine, NaCl, sodium dodecyl sulfate, and bovine serum albumin (BSA) were obtained from Sigma Chemical. Gemcitabine (Gemzar; kindly supplied by Eli Lilly) was stored at 4°C and dissolved in sterile PBS on the day of use.
Cell lines
The pancreatic cancer cell lines AsPC-1, BxPC-3, MIA PaCa-2, and PANC-28 were obtained from American Type Culture Collection. AsPC-1, PANC-28, BxPC3, and MIA PaCa-2 exhibit K-Ras and p53 mutations, but BxPC-3 harbors wild-type K-Ras. AsPC-1 and BxPC-3 cells were cultured in RPMI 1640 supplemented with 10% FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin. MIA PaCa-2 was cultured in DMEM supplemented with 12% FBS, and PANC-28 was cultured in DMEM supplemented with 10% FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin.
Proliferation assay
The effect of AKBA on cell proliferation was determined by the MTT uptake method as follows. The cells (2,000 per well) were incubated with AKBA in triplicate in a 96-well plate and then incubated for 2, 4, or 6 days at 37°C. An MTT solution was added to each well and incubated for 2 hours at 37°C. An extraction buffer (20% SDS and 50% dimethylformamide) was added, and the cells were incubated overnight at 37°C. The absorbance of the cell suspension was measured at 570 nm with an MRX Revelation 96-well multiscanner (Dynex Technologies).
Apoptosis assay
To determine whether AKBA potentiates the apoptotic effects alone or in combination with gemcitabine in PaCa cells, we used a LIVE/DEAD cell viability assay kit (Invitrogen), which measures intracellular esterase activity and plasma membrane integrity. This assay uses calcein, a polyanionic, green fluorescent dye that is retained within live cells, and a red fluorescent ethidium homodimer dye that can enter cells through damaged membranes and bind to nucleic acids but is excluded by the intact plasma membranes of live cells [42]. Briefly, cells (5,000 per well) were incubated in chamber slides, pretreated with AKBA for 12 hours, and treated with gemcitabine for 24 hours. Cells were then stained with the assay reagents for 30 minutes at room temperature. Cell viability was determined under a fluorescence microscope by counting live (green) and dead (red) cells.
Animals
Male athymic nu/nu mice (4 weeks old) were obtained from the breeding colony of the Department of Experimental Radiation Oncology at University of Texas MD Anderson Cancer Center. The animals were housed in standard Plexiglass cages (five per cage) in a room maintained at constant temperature and humidity and in a 12-hour light/12-hour dark cycle. Their diet consisted of regular autoclave chow and water ad libitum. None of the mice had any lesions, and all were tested pathogen free. Before initiating the experiment, we acclimatized all of the mice to a pulverized diet for 3 days. Our experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee at University of Texas MD Anderson Cancer Center.
Orthotopic implantation of pancreatic tumor cells
PANC-28 cells were stably transfected with luciferase as previously described [43]. Luciferase-transfected PANC-28 cells were harvested from subconfluent cultures after a brief exposure to 0.25% trypsin and 0.2% EDTA. Trypsinization was stopped with medium containing 10% FBS. The cells were washed once in serum-free medium and resuspended in PBS. Only suspensions consisting of single cells, with >90% viability, were used for the injections. After mice were anesthetized with ketamine-xylazine solution, a small incision was made in the left abdominal flank, and PANC-28 cells (1×106) in 100 µL PBS were injected into the subcapsular region of the pancreas with a 27-gauge needle and a calibrated push button–controlled dispensing device (Hamilton Syringe Co.). A cotton swab was held cautiously for 1 minute over the site of injection to prevent leakage. The abdominal wound was closed in one layer with wound clips (Braintree Scientific, Inc.).
Experimental protocol
One week after implantation, the mice were randomized into the following treatment groups (n = 5 per group) based on the bioluminescence first measured with an in vivo imaging system (IVIS 200, Xenogen Corp.): (a) untreated control (corn oil, 100 µL daily), (b) AKBA (100 mg/kg once daily p.o.), (c) gemcitabine alone (25 mg/kg twice weekly by i.p. injection), and (d) combination (AKBA, 100 mg/kg once daily p.o., and gemcitabine, 25 mg/kg twice weekly by i.p. injection). Tumor volumes were monitored weekly with the bioluminescence IVIS, which includes a cryogenic cooling unit, and a data acquisition computer running Living Image software (Xenogen Corp.). Before imaging, the animals were anesthetized in an acrylic chamber with 2.5% isoflurane/air mixture and injected i.p. with 40 mg/mL d-luciferin potassium salt in PBS at a dose of 150 mg/kg body weight. After 10 minutes of incubation with luciferin, each mouse was placed in a right lateral decubitus position and a digital grayscale animal image was acquired, followed by the acquisition and overlay of a pseudocolor image representing the spatial distribution of detected photons emerging from active luciferase within the animal. Signal intensity was quantified as the sum of all detected photons within the region of interest per second. The mice were subjected to imaging on days 0, 7, 14, 21, and 28 of treatment. Therapy was continued for 4 weeks, and the animals were euthanized 1 week later. Primary tumors in the pancreas were excised, and the final tumor volume was measured as V = 2/3πr3, where r is the mean of the three dimensions (length, width, and depth). Half of the tumor tissue was fixed in formalin and embedded in paraffin for immunohistochemistry and routine H&E staining. The other half was snap frozen in liquid nitrogen and stored at −80°C. H&E staining confirmed the presence of tumors in each pancreas.
Preparation of nuclear extract from tumor samples
Pancreatic tumor tissues (75–100 mg/mouse) from mice in the control and treatment groups were minced and incubated on ice for 30 minutes in 0.5 mL of ice-cold buffer A [10 mmol/L HEPES (pH 7.9), 1.5 mmol/L KCl, 10 mmol/L MgCl2, 0.5 mmol/L DTT, and 0.5 mmol/L phenylmethylsulfonyl fluoride (PMSF)]. The minced tissue was homogenized using a Dounce homogenizer and centrifuged at 16,000×g at 4°C for 10 minutes. The resulting nuclear pellet was suspended in 0.2 mL of buffer B [20 mmol/L HEPES (pH 7.9), 25% glycerol, 1.5 mmol/L MgCl2, 420 mmol/L NaCl, 0.5 mmol/L DTT, 0.2 mmol/L EDTA, 0.5 mmol/L PMSF, and 2 µg/mL leupeptin] and incubated on ice for 2 hours with intermittent mixing. The suspension was then centrifuged at 16,000×g at 4°C for 30 minutes. The supernatant (nuclear extract) was collected and stored at −70°C until used. Protein concentration was measured by the Bradford assay with BSA as the standard.
NF-κB activation in pancreatic cancer cells
To assess NF-κB activation, we isolated nuclei from pancreatic cancer cell lines and tumor samples and carried out electrophoretic mobility shift assays (EMSA) essentially as described next. Briefly, nuclear extracts prepared from pancreatic cancer cells (1×106/mL) and tumor samples were incubated with 32P-end-labeled 45-mer double-stranded NF-κB oligonucleotide (4 µg of protein with 16 fmol of DNA) from the HIV long terminal repeat (5′-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGGGAGGCGTGG-3′; boldface indicates NF-κB–binding sites) for 15 minutes at 37°C. The resulting DNA-protein complex was separated from free oligonucleotide on 6.6% native polyacrylamide gels. The dried gels were visualized, and radioactive bands were quantitated with PhosphorImager and ImageQuant software (GE Healthcare).
Immunohistochemistry of COX-2, MMP-9, CXCR4, and VEGF in tumor samples
The expression of COX-2, MMP-9, CXCR4, and VEGF was determined via an immunohistochemical method described previously [44]. Briefly, pancreatic tumor samples were embedded in paraffin and fixed with paraformaldehyde. After being washed in PBS, the slides were blocked with protein block solution (Dako) for 20 minutes and then incubated overnight with rabbit polyclonal anti-human CXCR4 and anti-MMP-9 (1∶100 for each) or mouse monoclonal anti-human VEGF and anti-COX-2 antibodies (1∶50 and 1∶75, respectively). After incubation with the antibodies, the slides were washed and then incubated with biotinylated link universal antiserum followed by horseradish peroxidase-streptavidin detection with the LSAB+ kit (Dako). The slides were rinsed, and color was developed with 3,3′-diaminobenzidine hydrochloride used as a chromogen. Finally, sections were rinsed in distilled water, counterstained with Mayer's hematoxylin, and mounted with DPX mounting medium (Sigma) for evaluation. Pictures were captured with a Photometrics CoolSNAP CF color camera (Nikon) and MetaMorph software version 4.6.5 (Universal Imaging).
Ki-67 immunohistochemistry
Formalin-fixed, paraffin-embedded sections (5 µm) were stained with anti-Ki-67 (rabbit monoclonal clone SP6) antibody as described previously [42]. Results were expressed as the percentage of Ki-67 positive ± SEM per 40× magnification. A total of ten 40× fields were examined and stained cells were counted in three tumors from each of the treatment groups.
CD31 immunohistochemistry
Ethanol-fixed, paraffin-embedded sections (5 µm) were stained with rat anti-mouse CD31 monoclonal antibodies. Areas of highest microvessel density were then examined under high magnification (100×) and counted. Any distinct area of positive staining for CD31 was counted as a single vessel. Results were expressed as the mean number of vessels ± SEM per high power field (100× magnification). Twenty high-power fields were examined and counted for each of three tumors from each treatment group.
Western blot analysis
Pancreatic tumor tissues (75–100 mg/mouse) from mice in the control and treatment groups were minced and incubated on ice for 30 minutes in 0.5 mL of ice-cold whole-cell lysate buffer (10% NP40, 5 mol/L NaCl, 1 mol/L HEPES, 0.1 mol/L EGTA, 0.5 mol/L EDTA, 0.1 mol/L PMSF, 0.2 mol/L sodium orthovanadate, 1 mol/L NaF, 2 µg/mL aprotinin, and 2 µg/mL leupeptin). The minced tissue was homogenized with a Dounce homogenizer and centrifuged at 16,000×g at 4°C for 10 minutes. The proteins were then fractionated by SDS-PAGE, electrotransferred to nitrocellulose membranes, blotted with each antibody, and detected by enhanced chemiluminescence reagents (GE Healthcare).
Measurement of serum and tissue levels of AKBA
AKBA was determined by reverse-phase high performance liquid chromatography (HPLC) on a high-pressure chromatograph equipped with an ultraviolet detector and a 2.0×150-mm column (Gemini C18; particle size, 5 µm, 110A0). The column was eluted in an isocratic flow with a component acetonitrile:methanol:acetic acid (1%) ratio of 70∶20∶10 at an eluent flow rate of 0.35 mL/min. The sample volume was 10 µL. The target compounds were detected at 250 nM. The solution components were identified and quantitatively analyzed using the external standard method with linear regression equations, reliability of approximation (R2), and detection thresholds for AKBA by HPLC. The blood samples were taken from animals 4 h after the administration of AKBA or vehicle. The samples were centrifuged for 20 min at 2000 rpm to obtain the blood plasma, treated with acetone (1.5∶1) for deproteination, and centrifuged again for 15 min at 3000 rpm. The supernatant fraction was separated and extracted with chloroform (2∶1). The chloroform extract was separated, the solvent was removed, and the dry residue was dissolved in methanol and analyzed by HPLC.
Statistics
In vitro experiments were repeated twice, and statistical analysis was performed. The values were initially subjected to one-way ANOVA, which revealed significant differences between groups, and then later compared between groups with an unpaired Student's t test, which revealed significant differences between two sample means. In vivo experiments were done as at least three independent assays. The values were initially subjected to one-way ANOVA and then later compared among groups with an unpaired Student's t test.
We thank Walter Pagel for carefully editing the manuscript. Dr. Aggarwal is the Ransom Horne, Jr., Professor of Cancer Research.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by a grant from the National Institutes of Health (NIH CA-124787-01A2). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS PathogPLoS PathogplosplospathPLoS Pathogens1553-73661553-7374Public Library of Science San Francisco, USA 22072968PPATHOGENS-D-11-0107710.1371/journal.ppat.1002350Research ArticleBiologyMedicineFatal Prion Disease in a Mouse Model of Genetic E200K Creutzfeldt-Jakob Disease Fatal Disease in a Mouse Model of E200K CJDFriedman-Levi Yael
1
Meiner Zeev
1
Canello Tamar
1
Frid Kati
1
Kovacs Gabor G.
2
Budka Herbert
2
Avrahami Dana
1
Gabizon Ruth
1
*
1
Department of Neurology, The Agnes Ginges Center for Human Neurogenetics, Jerusalem, Israel
2
Institute of Neurology, Medical University Vienna, Austria
Westaway David EditorUniversity of Alberta, Canada* E-mail: [email protected] and designed the experiments: YFL ZM GGK HB DA RG. Performed the experiments: TC KF YFL GGK DA RG. Analyzed the data: GGK HB ZM RG YFL. Contributed reagents/materials/analysis tools: ZM GGK TC. Wrote the paper: YFL GGK RG HB.
11 2011 3 11 2011 7 11 e100235024 5 2011 18 9 2011 Friedman-Levi et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Genetic prion diseases are late onset fatal neurodegenerative disorders linked to pathogenic mutations in the prion protein-encoding gene, PRNP. The most prevalent of these is the substitution of Glutamate for Lysine at codon 200 (E200K), causing genetic Creutzfeldt-Jakob disease (gCJD) in several clusters, including Jews of Libyan origin. Investigating the pathogenesis of genetic CJD, as well as developing prophylactic treatments for young asymptomatic carriers of this and other PrP mutations, may well depend upon the availability of appropriate animal models in which long term treatments can be evaluated for efficacy and toxicity. Here we present the first effective mouse model for E200KCJD, which expresses chimeric mouse/human (TgMHu2M) E199KPrP on both a null and a wt PrP background, as is the case for heterozygous patients and carriers. Mice from both lines suffered from distinct neurological symptoms as early as 5–6 month of age and deteriorated to death several months thereafter. Histopathological examination of the brain and spinal cord revealed early gliosis and age-related intraneuronal deposition of disease-associated PrP similarly to human E200K gCJD. Concomitantly we detected aggregated, proteinase K resistant, truncated and oxidized PrP forms on immunoblots. Inoculation of brain extracts from TgMHu2ME199K mice readily induced, the first time for any mutant prion transgenic model, a distinct fatal prion disease in wt mice. We believe that these mice may serve as an ideal platform for the investigation of the pathogenesis of genetic prion disease and thus for the monitoring of anti-prion treatments.
Author Summary
Inherited prion diseases, such as genetic CJD, are dominant disorders linked to mutations in the gene encoding the prion protein, PrP. Since therapeutic intervention in all types of human prion diseases has failed, we propose that therapeutic efforts should be directed mostly to the development of preventive treatments for subjects incubating prion diseases, as is the case for asymptomatic carriers of pathogenic PrP mutations. These subjects will develop disease symptoms at some point in their adult life; therefore they should be treated before clinical deterioration. Candidate treatments will need to be tested for efficacy and safety first in animal models that mimic most properties of genetic CJD. In this work, we describe a new transgenic mouse model for E200K genetic CJD, presenting progressive neurodegenerative disease and age related prion disease pathology and biochemistry, as is the case in the human disease. Brain extracts from these mice also transmitted prion disease to wt mice, as shown before for parallel human samples. We propose that these animals will play a significant role in the development of novel anti-prion prophylactic treatments.
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Introduction
Inherited prion diseases, such as gCJD and Gerstmann–Sträussler–Scheinker (GSS), are autosomal dominant disorders linked to mutations in the gene encoding the prion protein (PrP), denominated PRNP
[1], [2]. The largest focus of gCJD was identified among Libyan Jews carrying a missense mutation in codon 200 of PRNP (substituting lysine for glutamate, E200K) [3], [4]. This same mutation was also found in other communities around the world [5].
As of today, therapeutic intervention in human prion diseases has failed [6]
[7]. Indeed, some protocols reduced the rate of patients' deterioration for short periods of time [8], but none could hope to reverse the severe neurological deficits apparent already at diagnosis. We therefore propose that efforts should be directed mostly to develop preventive treatments for subjects at risk, as is the case for asymptomatic carriers of genetic prion diseases. Candidate anti-prion reagents will need to be tested in transgenic models mimicking gCJD. Such transgenic mice should succumb spontaneously to neurological disease in a high attack rate and in a short time frame, allowing for long term treatments and measurable delay of onset well within the life span of the animals. The model mice should also present prion related biochemistry and pathology, and if possible transmit disease directly to wt animals, as is the case for humans suffering from gCJD [9]
[10].
Indeed, several animal models of genetic prion disease were generated in the past, thereby demonstrating that late onset and spontaneous genetic human prion diseases can be reconstructed in mice [11], [12]. While very useful in the study of prion disease pathogenesis, not all these models presented all the properties described above.
The first transgenic (Tg) mice imitating human genetic prion disease carried a P102L-PrP GSS mutation on a mouse background and succumb spontaneously to prion disease after about 4–6 months [13]. However these mice transmitted infectivity only to unique recipients [14], [15], and in addition presented poor PrP pathology. Tg lines mimicking the PrP insertional mutation [16], the A117V mutation [17], as well as both the CJD [18] and the FFI D178N [19] mutation presented prion-like clinical disease with low to marginal disease related PrP. The FFI D178N mice transmitted disease to mice overexpressing wtPrP as well as those expressing wtPrP with the 3F4 epitope, and the recipient mice developed prion-related neuropathology in the absence of disease related PrP [19]. Two Tg lines mimicking the E200K PrP mutation, one on a human PrP gene and another on a mouse PrP gene did not present disease or other prion related properties [20]
[21].
In this work, we describe a transgenic mouse model for E200K gCJD expressing a chimeric mouse/human PrP [15], [22] both on a wt and a null PrP background, hereby denominated TgMHu2ME199K/wt and TgMHu2ME199K/ko respectively. The line on the wt background mimics most gCJD patients, who are heterozygous for the PrP mutation [2]. Mice from both lines presented progressive neurodegenerative disease starting from 5 to 6 month of age, deteriorated and died several months thereafter. Their brains comprise age related pathology characteristic of prion disease, such as gliosis and accumulated disease related PrP, which was shown by immunoblots to be resistant to digestion by high concentrations of proteinase K (PK). Most important, brain extracts from both lines transmitted prion disease to wt mice. We believe that these animals will play a significant role in the investigation of genetic prion disease pathogenesis and most important, in the development of novel anti-prion prophylactic treatments.
Results
TgMHu2ME199K mice develop spontaneous progressive neurological disease
TgMHu2ME199K on both a wt and a PrP ablated background were constructed (as described in the methods) by inserting an E to K substitution at position 199 of a chimeric mouse human (MHu2M) PrP construct. As of today a total of 300 mice were generated (240 on an ablated background and 60 on a wt background), and used for the different experiments described in this manuscript. These include characterization of clinical disease as well as investigation of kinetic of disease progression. We also studied pathological and biochemical prion disease properties of the Tg mice at different time points before and throughout disease progression and collected samples for expression and transmission studies.
The most prominent symptom of disease, which appeared in all Tg MHu2M E199K mice already at 5–6 months of age, is an a-symmetric hind limbs weakness that develops with time to paraplegia. This sign was followed by leg clasping and lower body atrophy. Contrarily, some of the most characteristic clinical signs of prion symptoms, i.e. plastic tail and tremor were only apparent in some of the mice. Figure 1 depicts affected mice suffering from hind limbs plegia, lower body atrophy and leg clasping. While the mice in the figure are each from a different line (Tg/ko and Tg/wt), the different signs appear in all sick mice. The clinical symptoms of the TgMHu2ME199K mice by order of appearance are described in Table 1. Table 2 demonstrates the score we constructed out of these clinical symptoms for kinetic analysis of disease progression. The time point of death (score 5) was determined when a mouse was too paralyzed to reach food and water independently (according to local committee ethical requirements).
10.1371/journal.ppat.1002350.g001Figure 1 Clinical Characterization of spontaneous disease in TgMHu2ME199K mice.
Typical pictures of sick TgMHu2ME199K mice showing hind limbs plegia, kyphosis and leg clasping.
10.1371/journal.ppat.1002350.t001Table 1 Clinical signs by order of appearance.
Percentage of affected mice
A-symmetric hind limbs weakness 100%
A-symmetric hind limbs plegia 100%
Abnormal hind limbs posture 100%
Lower body atrophy 100%
Kyphosis 100%
Myoclonic jerks ∼10%
Tremor ∼20%
Plastic tail Rare (less than 5%)
Blindness Rare (less than 5%)
10.1371/journal.ppat.1002350.t002Table 2 Score of disease severity by clinical signs.
Hind limbs weakness 1
Hind limbs partial paralysis 2
Full paralysis in one limb 3
Full hind limbs paralysis 4
Death 5
Additional symptoms such as plastic tail (rare) or tremor added one point to the total score.
To evaluate the kinetics of disease progression in these mice, a designated group of 62 TgMHu2ME199K/ko and 14 TgMHu2ME199K/wt, (half male, and half female) was followed carefully from birth throughout disease progression to death. Figure 2a shows the average age of disease onset (score 1) and disease end point (score 5) in the transgenic mice. Figure 2 b presents the severity of disease as related to age (each point represent the average score in groups of 2–8 littermates, which were averaged together to avoid individual differences), while figure 2 c demonstrates disease prevalence in these same groups as related to age. As stated above, our results indicate that all TgMHu2ME199K mice demonstrate first disease symptoms between the ages of 5 to 7 months old. No significant differences were observed in clinical and kinetic parameters between male and female mice. Small differences (non significant) in disease presentation and progression were observed between mice expressing the chimeric mutant PrP on null as compared to wt PrP background (figure 2a), however this may result from the smaller numbers of mice in the TgMHu2ME199K/wt group. Additional transgenic mice used in time course experiments showed similar disease parameters.
10.1371/journal.ppat.1002350.g002Figure 2 Disease progression in spontaneous disease.
a: Average disease onset and death of mice in kinetic studies. b: Aggravation of clinical score of disease as related to the mice's age and gender. Groups of TgMHu2ME199K mice (male and female) were scored for clinical signs from birth to death. Average disease score for each group was plotted against the age elapsed since the mice birth. Closed circles: males, open circles: females. c: Percentage of sick mice in each age group. Groups of mice (as in fig b) in which the average score was at least 1 were plotted against the age of the mice. Closed circles: males, open circles: females d: Relative PrP mRNA levels, as determined by quantitative RT-PCR for wt and TgMHu2ME199K/ko mice. Each bar represents the average of PrP mRNA normalized against controls genes levels (see methods) in 4 male mice. Statistical bars represent standard error e: Brain homogenates from TgMHu2ME199K/ko, TgMHu2ME199K/wt, PrP ablated, wt C57BL/6 and RML-infected mice were immunoblotted with α PrP 6H4 mAb f: Relative intensities of the bands as measured by NIH Image J analysis software.
PrP expression in TgMHu2ME199K mice
We next investigated the levels of PrP expression in the brains of the TgMHu2ME199K mice. To this effect, mRNA samples purified from brains of wt and TgMHu2ME199K/ko 6 months old mice (4 for each group) were subjected to reverse transcriptase and subsequently to amplification by real time PCR of PrP and control genes (see methods). Figure 2d shows that while mRNA levels of PrP were 20 fold higher in the brains of the transgenic mice as compared to wt mice, the actual levels of the PrP protein, as tested by immunoblotting of brain homogenates with α PrP mAb 6H4, were only increased by 2 folds (figures 2e &f). Whether this discrepancy between the PrP mRNA and protein levels of mutant PrP in the Tg mice is of biological significance is unknown at this point.
Neuropathological evaluation of the spontaneous prion disease in TgMHu2ME199K mice
Four µm thick sections of formalin-fixed, paraffin-embedded brains and spinal cords of TgMHu2ME199K mice of different ages and gene array were evaluated for neuropathology and PrP immunoreactivity with different α PrP antibodies (see figure 3a for epitope description of all α PrP antibodies used in this project). Figure 3b depicts the results for 8 months old mice (at least 3 in each group) of the different lines (TgMHu2ME199K/ko, TgMHu2ME199K/wt, PrP0/0 and wt mice). Figure S1 presents results for TgMHu2ME199K/ko mice at different ages (3 in each group), which are also summarized in figure 4. None of the Tg mice brains exhibited inflammatory infiltrates, demyelination, axonal swellings, or abnormal neurites, in accordance with classical prion disease–related pathology [23].
10.1371/journal.ppat.1002350.g003Figure 3 PrP immunoreactivity in the brains of TgMHu2ME199K mice.
a: Epitope mapping of α PrP antibodies used in these experiments. b: Four µm thick sections of formalin fixed, paraffin embedded brains from 8 months old TgMHu2ME199K on wt and ablated background, as compared to wt and PrP ablated mice were tested for disease related PrP immunoreactivity in diverse brain regions with both α PrP pAb RTC and α PrP mAb 6H4. The figure represents at least 3 samples from each group, and depicts intracellular PrP staining with RTC for the sick Tg mice. Scale bar in the upper left panel indicates 20 µm.
10.1371/journal.ppat.1002350.g004Figure 4 Summary of Pathology of TgMHu2ME199K mice: time course.
This figure summarizes the pathological findings in TgMHu2ME199K/ko mice of different ages (at least 3 mice for each age group, see Figure S1) a: the score for gliosis/neuronal loss in different brain areas of the TgMHu2ME199K mice as a function of aging. b: intraneuronal PrP immunoreactivity score in different brain areas as a function of aging.
The predominant form of disease related PrP immunoreactivity in the TgMHu2ME199K mice was an intraneuronal dot-like and granular immunostaining in widespread distribution but mainly in neurons of the spinal cord, basal ganglia, thalamus, frontal cortex, and in brainstem nuclei. This was detected by the C-terminally directed αPrP pAb RTC, and less so by αPrP mAb 12F10 (shown for human in Figure S1). Plaque-like or coarse PrP immunoreactivity was not seen in any of the sick TgMHu2ME199K mice. These patterns of immunopositivity, in particular the intraneuronal staining, is strikingly reminiscent of recently described human E200K gCJD [24]. In addition to the intraneuronal PrP detected by RTC, a fine granular immunostaining reminiscent of the so-called synaptic immunoreactivity was observed by immunostaining for α PrP mAb 6H4 (figure 3b and figure S1). Interestingly, while the intraneuronal PrP immunoreactivity was prominent in many regions including the spinal cord, the synaptic type was rather seen in subcortical gray matter structures (see figure S1 and figure 4 for time course and summary of pathological results). Both forms of disease-associated PrP immunodeposits are present in humans affected by E200K linked gCJD [24], further supporting the similarity between the human disease and this animal model.
For technical reasons, the pathological examination of heterozygous E200K human patients [24] could not establish whether the intracellular staining was associated with E200K PrP, wtPrP or both PrP forms. However, the examination of our TG model, which allows for the comparison of Tg/ko with Tg/wt, provides a partial answer to this question. Since there was no apparent difference between the PrP immunoreactivity of both these lines with RTC and 6H4 (figure 3), we may conclude that mutant PrP thus accumulate intraneuronally in all sick Tg mice. To establish whether also wt PrP in the TgMHu2ME199K/wt mice can accumulate inside neurons or produce any form of disease related PrP, we need an antibody that will recognize only this PrP form. Regretfully, we could not allocate a reagent with exclusive specificity for wt PrP as opposed to MHu2M PrP that will also be suitable for pathological studies.
Spongiform changes, a common feature of scrapie RML strain in mice [25], were observed only focally at the end-stage of disease, mostly in the frontal cortex and in the basal ganglia (figure S1). Mild degree of neuronal loss and reactive astrogliosis was observed already in 3 months old Tg mice in the basal ganglia, thalamus, and circumscribed areas of the frontal cortex, as well as in the spinal cord. These alterations became more prominent in later stages (at 8 months old and at the end point of disease) concomitantly with the clinical symptoms described above (see figure S1 and figure 4).
Biochemical characterization of PrP in the spontaneous disease
As described above, intraneuronal PrP in the TgMHu2ME199K mice was visualized mostly with C-terminal αPrP antibodies, in particular RTC, a polyclonal antibody which detects the 201–205 PrP epitope [26], suggesting disease related PrP may accumulate in the Tg mice as an N-terminally truncated form. To further test this possibility by biochemical methods, as well as to evaluate other prion like biochemical properties of PrP in the Tg mice, we used diverse αPrP antibodies (see figure 3a for specific epitopes) to immunoblot brain homogenates from 8 months old mice from different genetic backgrounds (figure 5a). These include TgMHu2M E200K mice on both the ablated (lane 1) and wt (lane 2) PrP background, as well as from age matched wt TgMHu2M (wt chimeric human PRNP transgene mice (lane 3), which have not developed spontaneous neurological disease during their life span [15]. As additional controls, we also tested brain homogenates from normal (lane 4) and RML scrapie infected (lane 5) mice (also see insert in figure 5 for sample description). Panel 5a shows that while αPrP mAb IPC1 reacted preferentially with samples 2, 4, and 5 which express PrP from a wt mouse allele, mAb 3F4 reacted only with the samples expressing TgMHu2MPrP, regardless of the presence of the E200K mutation or of the additional expression of wt PrP. Contrarily to the antibodies with species selective immunoreactivity (IPC1 and 3F4), mAb 6H4 recognized PrP forms from all brain samples at comparable levels (see also figure 1c). Last, the C-terminal RTC antibody detected equally the established PrP bands in all samples, but in addition recognized some truncated PrP forms (of about 10 and 20 kDA) in the samples comprising a TgMHu2ME199K allele. These bands (see arrows for truncated forms) were absent from samples of both wt mice and TgMHu2M controls, suggesting they are specific for PrP in the TgMHu2ME199K mice.
10.1371/journal.ppat.1002350.g005Figure 5 Biochemical Characterization of PrP in TgMHu2ME199K mice.
a: To establish the PrP specificity of different samples, brain homogenates from 8 months old mice from 1: TgMHu2ME199K/ko, 2: TgMHu2ME199K/wt, 3: TgMHu2M, 4: wt C57BL/6, and 5: RML infected mice, were immunoblotted with several α PrP antibodies (See figure 3a for α PrP epitope mapping). Arrows demonstrate truncated PrP forms only present in brains of TgMHu2ME199K mice. b: Samples as in panel a were treated in the presence or absence of PNGase and immunoblotted with designated α PrP antibodies. As above truncated PrP forms are demonstrated only in the TgMHu2ME199K mice demonstrated with arrows. c: Samples as in panel a were extracted with sarkosyl and then centrifuged at 100000 g for 1 h, separated into pellets and supernatants. Otherwise, similar samples (denominated as total) were digested with 30 ug/ml PK for 30 min at 37°C. All samples were then immunoblotted with the designated anti PrP antibodies. Arrows demonstrate PK resistant bands in the TgMHu2ME199K samples. d: Samples from TgMHu2ME199K/ko mice at different ages and controls were digested in the presence or absence of 30 ug/ml PK for 30 min at 37°C and immunoblotted with α PrP pAb RTC. #1: 1 month old; #2: 3 months old; #3: 7 months old; #4: another 7 months old sample, #5 PrP ablated mouse; #6 wt mouse, #7 RML infected mouse.
To learn more about the PrP bands recognized by pAb RTC in the Tg mice brains, we subjected the samples presented in panel a to digestion by PNGase, an enzyme which removes N-linked sugars from proteins [27]. Figure 5b shows that while the 26 KDa band, representing deglycosylated full length PrP was detected by RTC in all samples, the TgMHu2ME199K samples presented additional and unique deglycosylated bands (see arrows), different also in their molecular weight from the 19 Kda band representing deglycosylated PK resistant PrP in scrapie brains (lane 5).
To investigate whether PrP forms present in the brains of sick TgMHu2ME199K mice are aggregated and PK resistant, properties established as the hallmark of disease related PrP, we subjected Sarkosyl extracted brain homogenates from sick TgMHu2ME199K mice and controls (same lane numbering as above) to centrifugation at 100,000 g. In parallel, similar homogenate samples were digested with 30 µg/ml PK for 30 minutes at 37°C. The samples generated by these experiments were immunoblotted with α PrP antibodies 6H4 and RTC. Figure 5c shows that a significant fraction of the PrP protein present in the sick mice (lanes 1&2) pelleted under these conditions, resembling the fraction of aggregated PrP in scrapie infected mice (lane 5). This was not the case for PrP in the samples from the control chimeric or from the wt mice (lanes 3&4). As in the previous panel, immunoblotting of the same samples with pAb RTC revealed additional lower bands of about 10 to 20 Kda, in both the pellet and the supernatant. Following digestion of the homogenates with PK, and consistent with the pathological results (figure 2); it was again RTC that could detect the PrP bands resistant to protease digestion.
To establish whether truncated PK resistant PrP in the brains of TgMHu2ME199K mice are a feature of the mutated PrP chimera at all ages or represent the onset of disease in older mice, we looked for their presence in the brains of young and asymptomatic TgMHu2ME199K mice. Figure 5d shows that PK resistant PrP was absent at 1 month of age (lane 1), barely present at 3 months of age (lane 2), but was clearly apparent at 8 months of age (lanes 3 and 4), when animals were severely sick. These results demonstrate that, consistent with the immunohistochemistry results described in figure 4, the appearance of PK resistant PrP forms correlate with age and disease progression, and are not an automatic feature of mutated PrP.
Oxidation properties of PrP in the spontaneous and the transmitted disease
We have recently shown that pAb RVC, a polyclonal antibody generated against reduced 203–214 human/mouse PrP peptides, could not detect Human PrPSc in brains of genetic or sporadic CJD patients [26]. This and other experiments demonstrated that Methionine residues (Met) in human PrPSc are present in an oxidized form. This was also the case for Met residues in recombinant human E200K PrP. To test the oxidation status of PrP in our sick TgMHu2ME199K mice, we subjected Sarkosyl extracted brain homogenates from wt and from TgMHu2ME199K/ko mice, as well as from mice infected with RML prions to 10–60% sucrose gradients. Subsequently, the gradient fractions were immunoblotted with both RTC and RVC α PrP antibodies. Figure 6 shows how PrP in the TgMHu2ME199K mice (both full length and truncated) was detected in all the gradients fractions when the blots were challenged with pAb RTC, indicating the mutant protein may be present in the brains of these mice at diverse aggregation states. In contrast, pAb RVC detected only full length PrP in the lighter fractions, suggesting that TgMHu2ME199K PrP may be oxidized and aggregated during its metabolic pathway in the Tg mice, as is also the case for PrPSc in the infected brains. These experiments indicate that most mutant PrP in the Tg mice is not oxidized immediately upon its generation, but becomes oxidized concomitantly with its aggregation during its metabolic pathway.
10.1371/journal.ppat.1002350.g006Figure 6 Oxidation of TgMHu2ME199K PrP in Tg mice.
PrPSc in mice, hamsters and human samples cannot be detected by αPrP RVC, demonstrating it is oxidized in it helix3 Met residues. To see if this is also the case for all or part of E200K PrP in the Tg mice, brain homogenates from (wt C57BL/6, TgMHu2ME199K/ko, and scrapie RML), were extracted with sarkosyl and subjected to ultracentrifugation in 10–60% sucrose gradients. Individual fractions of each of the gradients were immunoblotted respectively with α PrP antibodies RTC and RVC. The figure shows that aggregated forms of the mutant protein are not recognized by this antibody, as is the case for RML PrPSc.
Transmission of spontaneous disease from TgMHu2ME199K to wt mice
Brain samples from heterozygous patients carrying the E200K PrP mutation were shown to transmit prion disease to primates [9] as well as to both wt and TgMHu2M PrP mice [10], [28]. To test if our mice also produce infectious prions in addition to fatal spontaneous disease, we inoculated the brain homogenates from an asymptomatic TgMHu2ME199K/wt mouse, from a sick TgMHu2ME199K/wt mouse, as well as from a sick TgMHu2ME199K/ko to groups of wt (C57Bl/6) mice.
We speculated that the presence of a wt allele in the E199K PrP Tgs may induce the formation of some levels of wt PrPSc, thereby facilitating transmission of disease to wt mice following their infection with brains of the Tgs. To this effect, we inoculated the samples from the heterozygous mice only intraperitoneally (i.p.), which although resulting in a longer inoculation time is a less invasive pathway, while the brain homogenate from the sick TgMHu2ME199K/wt was inoculated both i.c. (intracerebrally) and i.p., to maximize the possibility of transmission. As control for the experiment, the brain homogenate of a wt Tg MHu2M mouse was inoculated i.p. into a C57B/6 group. These mice were shown previously to remain healthy for more then 640 days [15]. In addition, a wt C57B/6mouse brain homogenate was inoculated i.c. to a group of mice in the same room as a general control for contamination.
All inoculated animals were evaluated twice a week for clinical signs. Figure 7a shows a typical sick mouse infected with any of the TgMHu2ME199K brain samples, demonstrating lower body atrophy and hind limbs weakness, both properties reminiscence of the spontaneous disease of the donor Tg mice. The transmitted mice also showed “tip toe” walking, a rare feature described only in some prion related models [29]. Clinical signs present in other infectious prion strains, such as kyphosis and plastic tail, were also observed in these mice, as opposed to the donor Tgs. The transmitted disease affected the 6 mice of group 9.9 (inoculated with brain homogenate from a sick TgMHu2ME199K/wt mouse), 3 out of 5 mice of group 9.3 (inoculated with the brain extract from an asymptomatic TgMHu2ME199K/wt mouse), 2 out of the 5 mice inoculated i.c. and 1 out of 6 mice inoculated i.p with a brain extract of a sick TgMHu2ME199K/ko mouse. Disease signs appeared first in the mice infected i.p. with TgMHu2ME199K/wt at about 160–180 days and progressed to their death 2–3 months thereafter (see figure 7c for survival results). After infection with TgMHu2ME199K/ko, some mice became sick at 210 days (i.c) and 300 days (i.p.). While disease in these mice was apparently shorter than in the ones infected with TgMHu2ME199K/wt brains (2–3 weeks), no conclusions can be drown from this observation due to the small numbers. None of the control mice (Tg MHu2M and wt) develop any signs of disease for more than 400 days.
10.1371/journal.ppat.1002350.g007Figure 7 Transmission of TgMHu2ME199K prions to wt mice.
a: Picture of a wt C57BL/6 mouse infected with brain homogenates from TgMHu2ME199K/wt mice showing abnormal hind limb posture. Upper panel shows foot prints of the mice as compared to wt mice, demonstrating abnormal pattern of walking. b: Survival curves for wt C57BL/6 mice infected i.p with a TgMHu2ME199K/wt sick mouse brain (group 9.9, close square) or a TgMHu2ME199K/wt asymptomatic mouse brain (group 9.3, open square), as well as i.c (close circles) or i.p (open circles) infected with a TgMHu2ME199K/ko sick mouse brain, or i.p with the brain homogenate of TgMHu2M mice (close diamond) and i.c with wt brain homogenate (open triangle). c: Brain homogenates of RML infected and wt mice as well as mice (1&2) infected with TgMHu2ME199K/wt brain homogenate (group 9.3) immunoblotted in the presence and absence of PK digestion with α PrP pAb RTC. d: Brain homogenates digested in the presence or absence of PK and immunoblotted with α PrP pAb RTC of the brain samples described in table 3. Upper panel: −PK, lower Panel: +PK overdeveloped. Compare sample 1 in figures c and d to appreciate the overdevelopment factor.
Our results therefore indicate that, like in human E200K brains [10], [28], infectious prions are spontaneously formed in brains of TgMHu2ME199K mice. Most important, potential infectivity is generated in these mice brains before the appearance of clinical signs, as seen by the fact that brains of asymptomatic mice transmitted disease to some of the wt mice. The levels of infectious prions may further increase with disease progression, as seen by the fact that mice infected with the sample from a sick TgMHu2ME199K/wt mice (group 9.9) succumbed to disease in a relative short time, as compared to asymptomatic sample 9.3. Our results also suggest that the wt allele in heterozygous mice may facilitate the transmission of infectivity to naïve wt mice, since transmission from a sick TgMHu2ME199K/ko mice required a very long incubation time and occurred only occasionally, in particular after i.p. inoculation. Facilitation of disease transmission by a wt allele may result from the in-vivo generation of wt PrPSc in the TgMHu2ME199K/wt mice (even if at marginal levels), concurrently with the quantitative spontaneously generation of mutant disease related PrP. This may indicate that the “species barrier” between both forms of PrP may have been abrogated to some extent in the TgMHu2ME199K/wt mice. It also implies that while wt PrP has little or no effect on the actual presentation of spontaneous disease and its progression, low levels of wt PrPSc in these animals may be very central for the further passage of disease to naïve wt mice.
PK resistant PrP in the brains of transmitted wt mice
Figure 7c shows an α PrP immunoblot (pAb RTC) of brain homogenates from individual mice, either infected with scrapie RML, naïve C57B/6, or infected with a brain homogenate from an asymptomatic TgMHu2ME199K/wt mouse, 9.3 (see table 3 for the numbering of samples in figure 7c &d). As can be seen in the figure, sample 1 (derived from sick wt mouse 224 days post infection) presents a similar pattern of disease related PrP as in the RML infected sample, as opposed to, sample 2 (derived from a healthy wt mouse 413 days post infection) in which no PrPSc can be detected. Figure 7d presents an immunoblots in which individual PK digested samples from infected mice were overdeveloped to allow for the detection of low levels of PK resistant PrP. Consistent with the results in figure 7c, PrPSc was not be detected in the asymptomatic mice from group 9.3. Contrarily, figure 7d shows that PK resistant PrP forms could be detected in the brains of a selection of brains from mice that succumbed to disease, however the levels and pattern of disease related PrP differed significantly between individual samples (see summary in Table 3). This was true even for extracts of mice infected with the same inoculum, and presenting the same symptoms, as was the case for samples 5–8, which were infected with brain homogenate of a sick TgMHu2ME199K/wt mouse, and samples 9 and 10, both infected with a TgMHu2ME199K/ko brain extract. As opposed to the donor Tg mice, no truncated PrP forms were observed with this antibody in any of the samples. The different levels of PrPSc in wt mice inoculated with the same prion homogenate are consistent with results from experiments describing the transmission of BSE into wt mice [30]. In that case the presence of PrPSc in the direct transmission from cow brains could be detected only in about 50% of the mice, while fatal disease presented in all of the animals. PrPSc became apparent in all mice following adaptation of the new strain by additional mice to mice passages.
10.1371/journal.ppat.1002350.t003Table 3 Summary of transmission studies.
sample group PK resistant PrP Age of death (days)
1 Infected with Tg/wt 9.3 (i.p) +++ 224
2 Infected with Tg/wt 9.3 (i.p) − Sac at 413
3 Infected with Tg/wt 9.3 (i.p) − Sac at 413
4 Infected with Tg/wt 9.3 (i.p) − Sac at 413
5 Infected with Tg/wt 9.9 (i.p) +++ 224
6 Infected with Tg/wt 9.9 (i.p) +++ 252
7 Infected with Tg/wt 9.9 (i.p) + 254
8 Infected with Tg/wt 9.9 (i.p) + 257
9 Infected with Tg/KO (i.c) +++ 210
10 Infected with Tg/KO (i.c) +/− 210
11 TgMHu2ME199K/KO ++ Sac at 240
12 TgMHu2ME199K/wt ++ Sac at 240
13 wt C57Bl/6 − Sac at 240
14 Mouse RML +++ 180
This table summarizes the results of the transmission experiment as described in figure 7 b (survival of mice), as well as the presence of PK resistant PrP in infected samples (panels c &d).
Neuropathological evaluation of wt mice infected with TgMHu2ME199K brains
Sections of formalin-fixed, paraffin-embedded brains of mice infected with TgMHu2ME199K/wt, TgMHu2ME199K/ko and RML prions were examined for prion parameters. Figure 8a presents sections of the frontal cortex. Brains infected with TgMHu2ME199K samples present minor to moderate spongiform changes, distinctly different from the high levels of spongiform changes apparent in the RML strain [31]. The infected mice also showed severe astrogliosis and neuronal loss, as well as prominent diffuse synaptic type disease-related 6H4 PrP immunoreactivity, similar to the ones seen for the RML sections. As opposed to the spontaneous disease of TgMHu2ME199K mice (figure 3), only low levels of RTC related immunostaining were observed in the infected mice's brains, as shown in figure 8a for the sample infected with a TgMHu2ME199K brains. RTC immunostaining was not observed in the RML samples.
10.1371/journal.ppat.1002350.g008Figure 8 Transmission of TgMHu2ME199K prions to wt mice: pathology.
a: Frontal cortex sections of mice infected with RML or with TgMHu2ME199K prions on a wt or ablated background, analyzed for prion pathological properties, spongiosis, gliosis, and disease related PrP immunoreactivity with pAb RTC and mAb 6H4. Scale bar in upper left image indicates 50 µm. b: RTC immunoreactivity in the absence of formic acid for brains infected with TgMHu2ME199K/wt or RML, as well as for naïve PrP ablated mice. Picture shows different pattern of immunoreactivity for both infected samples. Scale bar in upper left image indicates 20 µm.
To test whether RTC related immunostaining can distinguish better between the RML and TgMHu2ME199K generated prions at a different experimental setup, we immunostained sections of TgMHu2ME199K and RML infected mice with RTC following a less harsh epitope revealing treatment (no formic acid after heating with citrate). Figure 8b shows that under these conditions, pAb RTC detected intracellular PrP aggregates in the mice infected with TgMHu2ME199K, but not in those infected with RML homogenates. Both brain samples presented a diffused immunoreactivity reminiscence of PrPC. No immunoreactivity of any kind was observed in brains of PrP ablated mice, indicating that the positive stain in the infected sample is indeed PrP.
In conclusion, clinical, biochemical and pathological results presented in this section demonstrate that brains from TgMHu2ME199K mice may generate de-novo prions with specific properties. These prions may readily transmit to wt mice, and are particularly infectious when in the brains of sick TgMHu2ME199K on a wt background. Whether other organs of these mice, and in particular blood and immune cells, may also transmit infectivity remains to be established. Results from such experiments may be very important to assess blood safety in the community.
Discussion
Constructing a clinically relevant mouse model has proven to be a hard task for most neurodegenerative diseases [32]. The existing models, in particular for Alzheimer's of Parkinson diseases present mostly pathological markers and in some cases behavioral changes, but not obvious clinical symptoms, or age dependent deterioration that correlates with those observed in human patients [33].
As opposed to models for the more common neurodegenerative conditions, several genetic prion diseases linked to pathological mutations in the PRNP gene have been reconstructed clinically in transgenic mice lines. Each of these models demonstrate several of the basic features of genetic fatal prion disease, as is the case for those mimicking GSS linked to the PrP P101L mutation [3]
[21], the D177N CJD or FFI mutations [18], [19] or the insertional PrP modification [34]. These mouse models were seminal in proving that spontaneous prion disease may indeed result from the presence of a pathological PrP mutation.
In this work, we describe the properties of a Tg line mimicking the most common genetic prion disease [3], i.e CJD linked to the human E200K PrP mutation. Our Tg line presents all prion relevant properties, spontaneous fatal disease, PrP pathology and transmission of prion disease to wt mice. This is particularly intriguing in view of the fact that two other models of this same mutation failed to generate disease in transgenic mice. While they may be other explanations for the different results in our case, we assume that the introduction of the E200K mutation into a chimeric mouse human PrP, as opposed to a mouse PrP [21] or a human PrP [20], is of biological importance. Chimeric PrP may constitute the bridge that allows human prion diseases to manifest in mice. Indeed, chimeric human mouse PrP was required to transmit at low incubation times genetic and sporadic human prion disease to mice [15]. Moreover, while Tgs expressing the GSS 102 mutation in human PrP did not present spontaneous disease, the same mutation in chimeric PrP did present neurodegenerative disease [28]. Whether the structure of chimeric PrP is more favorable for disease transmission or otherwise the chimeric form has the ability to bind a mouse component important for transmission of human prion diseases to mouse models remains to be established.
Another novel feature of our Tg line is the generation of de-novo infectious prions that could be transmitted to naïve wt mice. Indeed, E200K CJD is the genetic disease most similar to the sporadic forms, in both clinical appearance, age of onset and pathology [2], [10]. This may imply that E200K de-novo prions are more similar in structure to sporadic ones, which are highly transmissible [9]. Whether the chimeric background of E200K PrP in these mice is also a factor in the transmissibility of disease is unknown at this point, however it is important to state that chimeric mouse human PrP Tg mice are susceptible for infection with both mouse and human prions [28].
While the neuropathology features of our Tg mice were similar to E200K human patients with regard to reduced spongiosis and intracellular PrP accumulation [24], PK resistant PrP in the TgMHu2ME199K mice was detected mostly by the C-terminal pAb RTC, suggesting a considerable fraction of disease related PrP in the brains of these mice accumulates as a truncated form. Indeed, diverse truncated PrP forms were also described in brains of CJD patients [35], including those carrying the E200K mutation [36]. Interestingly, intraneuronal immunoreactivity in these CJD patients predominates in the brainstem and may be associated with alterations in the accumulation of other neurodegeneration-related proteins (e.g. phospho-tau, alpha-synuclein) [24]. Evaluation of concomitant protein pathology in our model is the objective of another ongoing study.
Because gCJD is a dominant genetic disorder, we investigated the properties of the TgMHu2ME199K mice not only on a PrP ablated but also on a wt PrP background. We first speculated that the presence of wt PrP may preclude some disease symptoms related to the absence of the elusive PrPC activity or to the putative toxicity of truncated PrP forms, as was shown previously in other systems [29]. However, this is probably not the case, as can be inferred from the fact that no significant differences were seen between both lines of Tg mice in clinical symptoms, kinetics and pathological examination. Finally, while the investigation of a small group of homozygous E200K CJD patients showed a moderate decrease in the age of disease onset for most patients, it also described a patient with a very slow progressive disease (96 months), who died in the absence of PrPSc accumulation [37]. Whether disease onset in one or both lines of TgMHu2ME199K mice may be modulated by oxidative stress or other pathogenic insults is under investigation in our laboratory.
In summary, we believe that our TgMHu2ME199K lines will play a central role both in the elucidation of genetic prion disease pathogenic mechanism as well as in the search for anti-prion compounds. The early presence of spontaneous disease followed by their sequential age related deterioration during several months until death will permit to study the long term effect of reagents that may delay disease onset in at risk subjects. Among those to be tested first are substances suggested to have a marginal but still encouraging result in already sick CJD patients, such as doxicyline [38] and flupirtine [7], as well as those believed to present significant therapeutic results in scrapie infected mice or infected cells, such as Quinacrine and Simvastatin [39], [40]. Novel approaches such as passive [41] or active immunization, as well as RNAi inhibition of mutant PrP expression [42], will also be tested in the near future.
Materials and Methods
Ethical statement
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Hebrew University Medical school (Permit Number: MD-11746-5). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.
Generation of TgMHu2ME199K mice
Transgenic mice harboring the E200K mutation were generated by one of us (ZM) in the Prusiner laboratory as follows. Using site-directed mutagenesis by PCR, the E200K mutation was inserted into the chimeric human/mouse PrP open reading frame (ORF) of an MHu2M construct that was previously prepared as described [15]. The 0.8 kb SalI-XhoI fragment containing the PrP ORF with the E200K mutation was inserted into cos.ShaTet and further injected into transgenic mice ablated for the PrP gene [43]. Creating and screening of the transgenic mice were done as described [44]. Several lines were produces at the time and at least 2 presented spontaneous prion disease (ZM, personal communication). For the present project, C57BL/6 female mice were impregnated with frozen sperm from one of these lines in the Jackson laboratories. The designated offspring were subsequently crossed either with wt or with PrP ablated C57BL/6 mice (Harlan laboratories, obtained by crossing of PrP0/0 FVB mice [43] with the C57BL/6 strain for 10 generations, and screened for the presence of the TgMHu2ME199K PrP, ablated or wt PrP allele, as required. The mice used in this project are of a mixed C57B/6/FVB background, ranging from 75- to 95% C57B/6.
Real time PCR
Total RNA from mice brains was isolated using TRI reagent (Sigma, Israel). cDNA was prepared from 2 µg of total RNA using MuLV reverse transcriptase and random hexamers (Promega) according to the manufacturer's instructions. Quantitative RT-PCR was carried out in 15 µl reactions containing 1 µl of cDNA, 0.3 µM of the appropriate primers (sigma, Israel), and 7.5 µl of the SYBR Green master mix (Finnzymes). Gene amplification was carried out using the GeneAmp 7500 Sequence Detection System (Applied Biosystems). Measurements were performed in triplicates and UBC (Ubiquitin C) and TBP (TATA-box binding protein) transcript levels were used to normalize between samples. The primers used were PrP, 5′-CAA GCA GCA CAC GGT CAC C-3′ (forward), 5′-GGC CTG GGA CTC CTT CTG G-3′ (reverse) TBP, 5′-TGT GCA CAG GAG CCA AGA-3′ (forward), 5′-CCC CAC CAT GTT CTG GAT-3′ (reverse); UBC, 5′-CAG CCG TAT ATC TTC CCA GAC T-3′ (forward); 5′-CTC AGA GGG ATG CCA GTA ATC TA-3′ (reverse). The primers used for PrP were chosen so that they can be used for both wt (mouse) PrP as well as for MHu2M PrP.
Spontaneous disease in the TgMHu2ME199K mice
Mutant TgMHu2ME199K mice from both lines (PrP ablated or wt background) were followed twice a week for the appearance of spontaneous neurological disease. Mice were scored for disease severity and progression according to the scale of clinical signs described in Table 2. This scale was designed by us to fit the clinical symptoms observed in the Tg mice and was proven to be parallel to the NNS (neurological severity score). Mice were sacrificed according to the ethical requirements of the Hebrew University Animal authorities (when too sick or paralyzed to reach food and water, or after loosing 20% body weight).
Clinical and behavioral evaluation
As described in table 2 mice were scored for disease severity and progression according to a scale of clinical signs designed by us to fit the clinical symptoms observed in the Tg mice. Hind limbs weakness was first evaluated by closely watching the mouse walking on a flat surface looking for sings of abnormal limb posture or abnormal walking pattern (high or low gait, leg dragging). Next, mice were tested for their ability to walk on a 3 cm beam in a straight line and maintain balance. Finally mice were lifted by their tail to check for leg clasping. Full paralysis was evaluated by total lack of movement in the limb. This scale of scoring was proven to be parallel to the NSS (neurological severity score) [45]. Blindness was tested by the lack of reaction of the mice to a paper slowly placed before its eyes.
Transmission of disease from TgMHu2ME199K mice to wt mice
10% brain homogenates of asymptomatic or sick TgMHu2ME199K/wt, sick TgMHu2ME199K/ko, as well as from control TgMHu2M and naïve wt mice were each inoculated i.p. or i.c, as designated in the text, into a group of 6 C57BL/6 mice (Harlan laboratories). The inoculated mice were scored twice a week for clinical signs of prion disease until the beginning of symptoms and more closely thereafter. Following termination of each experiment, mice were sacrificed and analyzed for pathology and for the presence of disease related PrP.
Pathology
Four µm thick sections of formalin fixed, paraffin embedded brains of TgMHu2ME199K mice as well as of C57BL/6 mice infected with TgMHu2ME199K brains, in addition to controls and PrP ablated mice were evaluated for the presence of disease related PrP, gliosis and spongiform changes as previously described [24]. A less harsh epitope retrieval method, with the avoidance of formic acid, was also applied in some cases.
Immunoblotting of brain homogenates from TgMHu2ME199K mice
Brains from TgMHu2ME199K mice on a wt or ablated background, normal mice, control TgMHu2M and scrapie RML infected mice were homogenate at 10% (W/V) in 10 mM Tris-HCl, pH 7.4 and 0.3 M sucrose. For Proteinase K digestions, 30 µl of 10% brain homogenates extracted with 2% sarkosyl were incubated with 30 µg/ml Proteinase K for 30 min at 37°C. Samples were subsequently subjected to SDS PAGE and immunoblotted with the diverse α-PrP antibodies, as described in Figure 2 a. Protein precipitation experiments, as the ones observed in Figure 5 c, were performed by ultracentrifugation of Sarkosyl extracted homogenates at 100000 g, and subsequently separating pellets from supernatant. Deglycosylation by PNGase was performed as previously described [46].
Sucrose gradient centrifugation experiments
Normal and prion infected Sarkosyl extracted brain homogenates were subjected to sucrose gradients as described [47]. Shortly, 140 µl of 10% brain homogenates extracted in the presence of 2% Sarkosyl were overlaid on a sucrose gradient composed of layers of increasing concentrations of sucrose (10–60%). Gradients were then centrifuged for 1 h at 55000 rpm in a Sorval mini-ultracentrifuge and subsequently 11 samples of 120 µl were collected from the top to the bottom. Gradient fractions were then immunoblotted with either α PrP pAb RTC or RVC.
Accession numbers
prnp mus musculus: ENSMUST00000091288
prnp homo sapiens: ENSG00000171867
UBC mus musculus: ENSMUSG00000008348
TBP mus musculus: ENSMUSG00000014767
Supporting Information
Figure S1
Time course histopathology and PrP immunoreactivity in the brain and spinal cord of TgMHu2ME199K mice.
a: Hematoxylin & Eosin (H&E) staining of frontal cortex: Spongiform change was noted only focally in end-stage animals (H&E staining, frontal cortex). b–d: GFAP staining: Reactive astrogliosis progressed with the age of the animals as represented here in the frontal cortex (b), thalamus (c), and the spinal cord (d). e & f: PrP immunoreactivity. PrP immunoreactivity at disease end point is presented with 2 α PrP antibodies, pAb RTC and mAb 6H4, in different brain areas. For comparison the right lower image is a representative micrograph of intraneuronal PrP immunoreactivity in the putamen in human genetic CJD associated with E200K mutation using αPrP mAb 12F10. Bar in upper left image represents 50 µm for all upper panel images and 20 µm for lower panel images.
(TIF)
Click here for additional data file.
We thank Dr Stanley Prusiner for the sperm of the transgenic E200K mice which made this entire project possible. Brain samples from terminal stage RML mouse were kindly provided by Dr. Till Voigtländer, Vienna, Austria.
The authors have declared that no competing interests exist.
This research was supported by The Legacy Heritage Biomedical Science Partnership Program of the Israel Science Foundation (grant No. 1860/08). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Mol VisMVMolecular Vision1090-0535Molecular Vision 2952010molvis0504Research ArticleEnhanced in vitro antiproliferative effects of EpCAM antibody-functionalized paclitaxel-loaded PLGA nanoparticles in retinoblastoma cells Mitra Moutushy 13Misra Ranjita 2Harilal Anju 1Sahoo Sanjeeb K 2Krishnakumar Subramanian 11 Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Tamil Nadu, India2 Laboratory of Nanomedicine, Institute of Life Sciences, Chandrasekharpur, Bhubaneswar, India3 CeNTAB, SASTRA University, Tanjore, IndiaCorrespondence to: Subramanian Krishnakumar, Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, No. 18 College Road, Nungambakkam, Chennai – 600006, India; Phone: 91-44-28271616 extn: 1302; FAX: 91-44-28254180; email: [email protected] 19 10 2011 17 2724 2737 18 11 2010 16 10 2011 Copyright © 2011 Molecular Vision.2011This is an open-access article distributed under the terms of the
Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original
work is properly cited.Background
To specifically deliver paclitaxel (PTX) to retinoblastoma (RB) cells, the anionic surface-charged poly(lactic-co-glycolic acid) (PLGA) NPs loaded with paclitaxel were conjugated with epithelial cell adhesion molecule (EpCAM) antibody for enhancing site-specific intracellular delivery of paclitaxel against EpCAM overexpressing RB cells.
Methods
PTX-loaded PLGA NPs were prepared by the oil-in-water single emulsion solvent evaporation method, and the PTX content in NPs was estimated by the reverse phase isocratic mode of high performance liquid chromatography. Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysuccinimide chemistry was employed for the covalent attachment of monoclonal EpCAM antibody onto the NP surface. In vitro cytotoxicity of native PTX, unconjugated PTX-loaded NPs (PTX-NPs), and EpCAM antibody-conjugated PTX-loaded nanoparticles (PTX-NP-EpCAM) were evaluated on a Y79 RB cell line by a dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, while cellular apoptosis, cysteinyl-aspartic acid protease (caspase)-3 activation, Poly (adenosine diphosphate-ribose) polymerase (PARP) cleavage, and cell-cycle arrest were quantified by flow cytometry. By employing flow cytometry and fluorescence image analyses, the extent of cellular uptake was comparatively evaluated.
Results
PTX-NP-EpCAM had superior antiproliferation activity, increased arrested cell population at the G2-M phase, and increased activation of caspase-3, followed by PARP cleavage in parallel with the induction of apoptosis. Increased uptake of PTX-Np-EpCAM by the cells suggests that they were mainly taken up through EpCAM mediated endocytosis.
Conclusions
EpCAM antibody-functionalized biodegradable NPs for tumor-selective drug delivery and overcoming drug resistance could be an efficient therapeutic strategy for retinoblastoma treatment.
GalleyStatusExport to XMLcorr-authorKrishnakumar
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Introduction
Advances in our knowledge of molecular biology of cancer and pathways involved in malignant transformation of cells are revolutionizing the approach to cancer treatment with a focus on targeted cancer therapy. The newer approaches to cancer treatment not only supplement conventional chemotherapy and radiotherapy but also aim to prevent damage to normal tissues and overcome drug resistance [1]. Nanoparticulate drug delivery systems using biodegradable polymeric carriers have attracted increasing attention in recent years. The major advantage of using these nanoparticles (NPs) is their sustained release property, and since the drug is encapsulated, it is unexposed to the cell membrane-associated efflux transporters [2-4]. In this way the efflux action of these transporters could be bypassed, resulting in greater cellular drug uptake than that with drug in solution. Polymeric NPs, primarily based on biodegradable poly (D,L-lactic-co-glycolic acid; PLGA) polymers, have been used for the administration of water insoluble anticancer agents, such as paclitaxel (PTX) [3,5-8]. Since PLGA NPs cannot be delivered to specific cells in a target-specific manner, using cell recognizable targeting ligands, such as monoclonal antibodies, endogenous targeting peptides, and low-molecular-weight compounds, such as folate, onto the surface of the NPs will enhance the intracellular delivery capacity of polymeric NPs to specific cells [9-13]. One possible approach of target-specific delivery could be using antibodies directed toward membrane protein overexpressed by cancer cells.
Earlier we showed that epithelial cell adhesion molecule (EpCAM), a transmembrane protein, is highly expressed in retinoblastoma (RB) primary tumors [14], and recently we demonstrated that EpCAM inhibition leads to decreased RB cell proliferation in vitro [15]. EpCAM is a 40,000 molecular weight, type I, transmembrane glycoprotein that consists of two epidermal growth factor-like extracellular domains, a cysteine-poor region, a transmembrane domain, and a short cytoplasmic tail. EpCAM is overexpressed in various epithelial cancers [16] and is an ideal therapeutic target because of the following reasons: (a) overexpression in cancer cells versus noncancerous cells, (b) apical expression in cancer cells and basolateral expression in normal epithelial cells [17], and (c) not shed into the circulation [18].
In this context, we made use of EpCAM membrane protein for targeted delivery of the chemotherapy drug paclitaxel to retinoblastoma cells that express high EpCAM. We formulated paclitaxel-loaded PLGA NP surfaces functionalized with EpCAM monoclonal antibody and tested their efficacy in the retinoblastoma Y79 cell line in vitro.
Methods
Preparation of PTX-loaded nanoparticles
PTX-loaded PLGA NPs were prepared by the oil-in-water, single emulsion, solvent evaporation method with little modifications. In this method, PTX (equivalent to 10% weight/weight [w/w] dry weight of polymer) was dissolved in 3 ml organic solvent (chloroform) containing 100 mg of polymer (PLGA) to form a primary emulsion. The emulsion was further emulsified in an aqueous poly vinyl alcohol (PVA) solution (12 ml, 2% w/volume [v]) to form an oil-in-water emulsion. The emulsification was performed using a microtip probe sonicator (VC 505; Vibracell Sonics, Newtown, CT) set at 55 W of energy output for 2 min over an ice bath. The emulsion was stirred overnight on a magnetic stir plate at room temperature to evaporate the organic solvent. The excess amount of PVA was removed the next day by ultracentrifugation at 8,500 ×g, 4 °C for 20 min (Kendro/Sorvall Ultraspeed Centrifuge, Artisan Scientific Corporation, Champaign, IL), followed by three washes with double distilled water. The recovered nanoparticulate suspension was lyophilized for 2 days (−80 °C and <10 mm Hg; LYPHLOCK; Labconco, Kansas City, MO) to obtain lyophilized powder for further use.
Particle size analysis and zeta potential measurement
To determine the particle size and zeta potential, 1 mg/ml of NP solution was prepared in double distilled water. The sample (100 μl) was diluted to 1 ml, sonicated in an ice bath for 30 s, and subjected to particle size and zeta potential measurement using a zetasizer (Zetasizer nano-zs ZEN3600; Malvern Instrument, Worcestershire, UK).
Transmission electron microscopic studies
NPs were also evaluated for size by transmission electron microscopy (TEM; Philips/FEI Inc., Barcliff Manor, NY). For this purpose, a sample of NPs (0.5 mg/ml) was suspended in water and sonicated for 30 s. One drop of this suspension was placed over a carbon-coated copper TEM grid (150 mesh; Ted PELLA Inc., Redding, CA) and negatively stained with 1% uranyl acetate for 10 min and then allowed to dry. Images were visualized at 120 kV under a TEM (Philips/FEI Inc., Barcliff Manor, NY).
Scanning electron microscopic studies
The surface morphology of NPs was characterized by scanning electron microscopy (SEM; JEOL JSM-T220A; Tokyo, Japan) operating at an accelerating voltage of 10–30 kV. The NPs were sputtered with gold to make them conductive and placed on a copper stub before the acquisition of SEM images.
Estimation of entrapment efficiency of Paclitaxel loaded nanoparticles
The PTX content in NPs was estimated by the reverse phase isocratic mode of high performance liquid chromatography (HPLC) with slight modification, using an Agilent 1100 HPLC (Agilent Technologies, Boblingen, Germany), which consists of a Zorbax Eclipse XDB-C18, 150×4.6 mm internal diameter, with an internal standard of dimethylphthalate. Briefly, 1 mg of lyophilized PTX-loaded NPs was dissolved in 1 ml of acetonitrile and kept in a shaker at 37 °C and 150 rpm (Wadegati Labequip, Mumbai, India) for 48 h for proper dissolution of the particulate system and better release of the entrapped drug. After 48 h, samples were removed from the shaker and centrifuged at 1,000× g for 10 min at 25 °C (SIGMA 3K30; Life-Sciences, Bremen, Germany) to extract the drug present in the solution. Five hundred microliters of the supernatant was collected, and 20 µl of this supernatant was injected manually in the injection port and was analyzed using a mobile phase of methanol–acetonitrile–water (60:5:35; v/v/v). Separation was achieved by isocratic solvent elution at a flow rate of 1 ml/min with a quaternary pump (Model No-G1311A, Agilent Technologies) at 10 °C with a thermostat (Model No-G1316A, Agilent Technologies). The PTX level was quantified by ultraviolet detection at 228 nm (with Diode Array Det [DAD], Model-G 1315A, Agilent Technologies). The amount of PTX in the NPs was determined from the peak area correlated with the standard curve. The standard curve of PTX was prepared under identical conditions. All analyses were performed in triplicate. Triplicate samples were analyzed, and the PTX encapsulation efficiency was calculated by dividing the amount of PTX entrapped by the total amount of PTX added, multiplied by 100.
In vitro release of paclitaxel from nanoparticles
In vitro release kinetics of PTX from NPs was determined in PBS buffer (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM KH2PO4; pH adjusted to 7.4 containing 0.1% Tween-80) at 37 °C. Ten milligrams of NPs was dispersed in 3 ml of the PBS buffer. The NP suspension was equally divided (1 ml each) in three tubes. These tubes were kept on a shaker at 37 °C and 150 rpm (Wadegati Labequip, Mumbai, India). At particular time intervals these tubes were taken from the shaker and centrifuged at 1,000 ×g, 4 °C for 10 min (Sigma 1–15K microfuge, Shropshire, UK). The supernatants were removed to estimate the amount of drug released at that particular time, using reverse phase (RP)-HPLC. The same amount of fresh PBS was added to the residue, which was placed back on the shaker for further in vitro release studies at different time points.
Conjugation of epithelial cell adhesion molecule antibody on the surface of nanoparticles
For covalent attachment of monoclonal EpCAM antibody onto the NP surface, EDC/NHS chemistry was employed. Briefly, 10 mg of PTX-loaded NPs was dissolved in 5 ml of PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM KH2PO4; pH adjusted to 7.4), followed by drop-wise addition of 250 µl of EDC solution (1 mg/ml) and 250 µl of NHS solution (1 mg/ml) in 0.02 M PBS to the NP suspension. The sample was left at room temperature under agitation for 4 h on a magnetic stirrer. The sample was then ultracentrifuged at 8,500× g at 4 °C for 20 min (Sorvall Ultraspeed Centrifuge; Kendro) to remove unreacted EDC and NHS. The process was repeated three times, and the sediment was washed each time with 1 ml PBS (0.02 M, pH 7.4). Finally, to dissolve the pellet obtained after centrifugation, 2 ml of PBS (0.02 M, pH 7.4) was added. We used EpCAM-fluorescein isothiocyanate (FITC) for conjugation to identify the amount of EpCAM conjugated to NPs. For coupling EpCAM-FITC, the EDC-activated NPs were suspended in 2 ml of PBS (0.02M) and 500 µl of EpCAM-FITC antibody (200 µg/ml in PBS, Santa Cruz Biotechnology, Santa Cruz, CA) was added drop wise and stirred for another 2 h at room temperature; this solution was incubated overnight at 4 °C. The next day unconjugated EpCAM-FITC was removed by ultracentrifugation and the supernatant was collected to calculate the conjugation efficiency. EpCAM-conjugated NP suspension was lyophilized to obtain the powder for further use. The collected supernatant was used to estimate the amount of EpCAM-FITC that was not conjugated to PTX-loaded NPs, using a fluorescence spectrophotometer (Synergy HT; BioTek® Instruments Inc., Winooski, VT), and the pellet was lyophilized for further studies. The above procedure was followed to conjugate EpCAM to NPs (EpCAM was used instead of EpCAM-FITC) for further experiments.
Cell lines and cell culture
The Y79 cell line (endogenously EpCAM-expressing cell line) was obtained from the cell bank, RIKEN BioResource Center (Ibaraki, Japan). Rosewell Park Memorial Institute (RPMI) 1640 media and fetal bovine serum (FBS) were purchased from Gibco-BRL (Rockville, MD). Y79 was cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 0.1% ciprofloxacin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 4.5% dextrose and grown in suspension at 37 °C in a 5% CO2-humidified incubator. The study adhered to the Declaration of Helsinki. This study was conducted at the Medical Research Foundation and Vision Research Foundation, Sankara Nethralaya, India, and was approved by the Vision Research Foundation ethics board.
Y79 cells treatment with native Free Paclitaxel (PTX)/Paclitaxel loaded Nanoparticles (PTX-NPs)/Epithelial cell adhesion molecule conjugated, paclitaxel loaded Nanoparticles (PTX-NP-EpCAM)
Y79 cells (1×105 cells/ml) were seeded on tissue culture treated flat-bottomed cell culture plates (Axygen, Inc. Union city, CA) containing RPMI media and allowed to grow overnight. The next day, 0.5 µg/ml media containing native PTX and an equivalent concentration of freeze-dried PTX-NPs/PTX-NPs-EpCAM was added to the wells and the plates were incubated for 48 h in a CO2 incubator (Hera Cell; Thermo Scientific, Waltham, MA). Void nanoparticle served as the control. After respective incubation periods, the cells were washed twice with PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM KH2PO4; pH adjusted to 7.4) and collected for mitogenic assay, apoptosis assay, cell-cycle analysis, cysteine-aspartic proteases (caspase)-3, and poly (ADP-ribose) polymerase (PARP) activity assay
Mitogenic assay
In a dose–response study, cell viability was determined after 5 days following treatment. A standard (3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based colorimetric assay was used to determine cell viability. Reagents were mixed and added to each well (10 µl/well), plates were incubated for 3 h at 37 °C in a cell culture incubator, and the color intensity was measured at 490 nm using a microplate reader (BioTek, Winooski, VT). The antiproliferative effect of different treatments was calculated as a percentage of cell growth with respect to the respective control.
Assessment of apoptosis by flow cytometry
The induction of apoptosis by PTX/PTX-NPs/PTX-NP-EpCAM was studied by flow cytometry, using the annexin FITC apoptosis detection kit (BD Biosciences, Mississauga, ON, Canada). For apoptosis study the cells were pelleted and then resuspended in 100 µl of 1× binding buffer (Clontech Laboratories, Inc., Palo Alto, CA). Thereafter, 5 µl annexin V-FITC (final concentration, 1 µg/ml; BD Biosciences) and 5 µl propidium iodide (10 µg/µl; MP Biomedicals Inc.) were added to the cells and incubated at room temperature in the dark for 20 min. Before flow cytometric analysis 400 µl of 1× binding buffer was added to the cells and the extent of apoptosis was determined by analyzing 15,000 ungated cells, using a FACScan flow cytometer and Cell Quest software (FACS Calibur; Becton-Dickinson, San Jose, CA). All experiments were performed in triplicate.
Cell-cycle analysis
Cell-cycle analysis was studied by flow cytometry using the BD cycle test plus DNA Reagent kit (BD Biosciences) according to the manufacturer’s protocol. In brief, the treated cells were centrifuged, cells were centrifuged for 5 min at 300× g at room temperature and 1 ml buffer (DMSO [DMSO] in sucrose-sodium citrate) solution was added to the supernatant. Cells were resuspended by gentle vortexing at low speed (procedure repeated twice). Solution A 206 (250 μl) was added to the cells re-suspended in buffer solution, and the solution was mixed by tapping and then incubated for 10 min. Next, 200 μl of solution B was added and mixed by tapping, followed by incubation for 10 min. Then, 200 µl of cold solution C (propidium iodide [PI]) was added to each tube, mixed by tapping, and then incubated for 10 min in the dark on ice. The sample was filtered through a 35-µm cell strainer and analyzed by flow cytometry.
Measurement of intracellular uptake
One day before treatment, Y79 cells were plated in a 24-well format with 1×105 cells/well containing 1 ml of RPMI 1650 medium with 10% FBS. On the day of treatment, Y79 cells were incubated with coumarin-labeled NPs (NPs or NP-EpCAM) for 2 days. Untreated cells were kept as a control. Following incubation, the cells were harvested, washed twice with PBS, centrifuged for 5 min at 500× g, and resuspended in ice-cold PBS. Intracellular uptake was determined by flow cytometry [19].
Microscopic studies
To study the intracellular retention of the drug, cells were treated with either dye, coumarin in solution, or freeze-dried dye-loaded NPs (unconjugated or conjugated with EpCAM antibody; 10 μg/ml media). Untreated cells were used as a control to account for the autofluorescence, if any. The medium was changed on day 2 after treatment and then every alternate day, and no further dose of the dye was added. At different time points, the cells were washed thrice with PBS to remove any uninternalized dye and then visualized using an Axio Observer fluorescent microscope (Carl Zeiss, Berlin, Germany) equipped with an argon laser with an excitation wavelength set at 488 nm and emission at 525 nm [20]. Detection was with a band-pass emission barrier filter. The images were processed using Axio Vision 4.7 software (Carl Zeiss, Bangalore, India).
Caspase-3 and poly (Adenosine Diphosphate-ribose) polymerase activity assay by flow cytometer
For intracellular staining, cells were fixed and permeabilized with 2% paraformaldehyde and 0.05% Tween-20 to allow intracellular labeling with the respective cleaved PARP antibody (ab32064; Abcam, San Francisco, CA), and cleaved caspase-3 antibody (Cell Signaling, Danvers, MA; 1:200 dilution) was added and incubated for 1 h. Following incubation, cells were washed twice with ice-cold PBS and incubated with secondary FITC-antirabbit immunoglobulin (Sigma, 1:1,000 dilution) for 30 min at 4 °C. Cells were then washed twice with ice-cold PBS, resuspended in FACS buffer, and analyzed by FACS.
Statistical analysis
All experiments were repeated at least three times. ANOVA (ANOVA) was used for statistical analysis. The differences were considered significant for p values of <0.05.
Results
Characterization of paclitaxel-loaded nanoparticles conjugated with epithelial cell adhesion molecule antibody
PTX-loaded NPs were prepared by the single emulsion method as described in the methodology. EpCAM was covalently conjugated to the carboxylic group of PLGA NPs by the EDC and NHS activation method (Figure 1). The amount of EpCAM-FITC antibody conjugated to the nanoparticle surface was determined by fluorescence spectrophotometry. Approximately 7.8 µg of EpCAM antibody was found to attach per milligram of the nanoparticle. Dynamic light scattering analysis revealed that the formulated nanoparticle had an average diameter of 272±1.6 nm (Figure 2) with a negative zeta potential of -14.8±2.2 mV (Figure 3). On measuring the size of EpCAM-conjugated NPs, a negligible increase in the size of NPs (272 to 313 nm) was observed after conjugation of EpCAM to the NP surface (Table 1). The zeta potential of NP-EpCAM was comparatively less than unconjugated NPs (-14.8±2.2 mV versus -13.1±2.5 mV; Table 1). TEM images showed a discrete spherical outline and monodispersed size distribution (~100 nm) of PLGA NPs (Figure 4). The topology of the NPs as observed by SEM analysis confirmed the smooth and spherical nature of PTX-NPs (Figure 5). The encapsulation efficiency of the NPs was around 83% (i.e., 83% of the drug added in formulation was entrapped in NPs) as estimated by RP-HPLC. After conjugation the encapsulation efficiency slightly decreased to about 79%.
Figure 1 Schematic presentation of epithelial cell adhesion molecule conjugated to paclitaxel-loaded nanoparticles. The free carboxyl groups of poly(lactic-co-glycolic acid) (PLGA) are covalently conjugated to amine groups of antibody through ethyl(dimethylaminopropyl) carbodiimide (EDC)/N-Hydroxysuccinimide (NHS) chemistry. Abbreviations: COOH represents carboxylic functional group; NH2 represents amine functional group; CO NH represents amide functional group.
Figure 2 This figure shows the size distribution of the paclitaxel loaded poly(lactic-co-glycolic acid) (PLGA). Nanoparticles in terms of the diameter in nanometers (272±1.6) as estimated by photon correlation spectroscopy.
Figure 3 This figure shows the measurement of zeta potential (surface charge) of paclitaxel loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles measured by photon correlation spectroscopy.
Table 1 Physico-chemical characteristics of nanoparticle formulations.
Formulation Sizea Zeta potentialb PDIc Encapsulation efficiencyd
PTX-NPs 272±1.6 −14.8±2.2 0.17 83
PTX-NP-EpCAM 313±3.3 −13.1±2.5 0.15 79
aSize in nm as measured by photon correlation spectroscopy. bZetapotential in mV measured by zetasizer. cPolydispersity index measured by photon correlation spectroscopy. dPercentage of encapsulation efficiency of NPs measured by RP-HPLC.
Figure 4 Characterization of paclitaxel loaded nanoparticles by transmission electron microscopy (TEM). TEM was used to analyze the size and size distribution of the paclitaxel loaded nanoparticles.
Figure 5 Characterization of paclitaxel loaded nanoparticles by scanning electron microscopy (SEM). SEM was used for imaging and to study the surface morphology of the paclitaxel loaded nanoparticles.
In vitro release of paclitaxel by nanoparticles
In the in vitro release study, NPs demonstrated a sustained release of the encapsulated drug, with approximately 38% cumulative drug release in 21 days (Figure 6). The NPs gave a comparatively faster release (~22% in 1 day) followed by a slow release (~38% in 21 days) of the drug from the polymeric matrix.
Figure 6 The line graph shows the cumulative percentage release of paclitaxel drug into the phosphate buffer solution (PBS) buffer from the paclitaxel loaded nanoparticles over a period of time (in days). Error bars represents that experiments were performed in triplicates.
Cytotoxicity assay
The ability of the PTX-NPs (conjugated or unconjugated) to deliver PTX and induce cell death was examined on the retinoblastoma cell line (Y79) by the MTT assay. Results demonstrated that cell viability of the Y79 cell line was affected by different concentrations of PTX as well as PTX-NPs/PTX-NP-EpCAM. Greater antiproliferative activity was observed for almost all doses of PTX-NP-EpCAM compared to that of the native PTX and PTX-NP (Figure 7A). PTX-NP-EpCAM-treated Y79 cells exhibited an inhibitory concentration 50 (IC50) value as low as 0.005 µg/ml, while native PTX and PTX-NP had an IC50 value as high as 0.05 and 3.5 µg/ml, respectively. Another important observation was the increase in the antiproliferative activity of the drug with incubation time (day 5) when cells were treated with low doses of the drug using PTX-NP-EpCAM (PTX dose=0.005 µg/ml; Figure 7B). Differences between the native PTX and PTX-NPs (conjugated or unconjugated) were significantly evident after 5 days of drug treatment as they showed significantly greater antiproliferative effect (42% inhibition by PTX-NP-EpCAM versus 71% and 60% inhibition by native PTX and PTX-NP, respectively). The Y79 cells were treated with native PTX and PTX-NP (conjugated and unconjugated) for 2 days in vitro. The cells were harvested on day 2 for an apoptotic assay using FACS. Following treatment with native PTX, Y79 cells showed 2.91% apoptosis (1.68% of early apoptosis, 1.23% of late apoptosis), and with PTX-NP, Y79 cells showed 8.06% of apoptosis (6.93% early apoptosis and 1.13% late apoptosis). In contrast, Y79 cells treated with PTX-NP-EpCAM showed increased late apoptosis (17.16% early apoptosis; 2.24% late apoptosis; Figure 8).
Figure 7 Dose and time-dependent cytotoxicity of paclitaxel (PTX) and PTX loaded nanoparticles (NPs) in Y79 cells. A and B: Different concentrations of PTX either as solution or PTX encapsulated in NPs or Epithelial cell adhesion molecule antibody-conjugated PTX-NPs were added to the wells with medium. The extent of growth inhibition was measured at 48 h and at day 5 by the (3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Inhibition was calculated with respect to respective controls. Experiments were performed in triplicates and the data are represented as mean±standard error (*p<0.001).
Figure 8 Apoptotic effects of paclitaxel loaded nano-conjugates on Y79 cells. Flow cytometry analysis showing the effect of control (A), native paclitaxel (PTX; B), PTX-loaded nanoparticles (PTX-NP; C), and PTX-NP conjugated with epithelial cell adhesion molecule antibody (PTX-NP-EpCAM; D) treatment on Y79 cells after 48 h incubation. The Y79 cells treated with PTX-NP-EpCAM showed significantly higher (p<0.001) early and intermediary apoptotic events compared to PTX-NP or native PTX. Abbreviation: PI represents propidium iodide.
Increased caspase-3 activation and Poly (adenosine diphosphate-ribose) polymerase cleavage in Y79 cells treated with epithelial cell adhesion molecule antibody conjugated paclitaxel loaded nanoparticles
To determine whether the apoptosis of Y79 cells induced by PTX is a specific caspase-dependent pathway, we analyzed the effect of native PTX, PTX-NPs, and PTX-NP-EpCAM on caspase-dependent apoptosis. Caspase activation followed by cleavage of PARP was enhanced in Y79 cells treated with PTX-NP-EpCAM compared to PTX-NP and native PTX. Active caspase-3 was positive in 23.76% cells, and 16.93% cells were positive for cleaved PARP after treatment with PTX-NP-EpCAM; 13.22% cells were positive for active caspase-3 and 7.11% cells for cleaved PARP with PTX-NPs; 9.28% of cells were positive for active caspase-3 and 4.66% cells for cleaved PARP with native PTX (Figure 9 and Figure 10).
Figure 9 Analysis of caspase-3 expression in paclitaxel loaded nano-conjugates. Flow cytometry analysis showing caspase-3 expression in Y79 cells after the treatment with control (A); native paclitaxel (PTX; B); PTX-loaded nanoparticles (PTX-NP; C), and PTX-NP conjugated with epithelial cell adhesion molecule antibody (PTX-NP-EpCAM; D). Increased caspase expression was noted in Y79 cells treated with PTX-NP-EpCAM compared to PTX-NP or native PTX.
Figure 10 Analysis of Poly (adenosine diphosphate-ribose) polymerase expression in paclitaxel loaded nano-conjugates. Flow cytometry analysis showing PARP activation in Y79 cells after the treatment with control (A); native paclitaxel (PTX; B); PTX-loaded nanoparticles (PTX-NP; C), and PTX-NP conjugated with epithelial cell adhesion molecule antibody (PTX-NP-EpCAM; D). Increased PARP activation was noted in Y79 cells treated with PTX-NP-EpCAM compared to PTX-NP or native PTX.
Enhanced G2-M phase halt in Y79 cells treated with epithelial cell adhesion molecule antibody conjugated paclitaxel loaded nanoparticles
We quantified the percentage of cells at various phases of the cell cycle and plotted them against the native PTX/PTX-NP/PTX-NP-EpCAM (Figure 11). In untreated Y79, 3.84% cells were at the G2-M phase. When exposed to native PTX and PTX-NP, the arrested population increased about 12.2% and 17.8%, and the increase continued progressively to 21.7% with PTX-NP-EpCAM.
Figure 11 Cell cycle analysis of Y79 cells treated with paclitaxel loaded nano-conjugates. Increased G2-M arrest was observed by flow cytometry analysis in Y79 cells treated with paclitaxel-loaded nanoparticles conjugated with epithelial cell adhesion molecule antibody (PTX-NP-EpCAM) at 48 h compared to paclitaxel-loaded nanoparticles (PTX-NP) or native paclitaxel (PTX). A: Untreated Y79 cells showing 3.8% of G2-M phase after doublet discrimination. B: Y79 cells treated with native paclitaxel showing increased G2-M cells (12.2%). C: Y79 cells treated with PTX-NP showing 17.8% of G2-M phase. D: Y79 cells treated with PTX-NP-EpCAM showing 21.7% G2-M phase.
Increased uptake of coumarin-6 labeled epithelial cell adhesion molecule antibody conjugated nanoparticles by Y79 cells
To explain the increased cytotoxicity of the PTX-NP-EpCAM formulation, the intracellular uptake of NP-EpCAM was quantitatively examined in an EpCAM-overexpressing Y79 cell line (Figure 12). In the case of NP-EpCAM, increased fluorescence intensity profiles to the right direction indicates that the cellular uptake was significantly enhanced due to EpCAM-mediated endocytosis.
Figure 12 Flow cytometry analysis of uptake of nanoparticles by Y79 cells Flow cytometry analysis showed significantly higher uptake of coumarin encapsulated nanoparticles conjugated with epithelial cell adhesion molecule antibody (COU-NP-EpCAM) by Y79 cells compared to that of unconjugated coumarin encapsulated nanoparticles.” EpCAM negative cell line HeLa cells were used as negative control which showed relatively less uptake (mean intensity -1144.44) compared to EpCAM positive Y79 cells (mean intensity -2090.80).
The extent of cellular uptake for NP-EpCAM was about twofold greater than that of EpCAM antibody unconjugated NPs and tenfold greater than the free coumarin-6 dye, (Figure 12). The fluorescence intensity was twofold less in the HeLa cell line, which does not express EpCAM compared to uptake in Y79 cells. This clearly shows that EpCAM on Y79 cells mediates the specific uptake of NP-EpCAM.
Prolonged retention of NP-EpCAM in Y79 cells
To further confirm the enhanced intracellular uptake of NP-EpCAM, fluorescence microscopic analysis was performed. Y79 cells incubated with free coumarin in solution demonstrated drug internalization within 4 h of incubation, but with increase in incubation time, the fluorescent intensity decreased slowly. EpCAM-conjugated coumarin-loaded NPs on the other hand were more strongly stained with clear visualization of the internalized TXNPs, and in addition they showed a significant increase in fluorescence intensity with incubation time, with strong fluorescence even after 5 days of treatment as compared to the unconjugated coumarin-loaded NPs and free coumarin (Figure 13). In this study we envisioned that the uptake of EpCAM-conjugated NPs via EpCAM-mediated endocytosis could have an intracellular disposition pathway different from that of unconjugated NPs. This could influence the intracellular retention of NPs and hence the therapeutic efficacy of the encapsulated drug in retinoblastoma. Our study shows that the EpCAM-conjugated PLGA NPs could be potentially applied to target-specific intracellular delivery of various hydrophobic anticancer agents.
Figure 13 Microscopic analysis of uptake of nanoparticles by Y79 cells. Fluorescent microscopic analysis showing the uptake of native coumarin (A-D), coumarin encapsulated nanoparticles (COU-NP; E-H), and coumarin encapsulated nanoparticles conjugated with epithelial cell adhesion molecule antibody (COU-NP-EpCAM; K-N) at 4 h (A, E, K), day 1 (B, F, L), day 2 (C, G, M), and day 5 (D, H, N). The Y79 cells showed increased COU-NP-EpCAM uptake compared to unconjugated COU-NP at all time points. Increased coumarin-NP retention was observed in the Y79 cells even at day 5 when compared to free coumarin.
Discussion
Functionalization of NPs with tumor-targeting ligands, such as antibodies or peptides, directed against overexpressed tumor markers not only enhances localization of the particles to the solid tumor mass but also allows the NPs to target early stage tumors and metastatic tumor cells. Treatment with monoclonal antibody (mAbs) is a viable therapeutic option in cancer. Recently, these mAbs, such as cetuximab and herceptin, have been used as targeting agents to selectively deliver chemotherapeutics to cancerous cells [21]. For example, Reddy et al. has used folic acid-coated polymeric NPs and demonstrated enhanced localization and internalization of NPs for drug delivery to breast cancer cells [22], whereas Zhang et al. has shown that folic acid-coated magnetite NPs demonstrated improved localization and internalization intended for tumor imaging of breast cancer cells [23]. Similarly, tagging anti-HER2 to the nanoparticle surface greatly improved cell internalization of gelatin/albumin [24]
Our objective in the present study was to examine the target-specific intracellular delivery capacity of PLGA NPs by coating EpCAM antibody on their surface. Although the prepared PLGA NPs with a nanoscale size distribution accumulated at the solid tumor site in a passive targeting manner by an “enhanced permeation and retention” effect [22], cell-specific targeting ability by EpCAM antibody conjugation is responsible for promoting their intracellular uptake within EpCAM-expressing cancer cells. PTX-loaded NPs were prepared by the single emulsion method, and EpCAM moieties were covalently conjugated to the carboxylic group of PLGA NPs by the EDC and NHS activation method for cell recognition. The enhancement of cellular uptake was gained via an EpCAM receptor-mediated intracellular delivery mechanism.
Fluorescent microscopy and flow cytometry analysis revealed increased uptake of NP-EpCAM by Y79 cells compared to unconjugated NPs or native coumarin-6 dye. Interestingly, PTX-NP-EpCAM showed an increase in the antiproliferative activity of the drug with incubation time when cells were treated with low doses of the drug using PTX-NP-EpCAM (PTX dose=0.005 µg/ml). PTX-NP-EpCAM showed higher apoptotic events in Y79 cells as evidenced by caspase-3 activation. The induction of apoptosis is considered to be one of the principle mechanisms by which PTX induces tumor regression in retinoblastoma [23,24]. Suarez et al. [23] has shown that paclitaxel administered through the subconjunctival route effectively inhibited the retinoblastoma tumor burden in the LH beta-Tag animal models. However, higher doses of native drug administration are associated with ocular tissue toxicities [23]. For improved sustain drug release, PLGA NPs were used to deliver drugs in diabetic rat models through subconjunctival administration [25]. This approach reduces the ocular tissue toxicity associated with native drugs and enhances sustained drug release for effective therapeutic response. Our present study has further modified the paclitaxel-containing PLGA NPs with EpCAM antibody for targeted and sustained drug release to RB cells.
One of the key initiation elements of the apoptotic pathway is the activation of caspases followed by cleavage of the caspase substrates [26]. The 113-kDa PARP-1, which is normally involved in DNA repair, DNA stability, and other cellular events, is cleaved by members of the caspase family during early apoptosis. PTX-NP-EpCAM showed increased G2-M phase arrest in Y79 cells compared to PTX-NP or native PTX. Previous studies have shown that PTX arrests cells at the G2-M phase of the cell cycle [27,28] and that defects of spindle assembly or the presence of detached chromosomes activates an internal signaling pathway that probably initiates the induction of PTX-induced apoptosis [29].
To conclude, this study demonstrates the proof of principle of using EpCAM antibody to specifically deliver the chemotherapy drugs to retinoblastoma cells. Further in vivo studies are warranted to use this formulation in clinical settings for retinoblastoma and other EpCAM-expressing cancer management.
Acknowledgments
Supported by DBT Grant: BT/PR7968/MED/14/1206/2006. Core laboratory facility staff, Vision Research Foundation, Sankara Nethralaya for the technical help in flow cytometry experiments.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22096605PONE-D-11-1051310.1371/journal.pone.0027630Research ArticleBiologyAnatomy and PhysiologyImmune PhysiologyCytokinesDevelopmental BiologyMolecular DevelopmentCytokinesImmunologyImmune SystemCytokinesImmunityInflammationMicrobiologyImmunityInflammationMolecular cell biologySignal transductionSignaling cascadesAkt signaling cascadeERK signaling cascadePKA signaling cascadeMedicineAnatomy and PhysiologyImmune PhysiologyCytokinesClinical ImmunologyAutoimmune DiseasesRheumatoid ArthritisImmune SystemCytokinesImmunityInflammationRheumatologyRheumatoid ArthritisInterleukin-6 Synthesis in Human Chondrocytes Is Regulated via the Antagonistic Actions of Prostaglandin (PG)E2 and 15-deoxy-Δ12,14-PGJ2
Effects of PGE2 and 15d-PGJ2 on IL-6 SynthesisWang Pu
1
Zhu Fei
1
Konstantopoulos Konstantinos
1
2
3
*
1
Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland, United States of America
2
Johns Hopkins Physical Sciences in Oncology Center and Institute for NanoBioTechnology, The Johns Hopkins University, Baltimore, Maryland, United States of America
3
Institute for NanoBioTechnology, The Johns Hopkins University, Baltimore, Maryland, United States of America
Zheng Song Guo EditorUniversity of Southern California, United States of America* E-mail: [email protected] and designed the experiments: PW KK. Performed the experiments: PW FZ. Analyzed the data: PW FZ KK. Wrote the paper: PW KK.
2011 11 11 2011 6 11 e2763014 6 2011 20 10 2011 Wang et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
Elevated levels of interleukin-6 (IL-6), prostaglandin (PG)E2, PGD2 and its dehydration end product 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) have been detected in joint synovial fluids from patients with rheumatoid arthritis (RA). PGE2 directly stimulates IL-6 production in human articular chondrocytes. However, the effects of PGD2 and 15d-PGJ2 in the absence or presence of PGE2 on IL-6 synthesis in human chondrocytes have yet to be determined. It is believed that dysregulated overproduction of IL-6 is responsible for the systemic inflammatory manifestations and abnormal laboratory findings in RA patients.
Methodology/Principal Findings
Using the T/C-28a2 chondrocyte cell line as a model system, we report that exogenous PGE2 and PGD2/15d-PGJ2 exert antagonistic effects on IL-6 synthesis in human T/C-28a2 chondrocytes. Using a synthesis of sophisticated molecular biology techniques, we determined that PGE2 stimulates Toll-like receptor 4 (TLR4) synthesis, which is in turn responsible for the activation of the ERK1/2, PI3K/Akt and PKA/CREB pathways that phosphorylate the NF-κB p65 subunit leading to NF-κB activation. Binding of the activated NF-κB p65 subunit to IL-6 promoter induces IL-6 synthesis in human T/C28a2 chondrocytes. PGD2 or 15d-PGJ2 concurrently downregulates TLR4 and upregulates caveolin-1, which in turn inhibit the PGE2-dependent ERK1/2, PI3-K and PKA activation, and ultimately with NF-κB-dependent IL-6 synthesis in chondrocytes.
Conclusions/Significance
We have delineated the signaling cascade by which PGE2 and PGD2/15d-PGJ2 exert opposing effects on IL-6 synthesis in human chondrocytes. Elucidation of the molecular pathway of IL-6 synthesis and secretion by chondrocytes will provide insights for developing strategies to reduce inflammation and pain in RA patients.
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Introduction
Rheumatoid arthritis (RA) is characterized by systemic and local inflammation, which results in cartilage and bone destruction. Nonsteroidal anti-inflammatory drugs (NSAIDs), which are used to treat RA, elicit their effects by inhibiting cyclooxygenase (COX) activity [1]. COX is known to exist in two isoforms: COX-1 and COX-2. Despite their similar active site structures, products and kinetics, only COX-2 is inducible and is primarily responsible for the elevated production of prostanoids in chondrocytes [2]. COX-2 catalyzes the rate-limiting step of prostaglandin (PG) synthesis. PGE2 and PGD2 are the major PGs synthesized by chondrocytes. PGD2 readily undergoes dehydration to yield the bioactive cyclopentenone-type PGs of the J2-series such as 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2).
Accumulating evidence suggests that the effects of PGE2 on chondrocyte function and cartilage tissue vary according to concentration levels. At the nano- to micro-molar concentrations produced by arthritic tissues [3], [4], PGE2 has been associated with catabolic effects because it suppresses the production of proteoglycans and stimulates the degradation of extracellular matrix [5], [6], [7]. In contrast, low (picomolar) concentrations of PGE2 exert anabolic effects [6], as evidenced by stimulation of proteoglycan (aggrecan) synthesis [8]. Elevated levels of PGE2 have been detected in the cartilage and synovial fluid from patients with RA [9]. It is believed that PGE2 plays a critical role in the generation and maintenance of edema and erosion of cartilage and juxtaarticular bone [1], [10].
Elevated levels of 15d-PGJ2 have also been detected in joint synovial fluids obtained from RA patients [11]. However, the role of 15d-PGJ2 in RA is still a matter of debate. 15d-PGJ2 has been reported to induce chondrocyte apoptosis in a dose- and time-dependent manner through a peroxisome proliferator-activated receptor-γ (PPAR-γ)-dependent pathway [11]. Although 15d-PGJ2 has also been shown to have a pro-apoptotic effect on other cell types, such as endothelial cells [12], tumor cells [13] and neurons [14], separate lines of evidence suggest that it may have chondroprotective effects. For instance, 15d-PGJ2 and PGD2 counteract the induction of matrix metalloproteinases in cytokine-activated chondrocytes [15], [16], which have a key role in cartilage degradation. 15d-PGJ2 have also been reported to block apoptosis of human primary chondrocytes induced by the NF-κB inhibitor Bay 11-7085 [12]. Taken together, the contributions of PGD2 and its metabolite 15d-PGJ2 to chondrocyte function remain controversial.
In addition to PGE2 and 15d-PGJ2, elevated levels of IL-6 have been detected in synovial fluid from patients with RA [17]. A positive association between PGE2 and IL-6 production has been suggested in many different cells, including astrocytes [18], macrophages [1], synovial [19] and gingival [20] fibroblasts, osteoblasts [21], and chondrocytes [22]. Moreover, we have recently reported that PGE2 induces IL-6 expression in human chondrocytes via cAMP/protein kinase A (PKA)- and phosphatidylinositol 3 kinase (PI3-K)-dependent pathways [23]. It is believed that dysregulated overproduction of PGE2 is responsible for inducing IL-6 synthesis in RA patients. In an animal model of adjuvant-induced arthritis, the administration of a neutralizing antibody against PGE2 to arthritic rats inhibited the edema, hyperalgesia and IL-6 production at sites of inflammation [23]. In contrast, the role of PGD2 and 15d-PGJ2 in IL-6 regulation is still a matter of debate. Although Thieringer, et al.
[24] support the notion that 15d-PGJ2 enhances IL-6 expression in LPS-treated human peripheral blood monocytes, most previous studies showed a negative relationship between 15d-PGJ2 and IL-6 production in different cell lines, such as intestinal epithelial cells [25] and rat pancreatic acinar AR42J cells [26]. However, the potential effects of 15d-PGJ2 in the presence and absence of PGE2 on IL-6 regulation in human chondrocytes have yet to be delineated.
Using the T/C-28a2 chondrocyte cell line as a model system, we herein report that PGE2 and 15d-PGJ2 exert antagonistic effects on IL-6 synthesis. Moreover, we delineate the signaling pathway of IL-6 regulation in human chondrocytes primed with exogenous PGE2 and/or 15d-PGJ2.
Results
PGE2 and PGD2/15d-PGJ2 exert antagonistic effects on IL-6 synthesis in human T/C-28a2 chondrocytes
Elevated levels of PGE2
[9], PGD2 (and its dehydration end product 15d-PGJ2) [27] and IL-6 [17] have been detected in joint synovial fluids obtained from RA patients. We and others have shown that PGE2 directly stimulates IL-6 production in human articular chondrocytes [22], [23], [28]. Prior work has shown that 15d-PGJ2 can positively or negatively regulate IL-6 synthesis in different cell types. However, the effects of PGD2 and 15d-PGJ2 on IL-6 synthesis in human chondrocytes have yet to be determined. It is believed that dysregulated overproduction of IL-6 is responsible for the systemic inflammatory manifestions and abnormal laboratory findings in RA patients. The human T/C-28a2 chondrocyte cell line was chosen as a model system, since T/C-28a2 cells have been shown to behave much like primary human chondrocytes when cultured under appropriate conditions [23], [29]. Our data reveal that prolonged (48 h) treatment of human T/C-28a2 cells with exogenous PGD2 (Fig. 1A) or 15d-PGJ2 (Fig. 1B) suppresses IL-6 mRNA and protein synthesis in a dose-dependent manner. Moreover, marked downregulation of IL-6 expression (data not shown) and spontaneous secretion (Fig. S1A) is detected after 24 h to 48 h treatment of T/C-28a2 cells with exogenously added 15d-PGJ2 (1 µM). 15d-PGJ2 (1 µM) also repressed IL-6 secretion in human primary articular chondrocytes (Fig. S1B). In marked contrast, and in agreement with previously published data [23], PGE2 rapidly induces IL-6 mRNA and protein synthesis in a dose-dependent fashion (Fig. 1C). Taken together, these data illustrate that PGE2 and 15d-PGJ2 exert opposing effects on IL-6 expression in human chondrocytes.
10.1371/journal.pone.0027630.g001Figure 1 Dose-dependent regulation of TLR4, caveolin-1 and IL-6 synthesis by PGE2 or PGD2 or 15d-PGJ2 in human chondrocytes.
T/C-28a2 chondrocytes were treated with either PGD2 (A) or 15d-PGJ2 (B) for 48 h, or PGE2 (C) for 2 h. TLR4, caveolin-1 and IL-6 protein (upper) and mRNA (lower) expression was determined by Western blotting or qRT-PCR, respectively. ß-actin and GAPDH served as internal controls in immunoblotting and qRT-PCR, respectively. The Western blots are representative of three independent experiments, all revealing similar results. Data represent the mean ± S.E. of 3 independent qRT-PCR experiments. * and ▴, p<0.05 with respect to the corresponding vehicle control.
PGE2 and 15d-PGJ2 differentially regulate TLR4 and caveolin-1 expression, which in turn modulate the IL-6 synthesis in human chondrocytes
Prior work showed that caveolin-1 diminishes the lipopolysaccharide (LPS)-mediated nuclear translocation of NF-kB p65 and IL-6 production in murine macrophage RAW264.7 cells via binding and inactivating TLR4 [30]. We therefore investigated the effects of exogenous PGE2 and 15d-PGJ2 on TLR4 and caveolin-1 expression as well as their roles in IL-6 synthesis in human T/C-28a2 chondrocytes. Our data reveal that PGE2 induces TLR4 synthesis in a dose-dependent manner without affecting caveolin-1 expression (Fig. 1C). In contrast, PGD2 or 15d-PGJ2 concurrently downregulates TLR4 and upregulates caveolin-1 mRNA and protein synthesis in a dose-dependent fashion (Figs. 1A, B). Interestingly, pre-treatment of T/C-28a2 cells with 15d-PGJ2 (1 µM) or PGD2 (8 µM) for 48 h abolishes the PGE2-dependent IL-6 and TLR4 synthesis at both transcriptional and translation levels (Figs. 2A, B). PGE2 does not alter the PGD2/15d-PGJ2-dependent upregulation of caveolin-1 (Fig. 2A).
10.1371/journal.pone.0027630.g002Figure 2 The antagonistic effects of PGE2 and PGD2/15d-PGJ2 on TLR4, caveolin-1 and IL-6 synthesis in T/C-28a2 chondrocytes.
T/C-28a2 cells were incubated with either PGE2 (10 µM) for 2 h, or PGD2 (8 µM) or 15d-PGJ2 (1 µM) for 48 h. In other experiments, T/C-28a2 cells were pre-treated with PGD2 (8 µM) or 15d-PGJ2 (1 µM) for 48 h before incubation with PGE2 (10 µM) for 2 h. In select experiments, T/C-28a2 cells were transfected with a siRNA oligonucleotide sequence specific for TLR4 (B, C) or caveolin-1 (D) or a plasmid containing the cDNA of TLR4 (D) or caveolin-1 (B, C) before PG treatment. TLR4 (A, C, D), caveolin-1 (A, C, D) and IL-6 (A, C, D) mRNA synthesis was determined by qRT-PCR. GAPDH served as internal control. Data represent the mean ± S.E. of at least 3 independent experiments. *, p<0.05 with respect to vehicle or mock transfected control. ▴, p<0.05 with respect to significantly regulated (*) groups. IL-6 (B) is shown by immunoblotting using specific Abs. Equal loading in each lane is ensured by the similar intensities of ß-actin. These western blots are representative of three independent experiments, all revealing similar results.
In view of our data showing that PGE2 upregulates TLR4 while leaving intact caveolin-1 expression, experiments were performed using T/C-28a2 cells transfected with either a siRNA oligonucleotide sequence specific for TLR4 or a plasmid containing the cDNA of caveolin-1. The efficacy of these genetic interventions is demonstrated at both the mRNA and protein levels (Fig. 2). Selective knockdown of TLR4 significantly inhibits IL-6 upregulation without altering caveolin-1 mRNA levels in PGE2-primed T/C-28a2 cells (Fig. 2C). Ectopic expression of caveolin-1 is sufficient to suppress the levels of IL-6 mRNA expression in control and PGE2-activated T/C-28a2 cells without impairing TLR4 synthesis (Fig. 2C).
In light of the effects of PGD2 or 15d-PGJ2 on TLR4 and caveolin-1 expression, experiments were carried out using cells transfected with a plasmid containing the cDNA of TLR4 or an siRNA oligonucleotide specific for caveolin-1. As shown in Fig. 2D, ectopic expression of TLR4 markedly increases IL-6 expression compared with untreated control T/C-28a2 chondrocytes in the absence of caveolin-1 regulation. Furthermore, TLR4 overexpression reverses the PGD2- or 15d-PGJ2-mediated IL-6 downregulation (Fig. 2D and Fig. S2). Similarly, caveolin-1 depletion increases IL-6 synthesis in both untreated control and PGD2 or 15d-PGJ2-treated T/C-28a2 cells (Fig. 2D and Fig. S2). Taken together, these data illustrate that PGE2 and 15d-PGJ2 differentially regulate TLR4 and caveolin-1 expression, which in turn modulate IL-6 expression in human chondrocytes.
In view of the key role of cAMP production in PGE2-mediated IL-6 synthesis [23], we examined the potential contribution of TLR4 and caveolin-1 to the regulation of cAMP accumulation in human T/C-28a2 chondrocytes. Our data reveal that selective TLR4 knockdown attenuates the intracellular cAMP levels in untreated control T/C-28a2 cells, as determined by the use of a cAMP enzyme immunoassay kit (Fig. 3A). In contrast, ectopic expression of TLR4 significantly augments cAMP levels (Fig. 3A). However, caveolin-1 depletion or overexpression does not impair the intracellular levels of cAMP (Fig. 3B). Cumulatively, these data suggest that TLR4, but not caveolin-1, regulates cAMP production in human chondrocytes.
10.1371/journal.pone.0027630.g003Figure 3 TLR4, but not caveolin-1, induces intracellular cAMP production and ERK1/2 inhibitors, PD98059 and U0126 inhibit IL-6 expression in human chondrocytes.
T/C-28a2 cells were transfected with an siRNA oligonucleotide sequence specific for TLR4 (A) or caveolin-1 (B). In select experiments, T/C-28a2 cells were transfected with a plasmid containing the cDNA of TLR4 (A) or caveolin-1 (B). In separate experiments, cells were incubated with PGE2 (10 µM) for 2 h in the presence or absence of the MEK1/2 inhibitors, PD98059 (20 µM) or U0126 (10 µM) (C). cAMP accumulation (A, B) was determined using a cAMP enzyme immunoassay kit. IL-6 mRNA synthesis (C right panel) was determined by qRT-PCR. GAPDH served as internal control. Data represent the mean ± S.E. of at least 3 independent experiments. *, p<0.05 with respect to scramble siRNA or empty vector transfected control. ▴, p<0.05 with respect to significantly regulated (*) groups. IL-6 and phosphorylated ERK1/2 (Thr202/Tyr204) (C left panel) are shown by immunoblotting using specific Abs. Equal loading in each lane is ensured by the similar intensities of total ERK1/2 and ß-actin. These western blots are representative of three independent experiments, all revealing similar results.
TLR4 and caveolin-1 differentially regulate PI3-K, PKA and ERK1/2 signaling pathways
We have recently reported that PGE2 induces IL-6 expression in chondrocytes via a cAMP/PKA- and PI3-K-dependent pathway [23]. Moreover, prior work has implicated ERK1/2 as a downstream target of PGE2 in myocytes [31]. The functional role of ERK1/2 in PGE2-mediated IL-6 synthesis in chondrocytes was documented via the use of the MEK1/2 inhibitors PD98059 and U0126, which both significantly inhibited IL-6 expression at both the transcriptional and translational levels (Fig. 3C). Therefore, we sought to determine how prostaglandins and its downstream effectors TLR4 and caveolin-1 regulate the activity of PI3-K, PKA and ERK1/2. In agreement with our previous work [23], exogenous PGE2 stimulates PI3-K and PKA activity at early time points (2 h), as evidenced by increased phosphorylation of Akt at Ser 473 and CREB at Ser-133, respectively, and returns to basal levels after prolonged (48 h) stimulation (Fig. 4A). The same temporal pattern is detected for the phosphorylation levels of ERK1/2 (Thr 202 and Tyr204) in PGE2-primed T/C-28a2 cells (Fig. 4A). Of note, the total ERK1/2, Akt and CREB levels are not impaired by PGE2 stimulation (Fig. 4A). Selective knockdown of TLR4 or ectopic expression of caveolin-1 suppresses the phosphorylation levels of Akt, CREB and ERK1/2 in PGE2-primed T/C-28a2 cells down to baseline controls (Fig. 4B).
10.1371/journal.pone.0027630.g004Figure 4 Exogenous PGE2 and 15d-PGJ2 differentially regulate TLR4 and caveolin-1, which are in turn responsible for the phosphorylation of ERK1/2, Akt and CREB in T/C-28a2 chondrocytes.
T/C-28a2 cells were incubated with either PGE2 (10 µM) for 2 h (A) or 15d-PGJ2 (1 µM) for 48 h (C, D). In other experiments, cells were pre-treated with 15d-PGJ2 (1 µM) for 48 h before incubation with PGE2 (10 µM) for 2 h (B). In select experiments, cells were transfected with an siRNA oligonucleotide sequence specific for TLR4 (B) or caveolin-1 (D) or a plasmid containing the cDNA of caveolin-1 (B) or TLR4 (D) before treatment with PGE2 or 15d-PGJ2. Phosphorylated ERK1/2 (Thr202/Tyr204), Akt (Ser 473) and CREB (Ser 133) are shown by immunoblotting using specific Abs. Equal loading in each lane is ensured by the similar intensities of total ERK1/2, Akt, CREB and ß-actin. TLR4 and caveolin-1 protein levels were also probed with an anti-TLR4 and an anti-caveolin-1 antibody, respectively. These western blots are representative of three independent experiments, all revealing similar results.
15d-PGJ2 attenuates the levels of Akt, CREB and ERK1/2 phosphorylation below those of untreated controls in human T/C-28a2 chondrocytes (Fig. 4C). Maximal downregulation is detected after 48 h of 15d-PGJ2 stimulation (Fig. 4C). Similarly, 15d-PGJ2 mitigates the enhanced phosphorylation levels of Akt, CREB and ERK1/2 in caveolin-1-knockdown or TLR4 overexpressing chondrocytes (Fig. 4D).
PGE2 and 15d-PGJ2 differentially regulate NF-κB activation in human chondrocytes
NF-κB was identified as the key transcriptional factor responsible for IL-6 synthesis in PGE2-primed chondrocytes [23]. Thus, we sought to determine the effects of 15d-PGJ2 on NF-κB activation induced by exogenous PGE2. As shown in Fig. 5A, 15d-PGJ2 blocked the PGE2-dependent transactivation of NF-κB p65 subunit, as evidenced by the inhibition of phosphorylation at Ser-276 and Ser-536. Moreover, exogenous 15d-PGJ2 exerted a pronounced inhibitory effect on IL-6 promoter activity (Fig. 6A) and reduced the levels of the NF-κB gel shift (Fig. 7A) and supershift (Fig. 7B) detected after T/C-28a2 chondrocyte stimulation with PGE2.
10.1371/journal.pone.0027630.g005Figure 5 Exogenous PGE2 and 15d-PGJ2 differentially regulate the phosphorylation of the NF-κB p65 subunit in T/C-28a2 chondrocytes.
T/C-28a2 cells were transfected with an siRNA oligonucleotide sequence specific for TLR4 (A) or caveolin-1 (B) or a plasmid containing the cDNA of caveolin-1 (A) or TLR4 (B) before treatment with PGE2 (10 µM) for 2 h (A) or 15d-PGJ2 (1 µM) for 48 h (B). In select experiments, cells were pretreated with 15d-PGJ2 (1 µM) for 48 h before incubation with PGE2 (10 µM) for 2 h (A). Phosphorylated p65 (Ser536 and Ser276) is detected by immunoblotting using specific Abs. Equal loading in each lane is ensured by the similar intensities of total p65 and ß-actin. These Western blots are representative of three independent experiments, all revealing similar results.
10.1371/journal.pone.0027630.g006Figure 6 Regulation of the IL-6 promoter activity in human T/C-28a2 chondrocytes by exogenous PGE2 or 15d-PGJ2.
T/C-28a2 cells were pre-treated with 15d-PGJ2 (1 µM) for 48 h before incubation with PGE2 (10 µM) for 2 h (A). In other experiments, cells were incubated with either PGE2 (10 µM) for 2 h (B, C, F) or 15d-PGJ2 (1 µM) for 48 h (D, E). In select experiments, T/C-28a2 cells were transfected with a plasmid containing an siRNA oligonucleotide sequence specific for TLR4 (B) or caveolin-1 (E) or the cDNA of caveolin-1 (C) or TLR4 (D) before PG treatment. In separate experiments, cells were incubated with PGE2 for 2 h in the presence or absence of the MEK1/2 inhibitors, PD98059 (20 µM) or U0126 (10 µM) (F). Cells were transfected with the indicated siRNAs or cDNA constructs along with the IL-6 promoter reporter construct pIL-6-luc651 or pIL-6-luc651 ΔNF-κB before PG stimulation, as described under “Experimental Procedures”. Luciferase activities were measured by using the Dual-Luciferase Reported Assay kit and normalized to sea pansy luciferase activity of co-transfected pRL-SV40. Data represent the mean ± S.E. of at least 3 independent experiments. *, p<0.05 with respect to the pIL-6-luc651 ΔNF-κB and vehicle or mock transfected control. ▴, p<0.05 with respect to significantly regulated (*) groups.
10.1371/journal.pone.0027630.g007Figure 7 Regulation of the binding of the NF-κB p65 subunit to the IL-6 promoter in T/C-28a2 chondrocytes.
T/C-28a2 cells were incubated with either PGE2 (10 µM) for 2 h or 15d-PGJ2 (1 µM) for 48 h. In other experiments, cells were pre-treated with 15d-PGJ2 (1 µM) for 48 h before incubation with PGE2 (10 µM) for 2 h. In select experiments, T/C-28a2 cells were transfected with an siRNA oligonucleotide sequence specific for TLR4 (A, B) or caveolin-1 (C, D) or a plasmid containing the cDNA of caveolin-1 (A, B) or TLR4 (C, D) before PG treatment. In separate experiments, cells were incubated with PGE2 for 2 h in the presence or absence of the MEK1/2 inhibitors, PD98059 (20 µM) or U0126 (10 µM) (E, F). Nuclear extracts were then isolated, and NF-κB-specific DNA-protein complex formation was determined by gel shift (A, C, E). Supershift assays (B, D, F) using an anti-p65 Ab were carried out as outlined in “Experimental Procedures”. Results of a competition experiment using 200-fold unlabeled NF-κB oligonucleotide (cold probe) are shown. These gels are representative of three independent experiments, all revealing similar results.
TLR4 depletion or caveolin-1 overexpression blocked the PGE2-dependent transactivation of NF-κB p65 subunit (Fig. 5A), IL-6 promoter activity (Figs. 6B,C) as well as the levels of NF-κB gel shift (Fig. 7A) and supershift (Fig. 7B). On the other hand, exogenous 15d-PGJ2 represses the phosphorylation of p65 (Fig. 5B) and its binding to the IL-6 promoter (Figs. 6D,E) as well as the NF-κB gel shift and supershift (Figs. 7C, D) induced by ectopic expression of TLR4 or knockdown of caveolin-1 in human T/C-28a2 chondrocytes. These data were further validated using chromatin immunoprecipitation assays (Fig. 8). Taken together, our data disclose that PGE2 and 15d-PGJ2 exert antagonistic effects on NF-κB activation, which are propagated via TLR4 and caveolin-1. Our data also reveal the critical role of ERK1/2, in addition to PI3-K and PKA [23], in the induction of IL-6 promoter activity (Fig. 6F) and increased levels of NF-κB gel shift (Fig. 7E) and supershift (Fig. 7F) detected after T/C-28a2 chondrocyte stimulation with PGE2.
10.1371/journal.pone.0027630.g008Figure 8 Regulation of the binding of the NF-κB p65 subunit to the IL-6 promoter in T/C-28a2 chondrocytes as monitored by ChIP assays.
T/C-28a2 cells were incubated with either PGE2 (10 µM) for 2 h or 15d-PGJ2 (1 µM) for 48 h. In other experiments, cells were pre-treated with 15d-PGJ2 (1 µM) for 48 h before incubation with PGE2 (10 µM) for 2 h (A). In select experiments, T/C-28a2 cells were transfected with an siRNA oligonucleotide sequence specific for TLR4 (B) or caveolin-1 (C) or a plasmid containing the cDNA of caveolin-1 (B) or TLR4 (C) before PG treatment. In separate experiments, cells were incubated with PGE2 for 2 h in the presence or absence of the MEK1/2 inhibitors, PD98059 (20 µM) or U0126 (10 µM) (D). Crosslinked chromatin was immunoprecipitated using an anti-p65 antibody. In ChIP assays, the anti-RNA polymerase II antibody was used as positive control, whereas the normal mouse IgG and anti-TLR4 antibodies were used as negative controls. DNA purified from both the immunoprecipitated (IP) and pre-immune (input) specimens were subjected to qRT-PCR amplification using primers for the GAPDH (control) and p65 promoter genes. Data represent the mean ± S.E. of at least 3 independent experiments. *, p<0.05 with respect to scramble siRNA or empty vector transfected control. ▴, p<0.05 with respect to significantly regulated (*) groups.
Discussion
The synovial fluid of RA patients relative to normal controls contains elevated levels of several soluble mediators including PGE2, PGD2/15d-PGJ2 and IL-6, which contribute to the systemic manifestations of the disease [9], [17], [27]. Although PGE2 has been reported to directly stimulate IL-6 production [22], [23], [28], the potential role of PGD2/15d-PGJ2 in the modulation of IL-6 synthesis in human articular chondrocytes has yet to be investigated. Here, we report that exogenous PGE2 and PGD2/15d-PGJ2 exert opposing effects on IL-6 expression and secretion. Specifically, PGE2 induces TLR4 synthesis, which is in turn responsible for the activation of ERK1/2, PI3-K and PKA pathways that act synergistically to activate NF-κB. Binding of the NF-κB p65 subunit to IL-6 promoter elicits IL-6 synthesis (Fig. 9). In contrast, exogenous PGD2/15d-PGJ2 concurrently downregulates TLR4 and upregulates caveolin-1 expression, which in turn suppress the PGE2-dependent activation of ERK1/2, PI3-K and PKA pathways and NF-κB dependent IL-6 production (Fig. 9).
10.1371/journal.pone.0027630.g009Figure 9 Proposed cascade of signaling events regulating IL-6 synthesis in human chondrocytes treated with PGE2 and 15d-PGJ2.
PGE2 stimulates TLR4 synthesis, which is in turn responsible for the activation of the ERK1/2, PI3K/Akt and PKA/CREB pathways that phosphorylate the NF-κB p65 subunit leading to NF-κB activation. Binding of the activated NF-κB p65 subunit to IL-6 promoter induces IL-6 synthesis in human chondrocytes. PGD2 or 15d-PGJ2 concurrently downregulates TLR4 and upregulates caveolin-1, which in turn inhibit the PGE2-dependent ERK1/2, PI3-K and PKA activation, and ultimately with NF-κB-dependent IL-6 synthesis in chondrocytes.
Prior work has shown that LPS binding to TLR4 induces mPGES-1 synthesis and PGE2 production in mouse osteoblasts [32]. However, we herein show that PGE2 is necessary and sufficient for induction of TLR4 at the transcriptional and translational level in human T/C-28a2 chondrocytes. We hypothesize that PGE2 induces TLR4 synthesis by an indirect manner in the view of its function as an autocrine regulatory factor. Our analysis also revealed that 15d-PGJ2 repressed TLR4 expression. In agreement with our data, Eun, et al.
[33] reported that treatment of human intestinal epithelial cells with 15d-PGJ2 attenuated LPS-induced TLR4 mRNA and protein expression, thereby providing evidence that 15d-PGJ2 may downregulate TLR4 expression. In contrast, Inoue, et al.
[34] showed that 15d-PGJ2 enhanced the expression of TLR4 in the LPS-induced acute lung injury mice. However, this observation regarding the potential stimulation of TLR4 expression by 15d-PGJ2 needs to be interpreted with caution, since 15d-PGJ2 does not exhibit any regulatory effect on TLR4 expression in the absence of LPS [34]. Of note, several lines of evidence suggest that 15d-PGJ2 is capable of suppressing TLR4 expression in different cell lines, such as mouse T lymphocytes [35], rat Schwann cells [36] and human intestinal epithelial cells [33].
Caveolin-1 is upregulated in osteoarthritic cartilage [37]. Moreover, caveolin-1 binding to CD26 has been reported to play a key role in T-cell-mediated antigen-specific response in RA [38]. Peroxisome proliferator-activated receptor γ (PPARγ) ligands, such as 15d-PGJ2, upregulate caveolin-1 expression in human carcinoma cells [39]. In agreement with this prior observation, our data reveal that 15d-PGJ2 induces caveolin-1 expression in human T/C-28a2 chondrocytes. Interestingly, caveolin-1 was recently shown to interact and inactivate TLR4-mediated IL-6 signaling in murine RAW264.7 macrophages [30]. Consistent with this observation, we found that TLR4 and caveolin-1 exert antagonistic effects on IL-6 synthesis in human T/C-28a2 chondrocytes.
The pro-inflammatory potential of TLR4 is associated with its ability to induce IL-6 production in diverse cell types such as human macrophages [40] and bladder epithelial cells [41], [42]. TLR4 activation by LPS induces IL-6 expression in bladder cancer cells via an ERK/p38/PI3-K-dependent pathway [41]. In line with our data, pharmacological inhibition of ERK attenuated LPS-induced IL-6 synthesis in bladder cancer cells [41]. In contrast to our results, use of a PI3-K inhibitor (LY294002) amplified IL-6 expression in LPS-primed bladder cancer cells [41]. Consistent with our observations, previous studies have shown that TLR4 can activate PI3-K, ERK or PKA pathways either by direct interaction with the PI3-K p85 regulatory subunit [43] or mitogen activated protein kinase kinase kinase 3 (which is upstream of ERK) [44] or by modulating intracellular cAMP level [45], respectively. Interestingly, caveolin-1 has been shown to suppress PI3-K, PKA and ERK1/2 activity by direct interaction [46], [47]. Cumulatively, our data along with previously published results suggest that TLR4 and caveolin-1 modulate the intracellular PI3-K, ERK1/2 and PKA pathways in a reverse, antagonistic manner.
We have recently demonstrated the key role of the NF-κB p65 subunit in the induction of IL-6 synthesis in shear-activated or PGE2 primed human T/C-28a2 chondrocytes via a PI3-K/PKA-dependent pathway [23], [29]. We herein extend these observations by showing the key role of ERK1/2 in the activation of the NF-κB p65 subunit and its binding to the IL-6 promoter in PGE2-stimulated human chondrocytes. We further report that inhibitory effects of 15d-PGJ2 in the activation of the PI3-K, PKA and ERK1/2 pathways and NF-κB-dependent IL-6 synthesis, which are mediated via the downregulation of TLR4 and upregulation of caveolin-1. TLR4 was also reported to trigger a rapid IL-6 response in LPS-stimulated bladder epithelial cells [42], which includes the sequential involvement of calcium, adenylyl cyclase 3-generated cAMP and the transcription factor CREB. Even though TLR4 stimulates intracellular cAMP production, which in turn plays a key role in PGE2-dependent IL-6 synthesis, we have found that CREB is not involved in the induction of IL-6 in human chondrocytes stimulated with exogenous PGE2
[23].
In summary, we have elucidated the signaling pathway by which PGE2 and PGD2/15d-PGJ2 exert opposing effects on IL-6 synthesis in human chondrocytes (Fig. 9), and demonstrated the key albeit antagonistic actions of TLR4 and caveolin-1 in this process. Understanding the signal transduction pathway of PG-regulated IL-6 synthesis in human chondrocytes will enable us to design therapeutic strategies to reduce inflammation and pain in arthritic patients.
Materials and Methods
Reagents
The PGD2, 15d-PGJ2 and PGE2 were obtained from Enzo Life Sciences International Inc (Plymouth Meeting, PA). The IL-6 promoter reporter constructs pIL6-luc651 (-651/+1) and pIL6-luc651 ΔNF-κB (NF-κB site mutation) were gifts from Dr. Eickelberg [48]. pRL-SV40 vector encoded with renilla luciferase gene was purchased from Promega (Madison, WI). The caveolin-1 and TLR4 cDNA plasmids were supplied from Origene Technologies (Rockville, MD), and subcloned to the pCMV6-XL vector. The MEK1/2 inhibitors U0126 and PD98059 were obtained from Sigma-Aldrich Corp. Antibodies specific for ß-actin, caveolin-1, Akt, p-Akt (Ser473), CREB, p-CREB (Ser133), ERK1/2, p-ERK1/2 (Thr 202/Tyr 204), NF-κB p65, p-p65 (Ser276) and p-p65 (Ser536) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibody specific for TLR4 was from Sigma-Aldrich Corp and monoclonal antibody specific for IL-6 as well as TLR4 and caveolin-1 siRNAs were from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). IL-6 and cAMP EIA kits were from Cayman Chemical, All reagents for qRT-PCR and SDS-PAGE experiments were purchased from Bio-Rad Laboratories. Reagents for EMSA were obtained from Pierce Chemical Company. The Dual-Luciferase Reporter Assay kit was purchased from Promega (Madison, WI). The EZ-ChIP kit was purchased from Upstate Biotechnology. All other reagents were from Invitrogen (Carlsbad, CA), unless otherwise specified.
Cell culture and Treatment
Human primary articular chondrocytes (Cell Applications, Inc) or T/C-28a2 chondrocytic cells (T/C-28a2 chondrocytic cells were kindly provided by Dr. Goldring at Harvard Medical School, Boston, MA, USA) [49] were seeded on 6-cm tissue culture dishes (106 cells per dish) in human chondrocyte growth medium (Cell Applications, Inc) or in DMEM/F12 medium supplemented with 10% FBS, respectively [23], [29], [50], [51], [52], [53]. 24 h later, human chondrocytic cells were grown in serum-free medium for another 24 h before being incubated with PGE2 (1–20 µM), 15d-PGJ2 (1 µM), PGD2 (0.5–8 µM) or vehicle (control) for prescribed periods of time in the presence or absence of pharmacological inhibitors.
Transient Transfection and Reporter Gene Assays
For ectopic expression of caveolin-1 or TLR4, T/C-28a2 chondrocytes were transfected with 1.6 µg/slide of plasmid containing cDNAs by using Lipofectamine 2000. In control experiments, cells were transfected with 1.6 µg/slide of the empty vector pCMV6-XL (OriGene Technologies). In select experiments, T/C-28a2 cells were transfected with 1.6 µg/slide of the IL-6 promoter reporter construct pIL-6-luc651 or pIL6-luc651 ΔNF-κB together with pRL-SV40 vector. In RNA interference assays, T/C-28a2 cells were transfected with 100 nM of a siRNA oligonucleotide sequence specific for caveolin-1 or TLR4. In control experiments, cells were transfected with 100 nM of scramble siRNA. Transfected cells were allowed to recover for at least 12 h in growth medium, and then incubated overnight in medium containing 1% Nutridoma-SP before their exposure to prostaglandins. In promoter activity experiments, luciferase activities were measured by using the Dual-Luciferase Reporter Assay kit (Promega), as previously described described [23], [29], [54].
Quantitative Real-Time PCR (qRT-PCR)
qRT-PCR assays were performed on the iCycler iQ detection system (Bio-Rad) using total RNA, the iScript one-step RT-PCR kit with SYBR green (Bio-Rad) and primers. The GenBank accession numbers and forward (F-) and reverse (R-) primers are as follows:
caveolin-1 (NM_001753), F- GGGCAACATCTAGAAGCCCAACAA,
R- CTGATGCACTGAATTCCAATCAGGAA;
TLR4 (NM_138554), F- TGCAATGGATCAAGGACCAGAGGC,
R- GTGCTGGGACACCACAACAATCACC;
The GenBank accession numbers and forward (F-) and reverse (R-) primers for IL-6 and GAPDH are provided in our previous publications [23], [29]. GAPDH was used as internal control. Reaction mixtures were incubated at 50°C for 15 min followed by 95°C for 5 min, and then 35 PCR cycles were performed with the following temperature profile: 95°C 15 s, 58°C 30 s, 68°C 1 min, 77°C 20 s. Data were collected at the (77°C 20 s) step to remove possible fluorescent contribution from dimer-primers [23], [29]. Gene expression values were normalized to GAPDH.
Western blot analysis
T/C-28a2 cells, from different treatment or transfection, were lysed in RIPA buffer (25 mM Tris•HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing a cocktail of proteinase inhibitors (Pierce Chemical Company). The protein content of the cell lysates was determined using bicinchoninic acid (BCA) protein assay reagent (Pierce Chemical Company). Total cell lysates (4 µg) were subjected to SDS-PAGE, transferred to a membrane, and probed with a panel of specific antibodies. Each membrane was only probed using one antibody. ß-actin was used as loading control. All Western hybridizations were performed at least in triplicate using a different cell preparation each time.
Preparation of cytosolic and nuclear extracts
Cytosolic and nuclear extracts were isolated using the NE-PER nuclear and cytoplasmic extraction kit (Pierce) following the manufacturer's instructions as previously described [23], [29], [54].
Gel-shift and supershift assay
A 5′-biotinylated oligonucleotide probe (5′-GGGATTTTCC-3′) was synthesized containing the NF-κB cis-element present on the IL-6 promoter. EMSAs were performed with a commercially available nonradioisotopic EMSA kit (LightShift Chemiluminescence EMSA kit; Pierce) as previous description [23], [29], [54].
Measurement of IL-6 and cAMP concentration in medium
The levels of IL-6 in medium and intracellular cAMP were determined using the corresponding kits, following the manufacturer's instructions. The total protein concentration in the medium was used as loading control, and the results were expressed as pg IL-6 or pmol cAMP per µg of total protein.
ChIP Assay
This assay was performed using the EZ ChIP kit following the manufacturer's instructions (Upstate Biotechnology) as previously described [23].
Statistics
Data represent the mean ± S.E. of at least 3 independent experiments. Statistical significance of differences between means was determined by Student's t-test or one-way ANOVA, wherever appropriate. If means were shown to be significantly different, multiple comparisons by pairs were performed by the Tukey test [49].
Supporting Information
Figure S1
Time-dependent regulation of IL-6 secretion by 15d-PGJ2-treated human chondrocytes. T/C-28a2 chondrocytes (A) or human primary articular chondrocytes (B) were treated with 15d-PGJ2 (1 µM) for the indicated time intervals. IL-6 production was determined by an IL-6 enzyme immunoassay kit. Data represent the mean ± S.E. of at least 3 independent experiments. *, p<0.05 with respect to vehicle control.
(TIF)
Click here for additional data file.
Figure S2
The effects of PGD2 on TLR4, caveolin-1 and IL-6 synthesis in T/C-28a2 chondrocytes. T/C-28a2 cells were transfected with a siRNA oligonucleotide sequence specific for caveolin-1 or a plasmid containing the cDNA of TLR4 before incubated with PGD2 (8 µM) for 48 h. TLR4, caveolin-1 and IL-6 mRNA synthesis was determined by qRT-PCR. GAPDH served as internal control. Data represent the mean ± S.E. of at least 3 independent experiments. *, p<0.05 with respect to vehicle or mock transfected control. ▴, p<0.05 with respect to significantly regulated (*) groups.
(TIF)
Click here for additional data file.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported, in whole or in part, by the National Institutes of Health NIAMS Grant RO1 AR053358. http://www.nih.gov/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22110582PONE-D-11-0150710.1371/journal.pone.0025933Research ArticleBiologyGeneticsHuman GeneticsGenetic Association StudiesPersonalized MedicineMedicineSurgeryTransplant SurgeryAssociation of MDR1 Gene SNPs and Haplotypes with the Tacrolimus Dose Requirements in Han Chinese Liver Transplant Recipients Haplotypes Associated with Tacrolimus RequirementYu Xiaobo
1
Xie Haiyang
1
Wei Bajin
1
Zhang Min
2
Wang Weilin
2
Wu Jian
2
Yan Sheng
2
Zheng Shusen
1
2
*
Zhou Lin
1
*
1
Key Lab of Combined Multi-Organ Transplantation, The First Affiliated Hospital, School of Medicine, Zhejiang University, Ministry of Public Health, Hangzhou, Zhejiang, China
2
Department of Surgery, Division of Hepatobiliary and Pancreatic Surgery, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China
Gregson Aric EditorUniversity of California Los Angeles, United States of America* E-mail: [email protected] (SZ); [email protected] (LZ)Conceived and designed the experiments: SZ. Performed the experiments: XY HX WW JW SY. Analyzed the data: XY MZ BW. Wrote the paper: XY LZ.
2011 14 11 2011 6 11 e2593318 1 2011 13 9 2011 Yu et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
This work seeks to evaluate the association between the C/D ratios (plasma concentration of tacrolimus divided by daily dose of tacrolimus per body weight) of tacrolimus and the haplotypes of MDR1 gene combined by C1236T (rs1128503), G2677A/T (rs2032582) and C3435T (rs1045642), and to further determine the functional significance of haplotypes in the clinical pharmacokinetics of oral tacrolimus in Han Chinese liver transplant recipients.
Methodology/Principal Findings
The tacrolimus blood concentrations were continuously recorded for one month after initial administration, and the peripheral blood DNA from a total of 62 liver transplant recipients was extracted. Genotyping of C1236T, G2677A/T and C3435T was performed, and SNP frequency, Hardy-Weinberg equilibrium, linkage disequilibrium, haplotypes analysis and multiple testing were achieved by software PLINK. C/D ratios of different SNP groups or haplotype groups were compared, with a p value<0.05 considered statistically significant. Linkage studies revealed that C1236T, G2677A/T and C3435T are genetically associated with each other. Patients carrying T-T haplotype combined by C1236T and G2677A/T, and an additional T/T homozygote at either position would require higher dose of tacrolimus. Tacrolimus C/D ratios of liver transplant recipients varied significantly among different haplotype groups of MDR1 gene.
Conclusions
Our studies suggest that the genetic polymorphism could be used as a valuable molecular marker for the prediction of tacrolimus C/D ratios of liver transplant recipients.
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Introduction
To lower the risk of rejection after allogenetic organ transplantation, immunosuppressive drugs are widely used to reduce the immune system activity. Tacrolimus, also named FK506, is a kind of immunosuppressive drugs, and able to inhibit the multiplication of T-cells [1]. Postoperative patients have to take tacrolimus all their lives to make a better graft survival, which results in heavy financial costs [1]. The optimal use of tacrolimus could not only lower the financial cost but also reduce the side effects caused by tacrolimus, which makes it a valuable therapy for liver transplant recipients. However, pharmacokinetic characteristics of tacrolimus vary dramatically among individuals. Pharmacokinetic characteristics could be influenced in many ways, one of which may be the genetic factors including single nucleotide polymorphism (SNP), haplotype and DNA methylation [2], [3], [4], [5], [6].
Human multidrug resistance (MDR1) gene, also named P-glycoprotein, is a member of the ATP-binding cassette superfamily. MDR1 protein anchors in cell membrane, and acts as an efflux transporter of various substrates for cell protection [4], [6]. It has been reported in the literature that tacrolimus is one substrate of MDR1 [5], [7], [8]. MDR1 is polymorphic, and at least 50 SNPs have been found so far [4], [9], [10], [11], [12], [13]. The functional consequences of reported SNPs are not completely understood and still controversial to date. SNPs occur as a result of single-nucleotide substitutions in coding region and non-coding region, which might influence mRNA expression [14] and protein translation and folding [6], [8], and finally affect drug pharmacokinetic characteristics. Moreover, the allelic frequency of MDR1 SNPs varies widely among ethnic groups [4], [5], [6]. Haplotype is a set of genetically associated SNPs [15], [16], [17], and can be mathematically calculated by software including PLINK and Haploview [18], [19]. Linkage studies showed that there is strong linkage disequilibrium among the highly frequent polymorphisms C1236T (rs1128503), G2677A/T (rs2032582) and C3435T (rs1045642) [6], [20], [21]. Furthermore, the effects of haplotype on drug response and disease outcome have been reported [20], [21], [22], [23]. Other studies on specific mechanism have demonstrated that haplotypes may alter mRNA stability [24], protein conformation and inhibitor efficiency [6].
Dose-adjusted trough concentration (concentration/dose [C/D], plasma concentration of drug divided by daily dose of drug per body weight) was used as the criteria for comparison among different SNP or haplotype groups in most of the previous studies [25], [26], [27], [28], [29]. We have already observed lower tacrolimus C/D ratios in liver transplant recipients of MDR1 C3435T C/C homozygotes previously [28]. Our new findings not only supported the previous observation, but also provided the evidence that MDR1 haplotype could affect tacrolimus C/D ratios.
Methods
Patients
The population in this study was Han Chinese, including 5 female and 57 male, aged from 21 to 64 years old (46.6±9.3), and weighed from 50 to 85 kg (66.4±8.4). For all the patients, tacrolimus-based immunosuppressive regimens were included. The oral administration of tacrolimus and steroid was introduced in our previous study [28].
Ethics statement
The research protocol was approved by the Institutional Review Board, Key Lab of Combined Multi-organ Transplantation, Ministry of Public Health. Informed written consent was obtained according to the Declaration of Helsinki.
Data Collection and Therapeutic Drug Monitoring
After the initial administration of tacrolimus, all patients received clinical evaluations and laboratory tests in the first month. The daily dose (mg) of tacrolimus was recorded, and the weight-adjusted dosage (mg/kg/d) was calculated. Drug blood levels were measured by immunoassay on the IMx analyzer (Abbott Diagnostics Laboratories, Abbott-Park, IL). Dose-adjusted trough concentrations were calculated by dividing tacrolimus trough concentrations by the corresponding dose on an mg/kg basis (concentration/dose [C/D] ratio).
Genotyping
Genomic DNA of patients was extracted from peripheral blood using QIAamp DNA Blood mini kit (QIAGEN, Hilden, Germany) following the manufacturer's instruction. RFLP (restriction fragment length polymorphism) PCR method was used to genotype the position C1236T, G2677A/T and C3435T. Primer pairs 5′ TTCACTTCAGTTACCCATC 3′ and 5′ CATAGAGCCTCTGCATCA 3′ and restriction enzyme BsuRI were used to distinguish T allele from C allele of C1236T, with primer pairs 5′ AGAGCATAGTAAGCAGTAGGGAGTA 3′ and 5′ GCAAATCTTGGGACAGGAATA 3′ and restriction enzyme RsaI for distinguishing A allele from G or T allele of G2677A/T, primer pairs 5′ AGTAAGCAGTAGGGAGTAACA 3′ and 5′ GATAAGAAAGAACTAGAACGT 3′ and restriction enzyme AclI for distinguishing T allele from G or A allele of G2677A/T, primer pairs 5′ GATCTGTGAACTCTTGTTTTCA 3′ and 5′ GAAGAGAGACTTACATTAGGC 3′ and restriction enzyme MboI for distinguishing T allele from C allele of C3435T. PCR and products digestion by restriction enzyme were performed as reported [30].
Statistical Analysis
Nonparametric tests, including Mann-Whitney test and Kruskal-Wallis test, were applied to assess significance test for comparisons of all group pairs, with a further confirmation by multiple test, max(T) permutation by 10000 times. Nonparametric tests were performed by Graphpad Prism 5.03 (Graphpad Software, San Diego, CA, USA). Hardy-Weinberg equilibrium, linkage disequilibrium, haplotype frequency analyses and max(T) permutation were performed by PLINK v1.06 (http://pngu.mgh.harvard.edu/purcell/plink/). The expectation-maximization (E-M) algorithm was used to estimate haplotype frequencies by PLINK. A p value<0.05 was considered statistically significant.
Results
Genotype Frequency of patients
All single SNP genotypes were recorded, and frequencies were calculated. No statistical significance was found among genotype groups related to gender, age and weight (Table 1). Results of Kruskal-Wallis tests were not shown. As mentioned in method, PLINK was used to analyze Hardy-Weinberg equilibrium, linkage disequilibrium and haplotype frequencies. G2677A/T has 3 alleles, however, according to the user manual, PLINK is unable to analyze SNPs with more than 2 alleles. Therefore, when one allele was compared with other two alleles, there had to be a new character to represent the two alleles. In accordance to the IUPAC (Union of Pure and Applied Chemistry) coding standards, ‘K’ was used as the abbreviation for T and G alleles, with ‘R’ for A and G alleles together and ‘W’ for A and T alleles together. So G2677A/T was also named as G2677A/T(A-K), G2677A/T(T-R) or G2677A/T(G-W). All three SNPs frequencies were in accordance with Hardy-Weinberg equilibrium, and the p value were >0.05 (Table 2).
10.1371/journal.pone.0025933.t001Table 1 Demographic characteristics of liver transplant patients.
SNP Genotype
N
Gender(M/F) Age (mean ± S.D.) Weight, kg (mean ± S.D.)
C1236T C/C 9 (14.5%) 9/0 47.9±9.5 69.3±8.4
C/T 25 (40.3%) 24/1 44.8±9.4 68.7±8.5
T/T 28 (45.1%) 24/4 47.9±9.1 63.4±7.5
G2677A/T A/A 2 (3.2%) 2/0 43.0±11.3 65.5±9.2
A/G 8 (12.9%) 8/0 49.6±6.5 71.1±7.7
A/T 6 (9.7%) 5/1 40.0±12.5 69.2±9.6
G/G 16 (25.8%) 15/1 48.4±6.4 63.1±8.7
G/T 22 (35.4%) 20/2 46.8±10.8 67.0±7.9
T/T 8 (12.9%) 7/1 45.4±8.8 62.1±7.0
C3435T C/C 7 (11.3%) 6/1 46.6±8.8 60.7±6.2
C/T 24 (38.7%) 24/1 44.8±10.8 66.3±8.0
T/T 31 (50%) 28/3 48.0±8.0 67.7±8.7
10.1371/journal.pone.0025933.t002Table 2 SNPs frequencies in liver transplant patients.
SNP position Alleles Frequency Genotypes Frequency Hardy–Weinberg equilibrium Comparison of C/D ratios
Allele N Genotype N O(HET)d
E(HET)e
p
C/D ratios
p
C1236T C 43 (34.7%) C/C 9 (14.5%) 0.4032 0.453 0.4048 143.50±37.99 0.6772
T 81 (65.3%) C/T 25 (40.3%) 142.73±39.23
T/T 28 (45.1%) 138.26±47.61
G2677A/T(A-K)a
A 18 (14.5%) A/A 2 (3.2%) 0.2258 0.2482 0.5985 146.20±35.66 0.4166
K 106 (85.5%) A/K 14 (22.6%) 139.01±44.86
K/K 46 (74.2%) 152.23±50.23
G2677A/T(T-R)b
T 44 (35.5%) T/T 8 (12.9%) 0.4516 0.4579 1 145.35±41.76 0.2677
R 80 (64.5%) T/R 28 (45.2%) 141.73±44.17
R/R 26 (41.9%) 122.77±38.51
G2677A/T(G-W)c
G 62 (50.0%) G/G 16 (25.8%) 0.4839 0.5 0.8025 148.33±45.48 0.5398
W 62 (50.0%) G/W 30 (48.4%) 137.76±42.17
W/W 16 (25.8%) 138.82±41.98
C3435T C 38 (30.6%) C/C 7 (11.3%) 0.3871 0.4251 0.5496 117.54±23.16 0.0419
T 86 (69.4%) C/T 24 (38.7%) 137.95±49.83
T/T 31 (50%) 150.20±48.05
C/C 117.54±23.16 0.0240
C/T+T/T 144.20±40.05
a the character ‘K’ is for T, thymine, or G, guanine.
b the character ‘R’ is for A, adenine, or G, guanine.
c the character ‘W’ is for A, adenine, or T, thymine.
d O(HET) is short for observed heterozygosity.
e E(HET) is short for expected heterozygosity.
Effect of SNPs on Tacrolimus Dose Requirement
Data of oral tacrolimus dose was collected, and the relationship between MDR1 SNP genotypes and C/D ratio was investigated. No statistically significant association was observed in position C1236T and G2677A/T, except C3435T (Table 2). Similar to the results of our previous study [28], we found that recipients with C/C genotype at C3435T would require a little higher dose of tacrolimus compared to those with C/T and T/T genotypes (Table 2).
It was reported that linkage disequilibrium existed in C1236T, G2677A/T and C3435T, and association among the three SNPs, also called haplotype, might influence drug pharmacokinetics. So we tested the linkage disequilibrium of all pairs of these three SNPs at the beginning. When C1236T combined with C3435T, or G2677A/T combined with C3435T, linkage disequilibrium was found (Table 3). C1236T also had linkage disequilibrium with G2677A/T(A-K) and G2677A/T(T-R), not with G2677A/T(G-W) (Table 3). And then according to the result of linkage disequilibrium, haplotype frequency analyses were performed, which do not include the combination between C1236T and G2677A/T(G-W). The haplotypes of individuals were recorded. No statistical significance was found in either of the 6 different pairs of combination (Table 4). But when C1236T and G2677A/T(T-R) were combined, patients with T-T/T-T, T-T/C-T and T-T/T-R haplotypes showed lower C/D ratios than those with T-R/T-R, T-R/C-R, T-T/C-R and C-R/C-R, which meant that patients carrying T-T haplotype and with an additional T/T homozygote at position C1236T or G2677A/T would require higher dose of tacrolimus (Table 5). Furthermore, it seemed that patients with T-C/T-C or T-C/R-T haplotypes showed lower C/D ratios than those with other haplotypes, which meant patients carrying T-C haplotype with the combination of G2677A/T(T-R) and C3435T required higher dose of tacrolimus to maintain serum concentration. However, after the max(T) permutation adjustment, no statistical significance was observed (Table 5).
10.1371/journal.pone.0025933.t003Table 3 Haplotype analysis of different pairs of the three SNPs.
Combined SNPs Haplotypes
N
LDa
C1236T vs G2677A/T(A-K) C-A 17 (13.5%) D′ = 0.897
T-A 1 (1%)
C-K 26 (21.1%)
T-K 80 (64.3%)
C1236T vs G2677A/T(T-R) C-T 4 (3.4%) D′ = 0.724
T-T 40 (32.1%)
C-R 39 (31.3%)
T-R 41 (33.2%)
C1236T vs G2677A/T(G-W) C-W 21 (17.3%) D′ = 0.000
T-W 41 (32.7%)
C-G 21 (17.3%)
T-G 41 (32.7%)
C1236T vs C3435T C-C 1 (1.1%) D′ = 0.895
T-C 37 (29.5%)
C-T 42 (33.6%)
T-T 44 (35.8%)
G2677A/T(A-K) vs C3435T A-C 0 (0%) D′ = 1.000
K-C 38 (30.6%)
A-T 18 (14.5%)
K-T 68 (54.8%)
G2677A/T(T-R) vs C3435T T-C 35 (28%) D′ = 0.868
R-C 3 (2.6%)
T-T 9 (7.4%)
R-T 77 (61.9%)
G2677A/T(G-W) vs C3435T W-C 34 (27.5%) D′ = 0.796
G-C 4 (3.1%)
W-T 28 (22.5%)
G-T 58 (46.9%)
a LD, linkage disequilibrium.
10.1371/journal.pone.0025933.t004Table 4 Tacrolimus concentration/dose (C/D) ratios of different haplotype groups.
Combined SNPs Haplotypes
N
C/D Combined SNPs Haplotypes
N
C/D
C1236T - G2677A/T(A-K) C-A/C-A 1 (1.6%) 188.07±0.00 G2677A/T(A-K) - C3435T A-T/A-T 2 (3.2%) 156.40±43.84
C-A/T-K 9 (14.5%) 147.86±44.63 A-T/K-C 4 (6.5%) 149.08±29.46
C-K/C-A 5 (8.1%) 128.09±40.17 K-T/A-T 10 (16.1%) 142.79±35.38
C-K/C-K 3 (4.8%) 116.71±40.46 K-T/K-T 19 (30.6%) 137.01±41.89
T-A/C-A 1 (1.6%) 177.61±0.00 K-T/K-C 20 (32.3%) 145.33±49.36
T-K/C-K 15 (24.2%) 145.89±47.77 K-C/K-C 7 (11.3%) 126.42±38.70
T-K/T-K 28 (45.2%) 137.47±39.28
C1236T - G2677AT-(T-R) C-R/C-R 8 (12.9%) 135.65±41.19 G2677AT-(T-R) - C3435T R-T/R-T 23 (37.1%) 139.23±36.19
C-R/C-T 1 (1.6%) 93.44±0.00 R-T/R-C 3 (4.8%) 192.31±46.51
C-R/T-T 12 (19.4%) 146.65±41.54 R-T/T-T 7 (11.3%) 149.24±49.34
T-R/C-R 11 (17.7%) 141.91±44.64 T-T/T-T 1 (1.6%) 97.17±0.00
T-R/T-R 7 (11.3%) 142.06±30.32 T-C/R-T 21 (33.9%) 139.10±42.89
T-R/T-T 15 (24.2%) 135.15±38.28 T-C/T-C 7 (11.3%) 126.42±38.70
T-T/C-T 2 (3.2%) 188.07±62.59
T-T/T-T 6 (9.7%) 138.83±53.93
C1236T - C3435T C-T/C-T 9 (14.5%) 130.97±42.39 G2677A/T(G-W) - C3435T G-T/G-T 13 (21.0%) 138.18±37.49
C-T/T-C 10 (16.1%) 147.61±44.29 G-T/G-C 3 (4.8%) 192.31±46.51
T-T/C-T 14 (22.6%) 148.35±49.13 G-T/W-T 13 (21.0%) 138.66±40.31
T-T/T-T 8 (12.9%) 137.06±36.82 W-T/W-T 5 (8.1%) 149.00±49.54
T-T/T-C 14 (22.6%) 137.15±36.54 W-T/W-C 4 (6.5%) 149.08±29.46
T-C/C-C 1 (1.6%) 143.82±0.00 W-C/G-T 17 (27.4%) 137.04±44.43
T-C/T-C 6 (9.7%) 138.83±43.93 W-C/W-C 7 (11.3%) 126.42±38.70
10.1371/journal.pone.0025933.t005Table 5 Statistical analysis of tacrolimus concentration/dose (C/D) ratios at 1 month after drug initiation between haplotype groups.
SNP position Haplotypes C/D
p
C1236T - G2677A/T(A-K) T-T/T-T+T-T/C-T+T-T/T-R 128.65±43.60 0.0156
T-R/T-R+T-R/C-R+T-T/C-R+C-R/C-R 148.14±40.40 0.034#
G2677A/T(T-R) - C3435T T-C/T-C+T-C/R-T 132.45±37.16 0.049
R-T/R-T+R-T/R-C+R-T/T-T 151.85±39.23 0.098#
# indicated the p value given by max(T) permutation.
Discussion
It has been reported that there are more than 50 SNPs in human MDR1 gene [9], [10], [11], [12], [13], [31]. SNPs spread from the 5′ start to the 3′ untranslated region in MDR1 transcript, resulting in both synonymous and non-synonymous mutations [4], [5], [6]. Three SNPs, C1236T, G2677A/T and C3435T, all locate in exons. Mutation of G2677A/T causes coding sequence missense, while the others are synonymous [5]. Missense substitutions in amino acid may result in abnormal protein folding, moreover, there has been a hypothesis that the presence of rare codons, marked by synonymous polymorphisms, may affect the insertion of MDR1 into the membrane and alter the structure of substrate interaction sites [8]. These SNPs have become research focus, which include effects of SNPs and haplotypes in different ethnic groups [7], [13], [32], [33], [34], [35], [36], [37], [38] on MDR1 mRNA stabilization [24], [39], [40], [41] or protein expression and folding [5], [10] in patients, and effects on substrates efflux in cell models [5], [8].
According to the literature mentioned above, both G2677A/T and C3435T have significant association with tacrolimus or cyclosporine pharmacokinetics, and their clinical behaviors exhibit significantly different requirements of drug dose among different SNP groups. Recipients with C/C homozygotes of MDR1 in position C3435T showed significantly lower dose-adjusted tacrolimus concentrations compared with the other groups [28], [37], [42]. Since the ethnic population was Han Chinese, the same population in our previous study [28], similar phenomenon was observed. Some other research groups also identified SNPs related to cyclosporine pharmacokinetics, still there are controversies. In some cases, recipients with C/C homozygote in position C3435T required higher dose of cyclosporine [43], [44], while others did not [45], [46], [47]. One of the explanations is that SNPs frequencies may vary quite differently depending on specific ethnic groups, for instance, homozygosity for T allele in position C1236T is 37.5%in Japanese [48], while 13.3% in Caucasians [11]. Different ethnic populations have different SNP frequencies at the same position, which may cause the controversial results.
Genetic association of SNPs, named haplotypes [15], [16], was also found to influence drug pharmacokinetics on MDR1 genotype-phenotype correlation in further studies [6], [20], [49]. Haplotypes analysis in this work provided the evidence that genetic association existed between each other among C1236T, G2677A/T and C3435T, and haplotypes of MDR1 influenced tacrolimus concentration/dose (C/D) ratios in liver transplant recipients. Our findings showed that recipients who carried T-T haplotype and an additional T/T homozygote at either SNPs required higher doses, when C1236T and G2677A/T were combined. The association between haplotypes for G2677A/T and C3435T and tacrolimus C/D ratio was weak after max(T) permutation adjustment.
Patients who have received a new liver, with a different genetic background, will metabolize drugs in different ways. Dose requirements of tacrolimus would be predicted much more precisely, if genetic polymorphism of MDR1 is investigated both in donors and recipients. And several research groups have obtained some helpful results [25], [28], [50].
The ultimate goal of human genetics and genomics studies is to understand the mechanism of gene interaction networks, which would finally explain how gene-drug interactions work [51]. Based on these efforts, pharmacologists and physicians hope that the individualized drug therapy would become reality one day. It is not difficult to identify genes contributing to some phenotype, such as drug pharmacokinetics. However, the phenotype is seldom monogenic. Lots of genes, including downstream molecules, are implicated in biological regulation. To facilitate the identification of these genes, new genome-wide research techniques have been developed. The Affymetrix or Illumina SNP chips are the newest human GWAS (genome wide association study) methods, which produce high throughput SNP data from big ethnic populations with high costs. For instance, by analyzing Affymetrix SNP chips data of a population suffering SLE (systemic lupus erythematosus), several susceptibility genes participating in network of immune response and signal regulation pathway were identified, including immune complex processing and immune signal transduction in lymphocytes [52]. However, only large research groups with enough budgets could afford it. For most research groups, it would be quite sensible to pick up some candidates from databases, and investigate in replicate populations followed by mechanism studies. For those SNPs, which have been proved clinically effective, genotyping with a cost of less than 1 US dollar for each site could significantly promote the development of individualized drug treatment.
In conclusion, our results provided new evidence of the association of MDR1 and tacrolimus dose requirements, which could be a great help to the individualized tacrolimus treatment of liver transplant recipients.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by National Basic Research Program of China (973 program) grant number 2009CB522403, National S&T Major Project grant number 2008ZX10002-026, National Science Fund for Distinguished Young Scholars grant number 30801327, and Nonprofit Technology and Application Research Program of Zhejiang Province grant number 2011C37013. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22110720PONE-D-11–278910.1371/journal.pone.0027684Research ArticleBiologyGeneticsEpigeneticsMolecular Cell BiologySignal TransductionMedicineGastroenterology and HepatologyPancreasOncologyCancers and NeoplasmsGastrointestinal TumorsPancreatic CancerBasic Cancer ResearchExpression of DNMT1 and DNMT3a Are Regulated by GLI1 in Human Pancreatic Cancer DNMT1 and DNMT3a Are Regulated by GLI1He ShanShan
1
Wang Feng
1
Yang LiJuan
1
2
Guo ChuanYong
1
Wan Rong
1
Ke AiWu
1
Xu Ling
1
Hu GuoYong
1
Xu XuanFu
1
Shen Jie
1
Wang XingPeng
1
2
*
1
Department of Gastroenterology, Shanghai 10th People's Hospital, Tongji University, Shanghai, People's Republic of China
2
Department of Gastroenterology, The First People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China
Guan Xin-yuan EditorThe University of Hong Kong, China* E-mail: [email protected] and designed the experiments: SSH FW LJY XPW. Performed the experiments: SSH FW LJY XFX GYH JS. Analyzed the data: SSH LX AWK. Contributed reagents/materials/analysis tools: XPW CYG RW. Wrote the paper: SSH FW AWK.
2011 14 11 2011 6 11 e276845 7 2011 21 10 2011 He et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background and Aims
GLI1, as an indispensable transcriptional factor of Hedgehog signaling pathway, plays an important role in the development of pancreatic cancer (PC). DNA methyltransferases (DNMTs) mediate the methylation of quantity of tumor-related genes. Our study aimed to explore the relationship between GLI1 and DNMTs.
Methods
Expressions of GLI1 and DNMTs were detected in tumor and adjacent normal tissues of PC patients by immunohistochemistry (IHC). PANC-1 cells were treated by cyclopamine and GLI1-siRNA, while BxPC-3 cells were transfected with overexpression-GLI1 lentiviral vector. Then GLI1 and DNMTs expression were analyzed by qRT-PCR and western blot (WB). Then we took chromatin immunoprecipitation (ChIP) to demonstrate GLI1 bind to DNMT1. Finally, nested MSP was taken to valuate the methylation levels of APC and hMLH1, when GLI1 expression altered.
Results
IHC result suggested the expressions of GLI1, DNMT1 and DNMT3a in PC tissues were all higher than those in adjacent normal tissues (p<0.05). After GLI1 expression repressed by cyclopamine in mRNA and protein level (down-regulation 88.1±2.2%, 86.4±2.2%, respectively), DNMT1 and DNMT3a mRNA and protein level decreased by 91.6%±2.2% and 83.8±4.8%, 87.4±2.7% and 84.4±1.3%, respectively. When further knocked down the expression of GLI1 by siRNA (mRNA decreased by 88.6±2.1%, protein decreased by 63.5±4.5%), DNMT1 and DNMT3a mRNA decreased by 80.9±2.3% and 78.6±3.8% and protein decreased by 64.8±2.8% and 67.5±5.6%, respectively. Over-expression of GLI1 by GLI1 gene transfection (mRNA increased by 655.5±85.9%, and protein increased by 272.3±14.4%.), DNMT1 and DNMT3a mRNA and protein increased by 293.0±14.8% and 578.3±58.5%, 143.5±17.4% and 214.0±18.9%, respectively. ChIP assays showed GLI1 protein bound to DNMT1 but not to DNMT3a. Results of nested MSP demonstrated GLI1 expression affected the DNA methylation level of APC but not hMLH1 in PC.
Conclusion
DNMT1 and DNMT3a are regulated by GLI1 in PC, and DNMT1 is its direct target gene.
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Introduction
Pancreatic cancer is a highly lethal disease, which is usually diagnosed in an advanced state for which there are little or no effective therapies. It has the worst prognosis of any major malignancy (3% 5-year survival) and is the fourth most common cause of cancer death yearly in multiple countries. Despite advances in surgical and medical therapy, little effect has been made on the mortality rate of this disease. One of the major hallmarks of pancreatic cancer is its extensive local tumor invasion and early systemic dissemination. So, it is an urgent need to reveal the underlying mechanisms by which pancreatic cancer cells become invasive and metastatic.
Hedgehog signaling cascade is aberrantly activated in a variety of human tumors including pancreatic cancer (PC) [1]. The activation of Hh pathway requires the binding of Hh ligands, such as Shh, Ihh and Dhh, to Hh receptor Patched (Ptch), thus releasing Hh signaling molecule Smoothened (Smo) from Ptch-induced inhibition. Smo in turn initiates the release of the transcription factor GLI from the cytoskeleton by a complex of proteins, thus facilitates its nuclear translocation, GLI activators then bind to the GACCACCCA-like motif for the transcriptional regulation of Hedgehog target genes, which are involved in the regulation of cellular proliferation, cell-fate determination, cellular survival, and epithelial-to mesenchymal transition(EMT) and etc. A membrane glycoprotein Human Hedgehog Interacting Protein (HHIP) can bind to all three Hh ligands and functions to negatively regulate the activity of Hh signaling pathway [2], [3].
DNA methylation change is a key contributor to human oncogenesis [4]. In human cancer cells, the normal somatic pattern of DNA methylation is altered. These changes include increased CpG island methylation, which mediates tumor suppressor gene silencing [4], and genomic DNA hypomethylation, which can lead to genomic instability [5], [6]. Cytosine DNA methylation is catalyzed and regulated by a small family of DNA methyltransferases (DNMTs), including DNMT1, DNMT3a, DNMT3b and DNMT3L [7]. Although cancer-specific mutations of DNMTs have not been reported, several studies suggest that DNMT genes are overexpressed in human cancer and during cellular transformation [8]–[11]. Several mechanisms seem to account for DNMTs over-expression, including aberrant cell cycle control, increased mRNA and protein stability, and E2F-mediated DNMTs promoter activation [11]–[14].
Although the evidences above indicate that DNMTs and active Hh signaling pathway are both involved in the development of pancreatic cancer, little is known about the correlation between DNMTs and members of the Hh pathway. Here, this study was undertaken to investigate the expression of GLI1 and DNMTs, and the correlation between them in human pancreatic cancer.
Materials and Methods
Ethics statement
Tissues of pancreatic cancer and corresponding non-cancerous pancreas were obtained from Shanghai Tenth People's Hospital, where we have obtained ethics approval from Medicine and Life Sciences Ethics Committee.
Cell cultures and drug treatment
Human pancreatic cancer cell line PANC-1 and BxPC-3 were grown in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), streptomycin 100 µg/ml, and penicillin 100 U/ml at 37°C in 5% CO2 and 95% air-humidified incubator. BxPC-3 cells were cultured for lentiviral transfection of overexpression-GLI1 lentiviral vector. PANC-1 cells were plated at a density of 4×104 cells/cm2 in a six-well plate, Cyclopamine (Sigma, St. Louis, MO) was dissolved in 100% ethanol and then diluted fresh on the day of testing for cell culture experiments. PANC-1 cells were treated with the final concentration of Cyclopamine at 10 µM for 24 hours.
Immunohistochemistry (IHC)
Twenty pairs of PC and corresponding non-cancerous pancreas tissues were obtained from Shanghai Tenth People's Hospital. Pancreatic patients' tumor sections were de-paraffinised, rehydrated, treated with 10 mM citrate buffer at 95°C to retrieve antigens, blocked with 5% BSA, and incubated with mouse anti-GLI1 (1∶100), rabbit anti-DNMT1 (1∶100) or rabbit anti-DNMT3a antibody (1∶100; all from Santa Cruz Biotech) overnight. The tissue sections were then incubated with secondary antibodies and DAB reagent (Gene Tech, Shanghai, China). The sections were then counterstained with hematoxylin, then were dehydrated and visualized with 3.3-diaminobenzidine(Gene Tech, Shanghai, China). Negative controls were performed in each case by replacing the primary antibody with PBS.
RT-PCR and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from PANC-1 cells using Trizol reagent (Invitrogen, California, USA), 1 µg RNA was reverse-transcribed to cDNA using PrimeScript RT reagent Kit (Takara Bio, Shiga, Japan). To determine the quantity of mRNA, the cDNA was amplified by real-time PCR with SYBR Premix Ex Taq RT-PCR kit (Takara Bio, Shiga, Japan), and the housekeeping gene β-actin was used as the internal control. The SYBR Green assays were performed in triplicate on a 7900HT real-time instrument (Applied Biosystems, CA, USA). Primers used for qRT-PCR were listed (Table 1). The relative expression levels were calculated using the 2−ΔΔCT method.
10.1371/journal.pone.0027684.t001Table 1 Oligonuleotides used for qRT-PCR.
Gene Accession No. Tm(°C) Size (bp) Sequence (5' to 3')
GLI1 NM_001160045.1 59 491 F - CCAACTCCACAGGCATAC
R - CTTACATACATACGGCTTCTC
DNMT1 NM_001130823.1 60 132 F - CCATCAGGCATTCTACCA
R - CGTTCTCCTTGTCTTCTCT
DNMT3a NM_022552.3 60 111 F - TATTGATGAGCGCACAAGAGAGC
R - GGGTGTTCCAGGGTAACATTGAG
Western Blot (WB)
Total cell lysate was prepared in a 1× sodium dodecyl sulfate buffer. Proteins in the same amount were separated by 6% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. After incubation with antibodies specific for DNMT1 (Santa Cruz Biotechnology, Santa Cruz, CA) or DNMT3a (Santa Cruz Biotechnology, Santa Cruz, CA) or GLI1 (Santa Cruz Biotechnology, Santa Cruz, CA) or β-actin (Cell Signaling Technology, Danvers, MA), the blots were incubated with goat anti-rabbit or anti-mouse secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and visualized with enhanced chemiluminescence.
Small interfering RNA mediated inhibition of GLI1 expression
Stealth small interference RNA (siRNA) sequences for GLI1 were designed and synthesized by GenePharma to target GLI1 mRNA. The coding strand for GLI1 siRNA was 5'-GGCTCAGCTTGTGTGTAAT-3'. An unrelated siRNA sequence was used as a control. In this experiment, cells were incubated for 12 h and transfected at approximately 60% confluency with 50 nm siRNA duplexes using Lipofectamine™2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. All the experiments were performed 72 hours after transfection.
Lentiviral Transfection of Overexpression-GLI1 Lentiviral Vector
Lentiviral transfection of overexpression-GLI1 lentiviral vector pGC-FU-GLI1 were performed as we reported previously [15]. Human GLI1 cDNA was purchased from Open-Biosystem (USA). The complete cDNA sequence of GLI1 was generated by PCR, and inserted into pGC-FU-3FLAG Vector (GeneChem Company, Shanghai, China) which was linearized with Age I and Nhe I (Fig. S1, S2). The resultant 3320-bp fragment was confirmed by sequencing (Fig. S3). Lentiviral vector were produced by co-transfected into 293T cells with helper construct. Titers of 2–5×107 TU/ml were routinely achieved. BxPC-3 cells were transfected with the lentiviral vector and GLI1 expression was established by real-time PCR and western blot analysis.
Chromatin Immunoprecipitation (ChIP)
DNA-GLI1-protein immune complexes were preparated as we reported previously [15], after reverse cross-linked, DNA was extracted with phenol/chloroform and precipitated. The presence of the DNMT1 and DNMT3a promoter domain containing GLI1 motifs in immunoprecipitated DNA was identified by PCR using primers (table 2). The PCR conditions for the DNMT1 and DNMT3a promoter region were: denaturation 30 seconds at 94°C, annealing 30 s, elongation 1 minute at 72°C. Annealing temperatures were listed in table 2. The amplification of the DNMT1 and DNMT3a promoter region was analyzed after 40 cycles. All experiments were repeated at least three times.
10.1371/journal.pone.0027684.t002Table 2 Oligonucleotides used for XChIP-PCR.
Gene Site Primer Tm(°C) Size (bp) Sequence (5' to 3')
DNMT1 1 DNMT1-A 53 317 F - GCTGAGGCATGAGAATCGCTTGAA
R - GGAGGATCGCTTGAGGTTAGGAGTT
2 DNMT1-B 54 243 F - AGGCTGGAATGTAGTGGTACAATCA
R - AGGGTGGGAGGATCGCTTGA
2 and 3 DNMT1-C 54 214 F - GTGATCTTCCTGCCTCAACCTCTG
R - CGCCTGTCATCCCAGCACTTT
4 DNMT1-D 55 123 F - CCAAAGTGCTGGGATGACAGG
R - GCTAGTACCAAGAATCTCACAGTGTA
5 DNMT1-E 53 400 F - GAGGTTGGATTGGAACTGAGGACTT
R - CATCTCGGAGGCTTCAGCAGAC
DNMT3a 1 DNMT3a-A 54 332 F - AGGCTGGAATGTAGTGGTACAATCA
R - AGGGTGGGAGGATCGCTTGA
2 and 3 DNMT3a-B 55 124 F - CCACCACCAACTCCAGCAATC
R - CTACTCAGCACTTCAGCTATATCACA
4 DNMT3a-C 55 194 F - GCCATGTCCTGTGCCAGTCA
R - CTCACTATGTGCTCATCTCACTCCT
5 DNMT3a-D 57 182 F - TGAGTGGCTGTGCTGGTGGAA
R - TGAGGTGGGAGGTTGAATGAAATGAC
6 DNMT3a-E 54 100 F - ATCTTTCAGTCTTCCAGTGCCCAAC
R - TCTCTGAGATGAGCTGCCTTGAAG
Supplementary figure legends.
DNA preparation
DNA were extracted by TIANamp Genomic DNA Kit (Tiangen, Beijing, China). Approximately 500 ng extracted DNA were bisulfite conversed and column-purified by EZ DNA Methylation-Gold™ Kit (Zymo Research, Orange, CA , USA) to make 10 µl sample.
Nested MSP
DNA methylation status on the promoter regions of the APC (adenomatous polyposis coli) and hMLH1 (human mutl homolog 1) were determined by the method of MSP further modified as a nested two-step approach with the primers described previously [16], [17]. In the step one of the nested MSP, primers were designed to amplify both methylated and unmethylated genomic regions. Products were equally diluted 1∶100 and subjected to the step two of the nested MSP with primers designed to recognize bisulfite-induced sequence differences between methylated and unmethylated genomic regions. The PCR conditions for step one were as follows: 95°C hot start×5 min, then 40 repetitive cycles of denaturation (95°C×30 s), annealing (56°C×30 s), extension (72°C×30 s) followed by a final 5 min extension at 72°C. And that for Step two were: 95°C hot start×5 min, then 30 repetitive cycles of denaturation (95°C×30 s), annealing (59°C×30 s for APC, 60°C×30 s for hMLH1), extension (72°C×30 s) followed by a final 5 min extension at 72°C. MSP products were separated electrophoretically on 2% agarose gels.
Statistical Analysis
Quantitative data are expressed as the mean ± standard deviation (SD). Real-time PCR data was analyzed according to the differences of target gene expression by the paired t-test and were 2−ΔΔCT transformed before analysis. IHC data was analyzed using the Chi-squared test. A p-value of less than 0.05 was considered statistically significant.
Results
GLI1, DNMT1 and DNMT3a were up-regulated in human pancreatic cancer tissues
To confirm the roles of GLI1 and DNMTs in the development of human pancreatic cancer, we first examined whether their expressions were altered in cancer tissues. Therefore, we studied GLI1, DNMT1 and DNMT3a expression in 20 paired biopsy tissues of PC patients by IHC. We found that GLI1, DNMT1 and DNMT3a expression were all higher in most PC compared with normal tissues (14/20 versus 5/20, p = 0.004; 15/20 versus 6/20, p = 0.004; 13/20 versus 5/20, p = 0.011; respectively; Figure 1). 14 of 20 PC cases had higher expression of GLI1 protein, among which 12 cases expressed higher levels of DNMT1 protein (p = 0.004) and 11 cases expressed higher levels of DNMT3a protein (p = 0.012).
10.1371/journal.pone.0027684.g001Figure 1 GLI1, DNMT1 and DNMT3a protein expression in PC tissues and adjacent normal tissues.
Immunohistochemical examination for GLI1, DNMT1, DNMT3a protein were performed in 20 pairs of PC and adjacent normal tissues. Representative pictures are shown. Adjacent normal tissues exhibited no or faint staining for GLI1, DNMT1 and DNMT3a, however, the incidence of all the three proteins nuclear immunoreactivity was much higher in PC tissues. All photomicrographs were obtained at ×200 magnification.
Cyclopamine and GLI1 siRNA both inhibited DNMT1 and DNMT3a expression
To determine whether Hh activity affected the expression of DNMTs, we used cyclopamine, a classical inhibitor of Hh signaling pathway, to decrease the expression of GLI1. PANC-1 cells, which were previously reported to express a high level of GLI1 [15],were treated with 10 µM cyclopamine for 24 h. Afterward, qRT-PCR and WB were taken to analyze the expression of GLI1 and DNMTs. DNMT1 and DNMT3a mRNA decreased by 91.6.0±2.2% and 83.8±4.8%, respectively, when GLI1 mRNA decreased by 88.1±2.2%. DNMT1 and DNMT3a protein decreased by 87.4±2.7% and 84.4±1.3% when GLI1 protein decreased by 86.4±2.2% (Figure 2).
10.1371/journal.pone.0027684.g002Figure 2 GLI1, DNMT1 and DNMT3a were inhibited in PANC-1 cell line after which treated by Cyclopamine.
PANC-1 cells were treated with 10 µM cyclopamine for 24 hours, then relative expression of GLI1, DNMT1 and DNMT3a mRNA was assessed by qRT-PCR (A, B, C), while the expression of GLI1, DNMT1 and DNMT3a protein was analyzed by Western blot (D). The inset shows a substantial decrease in GLI1, DNMT1 and DNMT3a expression. The results were normalized to that of β-actin expression. All data were presented as the mean ± SD of three independent experiments.
We further designed and synthesized GLI1 siRNA, then transfected into PANC-1 cell line. PANC-1 cells transfected with an unrelated siRNA sequence was used as a negative control [18], and PANC-1 those treated with Lipofectamine™2000 (Invitrogen, Carlsbad, CA) only was used as blank control. 72 hours after transfection, qRT-PCR and WB were taken to determine the expression of GLI1 and DNMTs. DNMT1 and DNMT3a mRNA decreased by 80.9±2.3% and 78.6±3.8%, respectively, when GLI1 mRNA decreased by 88.6±2.1%. DNMT1 and DNMT3a protein decreased by 64.8±2.8% and 67.5±5.6% when GLI1 protein decreased by 63.5±4.5% (Figure 3).
10.1371/journal.pone.0027684.g003Figure 3 DNMT1 and DNMT3a were suppressed after transfected by GLI1-siRNA in PANC-1 cells.
PANC-1 cells were transfected with GLI1 siRNA, 72 hours after transfection, relative expression of GLI1, DNMT1 and DNMT3a mRNA was assessed by qRT-PCR (A, B, C), while the expression of GLI1, DNMT1 and DNMT3a protein was analyzed by Western blot (D). The inset shows a substantial decrease in DNMT1 and DNMT3a expression after GLI1 interference. The results were normalized to that of β-actin expression. All data were presented as the mean ± SD of three independent experiments.
The expression of DNMT1 and DNMT3a were upregulated with GLI1 overexpression
To further confirm the regulation of DNMT1 and DNMT3a by GLI1, we designed and constructed a lentivirus vector that overexpressed GLI1, and transfected it into BxPC-3 with the lowest GLI1 expression in PC cell lines as we previous reported [15]. Cells transfected with empty lentivirus vector were used as negative controls, while cells without transfection were used as blank controls. 48 hours after transfection, qRT-PCR and WB were taken to determine the expression of GLI1 and DNMTs in the three cell lines. DNMT1 and DNMT3a mRNA increased by 293.0±14.8% and 578.3±58.5%, respectively, when GLI1 mRNA increased by 655.5±85.9%. DNMT1 and DNMT3a protein increased by 143.5±17.4% and 214.0±18.9%, respectively, when GLI1 protein increased by 272.3±14.4% (Figure 4).
10.1371/journal.pone.0027684.g004Figure 4 DNMT1 and DNMT3a were up-regulated after GLI1 over-expression.
BxPC-3 cells were transfected with pGC-FU-GLI1, relative expression of GLI1, DNMT1 and DNMT3a mRNA was assessed by qRT-PCR (A, B, C), while the expression of GLI1, DNMT1 and DNMT3a protein was analyzed by Western blot (D). The inset showed a substantial increase in DNMT1 and DNMT3a expression after GLI1 over-expression. The results were normalized to that of β-actin expression. All data were presented as the mean ± SD of three independent experiments.
Confirmation of GLI1 protein bound to promoter region of DNMT1 gene
We have, so far, proved the role of GLI1 in DNMT1 and DNMT3 expression. However, the mechanism underlying such regulation remains to be elucidated. To explore whether DNMTs are directly regulated by GLI1 or not, we searched the DNMT1 and DNMT3a promoter for potential GLI1 binding sites to the DNA consensus sequence 5'-GACCACCCA-3'
[19] or 5'-TGGGTGGTC-3'
[20], and five high-scoring candidate sites of GLI1 targets were found in DNMT1's promoter and six in DNMT3a's. Each site has only two nucleotide's difference in compare with 5'-GACCACCCA-3' or 5'-TGGGTGGTC-3' (Figure 5). ChIP was taken to confirm the bounding relationship between GLI protein and DNMT1/3a gene. DNA extracted from PANC-1 cells were sonicated into 100–1000 bp (Fig. S4) and as the ChIP-PCR template. The result of DNA electrophoresis showed the predicted DNA band in INPUT, GLI1-Ab, and postive control groups using human DNMT1 primer-C, and not in the IgG and negative control groups (Figure 6). Only INPUT and the positive control showed the predicted band using human DNMT1 primer-A, C-E and DNMT3a primer A-E but not in GLI-Ab, IgG, and negative groups (data not shown). As positive product amplified by DNMT1 primer-C contains candidate GLI1 binding site 2 and 3 (Table 2 and Figure 5), while product amplified by DNMT1 primer-B contains candidate GLI1 binding site 2 was negative, and results of sequence analysis showed that the sequences were the same as that of the DNMT1 gene promoter of site 3 (Fig. S5), suggested that GLI1 was bound to the DNMT1 gene promoter of site 3 (GGCCTCCCA).
10.1371/journal.pone.0027684.g005Figure 5 Potential GLI1 binding sites on the DNMT1's and DNMT3a's promoter.
Two parallel lines on top of the figure represented DNMT1 or DNMT3a DNA, respectively(A, B), within which gray frames represented exons, white frames represented introns, and black frames represented promoter. In the promoter, small gray frames marked with number 1 to 5 (A) or 1 to 6 (B) represented the potential GLI1 binding sites, which has only two nucleotides difference (underlined) from GLI1 consensus binding sequence, GACCACCCA. Primers were designed to amplify the DNMT1 (A) or DNMT3a (B) promoter region containing the putative GLI1-binding site. The position and length of products amplificated by each primer were shown.
10.1371/journal.pone.0027684.g006Figure 6 Identification of GLI1 binding to DNMT1 promoter by Chromatin Immunoprecipitation (ChIP).
Lysates from PANC-1 cells were subjected to Chromatin immunoprecipitation by anti-GLI1 antibody. Sonicated chromatin was used as INPUT DNA control (INPUT). RNA polymerase II was used as positive control (PC). IgG was used as random control (IgG) and β-actin Ab was used as negative control (NC). The band of ChIP-PCR products amplified by DNMT1 Primer-C (i) and by DNMT1 Primer-B (ii) were shown.
The DNA methylation levels of APC but not hMLH1 promoter regions changed with GLI1 expression
Amount of tumor-related genes were found to be silenced by DNA methylation in PC, including APC (adenomatous polyposis coli) and hMLH1 (human mutl homolog 1). To access whether inhibiting or increasing the expression of GLI1 could also lead to hypo- or hyper-methylation of APC and hMLH1 in PC, we used nested MSP to evaluate the methylation status of PANC-1 with or without GLI1 knockdown and BxPC-3 with or without GLI1 over-expression, respectively. Results showed that APC DNA methylation level increased in BxPC-3 cells transfected with Overexpression-GLI1 Lentiviral Vector in comparison with the negative control, and was inhibited in PANC-1 cells transfected with GLI1-siRNA in comparison with the negative control. However, the DNA methylation level of hMLH1 promoter region was not significantly changed after transfection (Figure 7). This probably because DNA methylation is coordinated by a family of DNMTs comprising DNMT1, -3a, -3b and -3L, maybe the expression change of only DNMT1 and -3a regulated by GLI1 was not sufficient to affect the DNA methylation levels of every tumor-related genes [21].
10.1371/journal.pone.0027684.g007Figure 7 Up- or under-expression of GLI1 affected DNA methylation levels of APC but not hMLH1.
Results of nested MSP of APC and hMLH1 in six kinds of PC cells respectively were shown. B represented BxPC-3 cells; B-G+ represented BxPC-3 transfected with pGC-FU-GLI1 to make GLI1 over-expression, and B-NC represented its negative control; P represented PANC-1; P-G-si represented PANC-1 transfected with GLI1-siRNA and P-NC represented its negative control. Positive bands under M and U represented the methylated and unmethylated DNA of the corresponding genes in the right panel, respectively.
Discussion
In this study, we found that GLI1, DNMT1 and DNMT3a are over-expressed in PC tissues compared with the corresponding non-cancerous pancreas tissues, then we showed that DNMT1 and DNMT3a expression changed according to the GLI1 expression in PANC-1 and BxPC-3 cell lines by specific GLI1 interference and gene transfection, as well as pharmacological method in vivo. More importantly, we proved beyond a reasonable doubt that GLI1 was able to bind to the DNMT1 gene promoter of site 3 (GGCCTCCCA) by the ChIP experiments. Finally, we used nested MSP to demonstrate that GLI1 expression affected the DNA methylation level of APC but not hMLH1 in PC. To the best of our knowledge, this is the first report demonstrated GLI1 as a transcriptional factor that regulated DNMT1 and -3a expression as well as APC methylation level in PC, and DNMT1 is its direct target gene.
GLI1, as a transcriptional factor of Hh signaling pathway, is upregulated in most digestive tumors including PC [22], [23]. Thus far, only a few downstream targets of GLI1 have been identified [24]. Recently, it was reported to be involved in PC invasion and metastasis, and has become a new target for treatment [25], [26]. However, little was known about the actual mechanism implied in its promotion of invasion and metastasis in PC. Moreover, we focused on accumulating evidence which demonstrated that carcinoma in various organs, including pancreas, is associated with aberrant DNA methylation, in which DNMTs is the key catalyst significantly correlated with accumulation of methylation of tumor-related genes, among which some were associated withcell proliferation such as APC, some were related with the reparation of DNA damage such as hMLH1, some were invasion- or metastasis-related, such as TIMP-3, SPARK, and CDH1, or cell death-related such as DAPK-1, thus playing an important role in multistage carcinogenesis of the pancreas from early precancerous stages to malignant progression [27]. Recently, it was found that tumor burden is significantly reduced with decreasing DNMT1 levels in vivo, suggesting that DNMTs mediated DNA methylation is involved in pancreatic carcinogenesis [28]. Based on this study and previous reports above, it's possible that GLI1-DNMTs cascade help to invasion or metastasis through promoting the methylation of some invasion- or metastasis-related genes, and may facilitate tumor growth by promoting the methylation of some cell death-related genes.
Our study showed that DNMT3a expression is regulated by GLI1 in human pancreatic cancer. However, the actual mechanism in the regulation of DNMT3a by GLI1 is still unknown. Recent years, many manuscripts have been reported that some microRNA families could target DNMTs in a diversity of human cancers [29]–[32]. On the other hand, it was reported that some microRNA such as microRNA-29 family was transcriptional suppressed by c-Myc, hedgehog and NF-kappaB [33]. Based on the evidence above, it is possible that Hh-GLI might regulate DNMT3a through some certain microRNAs, which remains to be explored.
In our study, ChIP assays showed GLI1 bind to DNMT1 but not DNMT3a. We also noticed that GLI1 elevated DNMT3a more folds than DNMT1. We thought there were some possible underlying mechanisms as follows: First, GLI1 might not regulate DNMT3a directly but through a certain gene, which might be a kinase or activin, and via cascade amplification so as to lead a higher regulative efficiency of DNMT3a by GLI1. Second, Hedghog-GLI1 might directly or indirectly regulate several genes involved in different signaling pathways, and two or more of these genes also regulate DNMT3a and have synergetic effects, so that despite GLI1 might not regulate DNMT3a directly, but would elevate DNMT3a more folds when it over-expresses. To solve this question, it's necessary to explore more target genes of Hedgehog-GLI1, and to probe into the crosstalk between various signaling pathways. We thought the regulative relationship between Hh-GLI1 and DNMTs would be not so simple as we already confirmed. Further studies are also needed to explore whether the biological behavior of GLI1 in PC may be achieved by regulating DNMTs.
The newly identified GLI1/DNMTs axis set a bridge between Hh signaling pathway and epigenetics, which would help to elucidate the underling molecular mechanism in the development of PC, and may provide new therapeutic targets or biomarkers for earlier diagnosis.
Supporting Information
Figure S1
Identificaton of positive clone products in overexpression-GLI1 lentiviral vector construction. The GLI1 cDNA products were inserted into linearized pGC-FU-3FLAG vector to construct pGC-FU-GLI1 plasmid after amplified and purified, then transformed into Competent Cells . Transformants were identified with PCR and electrophoresed on 1.5% agarose gel, transformants-1 and -4 were showed as a 731 bp band which proved to be positive clone.
(TIF)
Click here for additional data file.
Figure S2
Identification of GLI1 expression in pGC-FU-GLI1 clone by WB. pGC-FU-GLI1 is constructed as a lentivirus vector expressed GLI1, which was co-expressed with FLAG. (1) WB molecular Weight Marker, with 3-FLAG label, fused with GFP gene(48 KDa). (5–8) Sample after pGC-FU-GLI1 transfected 293T cells. (7) GLI1-FLAG fusion protein (122 KDa+2 KDa = 124 KDa), certificated GLI1 expression in pGC-FU-GLI1 plasmid.
(TIF)
Click here for additional data file.
Figure S3
Sequence analysis of positive clone products in GLI1-overexpression lentiviral vector construction. The resultant 3320-bp fragment was confirmed by sequencing which is the same with the sequence of the GLI1 gene expression region in GenBank (NM_005269.2).
(TIF)
Click here for additional data file.
Figure S4
Electropheretogram of sonicated chromatin solution. Sonicated chromatin solution in different conditions (100 W, 80 W and 60 W, respectively) were electrophoresed on 1.5% agarose gel containing ethidium bromied.
(TIF)
Click here for additional data file.
Figure S5
Sequence analysis of ChIP products which amplified by DNMT1 primer-C. The result showed that the sequence amplified with DNMT1 primer-C is the same as that of DNMT1 gene promoter region containing GLI1-binding site 2 and 3.
(TIF)
Click here for additional data file.
Competing Interests: The authors have declared that no competing interests exist.
Funding: National Natural Science Foundation of China (81072005, 81172312) and Shanghai Science and Technology Committee (08411963000, 09JC1412200). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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BMC ImmunolBMC Immunology1471-2172BioMed Central 1471-2172-12-632207438910.1186/1471-2172-12-63Research ArticleThe absence of MyD88 has no effect on the induction of alternatively activated macrophage during Fasciola hepatica infection Luo HongLin [email protected] Weiyi [email protected] Dongying [email protected] Haoju [email protected] Kui [email protected] Laboratory of Infection & Immunology Research, College of Animal Science & Technology, Southwest University, Chongqing, China2 Parasitology department, College of Animal Science & Technology, Guangxi University, Nanning, China3 ENVA, UMR BIPAR, Ecopham, Ecole Nationale Vétérinaire d'Alfort, Maisons-Alfort, France2011 11 11 2011 12 63 63 21 9 2011 11 11 2011 Copyright ©2011 Luo et al; licensee BioMed Central Ltd.2011Luo et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
Alternatively activated macrophages (AAMϕ) play important roles in allergies and responses to parasitic infections. However, whether signaling through toll-like receptors (TLRs) plays any role in AAMϕ induction when young Fasciola hepatica penetrates the liver capsule and migrates through the liver tissue is still unclear.
Results
The data show that the lack of myeloid differentiation factor 88 (MyD88) has no effect on the AAMϕ derived from the bone marrow (BMMϕ) in vitro and does not impair the mRNA expression of arginase-1, resistin-like molecule (RELMα), and Ym1 in BMMϕs. The Th2 cytokine production bias in splenocytes was not significantly altered in F. hepatica-infected mice in the absence of MyD88 in vitro and in the pleural cavity lavage in vivo. In addition, MyD88-deficiency has no effect on the arginase production of the F. hepatica elicited macrophages (Fe Mϕs), production of RELMα and Ym1 proteins and mRNA expression of Ym1 and RELMα of macrophages in the peritoneal cavity 6 weeks post F. hepatica infection.
Conclusions
The absence of MyD88 has no effect on presence of AAMϕ 6 weeks post F. hepatica infection.
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Background
Macrophages are highly plastic cells that respond to diverse environments by altering their phenotype and physiology [1,2] and play important roles in both innate and adaptive immunity. Currently, macrophages are classified under two phenotypes, classically activated macrophages (CAMΦ) and alternatively activated macrophages (AAMΦ). CAMΦ are induced by interferon-gamma (IFN-γ) and lipopolysaccharide (LPS), whereas induction of the AAMΦ phenotype is associated with various stimuli, such as IL-4/IL-13, IL-10, immunocomplexes, and glucocorticoids [2]. The most widely studied stimuli for generating AAMΦ is treatment with IL-4/IL-13 [1,3]. Although IL-4/IL-13 signaling are essential to the presence of AAMΦ and both cytokines have many overlapping activities on macrophages, they exhibit distinct functions because of their specific receptor subunits aside from their shared common alpha chain [4]. However, this does not alter the fact that a Th2-dominated environment is critical for AAMΦ induction [5-7]. All helminths have been demonstrated to induce profound Th2 responses, which are characterized by the production of IL-4, IL-5, IL-9, IL-10, and IL-13 by CD4+ T cells [8], and this Th2-dominated cytokine profile is associated with the presence of the AAMΦ phenotype (such as in Schistosoma mansoni [9], Taenia crassiceps [10], Brugia malayi [11], Heligmosomoides polygyrus [12], Nippostrongylus brasiliensis [13], and F. hepatica infection [14,15], and so forth). AAMΦ are increasingly recognized as a key effector arm of the Th2 immunity, but their real function in various helminth infections has not been illustrated and is likely to be diverse. However, discovery of molecular markers of AAMΦ, such as mannose receptor (CD206), IL-10, arginase -1 (instead of inducible nitric oxide), resistin-like molecule (RELMα), and Ym1 [6,16-19], made the identification of AAMΦ possible. Among them, three most abundant IL-4/IL-13 dependent genes: Ym1, a member of the family 18 chitinases family but with no chitinolytic activity [20], RELMα, was described as FIZZ1 [21], and is identified as a cysteine-rich molecule associated with resistin that is involved in glucose metabolism [22]. Arginase 1 plays a role in the regulation of nitric oxide (NO) production by competing with iNOS for substrate L-arginine [23], suppression of T cell responses via L-arginine depletion [24] and has been currently accepted as a molecular signature for AAMΦ. However, the functions of AAMΦ in helminth infections have not been fully illustrated. Questions such as whether AAMΦ promotes helminth killing or expulsion, whether alternative activation requires anti-worm effector function, or whether signaling TLRs play a role in AAMΦ induction, have not been fully answered.
In the current study, the role of TLRs in AAMΦ induction during F. hepatica infection, which has been observed to produce Th2-dominated responses in both mice and the natural ruminant hosts [14,15] is investigated using MyD88-deficient mice.
TLRs are pattern recognition receptors (PRRs) that recognize different pathogen-associated molecular patterns (PAMPs) [25]. TLR signaling is mostly MyD88-dependent [26,27] except for TLR3 signaling, which requires a TRIF adaptor [26,27]. Mice lacking MyD88 cannot respond to LPS [28]. The absence of MyD88 has been demonstrated to have no effect on the augmentation of Th2 responses [29,30], which indicates that Th2 responses are elicited in a MyD88-independent manner. However, contrary results have also demonstrated that TLR signaling plays a role in Th2 responses [31]. Aside from the required Th2 environment, especially with IL-4/IL-13, the determinants of the AAMΦ phenotype remain unclear. For F. hepatica infection, whether TLR signaling is required for AAMΦ induction is unknown. Therefore, whether AAMΦ could be induced without MyD88 and whether TLR signals affect AAMϕ activation were investigated.
Results
Lacking of MyD88 has no impact on the presence of AAMΦ derived from bone marrow
To determine whether MyD88 signaling is required for AAMΦ induction, investigations were made to determine whether BMMϕ, cultured from both WT and MyD88 knockout mice, could be activated through this approach. This is based on the fact that arginase-1 upregulation, which is generally associated with Th2 cytokines, such as IL-4 [32], is a feature of AAMΦs. As a control, iNOS, the enzyme associated with CAMΦs, was also detected [33]. BMMϕs were treated with LPS and IFN-γ, either together or separately, or with media alone for 20-24 h after overnight treatment with or without IL-4. Subsequently, arginase-1 and iNOS enzyme activity were measured (Figure 1). The results show both WT and MyD88-/- BMMϕ cells displayed arginase activity in response to IL-4 compared with the cells without IL-4 stimulation even though no statistically significant differences were found between groups (Figure 1A). WT BMMϕs treated with LPS also exhibited arginase activity and were absent in MyD88-/- mice. iNOS activity was monitored by measuring nitrite concentrations in the BMMϕ supernatant fluids using the Greiss reagent. The results show that WT BMMϕs (with or without IL-4 stimulation) produced nitrite when stimulated with LPS alone and with IFN-γ. A synergistic effect was observed on the iNOS activity with further upregulation when treated with both LPS and IFN-γ compared with LPS or IFN-γ treatment alone (Figure 1B). MyD88-/- BMMϕ showed similar trend with WT BMMϕ on NO production. However, almost no NO was detected upon stimulation with LPS alone compared with WT BMMϕs. Moreover, NO could be produced via pretreatment with IL-4 followed by stimulation with LPS alone. Treatment with IFN-γ alone had no effect on NO production (in both WT and MyD88-/- BMMϕ) even with IL-4 treatment (Figure 1B).
Figure 1 The absence of MyD88 has no effect on the AAMϕs derived from bone marrow in vitro. The BMMϕ were treated with or without IL-4 followed by the stimulation with LPS and IFN-γ together or separately for 12-24 h. (A). The urea concentration represents the arginase activity after the stimulation. (B). The nitrite concentration is shown as a measure of iNOS activity produced by the BMMϕs. Five mice were tested per group. Results are compiled from three independent experiments.
MyD88-deficiency of has no effect on arginase-1, RELMα, and Ym1 expression in BMMϕs
To ascertain further the phenotype of the AAMϕs elicited by the F. hepatica infection, the gene expression of AAMϕ markers arginase-1, RELMα, and Ym1 in BMMϕ were measured via real-time reverse transcriptase polymerase chain reaction (RT-PCR). The results show no deficiency in AAMϕgeneration in the MyD88-/- animals. Figure 2 shows that arginase-1 expression in MyD88-/- BMMϕ treated with IL-4 was lower than that of WT BMMϕ, and no expression on both untreated WT and MyD88-/- BMMϕ. The expression profiles of both RELMα and Ym1 in the BMMϕs show similar trends with higher levels on the MyD88-/- BMMϕ treated with IL-4; however, no statistically significant differences were found (Figure 2).
Figure 2 MyD88-deficiency did not impair arginase-1, RELMα, and Ym1 expression in BMMϕs. mRNA was extracted and real-time RT-PCR for arginase-1, RELMα, and Ym1 expression was carried out to examine AAMϕ generation. The black bars represent the mRNA expression in mice treated with IL-4. The mRNA expression is shown as a percent of the positive control sample and β-actin was used to normalize the experiments. Results are compiled from three independent experiments.
The Th2 cytokine bias is not significantly altered in F. hepatica-infected mice in the absence of MyD88 in vitro and in vivo
Ascertaining any effects on the overall Th2 response in F. hepatica-infected mice is essential before determining the effect of MyD88 knockdown on macrophage activation in vivo. Consequently, the Th2 cytokines IL-4, IL-5, IL-10, and IL-13, together with IFN-γ, a marker of Th1 response, were monitored in the supernatant fluids of cultured splenocytes treated with media, anti-CD3, or FhAg (Figures 3A-3E). The results showed that all Th2 cytokines were elevated on FhAg stimulation in the infected WT mice and were further increased in the MyD88-/- infected mice, although no statistically significant difference was found between the WT and MyD88-/- infected mice. In contrast, the Th1 response, as measured with IFN-γ decreased significantly upon stimulation with FhAg or anti-CD3 but not media in the infected MyD88-/- mice compared with the WT infected mice (Figure 3E). Meanwhile, the thioglycolate-injected WT and MyD88-/- mice exhibited low levels of both Th1 and Th2 compared with their infected counterparts. The local cytokine production in the PEC supernatant fluid (Figures 4A-4E) was also measured. Significantly higher IL-4 levels were observed in the MyD88-/- infected mice compared with both the WT and the WT-infected mice (Figure. 4 A). Similar trends were seen in cytokine IL-5 and IL-13 (Figures 4B and 4C). However, no statistically significant difference in IL-10 production was found between the WT and MyD88-/- mice in both the thio and infection treatments. In contrast, the IFN-γ in the both MyD88-/- thio and the MyD88-/- infected mice significantly decreased compared with that in THE F. hepatica-infected WT mice. Overall, these results show that the Th2 response is not impaired in F. hepatica-infected MyD88-/- mice both in vitro and vivo.
Figure 3 The Th2 cytokine production bias in splenocytes was not significantly altered in the F. hepatica-infected mice in the absence of MyD88 in vitro. Splenocytes collected from C57BL/6 mice were stimulated with media alone, anti-CD3, or F. hepatica antigen (FhAg) for 72 h. The levels of IFN-γ, IL-4, IL-5, IL-10, and IL-13 in the supernate were measured indirectly via ELISA. The statistical significant differences were determined via one-way ANOVA followed by a Kruskal-Wallis test. *p < 0.05. Five mice were tested per group. Results are compiled from three independent experiments.
Figure 4 The Th2 cytokine production bias in the pleural cavity lavage was not significantly altered in the MyD88-deficient F. hepatica-infected mice. Peritoneal exudate cells (PEC) were collected from the C57BL/6 mice and were treated with media alone, anti-CD3, or F. hepatica antigen (FhAg) for 72 h. The levels of IFN-γ, IL-4, IL-5, IL-10, and IL-13 in the supernate were measured indirectly via ELISA. Statistical significances were determined via one-way ANOVA followed by a Kruskal-Wallis test. * p < 0.05 and ** p < 0.01. Five mice were tested per group. Results are compiled from three independent experiments.
MyD88-deficiency did not affect the arginase production of the F. hepatica-elicited macrophages in PECs
To determine whether MyD88-deficiency affects production in macrophages recruited in PEC, the arginase activity levels were measured using the purified macrophages from the PEC (Figure 5). As shown, macrophages from F. hepatica-infected mice produced more arginase than macrophages from the mice treated with thioglycolate, and the MyD88-/- and WT infected mice produced significantly higher arginase than the MyD88-/- thio mice, although no difference was seen between the WT thio and the WT infected mice. In addition, no significant difference in arginase levels was observed between the WT and MyD88-/- infected mice.
Figure 5 MyD88-deficiency has no effect on arginase production of the F. hepatica-elicited macrophage (FeMϕ). Six weeks after F. hepatica infection, macrophages were purified from the PEC by adherence and the levels of arginase activity were measured. Thioglycolate (thio) was also injected as the control. Significant differences were determined via one-way ANOVA followed by a Kruskal-Wallis test. *p < 0.05 and ** p < 0.01. Results are compiled from three independent experiments.
The absence of MyD88 had no effect on RELMα and Ym1 production by cells in the peritoneal cavity after F. hepatica infection
We investigated whether the absence of MyD88 impairs RELMα and Ym1 expression in peritoneal lavage fluid using western blot analysis (Figures 6A and 6B) and real-time RT-PCR (Figure 7). As shown in the figure, although both the RELMα and Ym1 protein signals were detected in the WT and the MyD88-/- infected mice, no significant difference was found between these two groups. To determine whether the mRNA expression levels correlates with protein production, total RNA was extracted from purified macrophages in the peritoneal cavity and real-time RT-PCR was performed to detect RELMα, Ym1, and arginase1 mRNA expression (Figures 7A and 7B). Although WT and MyD88-/- infected mice produced detectable RELMα and Ym1, they could not be detected in the WT and MyD88-/- thio mice. No statistically significant difference in RELMα and Ym1 mRNA expression was observed between the WT and the MyD88-/- infected mice, which is consistent with the protein profiles of RELMα and Ym1 above.
Figure 6 The Absence of MyD88 had no effect on the production of RELMα and Ym1 proteins by cells in the peritoneal cavity after F. hepatica infection. Six weeks after F. hepatica infection of C57BL/6 mice, peritoneal cavity lavages were performed and western blots for RELMα (A) and Ym1 (B) were carried out on the lavage fluid. Image software was used to measure the relative concentrations of proteins on the blots.
Figure 7 The lack of MyD88 has no effect on Ym1 and RELMα mRNA expression of macrophages from the peritoneal cavity. Six weeks after F. hepatica infection of C57BL/6 mice, the peritoneal cavity lavages were collected and macrophages were purified. RNA extraction and real-time RT-PCR were used for detection of RELMα (A) and Ym1 (B). mRNA expression is shown as a percent of the positive control sample and β-actin was used to normalize the experiments.
Discussion
The data demonstrates that macrophages from a chronic infection, which consequently produce Th2 type cytokines at the stage wherein young F. hepatica penetrates the liver capsule and migrates through the liver tissue, do not require TLR signaling for AAMΦ induction.
Like many other helminths, to establish successful chronic infections, F. hepatica induces Th2 responses characterized by increased IL-4, IL-5, and IL-13, activation and expansion of eosinophils, CD4+cells, basophils, and mast cells [8-10,13,34-39]. Simultaneously, helminths release excretory-secretory proteins (ESP) to prevent dendritic cells and macrophages from acting on TLR2 Th1-stimulating ligands such as LPS and CpG during infections [40-42]. For example, cathepsin L1 cysteine protease released by F. hepatica suppresses the macrophage TLR recognition of LPS [43]. However, different infective stages may develop diverse immune responses. For instance, cytotoxic natural killer (CNK) cells dominate in the peritoneal fluid of F. hepatica-infected rats as early as 2 days post infection (p. i.). However, the cells decreased 4 days p.i. [44]. Therefore, the experimental set-up depends on the response outcomes needed. According to the life cycle of F. hepatica, the juvenile flukes penetrate the liver capsule and migrate through the liver tissue at 6 to 7 weeks before entering the bile ducts. This stage is rigorous for the host because of the violent penetration and migration of flukes. In addition, most activity detections of macrophages focus on the early stage of F. hepatica infection [14,45,46]; thus, little is known about the AAMΦ at 6 weeks post F. hepatica infection, which is the reason why the AAMΦ phenotype in MyD88 deficient mice at this stage needs to be addressed.
The data demonstrates that the absence of MyD88 does not impair the Th2 response in F. hepatica-infected mice compared with the infected WT mice when the splenocytes in vitro were stimulated with FhAg. Furthermore, a non-statistically significant increase toward the Th2 response was also found in between. Moreover, the in vivo experiments also show that IL-4, IL-5, and IL-13 on MyD88-/- infected mice were significantly higher compared with the WT and WT-infected mice. In contrast, the IFN-γ in both the MyD88-/- thio and the MyD88-/- infected mice were significantly decreased compared with that in F. hepatica-infected WT mice, which indicates that a Th2- dominant response was induced in vivo. This is consistent with the previous studies that provide evidence of elevated Th2 responses when MyD88-deficient mice were infected with Leishmania major [30,47], Chlamydia muridarum [48], or Schistosoma mansoni [49,50]. Similarly, MyD88 -/- mice infected with the gastrointestinal nematode Trichuris muris exhibited high resistance to infection and displayed an increase in IL-4 and IL-13 in cultured mesenteric lymph node cells with stimulation of T. muris specific antigen in vitro [51] compared with their WT counterparts. However, this was argued to be associated with powerful Th1 stimuli via a MyD88-dependent pathway because of the presence of commensal bacteria, which indicates the Th2 response to nematodes might be impaired because of increased Th1 response [52]. This is supported by experiments on S. mansoni showing that the absence of MyD88 supports Th2 responses [50]. However, in the present study, F. hepatica infection was not yet reported to carry any bacteria, which may mount a Th1 response. Therefore, no significant augmentation was seen in the MyD88 deficient mice. However, the Th2 response was clearly induced in the WT mice and mice lacking MyD88 with F. hepatica infection. As demonstrated by previous studies, Th2 response induced by helminth infections contribute to AAMΦ production (reviewed in [53]), F. hepatica infection may promote AAMΦ. This is supported by the fact that AAMΦ could be produced by FhAg combined with IL-4 and stimulation with FhAg together with LPS (as a stimulus for TLR4 activity) or purified protein derivative from Mycobacterium bovis (PPD-B, as a stimulus for TLR2 activity) in WT mice resulted in reduced NO or IFN-γ production, respectively [54]. Also, the thioredoxin peroxidase (TPX) secreted by F. hepatica induced the AAMΦ on cell lines in vitro [14,15]. Along with the present study, an implication that MyD88 deficiency is dispensable to the AAMϕ may be reached.
The present study implies that MyD88 is not required for Th2 response and AAMϕ activation. In WT BMMϕ, arginase production increased on treatment with LPS, which signals through the TLR4 pathway, which is consistent with the reports that LPS helps induce the production of both arginase isoforms (arginase-1 and arginase-2) [32,55]. Further, the arginase activity in MyD88-/- BMMϕ, treated with the media, LPS, IFN-γ, or both was almost absent, indicating that this activity is MyD88-independent. In both the WT and MyD88-/- BMMϕ, arginase mRNA increased upon treatment with IL-4, which is in agreement with the reports that arginase could be induced when stimulated with IL-4 [56] Similar trends were seen in the production of RELMα and Ym1 mRNA in WT and MyD88-/- BMMϕ in response to IL-4. These findings offering further evidence that AAMϕ is induced in MyD88 deficient mice. On the other hand, NO was produced synergistically by MyD88-/- BMMϕ when stimulated with both LPS and IFN-γ together, whereas it was produced by WT BMMϕ when treated with LPS alone. The NO produced by macrophages is essential to the suppression of host cytotoxicity and its production may be MyD88-dependent or -independent. In the present study, LPS signals through the TLR4 via the IRF-3 pathway during MyD88-deficiency, resulting in an increase in IFNβ instead of iNOS. IFNβ then induces IRF-1 production, which leads to the production of NO with the help of IFN-γ. This was supported by Koide et al. [57], who showed that the LPS-dependent increase in iNOS mRNA expression induced by IFN-γ is attributed to the IRF-1 upregulation induced by LPS. Moreover, iNOS cannot be induced by IFN-γ alone because of the lack of IRF-1 in the absence of MyD88. However, this speculation was not yet investigated.
Considering no significant difference was found in the Th2 cytokine profiles between the WT and MyD88-deficient mice, the lack of MyD88 may have affected the production of macrophages. However, the arginase activity in the macrophages from the PEC were at approximately the same level in the WT and MyD88-/- AAMϕs (Figure 5) despite both being significantly higher than those in MyD88 -/- thio AAMϕs. RELMα and Ym1 Protein expression in the peritoneal cavity were not impaired with the MyD88-deficiency (Figure 6) and the mRNA expression of both genes (Figure 7) retained the same profiles as the protein expression, respectively. However, some effects on the response to thioglycolate treatment of the MyD88-deficient mice were observed, which indicates the partial role of TLR stimulus in thioglycolate-induced macrophage phenotype. These findings may be related to the mixture of TLR ligands in thioglycolate, which might have been ignored when the actual function of thioglycolate in macrophage activation was analyzed. All of these findings support that MyD88 is not required for macrophage activation during F. hepatica infection.
Conclusion
In summary, MyD88 is not required for AAMϕ induction in vitro and in vivo. In addition, the Th2 cytokine profile remained intact in the MyD88-deficient mice infected with the F. hepatica 6 weeks post infection.
Methods
Mice and infection
All animal experiments were carried out in the Animal Care and Ethics Center. Female C57BL/6 mice (6-7 weeks old) were purchased from Slaccas Experimental Animal Company. MyD88-/- mice were bred at the experimental animal center and were in the 5th and 6th generation of backcross to C57BL/6 mice. All mice were raised with free access to tap water and standard rodent diet under a pathogen-free- environment. All mice were maintained based on the Institutional and National Institutes of Health guidelines.
The wild type (WT) and Myd88-/- mice with C57BL/6 background were infected orally with 45 Faciola hepatica metacercariae. The metacercariae were collected from miracidia-infected Galba truncatula snails. All mice were euthanized 10 weeks post infection and the peritoneal exudate cells (PEC) were harvested by lavaging the peritoneal cavity with 10 mL ice-cold Dulbecco's modified Eagle's medium (DMEM) (Gibco) for mRNA extraction, western blotting, and/or cytokine analysis. The spleen was used for cell culture, which was used for the detection of cytokines in vitro. Then, 0.6 mL of 5% thioglycolate medium (Becton Dickinson) per mice were injected as the non-Th2 polarized inflammation control.
Activation of BMMϕ
The BMMϕs from the C57BL/6 mice were harvested from the bone marrow in the femur and tibia. Macrophage differentiation was carried out based on previous literature [58]. Briefly, erythrocytes were treated with 3 mL red blood cell lysis buffer (Sigma-Aldrich) for 5 min. The cells were cultured at 5 × 106 cells per plate in DMEM with 20% Fetal Calf Serum (FCS) (GIBCO), 20% L929 supernatant, 2 mM L-glutamine, 0.25 U/mL penicillin, and 100 μg/mL streptomycin. The medium was replaced to obtain pure macrophages at six days post culture. The collected BMMϕ were stored in fresh Petri dishes for 20-24 h with or without IL-4 (25 ng/mL; BD Pharmingen) followed by treatment with LPS (100 ng/mL; Escherichia coli 0111:B4, Sigma-Aldrich) and IFN-γ (10 U/mL; BD Pharmingen) for 20-24 h together or separately.
Measurement of Nitric Oxide (NO)
NO was detected via nitrite accumulation in the macrophage culture media using Greiss Reagent (Sigma-Aldrich). Briefly, 100 μL of the supernatant fluid and 100 μL of 5.8% phosphoric acid (Sigma-Aldrich), 1% sulfanilamide (Sigma-Aldrich), 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride (Sigma-Aldrich) were briefly mixed. The absorbance was read at 540 nm on a microplate reader. The NO concentration was determined based on a standard sodium nitrite solution curve.
Determination of arginase activity
Arginase activity was measured according to the previous literature [23]. Briefly, 1 × 105 macrophage cells were treated with 100 μL 0.1% Triton X-100 (Sigma-Aldrich) and 100 μL of 25 mM Tris-HCL (Sigma-Aldrich). After a 30-min shaking incubation, 20 μL of 10 mM MnCl2 (Sigma-Aldrich) was added. Then, the cells were heated at 56°C for 10 min to activate the enzyme and 100 μL of this lysate with 100 μL of 0.5 M L-arginine (pH 9.7, Sigma-Aldrich) was incubated at 37°C for 60 min to examine the L-arginine hydrolysis. The reaction was stopped with addition of 800 μL of H2SO4 (96%)/H3PO4 (85%)/H2O (1/3/7, v/v/v), and 40 μL of 9% isonitroso-propiophenone (Sigma-Aldrich). The cells were then heated at 99°C for 30 min. The plates were read at 540 nm on a microplate reader. Arginase enzyme activity was determined based on a standard urea solution curve.
Preparation of F. hepatica antigens
Fresh F. hepatica adults from infected mice were washed with phosphate-buffered saline (PBS, pH 7.3) solution. The worms were homogenized with 10 mM Tris-HCl (pH 7.2), 150 mM EDTA at 4°C and sonicated on ice for 5 min. The homogenates were centrifuged at 12,000 × g at 4°C for 1 h. The supernatant fluids were harvested as F. hepatica extracted antigen (FhAg). The protein concentrations were detected using a BCA protein assay kit (Invitrogen, UK).
Cell culture and stimulation
Splenic cells and PECs were cultured in vitro. The spleens were crushed and the cells were centrifuged at 1100 × g for 5 min, and then resuspended at 5 × 106/mL. The supernatant fluid was abandoned after the addition of 3 mL of RBC Lysis Buffer (Sigma). The cells were centrifuged at 1100 × g for 5 min followed by a final re-suspension at 107/mL with the addition of 10 mL of RPMI 1640. The cells were dispensed at 5 × 105 cells per well. A final volume of 200 μL of splenic cells in triplicate were cultured with FhAg (10 μg/mL), RPMI 1640 media and anti-CD3 (1 μg/mL) at 37°C in 5% CO2 for 48 h, followed by the addition of 10% of total volume Alamar Blue (Invitrogen, UK) for another 24 h. The plates were read at 540 nm for cell proliferation and the supernatant fluids were harvested and kept at -20°C for further cytokine analyses after centrifugation at 1100 × g for 2 min. PEC culture and stimulation were performed similar to that for the splenocytes above.
Cytokine determination
IL-4, IL-5, IL-10, IL-13, and IFN-γ in the spleen and PEC supernatant fluid were detected by sandwich ELISA. The plates were coated with carbonate-buffered capture antibodies at 50 μL/well (the dilution factor was 1:500 for IL-4, IFN-γ and 1: 250 for IL-5, IL-10, and IL-13) and incubated overnight at 4°C. The plates were incubated in 4% BSA PBS (200 μL/well) for 2 h at room temperature in the dark, followed by addition of 50 μL/well 2-fold diluted standard antibodies (top concentration: IL-4 at 8 ng/mL; IL-5, IL-10, and IL-13 at 10 ng/mL; IFN-γ at 50 ng/mL diluted with 1% BSA PBS) in duplicate after washing. Then, 50 μL/well of the spleen or PEC supernatant samples were then added and incubated overnight at 4°C, followed by incubation in biotinylated antibodies (final dilution: IL-4, IL-5, IFN-γ at 1 μg/mL; IL-10 and IL-13 at 2 μg/mL in 1% BSA PBS) for 1 h at room temperature and AMDEX streptavidin-peroxidase (Sigma, France) at dilution 1: 6000 in 1% BSA PBS for 30 min. Finally, 50 μL/well of TMB (KPL) was added and reaction was stopped with the addition of 50 μL of 1 mM H2SO4. The absorbance was read at 450 nm.
Relative quantification of genes
The expression of arginase-1, RELMα, and Ym-1 genes were quantified via real-time RT-PCR. Total RNA was extracted according to the manufacturer's instructions. Approximately 1 μg of the total RNA was used to synthesize cDNA using MMLV reverse transcriptase (Stratagene). Then, the relative quantification of the genes was determined using a Light Cycler (Roche Molecular Biochemicals). A cDNA (FeMϕ)-positive control sample with five serial (1:4) dilutions was used as the standard curve in each reaction. β-Actin was used to normalize the expression of the test genes. A 10 μL reaction containing 1 μL cDNA, 4 mM MgCl2, 0.3 mM primers, and the Light Cycler-DNA SYBR Green I mix was carried out under the following conditions: denaturation at 95°C for 40 s, annealing at 54°C for 10 s, and elongation at 72°C for 15 s, 45-55 cycles. The annealing temperature for Ym1 was set to 62°C. The primers used for light cycler PCR analysis are listed in Table 1.
Table 1 Primers used for real-time PCR analysis
Gene Forward Reverse Annealing
Temp (°C)
Arginase1 GGTCCAGAAGAATGGAAGAGTCAG CAGATATGCAGGGAGTCACC 54
RELMα GGTCCCAGTGCATATGGATGAGACCATAGA CACCTCTTCACTCGAGGGACAGTTGGCAGC 54
Ym1 TCACAGGTCTGGCAATTCTTCTG TTTGTCCTTAGGAGGGCTTCCTCG. 62
β-Actin GAATCCTGTGGCATCCATGAAAC TAAAACGCAGCTCAGTAACAGTCCG 54
Western blot analysis
Up to 20 μL of the peritoneal cavity lavage fluid was mixed with LDS sample loading buffer (NuPAGE) and heated to resolve by SDS-PAGE using 4%-12% NuPAGE gel. The proteins were transferred from the gel onto a cellulose nitrate membrane by electrophoresis at 30 V for 1 h. Then, the membranes were incubated for 1 h in 5% skimmed milk in TBS and incubated with Anti-Ym1 (produced by immunization of the mice with recombinant protein, diluted in 5% skimmed milk in TBS blocking buffer: 1/3000) and anti-RELMα (produced by immunization of the mice with recombinant protein, 1/500) antibodies separately after 1 h block, followed by incubation with goat anti-mouse IgG alkaline phosphatase conjugate (1:5000) for 8 h. Then, it was incubated with the substrate (specify) Chemi Glow (luminol/enhancer solution: stable peroxide buffer = 1:1) until the color developed. The reaction was ceased by absorption of the rest of substrates. The results were recorded using the Gel Image System (Bio-Rad). Image software was used to measure the relative concentrations of proteins on the blots.
Statistical analysis
One-way ANOVA was applied to evaluate the statistical differences between groups. The non-parametric Kruskal-Wallis rank sum test along with Dunn's test was applied on all analyses. Differences with P < 0.05 were considered significant. The values are presented as mean ± SE unless otherwise stated. All graphs were made using PRISM software (version 5.0, GraphPad Software, Berkeley, CA).
List of Abbreviations
Abbreviations: AAMΦ: alternatively activated macrophages; CAMΦ: classically activated macrophages; MyD88: myeloid differentiation factor 88; TLRs: Toll-like receptors; PRRs: pattern recognition receptors; PAMPs: pathogen-associated molecular patterns; LPS: lipopolysaccharide; IFN-γ: interferon-γ; PEC: peritoneal exudate cells; FhAg: F. hepatica extracted antigen; ELISA: enzyme-linked immunosorbent assay; WT: wild type; MyD88-/-: MyD88 knockout.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
LHL conceived, designed and coordinated the study, performed the experimental work, data collection and drafted the manuscript. WDY participated in the design of the study, analysis of assay and data collection. WHJ collected parasite materials and performed biological assay, also developed methodologies for the antibody production of RELMα and Ym1. NK developed methodologies for biochemical assay and Western blotting optimization, also provided crucial reagents and advice. All authors read and approved the manuscript.
Acknowledgements
This work was supported by China Scholarship Council (CSC) and the Fundamental Research Funds for the Central Universities (XDJK2009B001). We are grateful to Nick Green for excellent technical assistance and Professor Wang hong for reading of the manuscript.
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J Med LifeJ Med LifeJMedLifeJournal of Medicine and Life1844-122X1844-3117Carol Davila University Press Romania 22514562JMedLife-04-320ReviewMinimally invasive treatment for female stress urinary incontinence – Romanian highlights Surcel C *Chibelean C *Iordache A *Mirvald C *Gîngu C *Margaritis S *Stoica R *Codoiu C *Savu C *Marksteiner R **Sinescu I ** "Fundeni” Clinic of Urology and Renal Transplantation, Bucharest, Romania
** Life Science Center Biotechnologie, Innsbruck, Austria
Correspondence to:Surcel Cristian, MD PhD
"Fundeni” Clinic of Urology and Renal Transplantation, Bucharest, Romania
Phone: +40 744 963 035, E-mail: [email protected]
14 11 2011 24 11 2011 4 4 320 323 20 4 2011 20 10 2011 ©Carol Davila University Press
2011This is
an open-access article distributed under the terms of the Creative
Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work
is properly cited.Rationale: Stress urinary incontinence is still a "battlefield" for many minimally invasive therapies, but, unfortunately, few can restore the anatomical and functional background of this disorder.
Objective: Assessing the latest minimally invasive procedures of intra and perisphincterian injection of autologous stem cells.
Method and Result: The first stem cell implantation (myoblasts and /or mature fibroblasts grown and multiplied in the laboratory from biopsy samples taken from the pectoralis muscle) in the urethral sphincter was performed on October 18, 2010, in “Fundeni” Clinic of Urology and Renal Transplantation, in Romania.
Discussion: The follow-up at six weeks with the quality of life questionnaires, micturition diary and clinical examination revealed a decrease of urine loss from six pads/ day at one per day, which significantly improved the patient’s quality of life according to visual analogue scale. Clinical and urodynamic evaluations will continue and will be future scientific topics.
Abbreviations: SUI = stress urinary incontinence; TVT = tension free vaginal tape; TVT-O = tension free vaginal tape obturator; QoL = quality of life
stem cellsmyoblaststransplantationurethral sphincter
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Introduction
Although not life threatening, stress urinary incontinence (SUI) is certainly a public health problem, affecting the quality of life, mainly of the female population. It is a symptom/sign/condition defined by involuntary loss of urine that occurs during physical activity, with the effort of coughing, sneezing, laughing, prolonged standing, sexual activity etc. [1] Its prevalence reaches alarming rates, about 20% of total female population being affected; percentages increasing to 35% for those aged over 60 years [2,3].
The most important risk factors for SUI are: female sex, childbirth, obstetric history, lifestyle, chronic cough (chronic bronchitis, asthma), advanced age, estrogen status, obesity and history of pelvic surgery [4–8].
The normal functioning of the lower urinary tract and that of the nervous system assures the urinary continence and the act of micturition. Two different muscular structures are mainly involved in controlling the act of micturition: the urethral sphincter, which controls continence and facilitates micturition and the bladder muscle layer, the detrusor, which has the unique properties of gradually distending to allow the filling of the bladder, with minimal pressure increase, followed by efficient contraction in order to void.
Various conditions lead to pelvic floor structures dysfunctions, and so the base of the bladder and the urethra, weaken, with urinary incontinence during increased abdominal pressure (coughing, laughing, sneezing, exercise).
Still, it has been described as a pathological entity in which the components of the pelvic floor are not affected and still the urine loss persists, the mechanism being described as intrinsic sphincter deficiency [1].
The management of SUI is not easy, as therapeutic approach varies from conservative methods including lifestyle changes, medication, pelvic floor muscles exercises, electro stimulation, to minimally invasive – injection of collagen, suburethral slings reserving invasive surgical treatment for complex, recurrent cases [9].
Method
The anatomical and functional restoration of the pelvic structures is a challenge for many minimally invasive therapies, and just a few have succeeded. Intrinsic urethral sphincter deficiency and abnormal mobility of the urethra emerged as a key mechanism underlying the occurrence of this condition, along with other pathogenic theories. More recently, according to the “trampoline” theory, any structural defect in the pelvic ligaments, bones, fascial structures may contribute to the impairment of the pelvic muscle cybernetic system [10]. However, clinical experience has shown that not all lesions have a proportional role in the development of SUI and the mechanism of urethral sealing - mainly muscular, contributes fundamentally to the achievement of urinary continence. It is not a lower density of skeletal muscle fibers in the urethral sphincter structure, involved in the appearance of SUI? Although there are controversies in the literature, many studies showed a reduction in muscle fibers density in a category of patients from which we have excluded all other pelvic pathologies [11,12]. Thus, theoretically an artificial increase of the number of muscle fibers in the structure of the urethral sphincter could represent an innovative solution. Among the latest minimally invasive procedures, the injection of autologous stem cell intra and around the intrinsic sphincter is one of the most anatomical and functional methods, as seen in Figure 1 (myoblasts and/or mature fibroblasts multiplied in the laboratory from biopsy samples taken from the pectoral muscles) [13]. This method has proven clearly superior to the injection of collagen, it is not associated with major side effects, has minimal morbidity rates, reduced mean hospitalization time and although still experimental, it stands to be a promising procedure in the near future. The use of stem cells (myoblasts) as implants is a paradigm shift of the current treatment for SUI, currently based upon using synthetic materials (such as polypropylene mesh), that although well tolerated, they can never replace the auto/allograft in terms of biocompatibility. In addition, the anatomical restoration of the defects means a return to the "restitutio ad integrum" principle and not just a simple adjustment in order to resolve a pelvic static problem.
Fig. 1 Theoretical scheme of stem cells implantation (with the permission of Prof. Marksteiner)
With over 30 years of clinical experience in renal transplantation performed in “Fundeni” Clinic of Urology and Renal Transplantation, with pioneers of urodynamic studies and surgical treatment of pelvic static disorders in Romania working in this Center, our specialists have actively participated in the clinical research of female pelvic disorders. In addition, the expertise gained in the most important Renal Transplantation Center from Central and Eastern Europe – the ”Fundeni” allows and provides the infrastructure needed for the development of this project.
For every study regarding SUI, selecting patients with stress urinary incontinence mainly caused by urethral sphincter deficiency is a difficult task.
Medical history, physical examination in conjunction with urodynamic studies are the necessary steps to recruit patients in the study group, which later will be compared to a control group to whom a standard minimally invasive procedure is performed according to the guidelines of the Romanian Association of Urology, European Association of Urology and International Society of Continence. Many variables, such as age, performance and estrogen status, medical and surgical history, the severity of SUI, previous therapies both medical and surgical performed for the treatment of pelvic disorders, have to be taken into account to create well balanced study groups. Based on clinical evidence and on the results obtained from patient’s follow–up, randomized, multicenter, well-managed studies will be designed in the future.
Stem cell implantation in the urethral sphincter was performed for the first time in Romania on October 18, 2010, in “Fundeni” Clinic of Urology and Renal Transplantation. The team was led by Professor Dr. Ioanel Sinescu and was made up of Dr. Cristian Surcel, Dr. Alexandru Iordache, Dr. Calin Chibelean, Dr. Cristian Mirvald, Dr. Carmen Savu, Nurse Liviu Andrei and Professor Rainer Marksteiner. Standard protocol was applied without incidents, the patient being discharged after 24 hours. The follow up at six weeks revealed a stunning improvement in the patient’s quality of life (QoL) and continence rate, certified by clinical examination, QoL questionnaires and frequency–volume chart. The leak was reduced from six to one pad/day. Clinical and urodynamic follow–up continues and will be a future scientific topic.
From a technical standpoint, the procedure involves four major stages:
1. Selecting patients with SUI with intrinsic sphincter insufficiency.
2. The collection myoblasts from the pectoris major muscle, a maneuver easy to perform, with a short learning curve.
3. Isolation of stem cells (myoblasts) and multiplying them in cell cultures at the Center of Excellence in Cell and Tissue Research in Innsbruck, Austria. (Figure 2 and Figure 3
Fig. 2 Myoblast growth in cell culture –200x, antiDesmine staining (With permission from Prof. Marksteiner).
Fig. 3 Myoblasts fusion to the myotubuli in cell culture –200x, antiDesmnine staining (with permission from Prof. Marksteiner).
4. The surgical procedure of stem cells implantation in the urethral sphincter.
It requires a special biopsy device – Sonoject – which includes a central piece through which the biopsy is being performed, that provides an adapter to a syringe containing the cell suspension and a channel for a 20 MHz circular ultrasound probe that is used to locate the external urethral sphincter and to guide the injections. The device is fixed to a metal arm that is attached to the surgical table (similar to that used in prostate brachytherapy) together with a metal cylinder that slides manually, on which the “Sonoject “ is locked on.
The procedure takes place with the patient in lithotomy position, under general anesthesia. The pubic region, internal thighs and perineal area are sterile draped. Before the procedure, the device is assembled and tested in a saline solution or sterile water. Thus the circular ultrasound probe is calibrated in order to detect the needle and its signal.
The bladder is filled with 200 mL of saline and the device is inserted through the urethra. The system is armed so it does not move during the maneuver and the surgeon identifies the bladder neck, urethra and urethral sphincter (Figure 4). Once established the injection site, the cell suspension solution is injected on the anterior side of the sphincter, in two different semi–circular quadrants. The procedure needs 20 different injection sites, with 100 μL of solution. At the end of the procedure, the device is withdrawn from the urethra and disassembled.
Fig. 4 Urethral sphincter ultrasound - intraoperative aspect
Discussion
At least three surgical specialties treat pelvic floor pathology. Posterior compartment prolapse and anal incontinence are evaluated by the general surgeon or proctologist, uterine and vaginal prolapse, dyspareunia by the gynecologist and cystocele and low urinary tract symptoms by the urologist. Urinary incontinence and pelvic floor prolapse are two pathological entities that gave birth to the fourth specialist: the urogynecologist. The pathogenesis of SUI is multifactorial and so, the management is difficult, requiring clinical and therapeutic experience and for this reason many patients receive an incomplete treatment, which often worsens the clinical background or trigger symptoms caused by other structures which at the time of presentation were compensated.
Suburethral slings, inserted transobturatory, were introduced in Europe several years ago. This procedure was carried out by urogynecologist despite the absence of long–term data regarding efficiency and the rate of healing. The same approach happened with TVT (tension free vaginal tape) when they were introduced and, although the medium and long term data were lacking, they were adopted and became today's gold–standard treatment for SUI in women.
According to Abdel–Fattah’s recently published series of reports [14,15] that assessed the preferences for minimally invasive treatment of SUI, one third of respondents considered that TVT–O (tension free vaginal tape obturator) is an up–to–date procedure and it must be immediately applied, while the others expect the medium and long term statistics. Thus, stem–cell– myoblasts therapy may represent an alternative in the future, every–day intervention, in the urologist’s armamentarium. At least for the group of patients, to whom, from the pathogenic point of view, the deficiency is limited to the urethral sphincter, we believe that the urologist’s interest should be maximal. The effectiveness of this treatment can change the course of therapy and last but not least, the accessibility to urological evaluation of patients with SUI.
Treatment is tailored to the patient’s suffering and not just treats the leak of urine. In other words, a successful therapy includes the main objective parameters (dry/wet) and the subjective quality of life which is assessed by questionnaires [16]. However, understanding the "results" and the statistical methods used in their quantification are not homogeneous and sufficiently clear in order to remove any controversy. Before we compare and decide which the most effective procedure is, we should reach a consensus regarding the definition of "results", how they should be measured, follow-up intervals, etc. Until these issues are clarified, the urologist will continue to choose one of the many existing procedures and will remain autonomous in his selection. Thus, the need for new therapeutic methods, that can restore the integrity of the pelvic structures as close as possible, is urgent. The Centers of Excellence in Urology must develop research programs and be partners in multicenter studies in order to obtain solid long term data. Thus, new standards will be created that will be approved by urologist everywhere.
Source of Funding:
„This paper is partially supported by the Sectoral Operational Programme Human Resources Development, financed from the European Social Fund and by the Romanian Government under the contract number POSDRU/89/1.5/S/64153”
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J Exp Clin Cancer ResJournal of Experimental & Clinical Cancer Research : CR0392-90781756-9966BioMed Central 1756-9966-30-1052207101910.1186/1756-9966-30-105ResearchScreening and identification of a renal carcinoma specific peptide from a phage display peptide library Tu Xiangan [email protected] Jintao [email protected] Wenwei [email protected] Liang [email protected] Liangyun [email protected] Jiquan [email protected] Chunhua [email protected] Shaopeng [email protected] Yuanyuan [email protected] Department of Urology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510700, Guangdong, PR China2 Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences, Winston-Salem, NC, 27157, USA2011 10 11 2011 30 1 105 105 30 5 2011 10 11 2011 Copyright ©2011 Tu et al; licensee BioMed Central Ltd.2011Tu et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
Specific peptide ligands to cell surface receptors have been extensively used in tumor research and clinical applications. Phage display technology is a powerful tool for the isolation of cell-specific peptide ligands. To screen and identify novel markers for renal cell carcinoma, we evaluated a peptide that had been identified by phage display technology.
Methods
A renal carcinoma cell line A498 and a normal renal cell line HK-2 were used to carry out subtractive screening in vitro with a phage display peptide library. After three rounds of panning, there was an obvious enrichment for the phages specifically binding to the A498 cells, and the output/input ratio of phages increased about 100 fold. A group of peptides capable of binding specifically to the renal carcinoma cells were obtained, and the affinity of these peptides to the targeting cells and tissues was studied.
Results
Through a cell-based ELISA, immunocytochemical staining, immunohistochemical staining, and immunofluorescence, the Phage ZT-2 and synthetic peptide ZT-2 were shown to specifically bind to the tumor cell surfaces of A498 and incision specimens, but not to normal renal tissue samples.
Conclusion
A peptide ZT-2, which binds specifically to the renal carcinoma cell line A498 was selected from phage display peptide libraries. Therefore, it provides a potential tool for early diagnosis of renal carcinoma or targeted drug delivery in chemotherapy.
Renal cell carcinomaPhage displayPeptideTargeting
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Introduction
Renal cell carcinoma (RCC) accounts for 3% of all adult malignancies and is the most lethal urological cancer. It accounted more than 57, 000 new cases and 13, 000 cancer-related deaths in the United States in 2009[1]. In China around 23, 000 new patients with RCC are diagnosed each year, and the incidence is increasing rapidly due to the aging population [2]. Approximately 60% of patients have clinically localized disease at presentation, with the majority undergoing curative nephrectomy. However, metastatic disease recurs in a third of these patients. The patients with metastatic RCC have a poor prognosis with a median survival time of 1 to 2 years [3]. Detection of RCC in early stages helps increase the life expectancy of the patient [4]. Two diagnosis methods, histopathology and image procedures (computed tomography scan, ultrasonography, or magnetic resonance imaging) provide increase the early detection of the RCC. Histopathologically, although several promising biomarkers such as Carbonic anhydrase IX, B7-H1 and P53 for RCC have been under investigation, none currently have been validated or are in routine use [5,6]. Therefore, some novel molecular markers must be screened and identified for improving early diagnosis and prognosis of RCC.
Phage display is a molecular diversity technology that allows the presentation of large peptide and protein libraries on the surface of filamentous phage. Phage display libraries permit the selection of peptides and proteins, including antibodies, with high affinity and specificity for all targets. An important distinctive mark of this technology is the direct link that exists between the experimental phenotype and its encapsulated genotype. Phage display technology is a powerful tool for the selection of cell-specific peptide ligands at present [7]. Some laboratories have applied this technology to isolate peptide ligands with good affinity and specificity for a variety of cell types. The specific ligands isolated from phage libraries can be used in diagnostic probe, therapeutic target validation, and drug design and vaccine development [8-10].
In the present study, we identified a specific novel peptide that bound to the cell surface of renal carcinoma cell line A498 generated in this laboratory by using in vitro phage-displayed random peptide libraries. Our results demonstrate that this biopanning strategy can be used to identify tumor-specific targeting peptides. One of our selected peptides, ZT-2 was most effective in targeting cells and tissues, indicating its potential for use in early diagnosis and targeted therapy of RCC.
Materials
Renal carcinoma line A498 and a normal renal cell line HK-2 were obtained from Medical Academy of China (Beijing, PR China). Fetal calf serum (FCS) and Dulbecco's modified eagle's medium (DMEM) were purchased from Gibco (Invitrogen, Carlsbad, USA). Phage DNA sequencing was performed by Shanghai Sangon Corp (Shanghai, PR China). Peptide ZT-2 (QQPPMHLMSYAG) and a nonspecific control peptide (EAFSILQWPFAH) were synthesized and labeled with fluorescein isothiocyanate (FITC) by Shanghai Bioengineering Ltd. Mass analysis of the peptides was confirmed by a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and all peptides were > 90% pure as determined by reverse-phase HPLC. Peptide stock solutions were prepared in PBS (pH 7.4). Horseradish peroxidase-conjugated sheep anti-rabbit antibody and rabbit anti-M13 bacteriophage antibody were purchased from Pharmacia (Peapack, NJ, USA). Trizol reagents were purchased from Gibco BRL (Gaithersburg, MD, USA) and the reverse transcriptase polymerase chain reaction (RT-PCR) system kits were purchased from Promega (Madison, WI, USA).
The Ph.D.-12 phage display peptide library kit (New England Biolabs, Beverly, MA, USA) was used to screen specific peptides binding to A498 cells. The phage display library contains random peptides constructed at the N terminus of the minor coat protein (cpIII) of the M13 phage. The titer of the library is 2.3 × 1013 pfu (plaque-forming units). The library contains a mixture of 3.1 × 109 individual clones, representing the entire obtainable repertoire of 12-mer peptide sequences that express random twelve-amino-acid sequences. Extensively sequencing the naive library has revealed a wide diversity of sequences with no obvious positional biases.
The E. coli host strain ER2738 (a robust F+ strain with a rapid growth rate) (New England Biolabs) was used for M13 phage propagation. The A498 and HK-2 cells were cultured in DMEM supplemented with penicillin, streptomycin, and 10% fetal bovine serum. Cells were harvested when subconfluent, and the total number of cells was counted using a hemocytometer.
In Vitro Panning
A498 cells were taken as the target cells, and HK-2 as the absorber cells for a whole-cell subtractive screening from a phage display 12-peptide library. Cells were cultured in DMEM with 10% FCS at 37°C in a humidified atmosphere containing 5% CO2. HK-2 cells were washed with PBS and kept in serum-free DMEM for 1 h before blocking with 3 mL blocking buffer (BF, PBS + 5% BSA) for 10 min at 37°C. Approximately 2 × 1011 pfu phages were added and mixed gently with the blocked HK-2 for 1 h at 37°C. Cells were then pelleted by centrifuging at 1000 rpm (80 g) for 5 min. HK-2 and phages bound to these cells were removed by centrifugation. Those phages in the supernatant were incubated with the BF-blocked A498 cells for 1 h at 37°C before cells were pelleted again. After that, the pelleted cells were washed twice with 0.1% TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20) to remove unbound phage particles. A498 cells and bound phages were both incubated with the E. coli host strain ER2738. Then, the phages were rescued by infection with bacteria while the cells died. The phage titer was subsequently evaluated by a blue plaque-forming assay on agar plates containing tetracycline. Finally, a portion of purified phage preparation was used as the input phage for the next round of in vitro selection.
For each round of selection, more than 1.5 × 1011 pfu of collected phages were used. The panning intensity was increased by prolonging the phage incubation period with HK-2 for 1.25 h or 1.5 h, shortening the phage incubation with A498 for 45 min and 30 min in the second and third rounds individually, and increasing washing with TBST for 4 times and 6 times in the second and third round individually.
Sequence Analysis of Selected Phages and Peptide Synthesis
After three rounds of in vitro panning, 60 blue plaques were randomly selected and their sequences were analyzed with an ABI Automatic DNA Analyzer (Shanghai Sangon Corp). A primer used for sequencing was 5'-CCC TCA TAG TTA GCG TAA CG-3' (-96 gIII sequencing primer, provided in the Ph.D.-12 Phage display peptide library kit). Homologous analysis and multiple sequence alignment were done using the BLAST and Clustal W programs to determine the groups of related peptides.
Cell-Based ELISA with Phage
A498 and HK-2 were cultured in DMEM with 10% FCS at 37°C in a humidified atmosphere containing 5% CO2, and the cells were seeded into 96-well plates (1 × 105 cells/well) overnight. Cells were then fixed on 96-well plates by 4% paraformaldehyde for 15 min at room temperature until cells were attached to the plates. Triplicate determinations were done at each data point. Selectivity was determined using a formula as follows [11]: Selectivity = ODM13 - ODC1/ODS2 - ODC2. Here, ODM13 and OD C1 represent the OD values from the selected phages and control phages binding to A498 cells, respectively. OD S2 and ODC2 represent the OD values from the selected phage and control phage binding to the control (HK-2 cell line), respectively.
Immunocytochemical Staining and Immunohistochemical Staining of Phage M13
Before staining with phage M13 [12], the cells in the different groups (A498 and HK-2) were cultured on coverslips and fixed with acetone at 4°C for 20 min. Then, about 1 × 1011 pfu of phage M13 diluted in PBS were added onto the coverslips and incubated at 4°C overnight. Coverslips were then washed for five times with TBST. The coverslips were blocked by H2O2 (3% in PBS) at room temperature for 510 min. After being washed by PBS for 5 min at 37°C, the coverslips were incubated with normal sheep serum for 20 min at 37°C. Subsequently, the coverslips were incubated overnight at 4°C with a mouse anti-M13 phage antibody at a dilution of 1:5000. The next day, the coverslips were rinsed for three times (10 min for each rinse) in PBS and incubated with a secondary antibody for 1 h at room temperature. Afterward, the coverslips were rinsed three times (5 min for each rinse) in PBS. The bound antibody was visualized using DAB. The coverslips were rinsed for three times (5 min for each rinse) using running tap water before staining by hematoxylin and eosin. Finally, the coverslips were rinsed for 10 min with running tap water before dehydration and mounting.
Frozen sections of human renal tissues with and without tumors were also prepared. The steps of immunohistochemical staining were similar to those for immunocytochemical staining described above. Instead of the selected phage clone M13, PBS and a nonspecific control phage with same titers were used for negative controls. The study protocol was reviewed and approved by the Institutional Review Board and Ethic Committee of the First Affiliated Hospital of Sun Yat-Sen University (NO.2011-137), and oral or written informed consent was obtained from all subjects prior to enrollment in the study.
Peptide Synthesis and Labeling
The ZT-2 peptide (QQPPMHLMSYAG) translated from the selected M13 phage DNA sequence and nonspecific control peptide (EAFSILQWPFAH) were synthesized and purified by Shanghai Bioengineering Ltd. Fluorescein isothiocyanate (FITC)-conjugated peptides were also produced by the same company.
Peptide Competitive Inhibition Assay for Characterization of Specific Phage Clones
The in vitro blue-plaque forming assay was performed to observe the competitive inhibition effect of ZT-2 peptide with its phage counterparts (M13). A498 cells were cultured in a 12-well plate overnight and then preincubated with blocking buffer to block nonspecific binding at 4°C for 30 min. The synthetic peptide (0, 0.0001, 0.001, 0.01, 0.1, 1 or 10 μM) was diluted in PBS and incubated with cells at 4°C for 1 h, and then incubated with 1 × 1011 pfu of phage M13 at 4°C for 1 h. The bound phages were recovered and titered in ER2738 culture. The phages binding to A498 cells were evaluated by blue plaque-forming assay, and the rate of inhibition was calculated by the following formula: Rate of inhibition = (number of blue plaques in A498 incubated with PBS - number of blue plaques in A498 with ZT-2 peptide)/number of blue plaques in A498 incubated with PBS × 100%. Nonspecific control phages (a synthetic peptide corresponding to an unrelated phage picked randomly from the original phage peptide library) were used as negative controls.
Immunofluorescence Microscopy and Image Analysis
Immunofluorescence microscopy was used to study the affinity of synthetic peptide (ZT-2) binding to A498 and renal carcinoma. A498 and HK-2 were digested with 0.25% trypsin and plated on coverslips overnight. Cells were washed three times with PBS and fixed with acetone at 4°C for 20 min before analysis. ZT-2 peptide labeled with FITC was incubated with cells. PBS and control peptides labeled with FITC were used as negative controls. After being washed for three times with PBS, the slips were observed using a fluorescence microscope.
Results
Specific Enrichment of A498 Cell-Bound Phages
Phages specifically bound to human A498 cells were identified through three rounds of in vitro panning. In each round, the bound phages were rescued and amplified in E. coli for the following round of panning, while the unbound phages were removed by washing with TBST. After the third round of the in vitro selection, the number of phages recovered from A498 cells increased 100-fold (Table 1). However, the number of phages recovered from HK-2 control cells decreased. The output/input ratio of phages recovered after each round of the panning was used to determine the phage recovery efficiency. These results indicated an obvious enrichment of phages specifically binding to A498 cells.
Table 1 Enrichment of phages for each round of selection from phage displayed peptide library
Rounds Selected Phage
(input) (cpu) Eluted Phage
(output) (cpu) Ratio(output/input)
1 1.5 × 1011 1.5 × 103 1 × 10-8
2 1012 105 10-7
3 1012 106 10-6
Verification of In Vitro Specific Binding by Cell-Based ELISA
A cellular ELISA was used to identify the affinities for the twenty selected phages binding to A498. To assess selectivity, the affinities of each phage binding to A498 cells and to the control HK-2 were compared. These phage clones bound more effectively to A498 cells compared with PBS and HK-2 control groups. Furthermore, the ZT-2 clone appeared to bind most effectively to A498 cells than the other clones (Figure 1). Therefore, we further analyzed the phage M13 and its displaying peptide ZT-2.
Figure 1 Evaluation by cell-ELISA of the binding selectivity of twenty phage clones. The selectivity values of five higher phage clone (ZT-2, ZT-4, ZT-8, ZT-9, and ZT-16), calculated by the formula mentioned in the text, were 3.15, 2.90, 2.95, 2.80, and 3.05, respectively. Therefore, clone ZT-2 appeared to bind more effectively than the other clones.
Affinity of the Phage M13 to A498 Cells and Renal carcinoma Tissues
To confirm the binding ability of the selected phage toward target A498 cells, the phage clone M13 (clone ZT-2) was isolated, amplified and purified for immunochemical assay. The HK-2 cell line, composed of human nontumor renal tissues, was included as a negative control. The interaction of the M13 phage and target cells (A498) was evaluated by immunocytochemical staining. A498 cells bound by the phage M13 were stained brown in contrast to the HK-2 cells. Negative results were also obtained when A498 cells bound with unrelated phage clone. However, A498 cells bound with phage clone ZT-2 were stained brown distinctively, demonstrating that ZT-2 was able to bind specifically to A498 cells (Figure 2). Subsequently, immunohistochemical stain was performed to observe the specific binding of the phage clone ZT-2 toward human renal carcinoma tissues. The cells in A498 tumor tissue sections when bound with phage clone ZT-2 were stained green fluorescence distinctively. When A498 tumor tissue sections bound by unrelated phage clone or the normal renal tissue sections when bound with phage clone ZT-2 showed negative staining. It is thus clear that the phage clone ZT-2 was able to bind specifically to A498 cells (Figure 3).
Figure 2 Immunocytochemical staining of A498 and control cells when bound with phage ZT-2. Cell-bound phages were detected using anti-M13 phage monoclonal antibody, secondary antibody, and ABC complex. The cells were stained with diaminobenzidine (DAB). (A) shows control cell (B) shows immunocytochemical staining of A498 cells when bound with phages without exogenous sequences (wild-type phage) (C) shows immunocytochemical staining of A498 cells when bound with unrelated phage (D) shows immunocytochemical staining of A498 cells when bound with phage ZT-2. Amplification × 200.
Figure 3 Immunohistochemical staining of renal carcinoma and nontumorous renal tissue sections when bound with ZT-2 peptide-fluorescein isothiocyanate. To investigate if the free ZT-2 peptide maintained its binding affinity to renal carcinoma cells, we made a synthetic peptide ZT-2 (QQPPMHLMSYAG) labeled with fluorescein isothiocyanate. (A) Immunohistochemical staining of renal carcinoma tissues when bound with phage ZT-2-FITC. The specific binding sites on tumor cells fluoresced green (B) Immunohistochemical staining of nontumorous renal tissues when bound with phage ZT-2 (C) a negative control section stained with random peptide-fluorescein isothiocyanate in renal carcinoma tissues. Magnification × 200.
Competitive Inhibition Assay
A peptide-competitive inhibition assay was performed to discover whether the synthetic peptide ZT-2 and the corresponding phage clone competed for the same binding site. When the synthetic peptide ZT-2 was pre-incubated with A498 cells, phage ZT-2 binding to A498 cells decreased in a dose-dependent manner. When the peptide ZT-2 concentrations increased, the titer of phages recovered from A498 cells was decreased and the inhibition was increased gradually. When the concentrations of peptide ZT-2 increased above 5 μM, the inhibition reached a flat phase. The control peptide (EAFSILQWPFAH) had no effect on the binding of the phage ZT-2 to A498 cells (Figure 4).
Figure 4 Competitive inhibition of binding of the phage ZT-2 to A498 cells by the synthetic peptide ZT-2 QQPPMHLMSYAG. The average inhibition rates at different concentrations of the peptide are shown. When the concentration of the peptide ZT-2 reached more than 0.001 μM, a significant inhibition occurred.
Discussion
Targeting specific ligand binding on specific tumor antigens is an efficient way to increase the selectivity of therapeutic targets in clinical oncology and helpful for the early detection and therapy of RCC. Tumor cells often display certain cell surface antigens such as tumor-associated antigens or tumor-specific antigens in high quantity, which are different from the antigens on normal tissues. To develop more biomarkers for the diagnosis of RCC, we used peptide phage display technology to identify potential molecular biomarkers of A498 carcinoma cells. After panning for three rounds, 20 clones were selected for further characterization. First, a cell-based ELISA assay was used to confirm the specific binding of the phage clones to A498 cells in vitro. ZT-2 was the best candidate phage clone with the highest specificity. Second, immunocytochemical and immunohistochemical staining were performed to confirm the selectivity of the phage ZT-2 to bind to A498 cells. Third, the results of the competitive inhibitory assays suggest that the peptide displayed by the phage M13-ZT-2, not other parts of this phage, can bind to the renal carcinoma cell surface. Under the same conditions, the normal renal cell line HK-2 did not show significant fluorescence when stained with ZT-2 peptide-FITC, which confirmed the targeting of ZT-2 to be A498 cells.
Monoclonal antibodies have become the most rapidly expanding class of drugs for treating kidney cancer, but poor tumor penetration, bone marrow toxicity and high immunogenicity of these antibodies have been limited in clinical applications [13,14]. Compared with monoclonal antibodies, peptide ligands, which have the advantages of rapid tissue penetration, faster blood clearance, easy incorporation into certain delivery vectors and low immunogenicity are being pursued as targeting moieties for the selective delivery of radionuclides cytokines, chemical drugs, or therapeutic genes to tumors [15]. This effect may open up diagnostic procedures and therapeutic options for the patient. Identification of the cancer cell receptors that binds the ZT-2 peptide would allow further improvement of the peptide for potential clinical use.
These preliminary experiments provide evidence that the ZT-2 peptide may be specific to A498 and therefore it would be useful for diagnosis of renal carcinoma or delivery of an antitumor therapeutic agent. Studies are continuing to identify the cellular receptors responsible for peptide binding and to apply the peptide to clinically relevant samples.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
TXA and ZYY designed the study. ZJT performed the cell-based ELISA and analyzed the data statistically. WWW performed immunocytochemical staining. ZL performed immunohistochemical staining. ZLY and ZJQ performed immunofluorescence microscopy and image analysis. DCH and QSP performed data analysis. TXA wrote the main manuscript. ZYY looked over the manuscript. All authors read and approved the final manuscript.
Acknowledgements
This work was supported by National Natural Science Foundation of China (No.81172432), The Project Supported by Guangdong Natural Science Foundation of the People's Republic of China (No.9151802904000002), Scientific and Technical Project of Guangdong Province of the People's Republic of China (2008B030301082), Doctoral Initiating Project, and Natural Scientific Foundation of Guangdong Province of the People's Republic of China (No.7301521)
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PLoS One
PLoS ONE
plos
plosone
PLoS ONE
1932-6203
Public Library of Science San Francisco, USA
22164206
PONE-D-11-04759
10.1371/journal.pone.0027093
Research Article
Biology
Molecular Cell Biology
Cell Death
Neuroscience
Cellular Neuroscience
Developmental Neuroscience
Molecular Neuroscience
Neurobiology of Disease and Regeneration
Nicotinamide Inhibits Alkylating Agent-Induced Apoptotic Neurodegeneration in the Developing Rat Brain
Effect of Nicotinamide against Thiotepa
Ullah Najeeb 1
Lee Hae Young 1
Naseer Muhammad Imran 1
Ullah Ikram 1
Suh Joo Won 2
Kim Myeong Ok 1 *
1 Division of Life Science, College of Natural Sciences (RINS) and Applied Life Science (Brain Korea 21), Gyeongsang National University, Chinju, Republic of Korea
2 Division of Bioscience and Bioinformatics, Myongji University, Namdong, Yongin, Kyonggido, Republic of Korea
Homayouni Ramin Editor
University of Memphis, United States of America
* E-mail: [email protected]
Conceived and designed the experiments: NU JWS MOK. Performed the experiments: NU MIN IU. Analyzed the data: NU HYL. Contributed reagents/materials/analysis tools: NU HYL. Wrote the paper: NU MOK.
2011
2 12 2011
6 12 e2709311 3 2011
10 10 2011
Ullah et al.
2011
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Background
Exposure to the chemotherapeutic alkylating agent thiotepa during brain development leads to neurological complications arising from neurodegeneration and irreversible damage to the developing central nerve system (CNS). Administration of single dose of thiotepa in 7-d postnatal (P7) rat triggers activation of apoptotic cascade and widespread neuronal death. The present study was aimed to elucidate whether nicotinamide may prevent thiotepa-induced neurodegeneration in the developing rat brain.
Methodology/Principal Findings
Neuronal cell death induced by thiotepa was associated with the induction of Bax, release of cytochrome-c from mitochondria into the cytosol, activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP-1). Post-treatment of developing rats with nicotinamide suppressed thiotepa-induced upregulation of Bax, reduced cytochrome-c release into the cytosol and reduced expression of activated caspase-3 and cleavage of PARP-1. Cresyl violet staining showed numerous dead cells in the cortex hippocampus and thalamus; post-treatment with nicotinamide reduced the number of dead cells in these brain regions. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) and immunohistochemical analysis of caspase-3 show that thiotepa-induced cell death is apoptotic and that it is inhibited by nicotinamide treatment.
Conclusion
Nicotinamide (Nic) treatment with thiotepa significantly improved neuronal survival and alleviated neuronal cell death in the developing rat. These data demonstrate that nicotinamide shows promise as a therapeutic and neuroprotective agent for the treatment of neurodegenerative disorders in newborns and infants.
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Introduction
Neurological dysfunction is a well-known adverse effect of cancer therapeutics [1]. Chemotherapy, for example, is associated with an increased occurrence of neurodegenerative disorders that impair the development of higher mental abilities, cognitive status and academic achievements in children [2], [3], [4]. Furthermore, the toxic effects of anticancer agents can lead to neurological disorders such as cerebral infarction, seizures, leukoencephalopathy and others [5]. Chemotherapeutic toxicity has been shown to induce neuronal cell demise through the activation of two well-known apoptotic cascades [6], [7], [8]. Under the influence of some anticancer drugs, cytochrome c is released into the cytosol; in the presence of ATP, such release causes oligomerization of Apaf-1 (apoptotic protease activating factor 1) and activation of caspase-9 and caspase-3 [9], [10], [11], [12]. One such drug is thiotepa (N,N′N′-triethylenethiophosphoramide), an alkylating agent used for treatment of breast, colon, lung, brain, gastric, bladder and ovarian cancers; administration of thiotepa can also lead to poly (ADP-ribose) polymerase (PARP-1) activation [13], [14].
Nicotinamide, an amide of vitamin B3, is the precursor of coenzyme β-nicotinamide adenine dinucleotide (NAD+). NAD+ is considered to be necessary for cellular functions and metabolism [15]. Nicotinamide is well known to exhibit preclinical efficacy and to protect against neurological damage, but the exact mechanism of neuroprotection remains enigmatic. It is known that severe cellular insult leads to increased activity of PARP-1, which causes NAD+ depletion and apoptosis [14]. In the presence of nicotinamide, an essential precursor to NAD+, cellular NAD+ stores are more effectively replenished and damaged DNA is more effectively repaired [15], [16]. Nicotinamide improves neuronal survival following a variety of insults, including free radical exposure and oxidative stress [17], [18]. Its protective function is thought to be based on its numerous and diverse pharmacological effects, which include inhibition of PARP-1, prevention of ATP depletion [19], [20], lipid peroxidation, anti-inflammatory activity, and prevention of apoptosis [18], [21]. Nicotinamide also modulates mitochondrial membrane potential and the formation of pores, prevents cytochrome c release into the cytosol, and inhibits caspase-9 and caspase-3 like activities through mechanisms that are independent of those involving the mitogen-activated protein MAP kinase p38 and the c-Jun N-terminal kinases JNK [17], [18], [19], [20], [21], [22].
Chemotherapy for cancer treatment is often a necessity, and people diagnosed with cancer frequently receive chemotherapy in spite of its severe neurotoxic effects. Because thiotepa is routinely used as a chemotherapeutic agent, improvement of the neurological outcome of neonates and infants who experience neurotoxicity following treatment with this drug depends on advancing understanding of the precise molecular mechanisms triggering thiotepa-induced neurodegeneration and the development of neuroprotective therapeutics. The present study aimed to examine the protective role of nicotinamide against thiotepa-induced neurodegeneration in developing rats. The results show that nicotinamide effectively inhibits thiotepa-induced apoptotic neurodegeneration in developing rats. However, more comprehensive research and clinical trials will be required to determine whether nicotinamide can be used in conjunction with chemotherapy for prophylaxis of neurodegeneration or for the treatment of anticancer drug-induced neurodegeneration.
Results
Effect of nicotinamide on thiotepa-induced upregulation of Bax mRNA expression in the developing rat brain
Overexpression of Bax leads to mitochondrial dysfunction and cell death [23]. RT-PCR analysis was performed to examine whether the thiotepa-induced upregulation of Bax mRNA levels could be reversed by nicotinamide. After administration of a single dose (30 mg/kg) of chemotherapeutic alkylating agent (thiotepa) [1] to developing rats after 4 h, a significant increase in Bax mRNA levels was found in the cortex and thalamus; no such change was seen in the hippocampus (data not shown). Treatment of the animals with nicotinamide shortly after thiotepa administration significantly decreased Bax mRNA expression level in the cortex and thalamus (Fig. 1A). No marked change was observed in the Bcl-2 mRNA levels in thiotepa-exposed developing rat cortex, hippocampus and thalamus either with or without nicotinamide treatment (Fig. 1B).
10.1371/journal.pone.0027093.g001 Figure 1 Co-treatment effect of nicotinamide (Nic) on thiotepa-induced Bax and Bcl-2 mRNA levels.
Representative RT-PCR analysis shows expression levels of (A) Bax and (B) Bcl-2 mRNA in the cortex and thalamus of 7-day-old rat pups. Treatment with thiotepa (30 mg/kg for 4 h) increased Bax mRNA levels in the cortex and thalamus. Treatment with (1 mg/g) of nicotinamide for 4 h significantly reduced thiotepa-induced up-regulation of Bax mRNA levels in the cortex and thalamus, as shown. Detailed procedures are described in the Materials and Methods section. The mRNA bands of RT-PCR were quantified using Sigma gel software; these differences are represented in the graph. GAPDH was used as mRNA loading control. Density values are expressed as mean ± SEM (n = 5 animals/group). The density values on the Y-axis are expressed as arbitrary units (AU). Statistical difference was determined using one-way analysis of variance (ANOVA) followed by Student's t-test. (aSignificantly different from CTL, [cortex P<0.01 and thalamus P<0.01]; bSignificantly different from Nicotinamide, [cortex P<0.001 and thalamus P<0.05]; cSignificantly different from Thiotepa + Nicotinamide, [cortex P<0.01 and thalamus P<0.05]; dSignificantly different from Thiotepa, [cortex P<0.01 and thalamus P<0.05]).
Effect of nicotinamide on thiotepa-induced upregulation of Bax protein in the developing rat brain
To explore the molecular mechanisms of thiotepa-induced apoptotic neurodegeneration and protection by nicotinamide, we monitored the change in the expression of apoptotic proteins in the mitochondrial pathway. The Bcl-2 family members, pro-apoptotic Bax and anti-apoptotic Bcl-2, are the critical regulators of the apoptotic pathway in mitochondria [24]. Therefore, we measured the levels of Bax and Bcl-2 protein in the cortex, hippocampus and thalamus of young rats after 4 h of thiotepa treatment with and without nicotinamide. Bax levels in the cortex and thalamus showed an upregulation on thiotepa treatment compared to those of control animals. Treatment with nicotinamide significantly reversed thiotepa-induced upregulation of Bax (Fig. 2A), while no marked difference was observed in the levels of Bax in hippocampus after nicotinamide treatment alone (data not shown). In agreement with the results of a previous investigation [25], the expression level of Bcl-2 in the cortex, hippocampus and thalamus of the drug-treated groups was unchanged as compared to control (Fig. 2B). These results show that treatment with thiotepa for 4 h results in an increase in Bax/Bcl-2 ratio, favoring neuroapoptosis, while nicotinamide supplementation significantly reduced Bax/Bcl-2 ratio, favoring neuroprotection.
10.1371/journal.pone.0027093.g002 Figure 2 Co-treatment effect of nicotinamide (Nic) on thiotepa-induced expression of Bax and Bcl-2 protein levels.
Representative Western blot analysis shows the expression levels of (A) Bax and (B) Bcl-2 protein in the cortex and thalamus of a 7-day-old rat. Treatment with (30 mg/kg) of thiotepa for 4 h increased Bax protein levels in the cortex and thalamus. Treatment with (1 mg/g) of nicotinamide for 4 h inhibited thiotepa-induced upregulation of Bax protein levels in the cortex and thalamus, as shown. The protein bands of the Western blot were quantified using Sigma gel software; their differences are represented in the graph. Actin was used as a protein loading control. Density values, expressed as mean ± SEM (n = 5–6 animals/group), of Bcl-2 and Bax proteins are presented. The density values on the Y-axis are expressed as arbitrary units (AU). Statistical difference was determined using one-way analysis of variance (ANOVA) followed by Student's t-test. (aSignificantly different from CTL, [cortex P<0.001 and thalamus P<0.05]; bSignificantly different from Nicotinamide, [cortex P<0.01 and thalamus P<0.001]; cSignificantly different from Thiotepa + Nicotinamide, [cortex P<0.01 and thalamus P<0.05]; dSignificantly different from Thiotepa, [cortex P<0.01 and thalamus P<0.05]).
Effect of nicotinamide on thiotepa-induced cytochrome c release from mitochondria into cytosol
The mitochondrial apoptotic cascade requires the release of the inter-mitochondrial membrane protein cytochrome c into the cytosol; an increased cytosolic level of cytochrome c induces activation of caspase-9 and caspase-3 and, subsequently, neuronal death [12]. Protection by nicotinamide against thiotepa-induced neurodegeneration might occur as a result of the inhibition of cytochrome c release from mitochondria into the cytosol. Because cytosolic cytochrome c is a marker for the activation of the mitochondrial apoptotic cascade, we measured the levels of cytosolic cytochrome c in the cortex, hippocampus and thalamus. Four hours after administration of thiotepa, we found that in the cortex and thalamus, increased levels of Bax correlated with increases in cytosolic cytochrome c levels; no significant difference in the cytosolic level of cytochrome c was observed in the hippocampus (data not shown). Administration of nicotinamide with thiotepa treatment resulted in significantly reduced cytochrome c release from mitochondria into cytosol compared to that in animals treated with thiotepa alone (Fig. 3).
10.1371/journal.pone.0027093.g003 Figure 3 Co-treatment effect of nicotinamide on thiotepa-induced cytosolic cytochrome c levels.
Representative Western blot analysis of cytochrome c levels in the cortex and thalamus of 7-day-old rats. Significant increases in the cytosolic level of cytochrome c in the cortex and thalamus after administration of thiotepa are shown. Treatment with nicotinamide for 4 h significantly reduced thiotepa-induced cytosolic cytochrome c levels in cortex and thalamus, as shown. The protein bands of the Western blot were quantified using Sigma gel software analysis; their differences are represented in the graph. Actin reactivity was used as a protein loading control. Density values, expressed as mean ± SEM (n = 5–6 animals/group), for cytochrome c and activated caspase-9 are presented. The density values on the Y-axis are expressed as arbitrary units (AU). Statistical difference was determined using one-way analysis of variance (ANOVA) followed by Student's t-test. (aSignificantly different from CTL, [cortex P<0.05 and thalamus P<0.05]); bSignificantly different from Nicotinamide, [cortex P<0.05 and thalamus P<0.05]; cSignificantly different from Thiotepa + Nicotinamide, [cortex P<0.01 and thalamus P<0.01]; dSignificantly different from Thiotepa, [cortex P<0.01 and thalamus P<0.01]).
Effect of nicotinamide on thiotepa-induced expression of active caspase-3 in the developing rat brain
Caspases are cysteine proteases that play a key role in apoptosis [26]. We therefore examined whether the prevention of mitochondrial membrane depolarization and cytochrome c release by nicotinamide occurs at the level of downstream cellular pathways such as the inhibition of activated caspase-3. We measured the levels of activated caspase-3 in the cortex, hippocampus and thalamus by western blotting. Compared to controls, levels of activated caspase-3 increased significantly in the cortex and thalamus after thiotepa administration (Fig. 4A); no marked difference was observed in the level of activated caspase-3 in the hippocampus (data not shown). Because we detected low levels of activated caspase-3 in the control group also, tissue extraction may induce low levels of caspase-3 activation; however, the levels were significantly lower than those in the experimental groups. Nicotinamide treatment with thiotepa significantly decreased the level of activated caspase-3 in both brain regions compared to that in animals treated with thiotepa alone.
10.1371/journal.pone.0027093.g004 Figure 4 Co-treatment effect of nicotinamide on thiotepa-induced expression of active caspase-3 and cleavage of PARP-1.
(A) Representative Western blot analysis of cleaved caspase-3 levels in the cortex and thalamus of rats. Thiotepa administration resulted in a significantly enhanced level of expression of active caspase-3. Treatment with nicotinamide significantly reduced thiotepa-induced activated caspase-3 levels in both cortex and thalamus as compared to control group. Detailed procedures are described in the Materials and Methods section. The protein bands of Western blots were quantified using Sigma gel software, and their differences are represented in the graph. Actin reactivity was used as a protein loading control. Density values, expressed as mean ± SEM (n = 5–6 animals/group), of the caspase-3 protein are presented. The density values on the Y-axis are expressed as arbitrary units (AU). Statistical difference was determined using one-way analysis of variance (ANOVA) followed by Student's t-test. (aSignificantly different from CTL, [cortex P<0.05 and thalamus P<0.05]; bSignificantly different from Nicotinamide, [cortex P<0.05 and thalamus P<0.01]; cSignificantly different from Thiotepa + Nicotinamide, [cortex P<0.05 and thalamus P<0.05]; dSignificantly different from Thiotepa, [cortex P<0.05 and thalamus P<0.05]). (B) Representative Western blot analysis of cleaved PARP-1 levels in the cortex and thalamus of rats. Thiotepa administration resulted in significantly enhanced expression levels of active caspase-3, and caspase-3 mediated a high level of cleaved 89-kDa apoptosis-related fragment of PARP-1. As shown, treatment with nicotinamide significantly reduced thiotepa-induced activated caspase-3 and cleaved PARP-1 levels in both the cortex and thalamus when compared to control group. Density values, expressed as mean ± SEM (n = 5–6 animals/group), for 89 Kda caspase-3 cleaved PARP-1 proteins are presented. Statistical difference was determined using one-way analysis of variance (ANOVA) followed by Student's t-test. (aSignificantly different from CTL, [cortex P<0.001 and thalamus P<0.01]; bSignificantly different from Nicotinamide, [cortex P<0.01 and thalamus P<0.05]; cSignificantly different from Thiotepa + Nicotinamide, [cortex P<0.01 and thalamus P<0.05]; dSignificantly different from Thiotepa, [cortex P<0.01 and thalamus P<0.05]). (C) Representative Photomicrographs showing immunohistochemical analysis 24 h after treatment with 30 mg/kg thiotepa in the absence or presence of nicotinamide. Light micrographs show active caspase-3-positive neurons in cingulate cortex and LDN of thalamus (Dd and Ff) after thiotepa treatment, but none in hippocampus (Ee). The arrows indicate dead neuronal cells with high active caspase-3 expression. Treatment of nicotinamide (1 mg/g) with thiotepa resulted in a marked reduction in the number of active caspase-positive neurons in the cortex and thalamus (Gg and Ii) compared to controls (Aa and Cc). Images are representative of staining obtained in sections (3–5/group) prepared from at least 5–6 animals/group. (a–i = 40×) are magnified views from panels (A–I = 20×), Scale bar = 50 µm. Statistical difference was determined using one-way analysis of variance (ANOVA) followed by Student's t-test. (aSignificantly different from CTL, [cortex P<0.001 and thalamus P<0.001]; bSignificantly different from Thiotepa + Nicotinamide, [cortex P<0.05 and thalamus P<0.01]; cSignificantly different from Thiotepa, [cortex P<0.01 and thalamus P<0.01]).
Effect of nicotinamide on thiotepa-induced cleavage of PARP-1 in the developing rat brain
Despite its function in DNA repair, overactivation of PARP-1 in neuronal excitotoxicity induces cell death [27]. In our experiments, western blot analysis was used to determine whether the elevated levels of activated caspase-3 observed in the cortex and thalamus of developing rats treated with thiotepa led to the cleavage of PARP-1 as occurs during apoptosis. The level of cleaved PARP-1 in the cortex and thalamus of thiotepa-treated animals was significantly enhanced compared to that in the control and nicotinamide-treated groups. Nicotinamide treatment after thiotepa resulted in a remarkable decrease in the level of full-length (116 Kda) PARP-1 and a concomitant decrease in its 89 Kda caspase-3 cleaved products in the cortex and thalamus (Fig. 4B). Taken together, our results demonstrate that an overall increase in PARP-1 cleavage occurs in the cortex and thalamus of the developing rat brain after exposure to thiotepa and that nicotinamide significantly reduces the levels of thiotepa-induced cleaved PARP-1 in both of these brain regions, such that the levels remain similar to those observed in control animals.
Effect of nicotinamide on caspase-3 expression in the developing rat brain 24 h after thiotepa treatment
Increased expression of active caspase-3 protein contributes to neuronal cell death. In order to investigate the role of activated caspase-3 in the pathogenesis of neuronal death after thiotepa treatment, RT-PCR and western blot findings were supplemented by immunohistochemical analysis. Expression of active caspase-3 was analyzed in the cortex, hippocampus and thalamus after 24 h of thiotepa treatment. In the cingulate cortex and LDN of thalamus of thiotepa-treated rats, numerous injured and dead neurons with strong active caspase-3 immunoreactivity were observed (Fig. 4C, panels Dd and Ff); however, no active caspase-3 immunoreactivity was observed in the CA1 region of hippocampus (Fig. 4C, panel Ee). The number of active caspase-3-positive cells in the cortex and thalamus progressively decreased when nicotinamide was administered with thiotepa (Fig. 4C, panels Gg and Ii).
Effect of nicotinamide on thiotepa-induced neurodegeneration in the developing rat brain
Nissl staining was performed after 24 h to examine the extent of neuronal cell death induced by thiotepa and to assess protection by nicotinamide in the cortex, hippocampus and thalamus of the developing rat brain. Nissl staining identifies all structures, particularly the nucleus and nucleic acids, which appear violet, while neurons appear faintly blue. This method allowed clear identification of dead neuronal cells (i.e., those with large or small condensed, fragmented and dark nuclei and apoptotic bodies) in the cingulate cortex, CA1 of hippocampus and LDN of thalamus. Significantly increased vacuolization, neuronal loss, and tissue breakdown were seen in the anterior cingulate cortex, CA1 of hippocampus and LDN of thalamus of thiotepa-treated animals (Fig. 5, panels F, G and H) as compared to control animals; in control animals these brain regions appeared morphologically normal (Fig. 5, panels C, D and E). The number of degenerated neurons was significantly higher in the cortex and thalamus than in the hippocampal region, which showed a low level of cell death. Treatment of nicotinamide with thiotepa was associated with significantly less vacuolization and neuronal loss in these brain regions (Fig. 5, panels I, J and K).
10.1371/journal.pone.0027093.g005 Figure 5 Co-treatment effect of nicotinamide on thiotepa-induced neuronal cell death.
Light micrographs of cresyl violet-stained neurons in tissue sections of developing rat brain after thiotepa with nicotinamide. The majority of thiotepa-induced degenerating neuronal cells (generally shrunken in appearance, as is commonly observed) are present in the anterior cingulate cortex, CA1 of hippocampus and LDN of thalamus (F, G, and H). The arrows indicate shrunken and damaged neurons. Almost complete protection was observed (I, J and K), when nicotinamide was administered with thiotepa. Images are representative of staining obtained in sections (3–5/group) prepared from at least 5–6 animals/group. (C–K) of Nissl-stained brain tissue at higher magnification with 40× objective field, Scale bar = 20 µm. Statistical difference was determined using one-way analysis of variance (ANOVA) followed by Student's t-test. (aSignificantly different from CTL, [cortex P<0.001, hippocampus P<0.05 and thalamus P<0.001]; bSignificantly different from Thiotepa + Nicotinamide, [cortex P<0.01, hippocampus P<0.05 and thalamus P<0.01]; cSignificantly different from Thiotepa, [cortex P<0.01, hippocampus P<0.05 and thalamus P<0.01]).
DNA damage is one of the hallmarks of apoptosis. Visualization of DNA damage is possible using TUNEL staining, an assay for DNA breaks based on enzymatic labeling of free 3′ DNA ends. The nuclear morphology of brain tissue was evaluated with TUNEL and DAPI staining. Thiotepa-induced neuroapoptosis in developing rat brain cortex and thalamus resulted in the presence of apoptotic bodies and nuclear fragmentation revealed by TUNEL and DAPI staining. Compared to control animals (Fig. 6, panels A and C), treatment with thiotepa significantly increased the number of TUNEL-positive shrunken cells with small multi-DNA masses (typical features of apoptosis) in the cortex and thalamus (Fig. 6, panels D and F) counterstained with DAPI (Fig. 6, panels G and I). TUNEL-positive cells were not detected in CA1 of hippocampus of thiotepa-treated rats (Fig. 6, panels E and H). Most of the TUNEL-positive cells were in the cortex and thalamus, and overlapping of TUNEL staining with DAPI staining showed apoptotic neuronal death (Fig. 6, panels G and I). Nicotinamide treatment with thiotepa significantly reduced the number of TUNEL-positive cells in these brain regions (Fig. 6, panels J and L).
10.1371/journal.pone.0027093.g006 Figure 6 Co-treatment effect of nicotinamide on thiotepa-induced apoptotic neurodegeneration.
Representative photomicrographs of TUNEL staining show apoptotic dead neuronal cells after thiotepa administration with nicotinamide. The arrows indicate thiotepa-induced TUNEL stained apoptotic dead neurons (D and F) counterstained with DAPI (G and I) in the cortex and thalamus. Nicotinamide treatment effectively blocked thiotepa-induced apoptosis, as evident from the lack of TUNEL-positive cells (J and L). Images are representative of staining obtained in sections (3–5/group) prepared from at least 5–6 animals/group. (A–L) of TUNEL-stained brain tissue at higher magnification with 40× objective field, Scale bar = 20 µm. Statistical difference was determined using one-way analysis of variance (ANOVA) followed by Student's t-test. (aSignificantly different from CTL, [cortex P<0.001 and thalamus P<0.001]; bSignificantly different from Thiotepa + Nicotinamide, [cortex P<0.01 and thalamus P<0.01]; cSignificantly different from Thiotepa, [cortex P<0.01 and thalamus P<0.01]).
Discussion
Neurotoxicity is a common and often dose-limiting complication of chemotherapy [28]. Despite intensive efforts at management of the neurological side effects of chemotherapy in patients and the development of neuroprotective agents, there is no generally accepted therapy at the present time [29]. The use of neuroprotective agents can reduce neurotoxicity resulting from anticancer chemotherapy. Intensive research is therefore currently being focused on the identification and understanding of molecular and cellular mechanisms of neuroprotectants against such toxicity [30]. A mitochondria- dependent apoptotic cascade plays an important role in thiotepa-induced neurodegeneration [31], [32].Specifically, the normal functions of the central nervous system depend on mitochondrial activity for regulating calcium homeostasis, which itself serves as a regulator of several enzymatic activities and cell processes including apoptosis [33].
Due to its high metabolic rate and relatively reduced capacity for cellular regeneration, the brain is particularly susceptible to the damaging effects of oxidative stress [34]. The chemotherapeutic alkylating agent thiotepa has neurodegenerative effects in the infant rat brain that appear to be associated with disruption of mitochondrial energy metabolism, oxidative stress, and activation of cell death cascades [1]. Our results show that the neuronal cell death induced by single dose of alkylating agent thiotepa in the developing rat brain is apoptotic, as revealed by the occurrence of apoptotic bodies, nuclear fragmentation and activation of caspases. Apoptotic signals amplify through mitochondria due to changes in mitochondrial membrane permeability, which facilitate the release of the apoptogenic factor cytochrome c, regulated by the Bax and Bcl-2 proteins [35]. Activation of PARP-1 is a critical event that directs toxic signaling towards apoptosis or necrosis. Its cleavage facilitates DNA alteration and nuclear disassembly and ensures completion of the energy-dependent cell death process [36]. Activated caspase-3 cleaves PARP-1, which is an important protein in DNA repair, thus promoting apoptosis; in contrast, excessive activation of PARP-1 depletes NAD+ and ATP, resulting in necrosis [27], [37]. Our results show that after 4 h of thiotepa treatment, the level of expression of anti-apoptotic Bcl-2 was not changed; this confirms the results of a previous study [25]. In contrast, thiotepa induced upregulation of Bax and release of cytochrome c into the cytosol. Activation of caspase-3 and cleavage of PARP-1 by caspase-3 were analyzed in order to assess the involvement of the mitochondrial apoptotic pathway [31] in the neuronal degeneration caused by thiotepa. The expression of Bax and Bcl-2 proteins exhibited trends similar to those of the corresponding mRNA levels which is control by p53 [38]. Thiotepa-induced insult has been shown to increase mitochondrial membrane permeability and lead to the release of cytochrome c, the activation of caspase-9 and the subsequent activation of caspase-3, all of which play key roles in apoptosis [12], [26], [39].
Nicotinamide is a necessary nutrient that acts as a protective agent against neurodegeneration induced by a variety of insults including oxidative stress [40], [41], [42]. In developing rats, its systemic use provides significant protection against thiotepa-induced apoptotic and necrotic cell death. The results presented here show that treatment with nicotinamide inhibits thiotepa-induced activation of the apoptotic cascade through downregulation of proapoptotic Bax, thus counteracting thiotepa-induced increases in Bax levels. As a result, mitochondrial membrane potential is stabilized, release of cytochrome c is inhibited, and the levels of active caspase-3 and the cleavage of PARP-1 are reduced, which are involved in the activation of DNAses and the formation of apoptotic bodies [43], [44]. Our results clearly show that the protective effect of nicotinamide against thiotepa involves the maintenance of mitochondrial integrity and the concomitant repression of Bax, inhibition of the mitochondrially-mediated release of cytochrome c into the cytosol, inhibition of activated caspase-3 and maintenance of NAD+ levels, all of which contribute to the inhibition of apoptotic and possible necrotic cell death in the developing rat brain (Fig. 7).
10.1371/journal.pone.0027093.g007 Figure 7 A schematic diagram representing the hypothetical mechanism by which nicotinamide protects against thiotepa-induced neurodegeneration in the brain of the developing rat.
Thiotepa-induced neurodegeneration is caused by the overactivation of mitochondria-dependent apoptosis, beginning with the down-regulation of Bax, an increase in cytochrome c release from mitochondria to cytosol, expression of activated capase-3, cleavage of PARP-1 and necrosis by overactivation and cleavage of PARP-1 and depletion of NAD+ (blue arrow). PARP-1 activation and formation of Poly ADP-ribose PAR in the nucleus, which translocates to the cytosol to induce caspase-independent cell death (black arrow). Nicotinamide, as indicated by the X sign, inhibits several key elements in the apoptotic cascade beginning with the down-regulation of Bax, a decrease in cytochrome c release from mitochondria to cytosol, inhibition of activated caspase-3 and cleavage of PARP-1 and necrosis by the inhibition of PARP-1 activation and prevention of ATP and NAD+ depletion (red arrow), resulting in protection against thiotepa-induced apoptotic and possible necrotic cell death.
Modulation of the PARP-1 cascade is a well-known strategy used by cells to prevent ATP and NAD+ depletion; such modulation protects the developing brain against ischemia and excitotoxic insults involving DNA damage. Pharmacological inhibition or genetic disruption of PARP-1 can markedly reduce cell death resulting from oxidative stress [45], [46]. Under normal physiological conditions, PARP-1 is thought to play a key role in DNA repair, thereby contributing to the maintenance of cellular genomic integrity [47]. However, high metabolic and oxidative stress activates PARP-1 and depletes intracellular NAD+ stores [48], [49]. Rapid depletion of NAD+ and ATP is a suicide response that can result in cell death by apoptosis or necrosis if broken DNA strands are not repaired [27], [50], as degree of reduction in the level of ATP that represents the threshold between apoptotic and necrotic cell death [51], [52], [53]. Treatment of developing rats with nicotinamide after thiotepa inhibits induction of the apoptotic cascade due to inhibition of caspase-3, which is responsible for cleavage of PARP-1; such treatment thereby interferes with NAD+ depletion and may inhibits necrosis (Fig. 7) and other non-apoptotic cell death mechanisms [20], [54], [55].
Immunohistochemical and TUNEL findings were consistent with the molecular results; both showed that thiotepa treatment significantly increased expression of active caspase-3 in the cortex and thalamus while there was no significant change in the hippocampus. Histomorphological analysis by Nissl staining showed low levels of neuronal cell death in the hippocampal region as well. The exact reason for this discrepancy is unknown; one possible explanation is the involvement of neuronal apoptosis inhibitory protein (NAIP), in the hippocampal region [56], [57]. Whether or not NAIP is involved in the inhibition of alkylating agent-induced degeneration of developing brain in hippocampus remains to be determined. Histomorphological analysis also showed that the cell death induced by thiotepa is more pronounced and severe in the cortex and thalamus than in the hippocampus, indicating that, in the developing rat, these brain regions are more sensitive to thiotepa-induced apoptotic insult. Here, we speculate that thiotepa-induced cell death in the cortex and thalamus is mainly apoptotic and caspase-dependent, while in the hippocampus, caspase-independent or non-apoptotic cell death processes might be involved (Fig. 6). A relationship between histomorphological neuronal death and cognitive deficits could not be established; however, it is reasonable to suggest that protection by nicotinamide against thiotepa might be beneficial for mental abilities, cognitive skills, and academic achievements as well as for other behavioral outcomes.
In the present study, we found that nicotinamide treatment of the developing rat brain with thiotepa was associated with a decrease in thiotepa-induced apoptotic and necrotic cell death and improved brain histomorphological outcomes. Because nicotinamide can easily reach the brain [58], [59], it might be able to function as a critical protective agent in various neurodegenerative paradigms in which the mitochondrial apoptotic pathway is involved. Our data suggests that stabilization of the mitochondrial apoptotic pathway by nicotinamide may protect developing neurons treatment with anticancer drugs or other forms of excitotoxic insult. These findings open new avenues for examining the role of nicotinamide as a promising and safe neuroprotective agent for the treatment of neurodegenerative disorders [59]. Other neuroprotectants that have similar modes of action might also be developed to stabilize the apoptotic cascade caused by various toxic agents in infants.
Conclusions
Our results support the hypothesis that nicotinamide treatment protects against thiotepa-induced neurodegeneration in the developing rat brain, interfering with apoptotic cell death. Nicotinamide could be a potential remedy for neurodegenerative conditions caused by toxic effects of various neurotoxic drugs in newborns or infants. More work is clearly needed to comprehensively assess the neuroprotective role of nicotinamide.
Materials and Methods
Animals
Seven-day-old Sprague-Dawley rat pups (average body weight 15 g) were used in all experimental paradigms and were equally distributed into four different groups (control, thiotepa, nicotinamide, and thiotepa + nicotinamide). All efforts were made to minimize the number of animals used and their suffering. All the experimental procedures were approved (Approval ID: 125) by the animal ethics committee (IACUC) of the Division of Applied Life Sciences, Department of Biology, Gyeongsang National University South Korea.
Drug treatment
Protective effect of nicotinamide against thiotepa was observed at different time points i.e. (2 h) pre-treatment of thiotepa, Co-treatment of thiotepa and (2 h) post-treatment of thiotepa. At all time periods nicotinamide showed its effects but the strongest effect was observed when used with thiotepa treatment. In our experiments developing rats were injected subcutaneously with (30 mg/kg) thiotepa with (1 mg/g) nicotinamide in 0.9% saline solution. Animals were sacrificed 4–24 h after drug treatment. Saline injections of equal volume were used as controls.
Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis
The cerebral cortex, hippocampus and thalamic cortex were rapidly collected, separated and snap-frozen in liquid nitrogen. Samples were kept at −80°C until further processing. Total cellular RNA was isolated by acidic phenol/chloroform extraction. The RNA was aliquoted and stored at −80°C until further use. Then, 2 µg of total RNA was reverse-transcribed to single-stranded cDNA using Oligo(dT)12–18 primer (Invitrogen, Carlsbad, CA, USA) with M-MLV reverse transcriptase (Promega, Madison, WI, USA). For PCR reactions, 4 µl of cDNA was incubated with 20 pmol each of the forward and reverse primers (Bioneer Corporation, Seoul, South Korea), and GoTaq®Green Master Mix 2× containing PCR buffer, 25 mM magnesium chloride, 10 mM dNTP mix and Taq enzyme (Promega, Madison, WI, USA) and amplified. The primers of each transcript were as follows: Bcl-2, 5/-CGACGACTTCTCCCGCCGCTACCGC-3/ (forward) and 5/-CCGCATGCTGGGGCCGTACAGTTCC-3/ (reverse), Bax, 5/-GTGCACCAAGGTGCCGGAC-3/ (forward) and 5/-TCAGCCCATCTTCTTCCAGA-3/ (reverse), and β-actin (as loading control), 5/-GTGGGGCGCCCCAGGCACCA-3/ (forward) and 5/CTCCTTAATGTCACGCACGATTTC-3/ (reverse). The conditions for PCR were: initial denaturation at 94°C for 5 min; 25 cycles of denaturation at 94°C for 1 min, annealing for 1 min (Bcl-2, 68°C; Bax, 53°C; Caspase-3, 55°C; β-actin, 63°C), elongation at 72°C for 1 min and a final extension step at 72°C for 10 min on a PC-812 Thermal Cycler (Astec, Fukuoka, Japan). The PCR products were electrophoresed in 1% agarose gels containing ethidium bromide for 25 min and exposed to UV light for photography of the bands. The molecular sizes of the amplified products were determined by comparison with molecular weight markers (100 bp DNA ladder, Promega, Madison, WI, USA) run in parallel with the PCR products. The densities of the mRNA bands were analyzed using Molecular Analyst™, version 1.4.1 (Bio-Rad, Hercules, CA, USA).
Western blot analysis
In order to assess whether changes in the mRNA expression profiles of Bax and Bcl-2 were matched by corresponding changes in protein levels in the developing rat pups, the amounts of these proteins were determined in the control, thiotepa, nicotinamide and thiotepa-plus- nicotinamide treated animals. Seven-day-old rats were killed after treatment with (1 mg/g) nicotinamide immediately after administration of (30 mg/kg) of thiotepa for 4 h; the brains were rapidly removed, the cortex, hippocampus and thalamus were carefully dissected and the tissue was frozen in dry ice. The brain tissues were homogenized in 0.2 M PBS with protease inhibitor cocktail. The protein concentration was measured using Bio-Rad protein assay solution. Equivalent amounts of protein (40 µg per sample) were electrophoresed on 10–15% SDS-PAGE gels under reducing conditions and transferred to a polyvinylidene difluoride (PVDF) membrane (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Prestained protein markers, broad range (6–175 kDa, New England Biolabs Inc., Ipswich, MA, USA) were run in parallel for detection of the molecular weights of the proteins. The membrane was blocked with 5% (w/v) skimmed milk in order to reduce non-specific binding and immunoblotting was performed using rabbit-derived anti-Bcl-2, anti-Bax, and anti-caspase-3, anti-PARP-1 antibodies and goat-derived polyclonal anti-cytochrome c (1∶500; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Anti-β-actin antibody (1∶500; Sigma, St. Louis, MO, USA) was used as a control to confirm uniform loading. Membranes were probed with a goat-derived horseradish peroxidase-conjugated anti-rabbit IgG (1∶1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and immunocomplexes were visualized using enhanced chemiluminescence ECL-detecting reagent (Amersham Pharmacia Biotech, Western blotting detection reagents). The X-ray films were scanned and the optical densities of the Western blots were analyzed by densitometry using the computer-based Sigma Gel, version 1.0 (SPSS, Chicago, IL, USA).
Tissue collection and sample preparation
Animals were sacrificed 24 h after drug treatment. Brain sections from control rats and rats subjected to thiotepa followed by nicotinamide for 24 h were analyzed. For tissue analysis (n = 5–6 per group), developing rat pups were perfused transcardially with 4% ice-cold paraformaldehyde followed by 1×PBS; brains were post-fixed in 4% paraformaldehyde overnight and then transferred to 20% sucrose until they sank to the bottom of the tubes. Brains were frozen in O.C.T compound (A.O. USA) and 16 µm sections were made in the coronal planes using a Leica cryostat (CM 3050C, Germany). Sections were thaw-mounted on probe-on plus charged slides (Fisher).
Immunohistochemical staining
Immunohistochemistry was performed as previously described by [60], with some modifications. The slides were washed in 0.01 M PBS, quenched for 10 min in a solution of methanol containing 3% hydrogen peroxide, and then incubated for 1 h in blocking solution (2% BSA/0.2% milk/0.1% Triton X-100 in PBS), followed by incubation overnight in rabbit anti-active caspase-3 antiserum (Cell Signaling Technology, Beverly, MA) diluted 1∶1000 in blocking solution. Following incubation with primary antiserum, the sections were incubated for 90 min in secondary antiserum (goat anti-rabbit, 1∶200 in blocking solution), and then reacted in the dark with ABC reagents (standard Vectastain ABC Elite Kit; Vector Laboratories, Burlingame, CA) for 90 min. The sections were then washed twice with PBS and incubated with VIP reagent (Vector VIP substrate kit for peroxidase, Vector Labs, Burlingame, CA) to develop a purple color. Images were viewed with a fluorescence light microscope. Active caspase-3-positive cells in the different regions of each section were counted by observers blinded to the treatment conditions.
Cresyl violet staining
Cresyl violet was used to stain tissue sections for histological examination and measurement of neuronal loss. Nissl histology of developing rat brain and the presence and absence of dead and injured neurons were analyzed on microscope slides mounted 16 µm thick brain sections. Sections derived from all investigated rat pups were defatted in ascending alcohols (70–100%), hydrated in descending alcohols (95–70%), washed in acetate buffer pH 5.0 and subsequently stained with a 0.25% cresyl violet for approximately 15 min. Section were then washed with distilled water and dehydrated in graded ethanol. Images were viewed with a fluorescence light microscope. Neurons in the different regions of each section (5–6/group) were counted manually by observers blinded to the treatment conditions.
TUNEL and DAPI staining
To detect typical features of apoptosis, nuclear DNA was stained with TUNEL (GenScript Corporation, USA) and counterstained using 4′,6-diamidino-2-phenylindole (DAPI). In situ detection of apoptotic cell death was performed using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) on cryosections (16 µm) of aggregates. TUNEL staining was performed according to supplier recommendations using the In Situ Cell Death Detection kit Fluorescein (Genescript, NJ, USA). Aggregate cryosections (16 µm) were incubated with DAPI (Molecular Probes, Eugene, OR, USA) for 10 min at room temperature and then rinsed with distilled water. Glass cover slips were mounted on glass slides with mounting medium. A DAPI filter was used to detect the DAPI staining (blue color) and an FITC filter was used was to detect TUNEL staining (green color). TUNEL-positive (green) and DAPI-positive (blue) staining patterns were acquired by use of a confocal laser scanning microscope (Fluoview FV 1000, Olympus, Japan). TUNEL-positive cells in the different regions of each section were counted by observers blinded to the treatment conditions.
Data analysis and statistics
Bands from RT-PCR and Western blots were scanned and analyzed by densitometry using the Sigma Gel System (SPSS Inc., Chicago, IL). Density values were expressed as mean ± SEM. Statistical difference was determined using one-way analysis of variance (ANOVA) followed by Student's t-test. P values less than 0.05 were considered significant.
We thank Moon Seok Park for statistical analysis.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by a grant from the Next Generation Biogreen 21 program (PJ008075) and Agenda PJ007361 of Rural Development Administration. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Environ Health PerspectEnviron. Health PerspectEHPEnvironmental Health Perspectives0091-67651552-9924National Institute of Environmental Health Sciences 21684833ehp.100333910.1289/ehp.1003339ResearchThe Role of the Extracellular Matrix Protein Mindin in Airway
Response to Environmental Airways Injury Frush Sarah 1*Li Zhuowei 1*Potts Erin N. 1Du Wanglei 1Eu Jerry P. 1Garantziotis Stavros 2He You-Wen 3Foster W. Michael 1Hollingsworth John W. 131 Division of Pulmonary, Allergy, and
Critical Care Medicine, Department of Medicine, Duke University, Durham,
North Carolina, USA2 National Institute of Environmental Health
Sciences, National Institutes of Health, Department of Health and Human
Services, Research Triangle Park, North Carolina, USA3 Department of Immunology, Duke University,
Durham, North Carolina, USAAddress correspondence to J.W. Hollingsworth, Division of
Pulmonary, Allergy, and Critical Care Medicine, Duke University Medical Center,
Box 103004, Durham, NC 27710 USA. Telephone: (919) 684-4588. Fax: (919)
684-3067. E-mail: [email protected]* These authors contributed equally to this work.
17 6 2011 10 2011 119 10 1403 1408 13 12 2010 17 6 2011 2011This is an open-access article distributed under the terms of the Creative
Commons Attribution License, which permits unrestricted use, distribution,
and reproduction in any medium, provided the original work is properly
cited.Background: Our previous work demonstrated that the extracellular matrix protein
mindin contributes to allergic airways disease. However, the role of mindin in
nonallergic airways disease has not previously been explored.
Objectives: We hypothesized that mindin would contribute to airways disease after
inhalation of either lipopolysaccharide (LPS) or ozone.
Methods: We exposed C57BL/6J and mindin-deficient (–/–)
mice to aerosolized LPS (0.9 μg/m3 for 2.5 hr), saline, ozone
(1 ppm for 3 hr), or filtered air (FA). All mice were evaluated 4 hr after
LPS/saline
exposure or 24 hr after ozone/FA exposure. We characterized
the physiological and biological responses by analysis of airway
hyperresponsiveness (AHR) with a computer-controlled small-animal ventilator
(FlexiVent), inflammatory cellular recruitment, total protein in bronchoalveolar
lavage fluid (BALF), proinflammatory cytokine profiling, and ex
vivo bronchial ring studies.
Results: After inhalation of LPS, mindin–/– mice
demonstrated significantly reduced total cell and neutrophil recruitment into
the airspace compared with their wild-type counterparts.
Mindin–/– mice also exhibited reduced
proinflammatory cytokine production and lower AHR to methacholine challenge by
FlexiVent. After inhalation of ozone, mice had no detectible differences in
cellular inflammation or total BALF protein dependent on mindin. However,
mindin–/– mice were protected from increased
proinflammatory cytokine production and AHR compared with their C57BL/6J
counterparts. After ozone exposure, bronchial rings derived from
mindin–/– mice demonstrated reduced constriction in
response to carbachol.
Conclusions: These data demonstrate that the extracellular matrix protein mindin
modifies the airway response to both LPS and ozone. Our data support a conserved
role of mindin in production of proinflammatory cytokines and the development of
AHR in two divergent models of reactive airways disease, as well as a role of
mindin in airway smooth muscle contractility after exposure to ozone.
airway smooth muscleendotoxininnate immunitylipopolysaccharideLPSlungmindinozoneTlr4toll-like receptor
==== Body
Asthma is a common disorder affecting 7–11% of the U.S. and European populations
and is associated with substantial morbidity and health care costs (Bell et al. 2004; Dockery et al. 1993; Gryparis et al.
2004; Katsouyanni et al. 1995). The
severity of asthma can be worsened by inhalation of commonly encountered environmental
toxicants, including bacterial endotoxin and ambient air pollutants. Activation of
innate immunity, in turn, can contribute to exacerbations of reactive airways disease.
We previously reported the role of a prototypic gene of innate immunity, toll-like
receptor 4 (Tlr4), in mediating the airway response to exposures to the
environmental toxicants aerosolized lipopolysaccharide (LPS) and ozone (Hollingsworth et al. 2004).
LPS, also known as endotoxin, is found on the cell membranes of gram-negative bacteria
and induces inflammation. LPS is ubiquitous in the environment. Experimental data from
both mice (Schwartz 1996; Schwartz et al. 1994) and humans (Jagielo et al. 1996; O’Grady et al.
2001) show that LPS can cause airway obstruction that lasts as long as 48 hr
immediately after a single exposure (Kline et al.
2000). Tlr4 is the transmembrane surface receptor for bacterial LPS. However,
many genes likely can modify the response to inhaled endotoxin. We hypothesized that the
gene mindin (spondin 2, extracellular matrix protein; Spon2) may
contribute to the development of environmental airways disease. Mindin is an
extracellular protein that contributes to pulmonary innate immune response (He et al. 2004) and allergic airways disease (Li H
et al. 2006; Li Z et al. 2009).
Prior studies have demonstrated that, similar to Tlr4 (Hoshino et al. 1999; Poltorak et al.
1998), mindin is necessary for the biological response to LPS (He et al. 2004). For example, mindin regulates
macrophage-dependent inflammatory response to LPS. Additionally, mindin contributes to
integrin-dependent migration of macrophages (He et al.
2004; Jia et al. 2005). Therefore,
deciphering a potential role for mindin in airway response to inhaled LPS would provide
insight into mechanisms and establish more completely the innate immune response to
LPS.
Increasing evidence supports the role of innate immunity in environmental airways
disease. We now recognize that an endogenous ligand of surface receptor Tlr4 contributes
to the biological response to ambient ozone (Garantziotis
et al. 2009, 2010). Understanding the
mechanisms that regulate the response to ambient ozone is of considerable interest to
human health. Inhalation of ozone has been shown to contribute to increased morbidity
and mortality in human populations (Bell et al.
2004; Gryparis et al. 2004; Ito et al. 2005; Levy et al. 2005; Parodi et al.
2005). Clear understanding of the host factors that contribute to the response to
ozone is important for several reasons, including identification of susceptible
individuals and the potential development of novel therapeutic interventions in reactive
airways disease. Previous work supports that both mice (Kleeberger et al. 1997, 2000) and
humans (Balmes et al. 1996; Weinmann et al. 1995a, 1995b) exhibit varying biological responses to ozone exposure, indicating that
susceptibility to this toxicant may have a genetic basis. Genetic approaches to
understanding the mechanisms that regulate response to ozone have highlighted the role
of innate immunity. For example, previous studies support the role of innate immune
proinflammatory factors in the biological response to inhaled ozone, including tumor
necrosis factor-α (TNFα) (Cho et al.
2001; Kleeberger et al. 1997; Shore et al. 2001; Yang et al. 2005), interleukin (IL)-1β (Park et al. 2004), IL-6 (Johnston
et al. 2005b), and KC (cytokine-induced neutrophil chemoattractant) (Driscoll et al. 1993; Johnston et al. 2005b). Cumulatively, these data support a role for
innate immunity in the response to ambient ozone. However, the extracellular factors
that contribute to the complete innate immune response to ambient ozone remain poorly
understood.
The role of mindin in nonallergic airways disease has not previously been studied.
Therefore, the goal of the present study was to determine the role of mindin in
environmental airway injury to both inhaled LPS and ambient ozone using a mouse model.
Our in vivo approach demonstrated that mindin was necessary for the
complete response to both inhaled endotoxin and ambient ozone. Our observations build on
the growing body of evidence supporting a fundamental role of the innate immune system
in host response to noninfectious injury. We report that mindin-deficient
(mindin–/–) mice have attenuated proinflammatory cytokine
response and airway hyperresponsiveness (AHR) to both inhaled endotoxin and ambient
ozone. These results support an essential role of mindin in host response to both of
these models of airways disease and highlight the potential importance of host factors
that modulate the complete innate immune response to commonly encountered environmental
toxins.
Materials and Methods
Inbred mice. C57BL/6J mice were purchased from Jackson Laboratory
(Bar Harbor, ME). Mindin–/– mice on a C57BL/6J background
were generated as previously reported (He et al.
2004). All experimental protocols were reviewed and approved by the
Institutional Animal Care and Use Committee at Duke University Medical Center and
performed in accordance with the National Institutes of Health guidelines (Institute for Laboratory Animal Research 1996).
Animals were treated humanely and with regard for alleviation of suffering. Each
experimental group consisted of four or five male mice 6–8 weeks of age.
Inhaled LPS protocol. Animals were challenged with aerosolized LPS
purified from 0111:B4 Escherichia coli (Sigma Aldrich, St. Louis,
MO) for 2.5 hr. All animals were evaluated 4–7 hr after the initiation of LPS
exposure. LPS at 0.9 μg/m3 in phosphate-buffered saline (PBS) was
placed in a TSI jet nebulizer in a 55-L Hinners-style exposure chamber as previously
reported (Hollingsworth et al. 2004). The
dosage is similar to that experienced by workers in grain mills, swine confinement
facilities, and the textile industry during a typical 8-hr work day and results in
an inflammatory response in the lower respiratory tract (Simpson et al. 1999). C57BL/6J and
mindin–/– mice were exposed to either inhaled LPS or
control saline solution (PBS). Data presented are representative of three individual
experiments.
Ozone protocol. Animals were placed into separate caging and exposed
in a chamber to filtered air (FA) or 1 ppm ozone for 3 hr and then allowed to
recover for 24 hr under normal housing conditions. The level of ozone exposure (1
ppm for 3 hr) used in this protocol produced a minimum lung injury in terms of total
and inflammatory cell counts in bronchoalveolar lavage fluid (BALF) compared with
the more typical murine ozone injury model (2 ppm for 3 hr). Our selection of ozone
concentration levels was based on similar biological responses observed in human
exposure studies and published deposition fraction data for ozone in rodent models
(Hatch et al. 1994; Wiester et al. 1988). This dose of ozone in mice is used to
model the level of ozone encountered by humans during a “red”
cautionary day as determined by the U.S. Environmental Protection Agency during the
summer in many urban U.S. environments. Animals were exposed in the chamber with air
at 20–22°C and 50–60% relative humidity supplied at a rate of
20 exchanges per hour. Ozone generated by directing 100% oxygen through an
ultraviolet (UV) ozone generator was supplied after mixing with FA. The
concentration of ozone in the exposure chamber was monitored continuously by an
ozone UV light photometer (model 400E; Teledyne Technologies Inc., Thousand Oaks,
CA). C57BL/6 and mindin–/– mice were exposed to either
ozone (1.0 ppm for 3 hr) or FA. Data presented are representative of three
individual experiments.
Lung lavage, cell counts, and analysis of supernatant. As previously
described by Garantziotis et al. (2009), mice
were euthanized with CO2, and the lungs were exposed and fully inflated
three times serially to 25 cm H2O with 0.9% NaCl. Cell counts were
performed using a hemocytometer, and differentials were performed using hematoxylin
and eosin–stained cytospins. Cell-free lavage supernatants were stored at
–70°C. Cytokine/chemokines IL-1β, KC, MCP-1 (monocyte
chemotactic protein-1), and TNF-α were determined by Luminex (Bio-Rad,
Hercules, CA) using 5-plex reagents from Millipore (Billerica, MA). Assay
sensitivities are 2.0 pg/mL for IL-1β, 1.4 pg/mL for KC, 5.3 pg/mL for MCP-1,
and 1.0 pg/mL for TNF-α. Total protein concentrations in lung lavage fluid
were measured by the Lowry Assay (Bio-Rad).
AHR analysis. Anesthesia was achieved with 60 mg/kg of pentobarbital
sodium injected intraperitoneally. Mice were then given neuromuscular blockade (0.8
mL/kg pancuronium bromide) and ventilated with a computer-controlled small animal
ventilator (FlexiVent; SCIREQ, Montreal, Quebec, Canada), with a tidal volume of 7.5
mL/kg
and a positive end-expiratory pressure of 3 cm H2O.
Measurements of respiratory mechanics were made by the forced oscillation technique.
Response to aerosolized methacholine (0, 10, 25, and 100 mg/mL) was determined by
resistance measurements every 30 sec for 5 min, ensuring that the parameters
calculated had peaked. The lungs were inflated to total lung capacity after each
dose of methacholine, maintaining open airways and returning the measurements back
to baseline. The resistance measurements were then averaged at each dose
(RT, measured in centimeters of water per milliliter per second)
along with the initial baseline measurement.
Bronchial ring protocol. Two bronchial rings from each mouse were
studied simultaneously for their contractile response, as previously described
(Du et al. 2005). Each bronchial ring was
mounted horizontally on two tungsten triangles and submerged in modified Krebs
buffer (118 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4, 1.2 mM
KH2PO4, 2.5 mM CaCl2, 25 mM NaHCO3,
11 mM glucose; pH 7.4) in a thermostated organ bath constantly bubbled with a
premixed gas consisting of 20% O2, 5% CO2, and balanced
N2 at a constant temperature of 37°C. An optimal resting
tension of 0.2 g was applied to each ring during the initial equilibration period of
30 min; the bronchial rings were then constricted with 80 mM KCl for 20 min to
establish references for comparison. After washing off the excess KCl, the bronchial
rings were treated stepwise with 100 nM to 3 μM (cumulative doses) carbachol
at 5-min intervals, which allowed the contractile responses (isometric tensions) of
bronchial rings to reach steady state after each carbachol dose. The increases in
steady-state isometric tension of each bronchial ring caused by each carbachol dose
were normalized against the length of the ring.
Statistical analysis. Data are expressed as mean ± SE.
Significant differences between groups were identified by analysis of variance, and
individual comparisons are made using unpaired two-tailed Student
t-tests. A p-value < 0.05 was considered
statistically significant.
Results
Mindin-dependent response to inhaled LPS. We first investigated
whether the absence of mindin affects the airway response to methacholine in control
mice after exposure to saline aerosol. At 4–7 hr postexposure, we observed no
detectable differences induced by saline in a mindin-dependent manner (Figure 1A). However, after exposure to LPS,
C57BL/6J mice exhibited significantly higher airway responsiveness than did
mindin–/– mice. Similarly, we found a significant
difference between saline-challenged mindin–/– mice and
LPS-exposed mindin–/– mice. The LPS-challenged
mindin–/– mice exhibited significantly lower airway
responsiveness than did LPS-challenged C57BL/6J mice (Figure 1B).
Figure 1 Airway responsiveness in C57BL/6J and mindin–/– mice before
(A) and after (B) challenge with
inhaled LPS (n = 5/group). Baseline values with
methacholine challenge were similar in unexposed animals
(A); however, after LPS exposure (B),
C57BL/6J mice showed significantly increased airway responsiveness compared
with LPS-exposed mindin–/– mice. *p <
0.05.
Next, we characterized cellular recruitment into the airspace in C57BL/6J and
mindin–/– mice exposed to either saline or LPS. LPS
exposure in both C57BL/6J and mindin–/– mice resulted in an
increase in total cells and a higher absolute number of neutrophils in whole-lung
lavage fluid compared with saline (Figure 2A).
However, LPS-exposed mindin–/– mice demonstrated
significantly fewer total cells and neutrophils compared with LPS-exposed C57BL/6J
mice (Figure 2B).
Figure 2 Cellular recruitment into the airspace (A,B) and total
protein level (C) measured in whole-lung lavage fluid from
C57BL/6J and mindin–/– mice after exposure to saline or LPS
(n = 5/group). Cellular recruitment into the airspace
was evaluated by the number of total cells (A) and the
number of neutrophils (B). Mindin–/– mice
demonstrate reduced cellular inflammation after exposure to LPS compared
with C57BL/6J mice. Mindin–/– mice do not have a detectable
increase in total protein after exposure to LPS (C).
*p < 0.05 for LPS-exposed C57BL/6J mice compared
with either group of saline controls. **p < 0.05 for
LPS-exposed C57BL/6J mice compared with LPS-exposed mindin–/–
mice.
Total protein in whole-lung lavage fluid indicates epithelial permeability or lung
injury. After exposure to LPS, C57BL/6J mice demonstrated significantly higher
levels of total protein in whole lung lavage fluid compared with saline-challenged
genetic counterparts (Figure 2C). In contrast,
we found no significant difference between LPS-exposed
mindin–/– mice and their saline-exposed counterparts
(Figure 2C). We observed a trend toward
reduced level of total protein after exposure to LPS in
mindin–/– mice compared with C57BL/6J mice
(p = 0.07).
We measured the level of nuclear factor κB–dependent cytokines in the
BALF as a marker of activation of innate immunity. After LPS exposure, C57BL/6J mice
had notable increases in KC, IL-1β, MCP-1, and TNF-α in BALF compared
with saline-exposed mice (Figure 3). Cytokines
in LPS-exposed mindin–/– mice were also increased compared
with saline counterparts, but cytokines were significantly reduced compared with
LPS-exposed C57BL/6J mice (Figure 3).
Figure 3 Proinflammatory cytokines KC (A), IL-1β
(B), MCP-1 (C), and TNF-α
(D) measured in BALF from C57BL/6J and
mindin–/– mice after exposure to saline or LPS
(n = 5/group). *p < 0.05 for
LPS-exposed C57BL/6J mice compared with either group of saline controls.
**p < 0.05 for LPS-exposed C57BL/6J mice compared
with LPS-exposed mindin–/– mice.
Mindin-dependent response to inhaled ozone. To determine whether
mindin contributes to airway response after inhaled ozone, we challenged mice
acutely with either FA or ozone (1 ppm for 3 hr), and at 20–24 hr
postexposure we characterized them for airway sensitivity to methacholine challenge.
We observed no baseline differences between C57BL/6J and
mindin–/– mice in methacholine sensitivity after
exposure to FA (Figure 4A). As anticipated, we
found a significant difference in AHR in C57BL/6J mice exposed to FA and ozone
(Figure 4B). However,
mindin–/– mice showed no significant differences in
sensitivity to methacholine between FA-exposed and ozone-exposed mice. Thus,
mindin–/– mice exhibited a significant reduction in AHR
after ozone compared with C57BL/6J mice.
Figure 4 Airway responsiveness in C57BL/6J and mindin–/– mice before
(n = 4/group; A) and after
(n = 5/group; B) challenge with ozone.
*p < 0.05 compared with ozone-exposed
mindin–/– mice.
We also characterized cellular recruitment into the airspace at 20–24 hr after
ozone exposure. We found increased numbers of total cells and neutrophils after
inhalation in both strains of mice, but there were no significant differences in
total cells or neutrophils by genotype (Figure
5A,B).
Figure 5 Cellular recruitment into the airspace, evaluated by the number of total
cells (A) and the number of neutrophils
(B), and total protein level (C) measured
in whole-lung lavage fluid from C57BL/6J and mindin–/– mice after
exposure to FA or ozone (n = 5 per group).
*p < 0.05 for ozone-exposed C57BL/6J mice compared
with either group of FA-exposed controls. #p
< 0.05 for ozone-exposed mindin–/– mice compared with either
group of FA-exposed controls.
The total protein assays yielded a significant difference in BALF total protein
between ozone-exposed wild-type mice and their FA-exposed counterparts (Figure 5C). After exposure to ozone, both the
C57BL/6J and mindin–/– mice exhibited higher levels of
total protein in whole-lung lavage fluid. The level of BALF total protein in
response to ozone appears to be independent of mindin.
To determine activation of innate immunity, we measured levels of specific
proinflammatory cytokines previously associated with ozone-induced AHR. As
anticipated, ozone-exposed C57BL/6J mice had significantly higher concentrations of
KC, IL-1β, MCP-1, and TNF-α compared with FA-exposed C57BL/6J mice. In
ozone-exposed mindin–/– mice, the concentrations of these
proinflammatory cytokines were increased compared with FA controls. However,
inflammatory cytokine levels in BALF collected from the
mindin–/– mice were significantly decreased compared
with those from ozone-exposed C57BL/6J mice (Figure
6).
Figure 6 Proinflammatory cytokines KC (A), IL-1β
(B), MCP-1 (C), and TNF-α
(D) measured in the BALF from C57BL/6 and
mindin–/– mice after exposure to FA or ozone
(n = 5/group). *p < 0.05 for
ozone-exposed C57BL/6J mice compared with either group of FA-exposed
controls. **p < 0.05 for ozone-exposed C57BL/6J mice
compared with ozone-exposed mindin–/– mice.
Mindin-dependent bronchial ring contractility. To determine whether
mindin contributes to airway smooth muscle contractility, we examined bronchial ring
contractile response to carbachol. We observed no baseline differences in bronchial
ring contractile response in unexposed (Figure
7A) or LPS-exposed mindin–/– or C57BL/6J mice
(Figure 7B). However, we did observe
significant mindin-dependent differences in bronchial ring contractility to
carbachol 24 hr after inhalation of ozone (Figure
7C), in agreement with previous data suggesting that inhalation of ozone
may enhance airway smooth muscle contraction (Yoshida et al. 2002). Together, these observations support that mindin
contributes to airway smooth muscle contractility after inhalation of ozone but not
at baseline or after inhalation of LPS.
Figure 7 Bronchial ring contractile response to carbachol in naive
(A), LPS-exposed (B), and ozone-exposed
(C) C57BL/6J and mindin–/–
mice.In A, n =
6 for C57BL/6J and n = 5 for mindin–/–; in
B, n = 6 for C57BL/6J and
n = 6 for mindin–/–; in C,
n = 9 for C57BL/6J and n = 8 for
mindin–/–. *p < 0.05 for ozone-exposed
C57BL/6J mice compared with ozone-exposed mindin–/– mice.
Discussion
Our findings support the conclusion that mindin plays a modifying role in innate
immune response to the inhaled toxicants endotoxin and ozone. We demonstrate that
mindin–/– mice have divergent degrees of protection
from the biological response to either inhaled LPS or ozone. Specifically,
mindin–/–mice have attenuation of AHR and
proinflammatory cytokines in response to both endotoxin and ozone. These
observations are strikingly similar to AHR and inflammatory phenotypes previously
observed in the Tlr4–/– mouse (Hollingsworth et al. 2004). Collectively, these observations
suggest that mindin contributes to activation of innate immunity in the context of
environmental airways injury. Our observations further highlight the importance of
genes that modify innate immunity in reactive airways disease and response to
commonly encountered inhaled environmental toxicants.
The apparent similarities between Tlr4–/– mice and
mindin–/– mice in responses to LPS and ozone are quite
remarkable. We previously observed complete protection in the
Tlr4–/– mouse (Hollingsworth et al. 2004), whereas in the present study we saw an
attenuated response to LPS and ozone in the mindin–/–
mouse, despite similar levels of Tlr4 mRNA expression in whole lung
[see Supplemental Material, Figure 1A,B
(http://dx.doi.org/10.1289/ehp.1003339)]. This finding suggests that
mindin functions as a modifier gene in response to endotoxin, an observation
consistent with our current understanding that mindin is located in the
extracellular compartment and facilitates interaction between the carbohydrate
domain of LPS with the Tlr4 surface receptor (He et
al. 2004). It is equally intriguing that both the Tlr4-dependent and
mindin-dependent responses to ozone similarly affect only the profile of
proinflammatory cytokines and the development of AHR. After exposure to ozone, total
BALF protein is largely independent of either Tlr4 or mindin (Hollingsworth et al. 2004).This observation suggests that
changes in permeability or lung injury are generally independent of these genes of
innate immunity in these models of environmental lung injury.
Although we observed differences in mRNA expression of mindin in whole lung after
exposure to LPS [see Supplemental Material, Figure
1C (http://dx.doi.org/10.1289/ehp.1003339)], we observed no such
differences after inhalation of ozone (see Supplemental Material, Figure 1D). It is therefore plausible that either
the level of mindin expression or posttranslational modifications of extracellular
mindin could contribute to interaction with Tlr4 surface recognition. However, the
specific mechanism by which mindin contributes to the response to ozone remains
unknown and will be a focus of future investigations.
Previous work supports that mindin, as part of the extracellular matrix, provides a
lattice required for integrin-dependent binding and recruitment of inflammatory
cells, including macrophages, neutrophils (Jia et
al. 2005), and eosinophils (Li et al.
2009). Mindin contributed to reactive airways disease in the ovalbumin
model, which was associated with a defect in eosinophil recruitment (Li et al. 2009). It remains unclear whether the
mindin-dependent differences in AHR in the allergic model of reactive airways
disease are indirectly related to observed differences in granulocyte recruitment.
We consistently observed that mindin–/– mice exposed to LPS
had impaired recruitment of inflammatory cells to the lung. Thus, mindin-dependent
differences in AHR after exposure to LPS are associated with defects in inflammatory
cell recruitment. From this standpoint, our results regarding ozone exposure may
provide additional insight into the biological role of mindin. After ozone,
recruitment of inflammatory cells to the airspace was mindin independent, yet we
observed mindin-dependent defects in both inflammatory cytokines and AHR. This
observation suggests that the mechanisms that contribute to ozone-induced AHR are
independent of cellular inflammation. We previously observed that direct
instillation of hyaluronan fragments into the lung can induce AHR in a manner
independent of cellular inflammation (Garantziotis et
al. 2009). Therefore, we considered that because mindin binding to LPS
can be blocked by simple sugars (He et al.
2004), mindin could be required for biological response to hyaluronan
fragments, which contribute to Tlr4-dependent response to ozone (Garantziotis et al. 2009, 2010). However, we determined that the in vivo
AHR response to hyaluronan fragments in the airspace is independent of mindin [see
Supplemental Material, Figure 2 (http://dx.doi.org/10.1289/ehp.1003339)]. Next, we considered the
possibility that mindin directly contributes to airway smooth muscle contractility.
Although we did not observe mindin-dependent differences in carbachol-induced
contraction of bronchial rings in either unexposed or LPS-exposed mice, we were
surprised to find that mindin–/– mice were protected from
carbachol-induced bronchial ring contraction after inhalation of ozone (Figure 7). This finding is quite interesting for
two reasons: First, very little is known about the impact of ozone inhalation on
airway smooth muscle contractility, and second, we identified a novel role for
mindin in airway smooth muscle function. However, the specific mechanism by which
mindin can modify airway smooth muscle contractility after inhalation of ozone
remains unknown and will be an area of future investigation.
Our results in the ozone model of AHR support a direct role of innate immune
activation in the severity of reactive airways disease. For this reason, we consider
the possible therapeutic implications of inhibition of pulmonary innate immunity
during exacerbations of existing airways disease. Intensity of innate immune
response is a double-edged sword—precise regulation is required to optimize
both normal inflammation and resolution of tissue injury. It appears that the
intensity of innate immune activation can produce divergent effects on the host. For
example, low-level Tlr4 signaling appears protective in some forms of oxidative lung
injury (Qureshi et al. 2006; Zhang et al. 2005, 2006), moderate Tlr4 signaling facilitates the clearance of
pathogens (Chassin et al. 2009; Wieland et al. 2005), and excessive prolonged
Tlr4 signaling can augment lung injury (Brass et al.
2008). The future challenge of therapeutic development will be controlled
attenuation of excessive and prolonged proinflammatory response without impairing
intact host antibacterial defense. Understanding the basic mechanisms that regulate
host innate immune response to both infectious and noninfectious lung injuries can
provide the insight required for successful therapeutic development. Previous work
supports that both Tlr4 and mindin play a fundamental role in antibacterial host
defense. It is unclear if targeting the extracellular matrix protein mindin and/or
Tlr4 receptors in airway disease with an AHR clinical phenotype would provide
significant clinical benefit. However, a clearer understanding of host factors in
response to common environmental exposures can provide insight for the future
development of personalized therapies with a basis in pharmacogenetics.
Conclusions
The extracellular matrix protein mindin is an important modifier of pulmonary innate
immune response to commonly inhaled environmental toxicants. Mindin plays a central
role in the biological response to both inhaled LPS and ambient ozone.
Mindin–/– mice demonstrated reduced AHR and production
of proinflammatory cytokines in these two divergent models of environmental airways
injury. Mindin contributed to bronchial ring contractility only after inhalation of
ozone. Clear understanding of the mechanisms that mindin contributes to the
development of reactive airways disease could provide an opportunity for development
of novel therapeutic strategies in human reactive airways disease.
Supplemental Material
(88 KB) PDF Click here for additional data file.
This work was supported by National Institutes of Health grants to J.W.H.
(ES016126, ES02046), W.M.F. (ES016347), and J.P.E. (HL081825).
The authors declare they have no actual or potential competing financial
interests.
==== Refs
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Sensors (Basel)Sensors (Basel, Switzerland)1424-8220Molecular Diversity Preservation International (MDPI) 10.3390/s110605835sensors-11-05835ArticleAuthenticated Key Agreement with Rekeying for Secured Body Sensor Networks Eldefrawy Mohamed Hamdy 1Khan Muhammad Khurram 1*Alghathbar Khaled 12Tolba Ahmed Saleh 1Kim Kyngn Jung 31 Center of Excellence in Information Assurance, King Saud University, P.O. Box 92144, Riyadh 11653, Saudi Arabia; E-Mails: [email protected] (M.H.E.); [email protected] (K.A.); [email protected] (A.S.T.)2 Information Systems Department, College of Computer and Information Sciences, King Saud University, Riyadh, 11653, Saudi Arabia3 Department of Child Development & Welfare, Woosuk University, Jeonbuk, 565-701, Korea; E-Mail: [email protected] (K.J.K.)* Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +966-1-4696457.2011 31 5 2011 11 6 5835 5849 9 3 2011 16 5 2011 26 5 2011 © 2011 by the authors; licensee MDPI, Basel, Switzerland.2011This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Many medical systems are currently equipped with a large number of tiny, non-invasive sensors, located on, or close to, the patient’s body for health monitoring purposes. These groupings of sensors constitute a body sensor network (BSN). Key management is a fundamental service for medical BSN security. It provides and manages the cryptographic keys to enable essential security features such as confidentiality, integrity and authentication. Achieving key agreement in BSNs is a difficult task. Many key agreement schemes lack sensor addition, revocation, and rekeying properties, which are very important. Our proposed protocol circumvents these shortcomings by providing node rekeying properties, as well as node addition and revocation. It proposes a key distribution protocol based on public key cryptography—the RSA (Rivest, Shamir and Adleman) algorithm, and the DHECC (Diffie-Hellman Elliptic Curve Cryptography) algorithm. The proposed protocol does not trust individual sensors, and partially trusts the base station (hospital). Instead of loading full pair-wise keys into each node, after installation our protocol establishes pair-wise keys between nodes according to a specific routing algorithm. In this case, each node doesn’t have to share a key with all of its neighbors, only those involved in the routing path; this plays a key role in increasing the resiliency against node capture attacks and the network storage efficiency. Finally we evaluate our algorithm from the BSN security viewpoint and evaluate its performance in comparison with other proposals.
body sensor networkRSADHECCrekeying
==== Body
1. Introduction
The term body sensor network (BSN) [1] was coined to represent human body sensing applications in which a number of intelligent physiological sensors are integrated into a wearable wireless BSN, which can be used for computer assisted rehabilitation and/or the early detection of medical conditions. Such applications imply that outpatients can be monitored from their homes, freeing up space in hospital beds. As there is a legal requirement to keep patients’ physiological data private, any implemented network must include strong security protocols. However, its physical characteristics make incorporating security a challenging task. The constraints on sensors make the design and operation of contemporary networks exceedingly different. The existing security mechanisms for wired and wireless networks cannot be applied to BSNs, because of the constrained energy, memory and computational capability of the latter.
Key management protocols are at the core of secure communications. The goal of key management is to establish secure links between neighbor sensors in networks to exchange their data in a multi-hop fashion. Public key schemes have many advantages, such as low communication overhead, and good storage capability and scalability. These schemes can provide simpler solutions with much stronger security strength. Several researchers [2,3] have shown that public key schemes are valid on sensor nodes. The computational cost is expected to fall faster than the cost to transmit and receive. Furthermore, next generation sensor nodes are expected to combine ultra-low power circuitry allowing for a continuous energy supply.
The protocol proposed in this paper will circumvent the shortcoming of needing to provide the rekeying of nodes that occurs with previous algorithms, as well as node addition and revocation [4,5]. Therefore, with the fast growing technology, public key schemes are no longer impractical and are expected to be widely used in the near future [3]. RSA, elliptic curve and public key cryptography are all viable options on an 8-bit CPU. The relative performance advantage of ECC point multiplication over RSA modular exponentiation is inversely proportional to processor word size and directly proportional to key size [6].
Gateways have considerably high energy resources compared to sensor nodes and are equipped with high performance processors and more memory. The base station performs network management functions in a centralized fashion; constructing the Routing-Path-Table [7] for each node, depending on the installation knowledge. In location based routing, nodes are addressed by means of their locations [8]. We assume that each sensor has direct communication with the base station during key management operations, i.e., the formation phase. Consequently, we will analyze the security strength of our proposal with many desirable security attributes to infer its success; our algorithm will also be compared with previous studies to evaluate its performance behavior.
The rest of this paper is organized as follows: Section 2 discusses the related work, Section 3 discusses the system architecture, Section 4 proposes our new algorithm, Section 5 evaluates the security properties, Section 6 analyzes our scheme’s performance, and finally, Section 7 concludes the paper and illustrates the future research.
2. Related Work
The following section discusses some of the published BSN authentication and key agreement schemes. Topics related to BSN efficiency and its shortcomings according to the desirable security attributes that will be discussed below will also be illustrated. Pre-loaded symmetric shared keys are used in large scale sensor networks for geographical region observations [9,10]. In these techniques, a certain key is loaded in each node and used to derive a shared secret key. Balfanz et al. [11] utilized a secure-limited channel (e.g., infrared) to exchange public-keys between parties prior to the authentication process. However, this approach requires huge resources, which would be difficult to provide in medical environments. Human confirmation of correct association is also difficult when based on public-keys and without visual cues. The resurrecting duckling protocol [12] establishes a master-slave relationship between devices whereby the first device in contact with a sensor becomes its master and can upload policies to the sensor that permits interactions with other devices. Sensors from previous patients have to be explicitly disassociated by the master before they can be reused by other patients, which may not always be practical in hospitals.
Jiang et al. [13] use self-certified keys (SCK) and Elliptic Curve Cryptography (ECC) to establish pair-wise keys for authentication. Each sensor agrees upon a secret with the user based on the secret information pre-loaded by a key distribution centre (KDC). Authentication is achieved if the user demonstrates knowledge of the shared secret-key with at least t sensors. To achieve a sensor to patient association, each patient’s BSN would require different ECC curve parameters (as each BSN is a domain), which would be impractical for the hundreds of BSN in a hospital. SNAP [14] also uses ECC to establish pair-wise keys between nodes and the gateway. It requires that each sensor be equipped with a biometric device to authenticate the patient and uses the shared secret to communicate with the base station. However, it does not establish group keys. Many studies have been conducted to address ECC-based public-key cryptography [15]. It has been found to be viable for resource constrained wireless sensor networks, providing better key distribution, management and authentication.
In a medical environment, those techniques are not sufficient, as the wireless domains of groups may overlap and only the correct sensors must be associated with each patient. Hence, the rekeying property regarding node addition and revocation is not applicable for these protocols. In addition, the pair-wise key agreement approach can’t provide resiliency against node capturing attacks and also has storage efficiency issues. From that regard our protocol has to provide the rekeying feature with key addition and key revocation options, as well as consider the storage efficiency and resiliency by the utilization of a specific routing algorithm in the key agreement process.
3. The System Architecture
The network topological structure is illustrated in Figure 1. The network includes gateway and sensor nodes. A gateway is less energy constrained and tamper resistant, as compared to other nodes. Sensor nodes can communicate with each other. The gateway is assumed to be secure and trusted by all nodes in the network, as soon as nodes have been loaded. Upon the knowledge of sensor nodes’ positions, the gateway begins the calculation of the routing path, considering the communication path for each node to reach the gateway hop by hop, conduct a data aggregation, and then position the network nodes with a distribution of shared session keys calculated with the cooperation of each node. The routing algorithm must be adaptive to locate alternative routing paths for each node to reach its destination, considering the trust and cost parameters of the route selections that avoid loops [16].
Involving the routing technique for the BSN provides a higher level of connectivity with a relatively small number of neighbor-shared keys. Compared with a pair-wise key distribution [17], this algorithm has to obtain an optimal routing path without considering the necessity of a mesh implantation topology, hence, by analogy, the fully mesh connected regular network is equal to the pair-wise key distribution BSN linking. According to a specified routing algorithm, each node doesn’t have to share a key with all radio-range neighbors, only those involved in the routing path.
4. Proposed Protocol Description
Some researchers have previously shown that with an accurate design, the widely used RSA public key cryptosystem [18] and DHECC key agreement techniques [19] can be deployed on even the most constrained of current sensor network devices. Watro et al. [20] proposed a mechanism for providing authentication and key exchange based on the well-known RSA cryptosystem, using e = 3 as the public exponent for MICA1 Motes [21]. These devices carry an Atmel ATmega 128L microcontroller with a CPU of 4 MHz of 4 KB RAM and 128 KB flash memory. The security properties of the Low Exponent variant of RSA have been studied thoroughly [22]. The proposed protocol notations are mentioned in Table 1.
The proposed key agreement protocol is provided in Table 2 which is explained as follows:
4.1. Key Pre-Loading Phase
The gateway is loaded with its public and private keys of the RSA cryptosystem, which means that m and e are the Gateway public keys, and d, such that ed ≡ 1modϕ(m) is the gateway’s private key. In addition, the gateway is loaded with the public keys of all sensor nodes yi. Each sensor node is loaded with its DHECC private key xi and the public keys of the gateway m and e.
4.2. Key Establishment Phase
Step 1: All sensors generate an ephemeral random key ri, concatenate it with idi and encrypt the result with the gateway public key to obtain (ri || idi)e mod m. We assume that each sensor begins the session with maximum transmission power to reach the gateway.
Step 2: The gateway decrypts the received vectors coming from all sensors with its private key (ri || idi)ed ≡ (ri || idi)mod m to obtain ri for each Si. According to the routing map, after the gateway has been calculated, it starts to send the session keys to each node, as shown in Figure 1. The nodes S1, S2, S3, and S4 are from the same cluster PI, and as such, they will obtain the same session key.
Step 3: The gateway responds to these nodes by sending 𝒵PI = G(rS1 + rS2 + rS3 + rS4) encrypted by the DH long term private key for each node. For example, node S1 will receive yS1(rS1 + rS2 + rS3 + rS4), from the gateway and then S1 will calculate 𝒵PI = (yS1/xS1) (rS1 + rS2 + rS3 + rS4). As such, we realize that many nodes can share more than one cluster, which also means that they share more than one key. This manner of key distribution primarily depends on the routing path established by the gateway.
4.3. The Steady State Phase
Considering node S1 that would like to send information to the gateway through
S1→PIS2→C1S5→PIIS8→PIIGW, it sends its information to node S2, encrypted with ZPI. S2 then forwards it to deliver S5 encrypted with ZC1, and S5 delivers it to S8 encrypted with ZPII. Finally, S8 delivers it to the gateway encrypted with ZPII. Node S1 has alternative routing paths, to be used as needed, and therefore, the established session key between nodes is used later for a symmetric encryption for secure data forwarding to the gateway.
After a random period of time, any node in the cluster could initiate the rekeying in the following two steps:
Step 1: Si generates another ephemeral random key ri′ and send it to the gateway in the same fashion as the second phase. In a parallel fashion, Si sends this new ephemeral key ri′ concatenated with its idi concatenated with the present ephemeral key ri to its neighbors encrypted with the shared session key between them E𝒵Pi (ri′ || idi || ri).
Step 2: The gateway establishes a new session key for this cluster and sends it to the cluster nodes, while the cluster nodes themselves have calculated this new key by decrypting the received vector from node Si. They can replace the old ri with the new ri′ and compare the new key that comes from the gateway with the key they have calculated to validate the integrity.
4.4. Key Revocation and Addition
The applicability of the additively homomorphic DHECC algorithms plays a key role in the nodes’ addition and revocation.
The revocation: In the case of node capturing, or when a predefined value of a node life time is reached, the gateway begins the revocation by removing the current ephemeral random key for this node from the session key shared with this node’s neighbors. The gateway then resends the new key to all neighbors encrypted with their static private key. Consequently, we consider that the gateway has the ability to detect the nodes’ capture [23].
The addition: Establishing a key for a new node loaded by its public and private keys, xi, yi and the gateway public key m = PQ, with the network sensor nodes is similar to Vector Exchanging and the Key Establishment Phases. We assume that the gateway is informed by the public key of the new node before node installation.
5. Security Analysis
The inherent security [24] relies on the difficulty of recovering this key via the factorization of large integers. It is generally accepted that RSA keys should be a minimum of 1,024 bits. Discrete logarithm cryptography (DLC) is another area of cryptography where security is provided by difficulty in solving logarithmic equations over large finite groups. Elliptical Curve Cryptography (ECC) is a subset of DLP, where the discrete logarithmic solutions occur over an equation of a plane curve. Wireless networks are more vulnerable to attacks than their wired counterparts, due to the nature of wireless transmission, resource limitations and uncontrolled environments that represent a great challenge in BSN security. BSNs have the following security requirements [25,26]:
Known Key Security: The protocol should still achieve its goal in the face of an adversary who has learned other session keys Zcj. Hence, each run of the protocol between the nodes and the gateway produce a unique session key that depends on the random ephemeral keys ri of nodes Si. The adversary, who learned some other session keys, can’t predict new or subsequent session keys Zcj (forward secrecy), and also can’t predict any earlier session keys (backward secrecy).
Key Control: Neither of the principles who share the key agreement process are able to force the key to be any chosen value, otherwise, one party could force the use of an old key, key disposable safety. One potential benefit is that each principle doesn’t have to rely on any other party to generate appropriate keys. As long as neither party is malicious, it can often be guaranteed that the session key Zcj is a sufficiently random input. A related benefit is that principals can often be sure that the session key is fresh by ensuring that their own input is fresh. Consequently, involving an ephemeral random key generated by each node ri to share the session key establishment is to grant the key freshness of the session key and provide the key control property.
Implicit Key Authentication: A key establishment protocol is said to provide implicit key authentication (of Si to the gateway) if the gateway is assured that no other node Si, aside from the specifically identified Si, can learn the value of a particular secret key. A key agreement protocol which provides implicit key authentication to both participating principals is called the authenticated key agreement (AK) protocol. Our algorithm implicitly authenticates the exchanged information sensor nodes and the gateway using the long term private keys of others.
Key Confirmation: A key agreement protocol satisfies key confirmation; if one party is assured that all other parties have possession of a particular secret key, through the rekeying process, then the new session keys established by the gateway are compared with their similar, node self generation, keys. The lack of termination means that each node has the correct key, thus, the gateway confirms that the sensor nodes possess the correct secret key. This property only covers the steady state (rekeying) phase, a very frequent process. If this isn’t the case, node addition and revocation, the sensor nodes, have to trust the gateway; this is referred to as the partial trust of the gateway.
Explicit Key Authentication: If both implicit key authentications and key confirmations are provided.
Node Capture: Each node is pre-loaded with a unique private and public key of itself, the gateway public key, and a random ephemeral session key, revealed by node capturing. In this case, they cannot provide any profit to the intruder about the network, or the rest of the nodes. Our proposal achieves a good degree of resilience against node capture, because of the key freshness.
Scalability: The ability to support larger networks by adding more nodes is already provided through this algorithm, as discussed previously. The key distribution mechanism supports large networks and is flexible against a substantial increase in the size of the network after installation.
Confidentiality: This aspect is ensured by using symmetric encryption to encrypt the exchanged traffic by the established session keys between sensors. The confidentiality is conducted using periodic key freshness to prevent long term attacks.
Key Freshness: The derived session key must be fresh, as opposed to the reuse of old keying material. Since sensors generate the random integer ri for each session, we guarantee the key freshness property. We can also refer to this property as the ability to resist predictable attacks.
6. Performance Analysis
In this section, we analyze the performance of our algorithm with respect to storage and computational costs.
6.1. Storage Analysis
The storage complexity is the amount of memory (RAM size) required to store security credentials. The storage complexity in turn affects the hardware cost of sensor nodes. Our proposal considers the base station as the resource-rich node. Other schemes depend on key distribution before installation, which involves a tradeoff between the numbers of keys each node must store (storage) and the number of secure links between the nodes in the resulting network (connectivity). Our proposal achieves a key agreement system after deployment, according to a routing strategy. The many-to-one traffic pattern dominates in typical sensor networks, where all sensors send data to one sink [27]. Because of the many-to-one traffic pattern, a sensor node only communicates with a small portion of its neighbors, e.g., neighbor sensors that are in the routes from itself to the sink [28,29]. This means that a sensor node does not require shared keys with all neighbors. By consulting the datasheet of the PIC Microcontroller PIC18F2550 [30], we find that it has RAM of 2 Kbytes and a ROM of 24 Kbytes, which is enough for key generation for RSA (2 Kbytes).
The implementation of the prime field algorithms used 635 bytes of RAM (data) memory and 4,072 bytes of ROM (program) memory. This accounts for approximately 31% of the RAM (data) memory and approximately 13% of the ROM (program) memory available on the microcontroller [30]. Table 3 comes from a Standard Datasheet for the microcontroller which we have used; it also shows a comparison of different microcontrollers from the same family, and highlights the most important characteristics RAM, ROM which is critical when implementing cryptography algorithms on a small microcontroller.
6.2. Computation Analysis
Considering the computational complexity, many studies have been conducted to address PKC for sensor networks. Gura et al. [31] established the elliptical curve cryptography signature verification needs of 1.62 s using 160-bit keys on ATmega 128 of 8 MHz CPU; the processor was used for a Crossbow motes platform [31]. These results illustrate that ECC-based algorithms are good to use. In addition, the protocol by Watro et al. [23] was implemented on MICA1 Motes [21]. These devices carry an Atmel ATmega 128L microcontroller running at 4 MHz with 4 KB of RAM and 128 KB of flash memory.
This implementation was conducted over six years ago. Currently, there are many sensor nodes with high resources [32] compared with MICA1. In our study, an implementation of an elliptic curve cryptosystem on a Microchip PIC18FXX [30] family microcontroller is outlined. We designed a simple prototype diagram for our study [33] (Figure 2).
Figure 2 shows the schematic diagram of a simple sensor node. Each node consists of an Amplitude Shift Key transmitter and Receiver with a carrier frequency of 433 MHz for transmitting/receiving digital measurement data like temperature, light, etc. The ASK sensor is connected through the Microcontroller by using the digital Port of the microcontroller. The Random Number Generator Circuit feeds the microcontroller with random sequences that can be used for cryptography algorithms and key exchange techniques.
We chose PIC18FXX [30] for many reasons [34]: it’s cheap, it’s widely used, it’s available at competitive prices, and it can be used easily in BSN. The 8-bit bus width along with the data memory and processor speed limitations present some additional challenges versus the implementation on a general purpose computer. All algorithms required to perform an elliptic curve Diffie-Hellman key have been implemented. To minimize the processing time, the hardware circuit was designed with a clock rate of 48 MHz, the maximum clock rate of the PIC18F2550 microcontroller. This microcontroller has an internal USB interface with minimal external parts required which led to a simplified design for our study.
As the PIC18F2550 microcontroller and most 8 bit microcontrollers used in BSN do not contain a random number generator, it is necessary to either obtain random numbers from another source or incorporate a hardware random number generator into the design. For cryptographic uses, it is very important that random numbers be truly random and cannot be guessed or predicted in any way. For this problem, we designed a simple circuit for generating random sequences based on the avalanche effect. The circuit shown in Figure 2 was tested to provide the random noise input to the microcontroller. The base to the emitter junction of Q1 is used as the avalanche diode in this implementation.
Figure 3 shows the circuit diagram that is used for generating random sequences using the semi-conductor characteristics of transistors. With that regard we tried to wire up a circuit diagram as shown at that figure.
The involved capacitors in those circuits are called bypass capacitors [35], to which bypass high frequency signals from the power supply 12 VDC, so that we can get a clean Direct Current voltage, because high frequency signals may affect that random circuit generator.
Results: The efficiency of each of the algorithms in the prime field was measured by counting the cycles used on a simulator and then verifying the results by running it in real hardware. The elliptic curve point addition, doubling, and multiplication results were calculated using the actual times from the prime field algorithms and the number required.
Assuming that the communications time is negligible, the PIC18F2550 microcontroller can perform a Diffie-Hellman key exchange in approximately 5.4 seconds (two elliptic curve point multiplications).
Table 4 shows our simulation results when we tried to implement a Diffie-Hellman Key Exchange, we have simulated each arithmetic operation like addition, subtraction to evaluate the performance on the chosen microcontroller PIC18FXX while implementing the key exchange. We computed the cycles that are consumed by the clock of the microcontroller and the time it takes to perform the arithmetic operations that are involved in the elliptic curve algorithm. After that we present a few techniques that can enhance the performance and increase the efficiency while implementing the elliptic on small microcontrollers that are usually used in body sensor networks.
Assembly Coding Improvements: Most of the computationally intensive sections of this implementation have been coded in assembly language, which reduces the number of inefficiencies caused by a C compiler. Additional speed and memory efficiency may be gained by hand coding the entire implementation. This is a trade off with available time and readability versus possible negligible performance gains.
Speeds versus Memory Tradeoff: Our implementation can be made faster by unrolling all of the loops in the software to eliminate the counts and compare those used for the looping operation. Conversely, it could also be made smaller (more memory efficient) by using recursion and additional looping. The tradeoff between speed versus memory should be considered in future implementations. The PIC18F2550 microcontroller is easily capable of performing an elliptical curve Diffie-Hellman key exchange. With a working time of 5.4 seconds per exchange, this type of cryptography is not suited to high speed data transfers for this particular device. For high-speed transfers, the exchanged secret may be used as the key in a symmetric cipher, such as Rijndael (as used in the Advanced Encryption Standard [37]).
RSA Implementation: For implementing the RSA Public-Key Encryption algorithm, we used the same configuration as in the Elliptical Curve Diffie-Hellman key: an 8 bit PIC18F2550 microcontroller, with a clock speed of 48 MHz. We measured the speed of a single block of data 512 bits key:
Encryption: 2 s.
Decryption: 120 s.
We also tested another microcontroller, the dsPIC30F3013, which is a digital signal processor microcontroller with a Microchip© with a clock speed of 30 MHz.
Encryption: 0.2 s.
Decryption: 15 s.
7. Conclusions
In this paper, considering the BSN security based on the public key mode, we have demonstrated a novel key management scheme with rekeying. This key agreement system establishes an ephemeral session key between sensor nodes with the participation of the gateway as a trusted third party. Group key distribution is very simple and the rekey messages are also authenticated. This facilitates the efficient renewal of group keys to cater for membership changes. The proposed protocol covers the rekeying property and considers the nodes addition and revocation from the viewpoint of secured key establishment. Rekeying, node addition and revocation features are the main shortcomings of this process as compared to previous algorithms.
We achieved the security requirements without the utilization of a secure-limited channel, like in Balfanz [11], which requires huge resources. In addition, we didn’t go to the master-slave relationship, which destroys the key control property. From another viewpoint, our protocol establishes pair-wise keys between nodes according to a specific routing algorithm, instead of loading full pair-wise keys into each node. In this way, each node doesn’t have to share a key with all of its neighbors, except those involved in the routing path, which is the key role of increasing the resiliency against node capturing and storage efficiency. We also analyzed the security strength of our proposal with many desirable security attributes to deduce its success; our algorithm has been evaluated according to its performance behavior implemented with respect to many previous studies.
Particularly, we consider some of the future areas in the study of security issues in BSN as follows. Many researchers have shown that public key operations may be practical in sensor nodes. However, private key operations are still too expensive to accomplish in a sensor node. As public key cryptography can greatly ease the design of security in BSN, improving the efficiency of private key operations on sensor nodes is highly desirable [36]. The mobility of sensor nodes has a great influence on sensor network architecture and sequentially on the routing protocols. New secure routing protocols for mobile BSN are needed to be created. Multimedia sensors might not be widely utilized for BSNs now, but will likely be in the near future. Substantial differences in authentication and encryption exist between discrete applications and continuous real time application, indicating that there will be distinctions between continuous stream security and the current protocols used in BSNs. Current studies on security in WSNs focus on individual topics such as key establishment, secure routing, secure data aggregation, and intrusion detection. QoS and security services need to be evaluated together in BSN.
Figure 1. The Proposed Key Agreement Protocol in Body Sensor Network.
Figure 2. The Schematic Diagram with the Interfaces Consideration.
Figure 3. The Schematic Diagram for the Random Generator Circuit.
Table 1. The Proposed Protocol Notation.
Notation Description
P,Q Two large and distinct random primes.
m PQ multiplication such that ϕ(m) = (P – 1)(Q – 1).
e 1 < e < ϕ(m), and 1 < e < ϕ(m).
d The multiplicative inverse of e mod ϕ(m).
G The generating element of DHECC.
n The order of G.
Si Sensor node number i.
ri Random integers chosen by Si.
ti Ephemeral public keys: ti ≡ G × ri.
xi The private long-term keys of Si.
yi Si long-term public keys: yi ≡ G × xi.
idi The identification of Si.
𝒵Pi The shared secret for the patent number i.
𝒵Cj The shared secret for the shared cluster number j.
Table 2. The Proposed Key Agreement Protocol.
Before Installation: Key Pre-loading Phase
GW m = PQ, e, d, yi
Si xi, yi, e, m = PQ
After Installation: Vector Exchanging Phase
Si → GW (ri || idi)e mod m
GW → Si xi𝒵Cj
Key Establishment Phase
Si 𝒵Cj = xi𝒵Cj/xi
The Steady State (Rekeying) Phase
Si → GW (ri′ || idi)e mod m
Si→ S′i E𝒵Cj (ri′ || idi ||ri)
GW → Si xi𝒵′Cj
S′i ZCj=?Z′Cj
Table 3. The PIC Microcontroller PIC18F2550 Datasheet.
Device Program Memory Data Memory
Flash (bytes) # Single-Word Instruction SRAM (bytes) EEPROM (bytes)
PIC18F2455 24 K 12,288 2,048 256
PIC18F2550 32 K 16,384 2,048 256
PIC18F4455 24 K 12,288 2,048 256
PIC18F4550 32 K 16,384 2,048 256
Table 4. The Algorithm Execution Time Efficiency.
Algorithm Cycles Time
Addition 206 17.2 uS
Subtraction 273 22.75 uS
Multiplication 15,803 1,317 uS
Modulus p reduction 12,790 1,066 uS
Inverse 31,280 2,607 uS
Elliptic Curve Point Addition (1 Inv, 6 Sub, 2 Mul) 64,524 5.4 mS
Elliptic Curve Point Doubling (1 Inv, 5 Sub, 4 Mul) 95,857 8.0 mS
Elliptic Curve Point Multiplication (256 EC Dbl, 128 EC Add) 32,798,464 2.73 S
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Med Gas ResMedical Gas Research2045-9912BioMed Central 2045-9912-1-810.1186/2045-9912-1-8Research Carbon monoxide inhibits Fas activating antibody-induced apoptosis in endothelial cells Wang Xue [email protected] Yong [email protected] Seon-Jin [email protected] Hong Pyo [email protected] Augustine MK [email protected] Stefan W [email protected] Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Pittsburgh Medical Center, Pittsburgh, PA 15213, USA2 Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, Harvard Medical School, 75 Francis Street, Boston, MA, 02115, USA3 School of Biological Sciences, College of Natural Sciences, University of Ulsan, Ulsan, 680-749, Korea2011 18 5 2011 1 8 8 25 1 2011 18 5 2011 Copyright ©2011 Wang et al; licensee BioMed Central Ltd.2011Wang et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
The extrinsic apoptotic pathway initiates when a death ligand, such as the Fas ligand, interacts with its cell surface receptor (ie., Fas/CD95), forming a death-inducing signaling complex (DISC). The Fas-dependent apoptotic pathway has been implicated in several models of lung or vascular injury. Carbon monoxide, an enzymatic product of heme oxygenase-1, exerts antiapoptotic effects at low concentration in vitro and in vivo.
Methods
Using mouse lung endothelial cells (MLEC), we examined the antiapoptotic potential of carbon monoxide against apoptosis induced by the Fas/CD95-activating antibody (Jo2). Carbon monoxide was applied to cell cultures in vitro. The expression and/or activation of apoptosis-related proteins and signaling intermediates were determined using Western Immunoblot and co-immunoprecipitation assays. Cell death was monitored by lactate dehydrogenase (LDH) release assays. Statistical significance was determined by student T-test and a value of P < 0.05 was considered significant.
Results
Treatment of MLEC with Fas-activating antibody (Jo2) induced cell death associated with the formation of the DISC, and activation of caspases (-8, -9, and -3), as well as the pro-apoptotic Bcl-2 family protein Bax. Exposure of MLEC to carbon monoxide inhibited Jo2-induced cell death, which correlated with the inhibition of DISC formation, cleavage of caspases-8, -9, and -3, and Bax activation. Carbon monoxide inhibited the phosphorylation of the Fas-associated death domain-containing protein, as well as its association with the DISC. Furthermore, carbon monoxide induced the expression of the antiapoptotic protein FLIP and increased its association with the DISC.
CO-dependent cytoprotection against Fas mediated apoptosis in MLEC depended in part on activation of ERK1/2-dependent signaling.
Conclusions
Carbon monoxide has been proposed as a potential therapy for lung and other diseases based in part on its antiapoptotic effects in endothelial cells. In vitro, carbon monoxide may inhibit both Fas/caspase-8 and Bax-dependent apoptotic signaling pathways induced by Fas-activating antibody in endothelial cells. Strategies to block Fas-dependent apoptotic pathways may be useful in development of therapies for lung or vascular disorders.
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Background
Apoptosis, a form of programmed cell death, serves a critical function in the maintenance of tissue homeostasis under physiological conditions, as a component of developmental programs. Dysregulation of apoptosis may contribute to the progression of a number of disease states, including cancer, autoimmunity, and neurodegenerative disorders [1,2]. Furthermore, apoptosis has also been implicated in the pathogenesis of several pulmonary diseases, including acute lung injury/acute respiratory distress syndrome (ALI/ARDS) [3,4], and chronic obstructive pulmonary disease [5].
Apoptosis requires the regulated activation of proteases (ie., caspases) and nucleases within an intact cell membrane. Two apoptotic pathways have been identified by which cells can initiate and execute the cell death process: an intrinsic (mitochondria-dependent) pathway and an extrinsic (death receptor-dependent) pathway [6-8]. Intrinsic apoptosis involves the activation and mitochondrial translocation of pro-apoptotic Bcl-2 family members (e.g., Bax), leading to mitochondrial dysfunction and release of pro-apoptotic mediators (e.g., cytochrome-c).
Extrinsic apoptosis initiates with the plasma membrane assimilation of the death-inducing signaling complex (DISC), consisting of Fas, FADD, and caspase-8, by ligand-dependent (ie., Fas ligand, FasL) or independent mechanisms. Death receptors, a subset of type I transmembrane receptors of the tumor necrosis factor receptor family/nerve growth factor receptor family directly transduce apoptotic signals. Among these, Fas (Apo-1/CD95), is a transmembrane cell surface receptor containing three cysteine-rich extracellular domains at the amino-terminus, which are responsible for ligand binding, and an intracytoplasmic death domain (DD) of ~80 amino acids essential for transducing the apoptotic signal [9]. Binding of FasL to Fas causes a higher-order aggregation of the receptor molecules and recruitment of the adaptor molecule Fas-associated death domain (FADD) via DD-DD interactions. FADD also contains a death effector domain, which recruits pro-caspase-8 (FLICE) and/or pro-caspase-10 to the receptor. The resulting multimeric protein complex forms within seconds of receptor engagement [10]. Autoproteolytic activation of caspase-8 results in the processing of Bid to tBid, which assimilates into the mitochondria to trigger cytochrome c release, and may facilitate Bax activation [11].
FLIP, also known as Fas-associated death domain (FADD) interleukin-1β-converting enzyme (FLICE)-like inhibitory protein has been characterized as an inhibitor of apoptosis induced by death receptors such as Fas. Multiple splice variants of c-FLIP have been found. Of these, three could be detected at the protein level. These are designated as c-FLIP short (c-FLIPS), c-FLIP long (c-FLIPL), and c-FLIP Raji (c-FLIPR) [12-16]. While all these isoforms of FLIP interfere with caspase-8 cleavage, only FLIPL is cleaved at the DISC, whereas FLIPS and FLIPR inhibit caspase-8 by remaining in the DISC. Increased levels of FLIPL can confer protection against Fas-induced apoptosis [12-16].
We previously reported that the expression of FLIP protected against cell death in pulmonary epithelial and endothelial cells subjected to hyperoxia [17,18], or in endothelial cells subjected to hypoxia/reoxygenation [19].
Carbon monoxide (CO) occurs in nature as a product of the combustion of organic materials. CO also arises endogenously in cells and tissues as a product of heme oxygenase (HO) activity, which degrades heme to biliverdin-IXα and ferrous iron [20]. HO-1, the inducible form of HO, responds to transcriptional upregulation by multiple forms of cellular stress. HO-1 confers cytoprotection against oxidative stress in vitro and in vivo [21]. HO-derived CO acts as a vasorelaxant and inhibits other vascular functions such as platelet aggregation and smooth muscle proliferation [22]. When applied at low concentration, CO exerts potent cytoprotective effects mimicking those of HO-1 induction, which include anti-inflammatory effects in macrophages [23,24], as well as antiapoptotic effects in vascular cells [25,26]. HO-1 and/or CO provide tissue protection in a number of in vivo models, including vascular injury, ischemia/reperfusion (I/R) injury, oxidative lung injury [27-32], and organ transplantation [33]. The mechanisms of CO action potentially involve modulation of intracellular signaling pathways, including activation of p38 MAPK, and soluble guanylyl cyclase [23,34,35].
Exposure to low concentrations of CO prevents the initiation of apoptosis in various cultured cells [25,36,37]. Using mouse lung endothelial cells (MLEC), we have previously demonstrated that CO treatment inhibited apoptosis in a model of hyperoxia-induced oxidative stress [37]. In the current study, we demonstrate that CO prevented Fas-activating antibody (Jo2)-induced apoptosis in MLEC by inhibition of both extrinsic and intrinsic apoptotic pathways.
Methods
Chemical and Reagents
Antibodies: anti-Bax, anti-caspase-8, anti-caspase-9, anti-caspase-3, anti-Fas, anti-FLIP, and protein A-agarose were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-NF-κB p65, anti-P44/42, anti-JNK and anti-FADD were from Cell Signaling Technology, Inc. (Beverly, MA). Anti-Bax 6A7 antibody was purchased from BD PharMingen (San Diego, CA). The lactate dehydrogenase (LDH) assay kit was from Roche Diagnostics Corporation (Indianapolis, IN). JNK inhibitor-II and PD 98059 were from Calbiochem (San Diego, CA). Digitonin and all other reagent chemicals were from Sigma (St. Louis, USA).
Isolation and Culture of Murine Lung Endothelial Cells
Endothelial cells were isolated by an immunobead protocol as previously described [38]. Briefly, mouse lungs were digested in collagenase and filtered through 100-μm cell strainers, centrifuged, and washed twice with medium. Cell suspensions were incubated with a monoclonal antibody (rat anti-mouse) against platelet endothelial cell adhesion molecule-1 for 30 min at 4°C. The cells were washed twice to remove unbound antibody, and resuspended in a binding buffer containing washed magnetic beads coated with sheep anti-rat immunoglobulin G. Attached cells were washed four to five times in culture medium, and then digested with trypsin/EDTA to detach the beads. Bead-free cells were centrifuged and resuspended for culture. After two passages, the cells were incubated with fluorescent-labeled diacetylated low-density lipoprotein, which is only absorbed by endothelial cells and macrophages, and sorted to homogeneity by fluorescence-activated cell sorting.
Cell Culture and Treatments
The MLEC (passages 4-6) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 6.32 g/liter HEPES, and 3.3 ml of endothelial cell growth supplements in humidified incubators at 37°C. For Fas-activating antibody treatment, cultures were changed to serum-free medium, to which 200 ng/ml of anti-Fas antibody (Jo2) was added for the time indicated. PD 98059 from DMSO stock solution was applied at a final concentration of 25 μM for 30 min prior to Jo2 antibody treatment.
Carbon Monoxide Exposure
Cell cultures were treated in vitro with CO (250 ppm) in compressed air containing 5% CO2, in a modular exposure chamber as previously described [23]. A CO analyzer (Interscan Corp., Chatsworth, CA) was used to measure CO levels in the chamber. For all experiments involving exposure to CO, cells were pretreated with CO for 2 h.
Lactate Dehydrogenase (LDH) Release Assay
LDH release was measured using a commercially available assay (Cytotoxicity Detection Kit; Roche Molecular Biochemicals). After gentle agitation, 200 μl of medium was removed at various times to be used for the assay. The samples were incubated (30 min) with buffer containing NAD+, lactate, and tetrazolium. LDH converts lactate to pyruvate generating NADH. The NADH then reduces tetrazolium (yellow) to formazan (red), which was detected by absorbance at 490 nm.
Immunoprecipitation and Western Blot Analysis
Proteins were isolated from MLEC cultures in immunoprecipitation buffer (1 × PBS, 1% (vol/vol) Nonidet P-40, 0.5% (weight/vol) sodium deoxycholate, 0.1% (weight/vol) sodium dodecyl sulphate, 0.1 mg/ml phenylmethylsulfonyl fluoride, 30 μl/ml aprotinin, and 1 mM sodium orthovanadate). For immunoprecipitation, 1 μg of antibody (anti-Fas, anti-6A7) was added to 500 μg of total protein in 500 μl, rotated for 2 h at 4 °C, and then incubated with 20 μl of beads (protein A-sucrose, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 2 h, spun down at 500 × g, and washed three times with immunoprecipitation buffer. Then, 20 μl of loading buffer (100 mM Tris-HCl, 200 mM dithiothreitol, 4% SDS, 0.2% bromphenol blue, and 20% glycerol) was added. For SDS-PAGE, samples containing equal amounts of protein were boiled in the loading buffer and separated on SDS-PAGE, followed by transfer to polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat milk and stained with the primary antibodies for 2 h at the optimal concentrations. After five washes in phosphate-buffered saline with 0.2% Tween 20, the horseradish peroxidase-conjugated secondary antibody was applied, and the blot was developed with enhanced chemiluminescence reagents (Amersham Biosciences, Piscataway, NJ).
Statistical Analysis
All values are expressed as means +/- SE. Statistical significance was determined by student T-test and a value of P < 0.05 was considered significant.
Results
CO inhibits Fas activating antibody-induced apoptosis and caspase-9, -3 cleavage
The Fas-signaling pathway initiates upon stimulation with a specific ligand (Fas ligand) or an activating anti-Fas antibody, resulting in the activation of downstream caspases. We investigated the effect of CO on cell death induced by Fas-activating antibody (Jo2) in MLEC, using lactate dehydrogenase (LDH) release as a marker. As shown in Figure 1A, CO protected MLEC from Jo2-dependent cell death, relative to the air-exposed control. After 1 h of incubation with the activating anti-Fas antibody Jo2, caspase-9 and caspase-3 were cleaved in MLEC (Figure 1B). Carbon monoxide (CO) exposure delayed the activation (cleavage) of both caspases in response to Jo2 (Figure 1B), suggesting that CO inhibits Fas-dependent apoptosis in endothelial cells.
Figure 1 CO inhibits cell death and caspase activation induced by Fas activating antibody Jo2. MLEC cultures were pretreated in the absence or presence of CO (250 ppm) for 2 h prior to the addition of antibody Jo2 (200 ng/ml) for the indicated times. 200 μl of supernatant medium was removed for LDH assays as described in Methods (A). The data represent an average of two independent experiments with each sample in triplicate (n = 3). Data from Jo2 treated cells in the presence of CO were compared with control (Jo2 treatment alone) cells at each time point using Student's T-test (*P < 0.05). The total lysates were subjected to Western blot to detect caspase-9 and caspase-3. β-actin served as the standard (B). Westerns are representative of three independent experiments.
CO provides protection against extrinsic apoptosis
To investigate the mechanisms involved in the CO-dependent inhibition of Jo2-induced apoptosis, we tested the hypothesis that CO inhibits activation of extrinsic apoptotic signaling pathways. The treatment of MLEC with Jo2 induced DISC formation (Figure 2A) and activated caspase-8 (Figure 2B). When MLEC were treated with Jo2 in the presence of CO (250 ppm), the CO treatment inhibited Jo2-dependent DISC formation (Figure 2A) and caspase-8 activation (Figure 2B), relative to air-treated controls.
Figure 2 CO inhibited Jo2-dependent caspase-8 and Bax activation. MLEC cultures were pretreated in the absence or presence of CO (250 ppm) for 2 h prior to the addition of antibody Jo2 (200 ng/ml) for the indicated times. The total lysates were subjected to immunopreciptiation (IP) with anti-Fas, followed by immunoblotting (IB) to detect caspase-8. Total Fas served as the standard. The control lane (c) represents cell lysate taken immediately after Jo2 addtion (A). Lysates were subjected to Western blot analysis to detect caspase-8 (B). MLEC cultures were pretreated in the absence or presence of CO (250 ppm) for 2 h prior to the addition of antibody Jo2 (200 ng/ml) for the indicated times. Lysates were subjected to immunoprecipitation with antibody 6A7 that specifically recognizes the activated form of Bax, followed by immunoblotting with anti-Bax. Total Bax served as the standard (C). Westerns are representative of three independent experiments.
CO inhibits Bax activation
We next tested whether an intrinsic apoptosis pathway involving Bax may participate in apoptogenic signaling pathways activated by Jo2. The treatment of MLEC with Jo2 resulted in the activation of Bax (Figure 2C), as determined by immunoprecipitation with the anti-Bax monoclonal antibody 6A7, which specifically recognizes a conformational change in Bax protein associated with its activation [38]. Bax activation is a hallmark of the intrinsic apoptosis pathway, but may also be activated secondary to extrinsic apoptosis through the caspase-8 dependent activation of Bid [17]. CO decreased Jo2-inducible Bax activation (Figure 2C) relative to air-treated controls.
CO upregulates FLIP expression and increases FLIP in the DISC
We hypothesized that CO may protect MLEC from Jo2-dependent apoptosis by regulating the expression of FLIP. In MLEC stimulated with Jo2, CO treatment upregulated FLIPL (Figure 3A), and increased the amount of FLIP protein associated with the DISC relative to air-treated controls (Figure 3B). These results suggest that CO, by upregulating FLIPL and its association with the DISC, inhibits the recruitment and activation of caspase-8.
Figure 3 CO upregulated FLIP and increased DISC associated FLIP. MLEC cultures were pretreated in the absence or presence of CO (250 ppm) for 2 h prior to the addition of antibody Jo2 (200 ng/ml) for the indicated times. The total lysates were subjected to Western blot analysis to detect FLIP (A), or to immunoprecipitation (IP) with anti-Fas followed by immunoblotting (IB) to detect FLIP. β-Actin (A) or Total Fas (B) served as the standard. Westerns are representative of three independent experiments.
CO decreases FADD phosphorylation and its recruitment in the DISC via inhibition of JNK signaling
Treatment of MLEC with Jo2 increased the time-dependent phosphorylation of FADD (Figure 4A). Furthermore Jo2 treatment time-dependently increased the phosphorylation of FADD in association with the DISC in MLEC (Figure 4B), indicating that FADD phosphorylation may be necessary for DISC formation in MLEC. CO (250 ppm) decreased the phosphorylation of FADD in response to Jo2 stimulation, relative to air-treated controls (Figure 4A). CO also dramatically decreased FADD phosphorylation in the DISC (Figure 4B).
Figure 4 CO inhibited FADD phosphorylation via blockage of JNK signaling. MLEC cultures were pretreated in the absence or presence of CO (250 ppm) for 2 h prior to the addition of antibody Jo2 (200 ng/ml) for the indicated times. The total lysates were subjected to Western blot analysis to detect phosphorylated and total FADD (A), or to immunoprecipitation (IP) with anti-Fas followed by immunoblotting to detect phospho-FADD (B). Lysates were subjected to immunoblotting to detect phospho- and total JNK. (C). MLEC cultures were treated with antibody Jo2 (200 ng/ml) for the indicated times, in the absence or presence of JNK inhibitor (20 μM). The total lysates were subjected to immunoprecipitation with anti-Fas followed by immunoblotting to detect phospho-FADD (D). Total FADD (A, B, D) or total JNK (C) served as the standards. Westerns are representative of three independent experiments.
In the presence of Jo2, CO exposure (250 ppm) time-dependently downregulated the phosphorylation of c-Jun-NH2-terminal kinase (JNK) in MLEC, relative to that of air-treated controls (Figure 4C). Treatment with JNK inhibitor-II (JNKi-II), an inhibitor of JNK-1/2 decreased FADD phosphorylation in the DISC (Figure 4D). The data suggest that CO inhibits FADD phosphorylation, possibly via inhibition of JNK; and that JNK-1/2 may be necessary for the phosphorylation of FADD and/or its recruitment into the DISC induced by Jo2 antibody.
CO activates ERK1/2 and NFκB signaling
Next we tested the effect of CO on NF-κB and ERK1/2 activation in Jo2 treated MLEC. Jo2 treatment of MLEC stimulated the time-dependent phosphorylation of ERK1/2, with an apparent maximum at 12 hrs post treatment (Figure 5). Exposure to CO (250 ppm) further increased ERK1/2 activation with similar kinetics. Jo2 treatment alone did not appreciably induce NF-κB p65 phosphorylation in cells pretreated with air. On the other hand, 2 hours pretreatment with CO (250 ppm) caused a dramatic upregulation of NF-κB p65 phosphorylation at the time of Jo2 addition (0 hr), which persisted for 12 hrs (Figure 5).
Figure 5 CO activated ERK and NF-κB signaling. MLEC cultures were pretreated in the absence or presence of CO (250 ppm) for 2 h prior to the addition of antibody Jo2 (200 ng/ml) for the indicated times. The total lysates were subjected to Western blot to detect ERK or NF-κB p65 phosphorylation. Total p65 served as the standard. Westerns are representative of three independent experiments.
We next tested the role of the ERK1/2 MAPK pathway in Jo2-mediated cell killing and in the cytoprotection afforded by CO. MLEC were treated with the ERK1/2 pathway inhibitor PD 98059, which specifically targets MEK1, the upstream regulator of ERK1/2. Treatment with PD 98059 sensitized MLEC to Jo2 mediated cell killing, and partially compromised the ability of CO to confer protection against Jo2 toxicity, as determined by LDH release assays (Figure 6A). The requirement for ERK1/2 pathway in CO-dependent suppression of apoptosis was demonstrated by Western blot analysis of caspase-3 activation. The caspase-3 cleavage induced by Jo2 in MLEC was reduced to control values by co-treatment with CO, whereas inclusion of PD 98059 abolished this effect (Figure 6B). These experiments imply a cytoprotective role for the ERK1/2 pathway against activation of the extrinsic apoptotic pathway, which is required for CO-dependent therapeutic effects (Figure 7).
Figure 6 CO-dependent cytoprotection requires ERK1/2 pathway. MLEC cultures were pretreated with room air (RA) or CO (250 ppm) for 2 h prior to the addition of antibody Jo2 (200 ng/ml). Additionally, PD 98059 (25 μM) or vehicle (DMSO) was added to the cultures for 30 min prior to Jo2 addition. Cells were then incubated an additional 24 hours in the absence or presence of CO. 200 μl of supernatant medium was removed for LDH assays as described in Methods (A). The data represent triplicate (n = 3) determinations. Comparisons between the treatment groups were performed using Student's T-test (*P < 0.05). The total lysates were subjected to Western blot to detect caspase-3. β-actin served as the standard (B).
Figure 7 Proposed pathways of the antiapoptotic effects of CO in Jo2-dependent apoptosis.
Discussion
The antiapoptotic potential of CO was first demonstrated in cell culture studies using fibroblasts or endothelial cells. Both the exogenous administration of CO or the over-expression of HO-1, inhibited TNFα-induced apoptosis in murine fibroblasts [36]. In the endothelial cell model, the inhibitory effect of CO on TNFα-induced apoptosis could be abolished with the selective chemical inhibitor, SB203580, or a p38 MAPK dominant negative mutant, implying a critical role for the p38 MAPK pathway [25]. Furthermore, HO-1 or CO co-operated with NF-κB-dependent anti-apoptotic genes (c-IAP2 and A1) to protect against TNFα-mediated endothelial cell apoptosis [39]. Exogenous CO inhibited anoxia/reoxygenation-induced apoptosis in pulmonary artery endothelial cells. This effect was associated with the activation of MKK3 and p38α MAPK, and with inhibition of Fas and FasL expression [27,30]. We have recently demonstrated that CO inhibited hyperoxia-induced endothelial cell apoptosis by downregulating ROS production, and DISC formation in MLEC [37]. In Jurkatt T cells induced to apoptosis by FasL stimulation, CO promoted, rather than inhibited FasL-dependent apoptosis, by inhibiting ERK1/2 activation [40].
The Fas-dependent apoptotic pathway has been implicated in several models of lung or vascular injury, including acute lung injury and I/R injury [41-43]. In the current study, we treated MLEC with activating anti-Fas antibody (Jo2) as a model of Fas-dependent vascular cell apoptosis. Fas is a prototype death receptor characterized by the presence of an 80 amino acid death domain in its cytoplasmic tail. This domain is essential for the recruitment of a number of signaling components upon stimulation by either activating anti-Fas antibodies or cognate FasL that initiate apoptosis. The DISC consists of an adaptor protein (such as FADD) and initiator caspases (such as caspase-8) and is essential for induction of apoptosis [9,10]. A number of proteins have been reported to regulate formation or activity of the DISC [10].
The adaptor protein FADD is essential for apoptosis induced by death receptors, mediating aggregation and autocatalytic activation of caspase-8. FADD has been shown to undergo phosphorylation at Ser 194 at the G2/M transition of the cell cycle [44]. When cells are arrested in the G2-M phase of the cell cycle, they may either undergo cell death by apoptosis or necrosis or overcome the G2-M block and continue in the division cycle, often toward a post-mitotic cell death [45,46]. FADD phosphorylation plays a role in the regulation of cell growth and proliferation [44,47], and also in cellular sensitivity to apoptosis induced by anticancer drugs [48]. Currently, it remains unclear whether FADD phosphorylation is necessary for the assembly and activity of the DISC [11]. Our data suggest that FADD phosphorylation and its accumulation in the DISC is necessary for Jo2-induced apoptosis in MLEC. The kinase(s) regulating FADD phosphorylation remain unidentified [47]. In human prostate cancer cell lines, c-Jun NH2-terminal kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) family, was associated with FADD phosphorylation [48]. Our results clearly show that CO inhibited the phosphorylation of FADD and also inhibited the activation of JNK during Jo2 stimulation. We also find that CO inhibited the apparent phosphorylation of FADD in association with Fas. It remains unclear if the phosphorylation of FADD in this context was a prerequisite for DISC formation or occurred in the context of a pre-assembled DISC. Previous studies have suggested that the phosphorylation of FADD at Ser-194 is important in the regulation of the non-apoptotic roles of FADD in regulating cell proliferation and cell cycle progression [49,50]. Nevertheless, in MLEC stimulated with Jo2 we show that the JNK inhibitor JNKi-II interfered with the detection of phospho-FADD in association with Fas in the DISC. Consistent with our observations, JNK activation has been previously implicated in the stimulation of Fas-dependent apoptosis by vanadate. In this study, JNK activation was shown to be required for Fas/FADD association [51]. Holmstrom et al., using the MEK1 inhibitor, have shown that ERK1/2 MAPK pathways can inhibit extrinsic apoptosis at the level of caspase-8 activation but do not inhibit Fas trafficking or DISC assembly in T cells [52]. While we propose here that CO inhibited FADD phosphorylation and DISC assembly in part through downregulation of JNK, we cannot exclude the possibility that CO also downregulated other kinases involved in FADD phosphorylation. We also cannot exclude the possibility that the effects of CO specifically on JNK phosphorylation are relevant to the activation of the non-apoptotic functions of FADD, and/or other downstream cellular targets.
The results of our study also suggest the CO inhibited extrinsic apoptosis at the level of caspase-8 processing through FLIP activation. FLIP, an endogenous caspase-8 inhibitor, can block Fas-mediated apoptosis through association with the DISC [53]. Increased levels of FLIP can confer protection against Fas-induced apoptosis. FLIPL, FLIPS, FLIPR can be recruited to the DISC but they function differently: FLIPS/R prevent the initial cleavage step of caspase-8 activation between the p20 and the p10 subunit of the caspase homology domain; whereas FLIPL inhibits the final cleavage between the pro-domain and the p20 subunit of the p43/41 intermediate [14]. Recent studies show that PKCα/β-dependent phosphorylation regulates the ubiquitination of all c-FLIP proteins, whereas selectively prolongs the stability of the short isoforms [16]. CO upregulated FLIPL expression and increased the association of FLIPL with the DISC. Currently, we do not understand how CO upregulates FLIP expression in MLEC. We report that simultaneous application of CO during Fas-stimulation promoted the upregulation of ERK1/2 and NF-κB signaling pathways in MLEC, both of which have been implicated in FLIP regulation in other cell types [54-56]. The ERK1/2 pathway was at least in part involved in CO-dependent cytoprotection against Jo2-induced toxicity. Chemical inhibition of ERK1/2 reversed the ability of CO to downregulate Jo2-induced caspase-3 activation.
No direct evidence has been found to date that Fas-activating antibody can activate the intrinsic (Bax/mitochondria) apoptotic pathway. Injection of Jo2 antibody into survivin+/- and survivin+/+ mice showed that survivin+/- mice appeared normal, but liver lysates revealed a low-level activation of caspases, with accumulation of Bax, indicating a pro-apoptotic state [57]. Here, we found that Jo2 activated Bax in MLEC. It is possible that CO inhibits Bax activation through the upregulation of FLIP, which blocks the extrinsic apoptotic pathway at the level of caspase-8 activation. We have previously shown that the vector-driven overexpression of FLIP inhibited Bax activation in MLEC during hyperoxic or hypoxia/reoxygenation stress [18,19].
Conclusions
In summary, exogenous CO, when applied at low concentration, provides protection against Fas-activating antibody (Jo2)-induced apoptosis in MLEC by inhibiting Fas/caspase-8 and subsequently, Bax signaling pathways. FADD phosphorylation is important for DISC formation induced by Jo2. CO inhibits FADD phosphorylation and association of phospho-FADD with Fas. Although CO delayed JNK activation, the relative importance of JNK in regulating the Fas-dependent pathway remains unclear. CO increases the level of FLIP protein expression as well as its assimilation in the DISC, which inhibits the cleavage of caspase-8 (Figure 7). These results, taken together indicate that CO can protect against Fas-activating antibody-induced apoptosis by inhibiting both extrinsic (Fas/caspase-8)-dependent and intrinsic (Bax)-dependent apoptotic signaling pathways. The protection afforded by CO against activation of executioner caspase-3 was dependent on ERK1/2 pathway activation. Strategies to inhibit extrinsic apoptosis may provide therapeutic options in diseases where Fas-dependent apoptosis has been implicated in pathogenesis.
List of Abbreviations
The abbreviations used are: CO: Carbon monoxide; DISC: Death-inducing signal complex; ERK1/2: Extracellular regulated kinase-1/2; FADD: Fas-associated death domain; FBS: fetal bovine serum; c-FLIP: Fas-associated death domain interleukin-1β-converting enzyme-like inhibitory protein; c-FLIPS: c-FLIP short form; c-FLIPL: c-FLIP long form; c-FLIPR: c-FLIP Raji; HO-1: heme oxygenase-1; IB: immunoblotting; IP: immunoprecipitation; JNK: c-jun-NH2-terminal kinase; JNKi-II: inhibitor of JNK1/2; Jo2: Fas-activating antibody; LDH: lactate dehydrogenase; MAPK: mitogen activated protein kinase; MLEC: mouse lung endothelial cells; PD 98059: mitogen activated protein kinase kinase (MEK)-1 inhibitor.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
All authors have read and approved this manuscript. XW conceived and designed the studies, and conducted experiments; YW performed immunoblot and immunoprecipitation experiments, SJL performed revision experiments, HPK analyzed the data, AMKC supervised the studies and analyzed the data, SWR analyzed the data and wrote the manuscript.
Authors' Information
XW performed this research as a faculty member at the University of Pittsburgh, and is currently employed by the US Food and Drug Administration; SWR is currently employed by Brigham and Women's Hospital, Boston, and is an adjunct scientist of the Lovelace Respiratory Research Institute.
Acknowledgements
We thank Ms. Qing Dong for technical assistance. This work was initially supported by awards from the American Heart Association to S. W. Ryter, (AHA #0335035N), and H.P. Kim (AHA #0525552U) and NIH grants R01-HL60234, R01-HL55330, R01-HL079904, P01-HL70807, and a FAMRI clinical innovator award to A. M. K. Choi. S. W. Ryter received salary support from the Lovelace Respiratory Research Institute. None of these funding agencies had any role in the acquisition of data, interpretation, or preparation of this manuscript.
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PLoS BiolPLoS BiolplosplosbiolPLoS Biology1544-91731545-7885Public Library of Science San Francisco, USA 22162948PBIOLOGY-D-11-0148310.1371/journal.pbio.1001212Research ArticleBiologyImmunologyMicrobiologyModel OrganismsMedicineEndocrinologyMetabolic DisordersGut Microbiota Is a Key Modulator of Insulin Resistance in TLR 2 Knockout Mice TLR2, Gut Microbiota, and Insulin ResistanceCaricilli Andréa M.
1
Picardi Paty K.
1
de Abreu Lélia L.
2
Ueno Mirian
1
Prada Patrícia O.
1
Ropelle Eduardo R.
1
Hirabara Sandro Massao
3
Castoldi Ângela
4
Vieira Pedro
4
Camara Niels O. S.
4
Curi Rui
3
Carvalheira José B.
1
Saad Mário J. A.
1
*
1 Department of Internal Medicine, State University of Campinas, Campinas, Brazil2 Department of Nursing, State University of Campinas, Campinas, Brazil3 Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil4 Department of Immunology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, BrazilVidal-Puig Antonio J. Academic EditorUniversity of Cambridge, United Kingdom* E-mail: [email protected] author(s) have made the following declarations about their contributions: Conceived and designed the experiments: AC MS. Performed the experiments: AC PKP LLA ER MU PP SH AC PV NOSC. Analyzed the data: AC MS JBC. Contributed reagents/materials/analysis tools: RC NOSC JBC MS. Wrote the paper: AC MS.
12 2011 6 12 2011 6 12 2011 9 12 e100121211 4 2011 27 10 2011 Caricilli et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
Gut Bacteria May Override Genetic Protections against Diabetes
A genetic and pharmacological approach reveals novel insights into how changes in gut microbiota can subvert genetically predetermined phenotypes from lean to obese.
Environmental factors and host genetics interact to control the gut microbiota, which may have a role in the development of obesity and insulin resistance. TLR2-deficient mice, under germ-free conditions, are protected from diet-induced insulin resistance. It is possible that the presence of gut microbiota could reverse the phenotype of an animal, inducing insulin resistance in an animal genetically determined to have increased insulin sensitivity, such as the TLR2 KO mice. In the present study, we investigated the influence of gut microbiota on metabolic parameters, glucose tolerance, insulin sensitivity, and signaling of TLR2-deficient mice. We investigated the gut microbiota (by metagenomics), the metabolic characteristics, and insulin signaling in TLR2 knockout (KO) mice in a non-germ free facility. Results showed that the loss of TLR2 in conventionalized mice results in a phenotype reminiscent of metabolic syndrome, characterized by differences in the gut microbiota, with a 3-fold increase in Firmicutes and a slight increase in Bacteroidetes compared with controls. These changes in gut microbiota were accompanied by an increase in LPS absorption, subclinical inflammation, insulin resistance, glucose intolerance, and later, obesity. In addition, this sequence of events was reproduced in WT mice by microbiota transplantation and was also reversed by antibiotics. At the molecular level the mechanism was unique, with activation of TLR4 associated with ER stress and JNK activation, but no activation of the IKKβ-IκB-NFκB pathway. Our data also showed that in TLR2 KO mice there was a reduction in regulatory T cell in visceral fat, suggesting that this modulation may also contribute to the insulin resistance of these animals. Our results emphasize the role of microbiota in the complex network of molecular and cellular interactions that link genotype to phenotype and have potential implications for common human disorders involving obesity, diabetes, and even other immunological disorders.
Author Summary
An intricate interaction between genetic and environmental factors influences the development of obesity and diabetes. Previous studies have shown that mice lacking an important receptor of the innate immune system, Toll-like Receptor 2 (TLR2), are protected from insulin resistance. Given that the innate immune system has emerged as a key regulator of the gut microbiota, we undertook to investigate in this study whether the gut microbiota have a role in modulating the response to insulin. By rearing these TLR2 mutant mice in conventional facilities (as opposed to “germ-free” conditions) we figured that they would develop an altered gut microbiota. In contrast to previous studies, our results show that these TLR2 mutant mice now develop a diseased phenotype reminiscent of metabolic syndrome, including weight gain, and end up with gut microbiota similar to that found in obese mice and humans. These mice could be rescued by treatment with broad-spectrum antibiotics, which decimated the microbiota. Conversely, transplantation of the gut microbiota from these mice to wild-type mice induced weight gain and the metabolic syndrome phenotype. Our results indicate that the gut microbiota per se can subvert a genetically predetermined condition previously described as being protective towards obesity and insulin resistance into a phenotype associated with weight gain and its complications, such as glucose intolerance and diabetes.
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Introduction
The recent epidemics of obesity and type 2 diabetes mellitus (T2DM) in the past 20 years have stimulated researchers to investigate the mechanisms that are responsible for the development of these diseases. The general view is that obesity and T2DM have a genetic background and are strongly influenced by the environment and that insulin resistance is an early alteration in these diseases [1]–[5]. In addition, studies over the past 10 years have also shown that subclinical inflammation has an important role in the molecular mechanism of insulin resistance in obesity and T2DM [6]–[10]. During the past five years, an increasing body of literature has suggested other components of the mechanisms of these diseases that lie between the genetic and the environment factors, where the gut microbiota are now considered to make an important contribution to these mechanisms [11]–[16]. Then, it is now clear that environmental factors and host genetics interact to control the gut microbiota, which may have a role in the development of obesity and insulin resistance [17].
Metagenomic studies demonstrated that the proportion of Firmicutes is higher in obese animals and in humans, compared with lean controls, and this correlates with a higher number of genes encoding enzymes that break down otherwise indigestible dietary polysaccharides, with more fermentation end products and fewer calories remaining in the faeces of obese individuals [18],[19]. Another mechanism by which the microbiome may contribute to metabolic disorders is by triggering systemic inflammation [20]. The immune system coevolves with the microbiota during postnatal life, which allows the host and microbiota to coexist in a mutually beneficial relationship [21],[22]. In particular, the innate immune system has emerged as a key regulator of the gut microbiota. Innate immune recognition of microbe-associated molecular patterns is executed by families of pattern-recognition molecules with a special role for Toll-like receptors (TLRs) [23],[24]. Recent findings indicate that TLRs, which are up-regulated in the affected tissue of most inflammatory disorders, can mediate crosstalk between the immune systems and whole body metabolism [23]. It has been demonstrated that TLR4, a sensor for lipopolysaccharides on Gram-negative bacteria, is involved in the induction of proinflammatory cytokine expression in macrophages, adipocytes, and liver [13],[25]. We and others have demonstrated that TLR4 genetically deficient mice or mice with an inactivating mutation for this receptor are substantially protected from obesity-induced insulin resistance [26],[27]. Similarly, TLR2 genetically deficient mice are protected from high-fat-induced insulin resistance [28],[29]. On the other hand, TLR5-deficient mice exhibit hyperphagia and develop hallmark features of metabolic syndrome, including hyperlipidemia, hypertension, insulin resistance, and increased adiposity [30], and these alterations are the consequence of alterations in the gut microbiota. It is important to emphasize that the studies that investigated TLR4- and TLR5-deficient mice were performed without germ-free conditions [26],[27],[30], suggesting that the microbiota have an important influence on TLR5-deficient mice phenotype, inducing obesity and insulin resistance; however, in the TLR4-deficient mice, the microbiota do not have a role in these phenomena because these animals are protected from diet-induced insulin resistance, independently of germ-free conditions [26],[27]. Taken together, these findings suggest that the interaction of the innate immune system with gut microbiota may determine the insulin sensitivity of an animal and that TLRs may have different roles in this process. Since in most studies with TLR2-deficient mice the microbiota were not investigated, we cannot predict the influence of microbiota in the protection or in the development of insulin resistance in these mice. It is possible that the presence of a diverse gut microbiota could completely reverse the phenotype of an animal, inducing insulin resistance in an animal genetically determined to have increased insulin sensitivity, such as the TLR2 KO mice. The aim of the present study was to investigate the influence of gut microbiota in metabolic parameters, glucose tolerance, insulin sensitivity, and signaling of TLR2-deficient mice.
Results
Animal Characteristics
TLR2 KO mice did not present any difference in weight gain, compared with their controls up until 12 wk. However, after 12 wk, TLR2 KO mice were heavier than their controls (p<0.05; Figure 1A). No significant differences were observed with regard to food intake between the groups after either 8 or 16 wk (Figure 1B). The food intake was also normalized for body weight and no difference was observed between groups at 8 wk old (WT = 0.22±0.035 g/g animal/day; TLR2 KO = 0.21±0.021 g/g animal/day). However, after 16 wk, TLR2 KO mice presented increased epididymal fat weight (Figure 1C). After 12 wk, the amount of adipose tissue is visually increased in TLR2 KO mice (Figure 1D). It is interesting that TLR2 KO mice at 8 wk old had decreased glucose tolerance compared to their controls (p<0.05; Figure 1E), but no difference was observed in fasting serum insulin between the groups (Figure 1F). We next submitted these animals to a hyperinsulinemic euglycemic clamp to investigate insulin sensitivity; results showed that TLR2 KO mice presented a significant decrease in the rate of glucose uptake under high insulin stimulus (50% of control, p<0.05; Figure 1G), indicating a clear situation of insulin resistance.
10.1371/journal.pbio.1001212.g001Figure 1 Metabolic parameters of TLR2 knockout (TLR2−/−) and WT mice during 16 wk.
(A) Weight gain after 16 wk. (B) Food intake after 8 and 16 wk. (C) Epididymal fat pad weight after 8 and 16 wk. (D) WT and TLR2−/− mice after 20 wk. (E) Glucose tolerance test. (F) Serum insulin concentration. (G) Glucose uptake obtained from euglycaemic hyperinsunaemic clamp. (H) Oxygen consumption and (I) respiratory exchange rate. (J) UCP-1 expression in the brown adipose tissue. Equal protein loading in the gel was confirmed by reblotting the membrane with an anti-β-actin antibody (J, lower panel). All evaluations were made with mice on standard chow. Data are presented as means ± S.E.M from six to eight mice per group from experiments that were repeated at least three times. *p<0.05 between TLR2−/− mice and their controls.
We next analyzed the oxygen consumption from both groups and observed that TLR2 KO mice presented decreased oxygen consumption (Figure 1H), suggesting decreased energy expenditure when compared with their controls. However, the respiratory exchange ratio, approximately 0.85, was similar between the groups (Figure 1I). As the oxygen consumption was decreased in TLR2 KO mice, we evaluated a marker of thermogenesis in the brown adipose tissue of both groups. The expression of the thermogenesis-inducing protein, UCP1, was significantly decreased in TLR2 KO mice (Figure 1J), suggesting reduced energy expenditure in these animals, in accordance with the reduced oxygen consumption observed.
In order to characterize the gut microbiota of TLR2 KO mice, we pyrosequenced the 16S ribosomal RNA (rRNA) from the stools of these animals. TLR2 KO mice presented a different gut microbiota, compared with their controls. The major difference concerns the proportion of Firmicutes, which was approximately 47.92% in TLR2 KO mice, while the controls presented a proportion of 13.95%. Moreover, TLR2 KO mice presented 47.92% Bacteroidetes and 1.04% Proteobacteria, while their controls presented approximately 42.63% and 39.53%, respectively (Figure 2A,B). TLR2 KO mice presented other differences in regards to classes and families and these results are presented in the Supporting Information section (Figures S1 and S2).
10.1371/journal.pbio.1001212.g002Figure 2 TLR2 KO mice exhibit taxonomical alterations in gut microbiota.
Untreated WT (A) and TLR2 knockout (TLR2−/−) mice (B) stools were analyzed via 16S rRNA analysis. Data are presented from six to eight mice per group from experiments that were repeated at least three times. All evaluations were made with mice on standard chow.
However, it is important to notice that we have observed different proportions of these phyla between TLR2 KO mice and their controls in different ages of mice. From 4-wk-old to 1-y-old mice, we observed increased proportion of Firmicutes in TLR2 KO mice compared with the controls. We have also observed a tendency of decreasing the proportion of Bacteroidetes progressively as TLR2 KO mice get older (Figures S3, S4, S5).
Molecular Mechanisms of Insulin Resistance in TLR2 KO Mice
Next, we determined the serum concentration of IL-6 and TNF-α in both groups of animals and observed that TLR2 KO mice presented reduced levels of these cytokines compared with their controls (Figure 3A,B). We also investigated the serum concentrations of leptin, adiponectin, and LPS. No significant difference was observed between the groups with regard to leptin and adiponectin (Figure 3C,D). On the other hand, LPS serum concentration was increased in TLR2 KO mice (Figure 3E).
10.1371/journal.pbio.1001212.g003Figure 3 Measurements of cytokines, adipokines, and LPS.
Serum concentration of IL-6 (A), TNF-α (B), adiponectin (C), leptin (D), and LPS (E). Proportion of Bifidobacterium was obtained by 16S rRNA analysis of stools (F). Data are presented as means ± S.E.M from six to eight mice per group from experiments that were repeated at least three times. * p<0.05 between TLR2 KO mice and their controls; all evaluations were made with mice on standard chow.
As TLR2 KO mice presented increased serum LPS levels, and this alteration was previously described in an animal model of obesity in which there was a reduced proportion of Bifidobacterium
[31], we investigated the proportion of this group of bacteria. We observed that TLR2 KO mice presented a decrease in Bifidobacterium proportion compared with WT (Figure 3F).
In order to unravel the mechanism by which the insulin resistance occurs in the TLR2 KO mice, we studied important pathways involved in this phenomenon: phosphorylation of JNK, activation of ER stress, serine phosphorylation of the insulin receptor substrate (IRS)-1, and expression of IκB-α, which is involved in the inhibition of the IKK/NFκB pathway activation.
TLR2 KO mice presented increased phosphorylation of JNK in muscle, liver, and adipose tissue compared with controls (Figure 4A–C). Since the activation of ER stress leads to the phosphorylation of JNK, the increased phosphorylation of this protein in TLR2 KO mice could be due to this event. In fact, the phosphorylation of PERK was increased in the liver and adipose tissue of the KO mice, suggesting increased ER stress activation at least in these two tissues (Figure 4D–F).
10.1371/journal.pbio.1001212.g004Figure 4 Evaluation of pathways involved in the impairment of insulin signaling.
Phosphorylation of JNK in muscle (A), liver (B), and white adipose tissue (WAT) (C). Phosphorylation of PERK in muscle (D), liver (E), and WAT (F). Serine 307 phosphorylation of IRS-1 from muscle (G), liver (H), and WAT (I). Activation of TLR4 (studied by the immunoprecipitation of MyD88 and blotting with TLR4) in muscle (J), liver (K), and WAT (L). JNK, PERK, and IRS-1 protein expression in muscle, liver, and white adipose tissue (A–I, lower panels). Expression of IκB-α in muscle (M), liver (N), and WAT (O). Equal protein loading in the gel was confirmed by reblotting the membrane with an anti-β-actin antibody (M–O, lower panels). NFκB activation in muscle (P), liver (Q), and WAT (R). All evaluations were made with mice on standard chow. Data are presented as means ± S.E.M from six to eight mice per group from experiments that were repeated at least three times. * p<0.05 between TLR2−/− mice and their controls.
Next, we studied the inhibitory serine phosphorylation of IRS-1 in muscle, liver, and adipose tissue of TLR2 KO mice and observed that this phosphorylation was increased, compared with the controls, suggesting impairment of insulin signaling (Figure 4G–I). Since increased serum concentration of LPS, a TLR4 ligand, was observed in TLR2 KO mice, we investigated the activation of TLR4 in the muscle, liver, and adipose tissue of these mice. An increased activation of this receptor was observed in all tissues studied (Figure 4J–L), suggesting that, in the absence of TLR2, a compensatory action may lead to increased activation of TLR4, which may also contribute to the development of insulin resistance in TLR2 KO mice.
Then, we studied the activation of IKK/NFκB pathway, indirectly, by the expression of IκB-α. Curiously, the expression of IκB-α was increased in the muscle, liver, and white adipose tissue of TLR2 KO mice, compared with controls, suggesting a decreased activation of IKK/NFκB pathway (Figure 4M–O). In order to confirm this result, we studied the activation of NFκB and observed that this was decreased in all tissues studied from TLR2 KO mice (Figure 4P–R).
The insulin-induced tyrosine phosphorylation of the insulin receptor (IR) (Figure S6A–C) and of the insulin receptor substrate (IRS)-1 (Figure 5A–C), as well as the insulin-induced serine phosphorylation of AKT, was decreased in the muscle, liver, and adipose tissue of TLR2 KO mice (Figure 5D–F), compared with their controls, suggesting reduced insulin signaling in these tissues.
10.1371/journal.pbio.1001212.g005Figure 5 TLR2 knockout (TLR2−/−) mice present decreased insulin signaling.
Tyrosine 941 phosphorylation of the insulin receptor substrate (IRS)-1 in muscle (A), liver (B), and WAT (C). Serine phosphorylation of AKT in muscle (D), liver (E), and WAT (F). IRS-1 and AKT protein expression in muscle, liver, and white adipose tissue (A–F, lower panels). Equal protein loading in the gel was confirmed by reblotting the membrane with an anti-β-actin antibody (lower panels). All evaluations were made with mice on standard chow. Data are presented as means ± S.E.M from six to eight mice per group from experiments that were repeated at least three times. *p<0.05 between WT with and without insulin stimulus; ** p<0.05 between TLR2−/− mice and their controls with insulin stimulus.
Other proteins that are important in the modulation of insulin action were also investigated. Our data showed that the phosphorylation of AMPK (Figure S6D–F) and the expression of PGC-1α (Figure S6G–I) were similar between controls and TLR2 KO mice in the three tissues investigated.
As an increased phosphorylation of JNK was observed in TLR2 KO mice, we prevented the activation of this protein with a pharmacological inhibitor, SP600125, by treating mice with daily i.p. injections for 5 d. Subsequently, we observed an increased glucose uptake in these animals, suggesting that the activation of JNK is indeed relevant to the development of insulin resistance (Figure 6A). We also observed increased insulin-induced serine phosphorylation of AKT in the liver (Figure 6B), muscle, and white adipose tissue (unpublished data) of TLR2 KO mice after this treatment, suggesting increased insulin signaling, as well, associated with a reduction in JNK phosphorylation in the liver of TLR2 KO mice (Figure 6D).
10.1371/journal.pbio.1001212.g006Figure 6 Insulin sensitivity and signaling after treatment with selective inhibitors.
Glucose uptake obtained by the euglycaemic hyperinsulinaemic clamp from TLR2−/− mice treated or not with the drugs: SP600125 (SP), JNK inhibitor; 4-phenil butyric acid (PBA), endoplasmic reticulum stress inhibitor; TLR4 antisense oligonucleotide (ASO); (A) TAK-242, inhibitor of TLR4. (B) Serine phosphorylation of AKT after the treatment with SP600125 and PBA. (C) Serine phosphorylation of AKT after the treatment with TLR4 ASO and TAK-242. (D) Phosphorylation of JNK after the treatment with the drugs mentioned. Fasted TLR2 knockout mice and WT mice were gavaged by LPS (1.08, 10−8 g) diluted in water (100 µL) or without LPS. (E) Blood was collected from the cava vein 60 min after gavage and serum LPS was determined. (F) Zonula occludens (ZO)-1 expression in the ileum. (G) Frequency of CD4+Foxp3+ regulatory T cells in WT mice. (H) Frequency of CD4+Foxp3+ regulatory T cells in TLR2−/− mice. All evaluations were made with mice on standard chow. Data are presented as means ± S.E.M from six to eight mice per group from experiments that were repeated at least three times. * p<0.05 between WT mice with or without insulin stimulus; ** p<0.05 between WT and TLR2−/− mice with insulin stimulus ; $ p<0.05 between TLR2−/− mice with insulin stimulus, treated or not with SP; # p<0.05 between TLR2−/− mice with insulin stimulus, treated or not with PBA; § p<0.05 between TLR2−/− mice with insulin stimulus, treated or not with ASO; & p<0.05 between TLR2−/− mice with insulin stimulus, treated or not with TAK-242; a
p<0.05 between WT mice with or without LPS stimulus; ° p<0.05 between WT and TLR2−/− mice with LPS stimulus.
An increased activation of ER stress leads to the activation of JNK [32],[33]. Therefore, we studied whether preventing the activation of this phenomenon could improve the insulin sensitivity and signaling. For this purpose, we treated mice with a pharmacological inhibitor of ER stress, 4-phenyl butyric acid (PBA), using i.p. daily injections for 10 d. This treatment was found to lead to an increased glucose uptake in TLR2 KO mice (Figure 6A) and increased insulin-induced serine phosphorylation of AKT in the liver (Figure 6B), muscle, and white adipose tissue (unpublished data), suggesting an improvement in the insulin signaling as well. After this treatment, we also investigated the phosphorylation of JNK and observed a reduction in the liver (Figure 6D) of TLR2 KO mice. Results suggest that both the activation of ER stress and the activation of JNK are important contributors to the development of the phenotype observed in TLR2 KO mice.
Since TLR4 was more activated in the tissues of TLR2 KO mice, possibly constituting one of the mechanisms responsible for the development of insulin resistance, we inhibited its expression using a TLR4 antisense oligonucleotide (ASO; with two daily i.p. injections) for 5 d. After TLR4 ASO treatment, TLR2 KO mice were found to present a significantly increased glucose uptake during the euglycemic hyperinsulinemic clamp compared with their controls (Figure 6A). The insulin signaling was also increased, with increased insulin-induced serine phosphorylation of AKT in the liver (Figure 6C), muscle, and white adipose tissue (unpublished data) of TLR2 KO mice. After this treatment, decreased phosphorylation of JNK was observed in the liver (Figure 6D) of TLR2 KO mice.
Using another method to inhibit TLR4 signaling, a pharmacological inhibitor of TLR4, TAK-242, was administered daily by gavage during 5 d and confirmed the results seen with the TLR4 ASO treatment. The insulin sensitivity was increased in TLR2 KO-treated animals (Figure 6A), and the insulin-induced serine phosphorylation of AKT was also increased in the liver (Figure 6C) of these animals, suggesting an improvement in insulin signaling. The phosphorylation of JNK was decreased in the liver (Figure 6D) of TLR2 KO treated mice.
As the serum LPS levels were increased in TLR2 KO mice, and the changes in microbiota may not account for this increase, we tested whether the LPS absorption was also increased in these animals. For this purpose, we administered LPS orally to TLR2 KO mice and wild type mice and determined the circulating LPS levels 1 h later. We observed that all animals presented increased serum LPS concentration after the LPS administration. However, TLR2 KO mice presented a higher increase in serum LPS concentration after the treatment, compared with the wild type mice (Figure 6E). As this result suggested that TLR2 KO mice presented increased gut permeability, we investigated the expression of an important tight-junction protein of the ileum of these mice, zonula occludens (ZO)-1, and observed that it was indeed decreased, compared with the control mice (Figure 6F). Reduction of ZO-1 expression in TLR2 KO mice was also observed in other parts of the small intestine and in the colon (unpublished data).
Previous data showed that TLR2 KO mice have a decreased number of regulatory T cells in the circulation compared with control mice [34]. This can have a role in the modulation of intestinal barrier and also in insulin resistance. We next investigated the frequency of Foxp3+ CD4+ T regulatory cells in mesenteric adipose tissue. We observed that the frequency of these cells was decreased in TLR2 KO mice (Figure 6H), compared with the wild type mice (Figure 6G).
Treatment of TLR2 KO Mice with Antibiotics Changes the Composition of Their Gut Microbiota and Improves Insulin Sensitivity
As the gut microbiota from TLR2 KO mice was shown to differ from that of controls, we treated both groups with a mixture of antibiotics (ampicillin, neomycin, and metronidazole) in their drinking water for 20 d. Moreover, we characterized the gut microbiota of TLR2 KO mice using culture-based microbial analysis of cecal contents after the antibiotics treatment and the results showed that aerobic bacteria were almost suppressed, while anaerobic bacteria decreased its abundance to 40% compared to the control group (Figure S7A).
After the treatment with the mixture of antibiotics, we also observed changes in the relative abundance of three phyla of bacteria. The abundance of Bacteroidetes was reduced from 47.92% to 19.78% and Firmicutes abundance decreased from 47.92% to 11.76% in the TLR2 KO mice, while Proteobacteria abundance increased from 1.04% to 67.38% in these mice (Figure S7B,C). TLR2 KO treated mice presented other differences in regards to classes and families and these results are presented in the Supporting Information section (Figures S8 and S9). When TLR2 KO mice were treated with metronidazole, neomycin, and ampicillin individually, and not as an antibiotics mixture, we observed that ampicillin was the most effective one to exterminate more bacteria diversity. When treated with metronidazole, TLR2 KO mice presented 46.51% of Proteobacteria, 10.69% of Firmicutes, and 42.32% of Bacteroidetes. When treated with neomycin, TLR2 KO mice presented 44.18% of Firmicutes and 55.81% of Bacteroidetes. When treated with ampicillin, almost 100% of the sequenced bacteria left corresponded to Proteobacteria (Figure S10A–C). Since the treatment with ampicillin or metronidazole normalized glucose tolerance in TLR2 KO mice, and neomycin only mildly improved glucose tolerance in these mice (unpublished data), we can speculate that the changes in microbiota induced by ampicillin or metronidazole are more relevant than the changes induced by neomycin, although no specific genera of bacteria can be implicated in this response. However, in accordance with previous data on obese mice, the decrease in the proportion of the phylum Firmicutes, as observed in the groups that received ampicillin or metronidazole, correlates with the improvement in glucose tolerance.
TLR2 KO mice presented decreased epididymal fat pad and visceral adipose tissue weight after the treatment with antibiotics compared with non-treated TLR2 KO, while no difference was observed in the treated and non-treated control animals (Figure 7A,B). TLR2 KO mice also presented increased glucose tolerance (Figure 7C) and increased oxygen consumption (Figure 7D) after the treatment compared with non-treated TLR2 KO mice, but no significant difference was observed between treated and non-treated control mice. With regard to insulin sensitivity and signaling, we observed an improvement in insulin-induced glucose uptake, using the euglycemic hyperinsulinemic clamp, in TLR2 KO mice after antibiotics treatment (Figure 7E), with no difference in the treated and non-treated control mice. After the treatment, we also observed an increase in the UCP-1 expression in the brown adipose tissue of TLR2 KO mice, supporting the increased oxygen consumption observed in this condition (Figure 7F). We also observed an increased insulin-induced serine phosphorylation of AKT in the liver (Figure 7G), muscle, and white adipose tissue (unpublished data) of TLR2 KO mice after the treatment. Moreover, there was a decreased phosphorylation of JNK in the liver (Figure 7H), muscle, and white adipose tissue (unpublished data) of the knockout mice after the treatment. The antibiotics treatment also led to an increased expression of ZO-1 in TLR2 KO mice, with no difference in the treated and non-treated control mice (Figure 7I). These data suggest that, in TLR2 KO mice, the reduction in their gut microbiota associated with qualitative changes in composition, induced by antibiotics, was able to reverse the insulin resistance of these animals.
10.1371/journal.pbio.1001212.g007Figure 7 Alterations in the metabolic parameters and in insulin signaling and sensitivity after treatment with antibiotics.
(A) Epididymal fat pad weight. (B) Visceral adipose tissue weight. (C) Glucose tolerance test. (D) Oxygen consumption. (E) Glucose uptake obtained by the euglycaemic hyperinsulinaemic clamp. (F) UCP-1 expression in the brown adipose tissue. (G) Serine phosphorylation of AKT after the treatment with AB. (H) Phosphorylation of JNK after the treatment with AB. (I) Zonula occludens (ZO)-1 expression in the ileum. Equal protein loading in the gel was confirmed by reblotting the membrane with an anti-β-actin antibody (lower panels). Data are presented as means ± S.E.M from six to eight mice per group, from experiments that were repeated at least three times. All evaluations were made with mice on standard chow. # p<0.05 between WT mice with or without insulin stimulus; * p<0.05 between WT and TLR2−/− mice with insulin stimulus; ** p<0.05 between TLR2−/− and TLR2−/− treated with AB, with insulin stimulus.
Effect of Microbiota Transplantation from TLR2 KO Mice to Control Mice on Weight Gain and Insulin Sensitivity
In order to investigate whether the gut microbiota was responsible for triggering all the alterations seen in TLR2 KO mice, we transplanted the cecal microbiota from TKR2 KO mice and from WT mice in 4-wk-old-Bacillus-associated WT mice, which contain few species of the genus Bacillus, without any other genera, as obtained by 16S rRNA pyrosequencing, in the following proportion: Bacillus simplex (0.68%), Bacillus sp (1.1%), Bacillus sp Kaza-34 (6.28%), and uncultured Bacillus (91.96%). There was a non-significant increase in the epididymal adipose tissue fat pad weight, in the total body weight gain, in the fasting blood glucose, and in the oxygen consumption in Bacillus-associated mice transplanted with WT microbiota (Figure 8A,C,D,G). However, in Bacillus-associated mice transplanted with TLR2 KO microbiota, we observed a marked increase in the epididymal fat pad and visceral adipose tissue weight (Figure 8A,B); in the body weight gain (Figure 8C), with a trend towards increased fasting blood glucose (Figure 8D), as well as a decrease in the glucose tolerance (Figure 8E,F); in the oxygen consumption (Figure 8G); and in the insulin sensitivity, obtained by euglycemic hyperinsulinemic clamp, compared with those transplanted with WT microbiota (Figure 8H) 8 wk after the transplantation (p<0.05). Bacillus-associated mice transplanted with WT microbiota also presented decreased insulin sensitivity compared with the non-transplanted mice (p<0.05). Bacillus-associated WT mice transplanted with TLR2 KO or with WT microbiota also showed decreased expression of UCP-1 in the brown adipose tissue compared with the non-transplanted mice. Bacillus-associated mice transplanted with TLR2 KO microbiota showed marked decrease in UCP-1 expression compared with those transplanted with WT microbiota (Figure 8I). Moreover, these animals had decreased insulin signaling, as seen by the reduction in serine phosphorylation of AKT in liver, compared to mice transplanted with WT microbiota (Figure 8J). In the mice transplanted with TLR2 KO microbiota, there was increased phosphorylation of JNK in liver (Figure 8K), muscle, and white adipose tissue (unpublished data) compared with the mice transplanted with WT microbiota. The experiments described above had also been performed in few germ-free mice, but with very similar results (unpublished data).
10.1371/journal.pbio.1001212.g008Figure 8 WT mice reproduce TLR2 knockout (TLR2−/−) mice after cecal microbiota transplantation.
(A) Epididymal fat pad weight. (B) Visceral adipose tissue weight. (C) Weight gain of transplanted mice. (D) Serum glucose. (E) Glucose tolerance test. (F) Incremental area under curva (IAUC) obtained from the glucose tolerance test. (G) Oxygen consumption. (H) Glucose uptake obtained by the euglycaemic hyperinsulinaemic clamp. (I) UCP-1 expression in the brown adipose tissue. (J) Serine phosphorylation of AKT after the treatment with AB. (K) Phosphorylation of JNK after the treatment with AB. AKT and JNK protein expression in the liver of transplanted mice (J, K, lower panels). (L) Zonula occludens (ZO)-1 expression in the ileum. (M) Frequency of CD4+Foxp3+ regulatory T cells in Bacillus-associated mice. (N) Frequency of CD4+Foxp3+ regulatory T cells in Bacillus-associated mice transplanted with WT microbiota. (O) Frequency of CD4+Foxp3+ regulatory T cells in Bacillus-associated mice transplanted with TLR2−/− microbiota. Equal protein loading in the gel was confirmed by reblotting the membrane with an anti-β-actin antibody (lower panels). Data are presented as means ± S.E.M from six to eight mice per group from experiments that were repeated at least three times. All evaluations were made with mice on standard chow. * p<0.05 between Bacillus-associated mice transplanted with TLR2−/− microbiota (MA+TLR2−/−) and those transplanted with WT microbiota (MA+WT); ** p<0.05 between Bacillus-associated mice transplanted with WT microbiota (MA+WT) and Bacillus-associated mice (MA); # p<0.05 between Bacillus-associated mice with or without insulin stimulus.
Eight weeks after the transplantation, the expression of ZO-1 was evaluated in the 12-wk-old mice. We observed that it was decreased in mice transplanted with TLR2 KO microbiota compared to those transplanted with WT microbiota (Figure 8L). The same data were observed in other parts of the small intestine and in the colon (unpublished data).
We also investigated the frequency of CD4+Foxp3+ regulatory T cells in these animals and observed that they were decreased in mesenteric adipose tissue in mice transplanted with TLR2 KO microbiota (Figure 8O) compared with the mice transplanted with WT microbiota (Figure 8N) and non-transplanted Bacillus-associated mice (Figure 8M).
In summary, as expected, the transplantation of a wild-type microbiota in Bacillus-associated mice resulted in a moderate increase in adipose visceral fat and in a mild decrease in glucose tolerance. However, the effect of the transplantation of TLR2 KO microbiota in Bacillus-associated mice induced marked changes, and clearly indicates deleterious effects of this TLR2 KO microbiota on body weight and glucose metabolism.
Effect of High-Fat Diet (HFD) on Weight Gain and Insulin Sensitivity in TLR2 KO Mice
Next, we investigated the effect of high-fat diet (HFD) on metabolic parameters of TLR2 KO mice. The results showed that at 8 wk old TLR2 KO mice on a HFD presented increased body weight (Figure 9A), similar food intake (WT = 7.3 g per day, TLR2 KO = 6.1 g per day; WT = 0.25±0.055 g/g animal/day; TLR2 KO = 0.19±0.046 g/g animal/day) (Figure 9B), increased epididymal fat weight (Figure 9C), reduced glucose tolerance (Figure 9D), increased fasting serum insulin (Figure 9E), and reduced glucose uptake (Figure 9F) compared to the controls. The oxygen consumption of both groups was compared and TLR2 KO mice were seen to present decreased oxygen consumption (Figure 9G), suggesting decreased energy expenditure compared to the controls. However, the respiratory exchange ratio was similar in both groups, being around 0.75 (Figure 9H). In accordance with the reduced oxygen consumption observed, the expression of UCP1 was significantly decreased in TLR2 KO mice (Figure 9I). Similarly, insulin signaling was reduced in TLR2 KO mice fed on the HFD. The insulin-induced serine phosphorylation of AKT was reduced in the muscle, liver, and white adipose tissue of TLR2 KO mice, compared with controls (Figure 10A–C). Moreover, the phosphorylation of JNK was increased in all tissues studied of the TLR2 KO mice (Figure 10D–F), while the expression of IκB-α was increased (Figure 10G–I), suggesting that the IKK/NFκB pathway is decreased in TLR2 KO mice on a HFD, as observed for mice on a standard chow. These results suggest that the metabolic phenotype of the TLR2 KO mice characterized by insulin resistance is aggravated by HFD, which leads to the development of diabetes, as demonstrated by fasting blood glucose and glucose tolerance tests.
10.1371/journal.pbio.1001212.g009Figure 9 Metabolic parameters of TLR2 KO (TLR2−/−) mice fed a high-fat diet.
(A) Weight gain after 10 wk of high-fat diet (HFD). (B) Food intake. (C) Epididymal fat pad weight. (D) Glucose tolerance test. (E) Serum insulin concentration. (F) Glucose uptake obtained from the euglycaemic hyperinsulinaemic clamp. (G) Oxygen consumption and (H) respiratory exchange rate. (I) UCP-1 expression in the brown adipose tissue. Data are presented as means ± S.E.M from six to eight mice per group from experiments that were repeated at least three times. All evaluations were made with mice on standard chow. * p<0.05 between TLR2−/− mice and their controls.
10.1371/journal.pbio.1001212.g010Figure 10 Insulin signaling is impaired in TLR2 knockout (TLR2−/−) mice fed on a high-fat diet.
Phosphorylation of AKT in muscle (A), liver (B), and white adipose tissue (WAT) (C). AKT protein expression in muscle, liver, and WAT (A–C, lower panels). Phosphorylation of JNK in muscle (D), liver (E), and WAT (F). JNK protein expression in muscle, liver, and WAT (D–F, lower panels). IκB-α expression in muscle (G), liver (H), and WAT (I). Equal protein loading in the gel was confirmed by reblotting the membrane with an anti-β-actin antibody (lower panels). All evaluations were made with mice on standard chow. Data are presented as means ± S.E.M from six to eight mice per group from experiments that were repeated at least three times. * p<0.05 between TLR2−/− mice and their controls, with insulin stimulus; ** p<0.05 between WT with and without insulin stimulus.
Discussion
It is now considered that environmental factors and host genetics interact to control the acquisition and stability of gut microbiota. In turn, environment, host genetics, and microbiota interact to maintain the homeostasis of gut, weight control, and insulin sensitivity [17]. Clearly, the modification of one or more of these three components may trigger the development of insulin resistance and obesity. The results of the present study demonstrated that TLR2 KO mice in conventionalized conditions in our breeding center have insulin resistance and glucose intolerance associated with alterations in the composition of the gut microbiota, which displayed an increase in the relative abundance of Firmicutes and Bacteroidetes and decreased relative abundance of Proteobacteria, compared to their controls. The insulin resistance of TLR2 KO mice was accompanied by a down-modulation of insulin-induced insulin signaling in the liver, muscle, and adipose tissue, associated with an increase in endoplasmic reticulum stress. These metabolic alterations were characterized in 8-wk-old TLR2 KO mice, when they had similar body weights to the control animals. As demonstrated in other animal models [35],[36], this insulin resistance precedes the development of obesity, and an augmentation in body weight compared to controls is observed after the 12th wk of age.
However, previous studies [28],[37] have reported that TLR2 KO mice present decreased body weight and adiposity, are protected against insulin resistance, and gain less weight on a HFD than control mice and are also protected against related comorbidities [38],[39]. We believe that the main difference between these studies and our study may be related to gut microbiota. It should be taken into consideration that although the animals have the same genetic deficiency they were bred in different rooms and fed with food from different sources, which can certainly have a role in the establishment and maintenance of gut microbiota. Although in most of the previous studies the gut microbiota was not investigated, we can suggest that TLR2 deficiency associated with different environmental conditions can induce different phenotypes, probably induced by different microbiotas. Kellermayer et al. have shown that the proportion of Firmicutes found in TLR2 KO mice was lower than in WT, while the proportion of Bacteroidetes was increased [40]. In our study, we show that TLR2 KO mice present the opposite, with increased proportion of Firmicutes and decreased proportion of Bacteroidetes, compared with the WT. This way, it is possible that in the other published studies the proportions of this phyla might be different, compared with the proportions we have found, which might influence differently the phenotype observed. These results reinforce the importance of environment and of the innate immune system as key regulators of gut microbiota and suggest that a genetic condition, which by itself can prevent insulin resistance in some conditions, can also overcome the protective effect on insulin resistance in other environmental conditions inducing more weight gain, probably due to differences in the microbiota. In addition, these findings may help explain differences in the metabolic behavior of the same animal, when analyzed in distinct environments, and can contribute to explaining differences in metabolic behavior between animals with the same background or with the same genetic alteration.
The mechanisms by which the TLR2 KO mice presented insulin resistance and, later, obesity were also investigated. The gut microbiota of the TLR2 KO animal have some similarities to those found in obese animals and humans, with an increase in Firmicutes [41],[42]. This type of microbiota is usually associated with an increased capacity for energy harvesting from the diet [19]. This might contribute to explaining the obesity observed, but does not explain why these animals are clearly insulin resistant many weeks before they start to gain more weight than their controls. In addition, it was demonstrated that germ-free (that gain less weight on HFD) and conventionalized mice have similar energy contents in their feces, suggesting that other mechanisms may have an important role in gut microbiota-induced insulin resistance and obesity [43]. Additionally some studies suggest that the gut microbiota can contribute to obesity by inducing a reduction in fat oxidation and an increase in fat storage [43], associated with a relative reduction in the expression of PGC1 alpha and in AMPK phosphorylation. This mechanism is less probable in our animal model, because the RQ of TLR2 KO mice was identical to that of control mice, showing that they were oxidizing fat in the same proportion of controls, and also the tissue levels of PGC1alpha and also the phosphorylation of AMPK were similar in liver and muscle of controls and TLR2 KO mice.
Another possible mechanism that could induce insulin resistance in obesity is the increased level of LPS, which is observed in HFD mice [11],[44]. Notably, although TLR2 KO mice were fed on standard rodent chow, they presented higher circulating levels of LPS. Since the microbiota of these mice had a predominance of Firmicutes, which are gram-positive, and do not have LPS in the outer membrane, the increase of LPS circulating levels is certainly not the consequence of a microbiota that produces more LPS. However, the microbiota observed in obesity and also in TLR2 KO mice may increase gut permeability and LPS absorption [45]–[47]. Importantly, as observed in obese animals, which present a significant reduction in Bifidobacteria [48],[49], in the microbiota of lean TLR2 KO mice this genera was reduced compared with the controls. In this regard, the supplementation of Bifidobacteria has been linked to an improvement in the gut barrier function and to reduced levels of LPS [31],[50],[51]. In order to prove that the increased circulating LPS levels of TLR2 KO mice were related to gut permeability, we administered LPS orally to these mice and observed that, in addition to higher basal LPS levels, these animals also showed a higher peak of LPS 1 h after oral gavage of this lipopolysaccharide.
Previous data showed that TLR2 regulates tight junction (TJ)-associated intestinal epithelial barrier integrity and that TLR2 deficiency predisposes to alterations of TJ-modulated barrier function leading to perpetuation of mucosal inflammation [52],[53]. In this regard, our data also demonstrated that, in TLR2 KO mice, there is a reduction in ZO-1 in the small intestine and in the colon, reinforcing that there are alterations in epithelial integrity and gut permeability in these mice. Taken together, these results suggest that the interactions between the predisposition of TLR2 KO mice to alterations in barrier function and the microbiota may have an important role in the increased circulating LPS levels observed in these mice.
In accordance with alterations in gut permeability, Kellermayer et al. recently investigated the epigenomic and metagenomic consequences of Tlr2 deficiency in the colonic mucosa of mice in order to understand the biological pathways that shape the interface between the gut microbiota and the mammalian host. The results showed epigenomic and transcriptomic modifications associated with alterations in mucosal microbial composition and the abundance of many bacterial species were found to differ between WT and TLR2 KO animals. The expression of genes involved in the immune system was modified in the colonic mucosa of TLR2 KO mice, which correlated with DNA methylation changes. This pioneer study demonstrates that significant microbiota shifts associate with epithelial epigenetic changes influenced by the host genome [54].
In order to confirm that gut microbiota was inducing insulin resistance in TLR2 KO mice, we treated these mice with antibiotics for 15 d and showed that this treatment dramatically reduced the gut microbiota and also changed its composition. In parallel, there was an improvement in insulin action, characterized by an increased glucose infusion rate during the glucose clamp, and also an improvement in insulin signaling in the liver, muscle, and adipose tissue. In the TLR2 KO mice treated with antibiotics, we also observed a marked reduction in LPS levels. When we performed gut microbiota transplantation of TLR2 KO mice to Bacillus-associated WT mice, which are colonized only by the genus Bacillus and are capable of receiving a different microbiota from other mice, the complex composition of the transferred organism was preserved. The transplanted TLR2 KO mice microbiota conferred more weight gain, glucose intolerance, and reduced insulin sensitivity and signaling, associated with increased LPS circulating levels. These data reinforce the hypothesis that the TLR2 KO mice microbiota are able to induce changes in the gut permeability, in turn increasing serum LPS levels, associated with insulin resistance.
The increase in LPS may induce insulin resistance by counteracting insulin signaling, as previously demonstrated [11],[55],[56]. However, the insulin resistance observed in TLR2 KO mice has unique characteristics. There was activation of TLR4 in the liver, muscle, and adipose tissue, associated with ER stress and JNK activation, but no activation of the IKKβ-IκB-NFκB pathway. It was previously described that there is cooperation between TLR4 and TLR2 signaling. This cooperation is evident when LPS is injected in TLR2 KO mice. After the first bolus of LPS, TLR2 KO mice show a robust signal for genes encoding innate immune proteins in the brain. However, the second LPS infusion failed to trigger TNFalpha in TLR2 KO mice. These results indicate that TLR2 is involved in the second wave of TNFalpha expression after LPS and that there is an elegant cooperation between TLR2 and TLR4 [57]. Our results extended these data by showing that the chronic elevation in LPS levels in TLR2 KO mice was not able to increase IKK/IkB/NF-kB pathway and TNFalpha and IL-6 production, but induced an increase in JNK activation in liver, muscle, and adipose tissue of these mice. These data suggest that chronic activation of TLR4 by low doses of LPS is sufficient to increase JNK activation, but the activation of IKK/IkB/NF-kB pathway may also depend on the cooperation between TLR2 and TLR4.
The absence of activation of the NFκB pathway and the reduced levels of TNFα and IL-6 make the insulin resistance of TLR2 KO mice different from that observed in DIO mice or in ob/ob mice. We can, thus, suggest that the increase in LPS circulating levels caused activation of TLR4, induced ER stress and JNK activation accompanied by increased IRS-1 serine 307 phosphorylation in the liver, muscle, and adipose tissue, leading to a reduction in insulin sensitivity and signaling and conferring the phenotype observed in the TLR2 KO mice. Phosphorylation of IRS-1 on serine residues interferes with the subsequent insulin-stimulated tyrosine phosphorylation of IRS-1 by IR [58] and IRS-1 can also mediate inhibition of the insulin receptor tyrosine kinase activity [55], and also with downstream signaling as Akt phosphorylation. This insulin signaling pathway is crucial for the metabolic effects of insulin on glucose metabolism [59]. The pharmacological or genetic blockage of TLR4, of ER stress, or of JNK improved action and signaling of insulin in TLR2 KO mice, confirming that this sequence of events has an important role in the insulin resistance of these animals.
Regulatory T cells, a small subset of T lymphocytes, are thought to be one of the body's most important defenses against inappropriate immune responses [60],[61] and can influence the activities of cells of the innate immune system [62]–[64]. Previous data showed that regulatory T cells were highly enriched in the abdominal fat of control mice and reduced at this site in animal models of obesity. This reduction in obesity of regulatory T cells influenced the inflammatory state of adipose tissue and certainly contributes to insulin resistance. Our data showing that in TLR2 KO mice there is a reduction in regulatory T cell in visceral adipose tissue may suggest that this modulation may also contribute to the insulin resistance observed in these animals.
The development of obesity and insulin resistance in humans is thought to be promoted by a HFD. Feeding TLR2 KO mice with a HFD for 8 wk caused a marked increase in body weight and in fasting plasma glucose, with levels of over 400 mg/dl at 2 h during the glucose tolerance test, demonstrating that these animals developed not only a more severe form of insulin resistance but also diabetes. The alterations in insulin signaling in tissues also showed a marked down-regulation, in parallel with a higher activation of JNK compared to their controls on HFD. Interestingly, the absence of activation of the IKKβ-IκB-NFκB pathway, described in TLR2 KO mice on standard rodent chow, was also observed when these mice were on HFD. These results demonstrate that the insulin resistance, and later the increase in body weight observed in TLR2 KO mice, is exacerbated by HFD.
A recent report demonstrated that genetically deficient TLR5 mice exhibit hyperphagia, hyperlipidemia, insulin resistance, and increased adiposity [30]. These metabolic alterations correlated with changes in the composition of the gut microbiota. Our model, although showing similar features, presented different aspects that may suggest that different mechanisms may be operating in TLR5 or TLR2 KO mice. First, TLR2 KO mice did not present hyperphagia, and the difference in body weight starts only when these animals are 16 wk old. In the TLR5 KO mice, the insulin resistance is not dependent on TLR4, but in TLR2 KO mice there is an increase in circulating LPS and activation of TLR4. It is possible that these differences not only represent differences in genetic defects but also differences in gut microbiota between these mice.
In conclusion, we may suggest that the loss of TLR2 in conventionalized mice results in a reminiscent phenotype of metabolic syndrome, characterized by a clear difference in the gut microbiota, which induces insulin resistance, subclinical inflammation associated with ER stress, glucose intolerance, and later obesity, which is reproduced in WT by microbiota transplantation and can be reversed using antibiotics. Our results emphasize the role of microbiota in the complex network of molecular and cellular interactions that bridge genotype to phenotype and have potential implications for a wide array of common human disorders involving obesity, diabetes, and even other immunological disorders.
Materials and Methods
Materials
Human recombinant insulin was from Eli Lilly (Indianapolis, Indiana, USA). Reagents for SDS-PAGE and immunoblotting were from Bio-Rad. HEPES, phenylmethylsulfonyl fluoride, aprotinin, dithiothreitol, Triton X-100, Tween 20, glycerol, and bovine serum albumin (fraction V) were from Sigma. Protein A-Sepharose 6MB was from GE Healthcare, and nitrocellulose paper (BA85, 0.2 µm) was from Amersham Biosciences. The reagents for the chemiluminescence labeling of proteins in blots were from Amersham Biosciences. Sense and antisense oligonucleotides specific for TLR4 (sense, 5′-C TGA AAA AGC ATT CCC ACC T-3′ and antisense, 5′-A GGT GGG AAT GCT TTT TCA G-3′) were produced by Invitrogen Corp. (Carlsbad, CA). Antibodies against β-actin (mouse monoclonal, sc-8432), TLR4 (rabbit polyclonal, sc-30002), phospho [Ser307]-IRS-1 (rabbit polyclonal, sc-33956), phospho [Tyr941] (goat polyclonal, sc-17199), IRS-1 (rabbit polyclonal, sc-559), phospho [Ser 473]-AKT (rabbit polyclonal, sc-33437), AKT1 (goat polyclonal, sc-1618), phospho [Thr 981]-PERK (rabbit polyclonal, sc-32577), PERK (goat polyclonal, sc-9477), phospho-JNK (mouse monoclonal, sc-6254), JNK1 (mouse monoclonal, sc-1648), phospho[Tyr1162/1163]-Insulin Receptor (rabbit polyclonal, sc-25103), Insulin Receptor β (goat polyclonal, sc-31369), UCP1 (goat polyclonal sc-6529), and MyD88 (goat polyclonal, sc-8197) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibody against ZO-1 was from Abcam (AB96594) (Cambridge, MA). Antibodies against phospho [Thr172]-AMPKα (rabbit polyclonal, #2531), AMPKα (rabbit polyclonal, #2532), and IκB-α (rabbit polyclonal, #9242) were from Cell Signaling Technology (Beverly, Massachusetts, USA).
Mice
TLR2-deficient mice, also called TLR2 knockout (KO) mice, were obtained by Dr. Akira [65] and were kindly provided by Dr. Ricardo Gazzinelli [66] and maintained on a C57BL/6J genetic background. Studies were carried out using male TLR2 KO mice that were age matched with C57BL/6J and obtained from the University of Campinas Breeding Center. C57BL/6J and the TLR2 KO mice have the same origin and have been raised in the same institution (UNICAMP) and in the same room, at University of Campinas Breeding Center. The C57BL/6J strain was generated by backcrossing mice carrying the TLR2 KO mutation 10 times to C57BL/6J inbred mice [67]. TLR2-deficient mice are viable and fertile. The control and the knockout mice used for the experiments were littermates, obtained from a heterozygote × heterozygote cross, from the same mother, from the same cage, in order to have standard conditions for all animals. The investigation was approved by the ethics committee and followed the university guidelines for the use of animals in experimental studies, and experiments conform to the Guide for the Care and Use of Laboratory Animals, published by the U.S. National Institutes of Health (NIH publication no. 85–23 revised 1996). The animals were maintained on 12 h/12 h artificial light-dark cycles and housed in individual cages. Mice were randomly divided into two groups: control, fed on standard rodent chow (3.948 kcal/Kg−1), and HFD, fed on a rich-fat chow (5.358 kcal/Kg−1) ad libitum for 16 wk. The mice were bred under specific pathogen-free conditions at the Central Breeding Center of the University of Campinas.
Serum Analysis
Mice were fasted for 5 h, at which time blood was collected by the retrobulbar intraorbital capillary plexus. Hemolysis-free serum was generated by the centrifugation of blood using serum separator tubes (Becton Dickinson, Franklin Lakes, New Jersey). Serum insulin, cytokines, leptin, and adiponectin were analyzed by ELISA kits purchased from Linco Research Inc (St. Charles, Missouri).
Determination of NF-κB Activation
NF-κB p50 activation was determined in nuclear extracts from muscle and adipose tissue by ELISA (89858; Pierce Biotechnology), according to the recommendations of the manufacturer.
LPS Serum Determination
Serum LPS concentration was determined using a kit based on a Limulus amebocyte extract (LAL kit endpoint-QCL1000; Cambrex BioScience, Walkersville, Maryland), where samples were diluted 1/40 to 1/100 and heated for 10 min at 70°C. Internal control of recovery calculation was included in the assessment.
Glucose Tolerance Test
After 6 h fasting, mice were anesthetized by an i.p. injection of sodium amobarbital (15 mg/kg body weight), and the experiments were initiated after the loss of corneal and pedal reflexes. After collection of an unchallenged sample (time 0), a solution of 20% glucose (2.0 g/kg body weight) was administered into the peritoneal cavity. Blood samples were collected from the tail at 30, 60, 90, and 120 min for determination of glucose and insulin concentrations [68].
Euglycaemic-Hyperinsulinaemic Clamp
After a 6-h fast, a prime continuous (3.0 mU·kg−1·min−1) infusion of regular insulin was administered in the groups of mice for 2 h from time 0, to raise plasma insulin and maintain it at a steady-state plateau (90–120 min). A variable glucose infusion (10%) was started 5 min after the beginning of the experiment and was corrected, if necessary, to maintain euglycaemia between 5 and 6.1 mmol/l [69]. Blood samples for determination of plasma glucose were obtained at 5-min intervals throughout the study.
Oxygen Consumption/Carbon Dioxide Production and Respiratory Exchange Ratio Determination
Oxygen consumption/carbon dioxide production and respiratory exchange ratio (RER) were measured in fed animals through an indirect open circuit calorimeter (Oxymax Deluxe System; Columbus Instruments, Columbus, Ohio), as described previously [70].
Measurement of Food Intake
Standard chow or HFD was given and food intake was determined by measuring the difference between the weight of chow given and the weight of chow at the end of a 24-h period. This procedure was performed during 5 d, with 8-wk-old mice, using metabolic cages for a single mouse (Tecniplast, Italy), obtaining an average of food intake per cage per day. This average was also normalized for body weight.
4-Phenyl Butyric Acid (PBA) Treatment
PBA is a chemical chaperone and evidence suggests that it relieves endoplasmic reticulum stress [71]. For acclimation, mice received 100 µl phosphate buffered saline (PBS) twice daily (8 a.m. and 6 p.m.), by gavage, for 3 d. Following the acclimation period, PBA was administered twice daily in two divided doses (500 mg/kg at 8 a.m. and at 6 p.m., total 1 g/kg/day) by gavage for 10 d. Control groups received the same volume of vehicle instead of PBA at the same treatment points [33].
SP600125 Treatment
SP600125, a potent and selective inhibitor of JNK, was dissolved in a 7% (in PBS) Solutol HS-15 solution and administered intraperitoneally (30 mg/kg/day) for 5 d [72].
TLR4 Inhibition
In order to inhibit the expression of TLR4, two methods were used: pharmacological inhibition, using 2.4 mg/kg/day ethyl(6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242) (synthesized at the Chemistry Institute of the University of Campinas) [73], administered daily by gavage during 5 d, and 4 nmol TLR2 antisense oligonucleotide (ASO) inhibition, composed by 5′-AGGTGGGAATGCTTTTTCAG-3′ (sense) and 5′-CTGAAAAAGCATTCCCACCT-3′ (antisense), administered by two daily i.p. injections during 5 d, produced by Invitrogen Corp. (Carlsbad, California, USA).
LPS Absorption Test
An LPS tolerance test was performed as follows: Fasted mice were gavaged with LPS (300 µg/kg) diluted in water (100 µL) or with water (100 µL). Blood was collected from the cava vein 60 min after gavage. Plasma was separated and frozen [11].
Intracellular Cytokine Analysis and Foxp3 Staining
The cells were obtained from the adipose tissue and analyzed by flow cytometry. For the determination of the frequency of putative regulatory T cells, the adipose tissue mononuclear cells were stained for the surface marker CD4 (Percp) and after for intracellular transcription factor Foxp3 using APC anti-mouse/rat Foxp3 staining (eBioscience, San Diego, California). The cells were acquired in the FACS Calibur Flow cytometer (BD) and analyzed with FlowJo software.
Antibiotics Treatment
Four-week-old WT and TLR2 KO mice were placed on broad spectrum antibiotics (1.0 g/L ampicillin, 1.0 g/L metronidazole, and 0.5 g/L neomycin) in drinking water for 20 d. During this period mice were monitored for food intake and stool microbiota sequencing.
Culture-Based Microbial Analysis of Cecal Contents
Total aerobic and anaerobic bacteria were enumerated in selective media and incubation conditions according to Schumann et al. [74]. In brief, cecal samples were diluted in Ringer medium, and total aerobic and anaerobic bacteria were investigated by plating onto nonselective media: TSS medium (Biomerieux, Lyon, France) for 24 to 48 h at 37°C in aerobic and anaerobic conditions. Bacterial numbers were expressed as colony forming units (CFU)/mg cecal content [75].
Metagenome Profile
Faeces samples were collected in metabolic cages with separated waste collectors, frozen in liquid nitrogen, and kept at −80°C until use. DNA was then extracted using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) and quantified. Libraries were synthesized from 500 ng of total DNA following the Rapid Library Preparation Kit (Roche Applied Science, Mannheim, Germany) instructions. These libraries were analyzed in a Bioanalyzer with a High Sensitive DNA Kit (Agilent Technologies Inc., Santa Clara, California, USA), and equimolar pools were made, titrated, and submitted to large volume PCR, following the manufacturer's instructions (Roche Applied Science, Mannheim, Germany). Subsequently, samples were sequenced in GS FLX Titanium, using a GS FLX Titanium PicoTiterPlate Kit combined with a GS FLX Titanium Sequencing Kit XLR70 (Roche Applied Science, Mannheim, Germany). The data obtained from the sequencing were submitted to the MG-RAST server and compared by phylum prevalence among groups [76].
Microbiota Transplantation
Cecal contents were pooled from 3 TLR2 KO mice and age- and gender-matched WT littermates. Cecal extracts were suspended in PBS (2.5 ml per cecum) and were administered (0.1 ml per mouse) immediately to sterilely packed, 4-wk-old, Bacillus-associated, WT mice that were obtained from the Central Breeding Center of the State University of Campinas. Transplanted mice were maintained in sterile cages and monitored for body weight [30].
Tissue Extraction, Immunoprecipitation, and Immunoblotting
Mice were anesthetized by intraperitoneal injection of sodium thiopental and used 10–15 min later—i.e., as soon as anesthesia was assured by the loss of pedal and corneal reflexes. In some experiments, 3 or 5 min after insulin injection (3.8 units/kg, intraperitoneally), liver or muscle and white adipose tissue were removed, respectively, and homogenized immediately in extraction buffer at 4°C (1% Triton X-100, 100 mm Tris-HCl (pH 7.4), 100 mm sodium pyrophosphate, 100 mm sodium fluoride, 10 mm EDTA, 10 mm sodium orthovanadate, 2.0 mm phenylmethylsulfonyl fluoride, and 0.1 mg of aprotinin/ml) with a Polytron PTA 20 S generator (model PT 10/35; Brinkmann Instruments). Insoluble material was removed by centrifugation for 30 min at 9,000×g in a 70 Ti rotor (Beckman, Fullerton, California) at 4°C. The protein concentrations of the supernatants were determined by the Bradford dye binding method. Aliquots of the resulting supernatants containing 1.0 mg of total protein were used for immunoprecipitation with antibodies against MyD88 overnight at 4°C, followed by SDS-PAGE, transfer to nitrocellulose membranes, and blotting with anti-TLR4. In direct immunoblot experiments, 0.2 mg of protein extracts were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with anti-UCP1, anti-phospho-JNK, anti-IκBα, anti-phospho-PERK, anti-phospho-AKT, anti-phospho [Ser307]-IRS-1, anti-phospho [Tyr941]-IRS-1 (Tyr), anti-phospho-IR, anti-ZO-1, anti-PGC-1α, anti-phospho [Thr171]-AMPK, and anti-IκB-α. The homogeneity of gel loading was always evaluated by blotting the membranes with antibodies against β-actin, IRS-1, AKT, IR, JNK, PERK, and AMPK as appropriate.
Statistical Analysis
Specific protein bands present on the blots were quantified by densitometry. Mean ± S.E. values obtained from densitometric scans and from the other experiments were compared utilizing Student's t test for paired samples or by repeat-measure analysis of variance (one-way or two-way analysis of variance) followed by post hoc analysis of significance (Bonferroni test) when appropriate. When analyzing non-linear parameters, we used Mann-Whitney test. A p<0.05 was accepted as statistically significant.
Supporting Information
Figure S1 Taxonomical characterization of WT gut microbiota. Untreated WT stools were analyzed via 16S rRNA analysis.
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Figure S2 TLR2 KO mice exhibit taxonomical alterations in gut microbiota. TLR2 knockout (TLR2−/−) mice stools were analyzed via 16S rRNA analysis.
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Figure S3 Bacterial phyla distribution in 4-wk-old-WT (A) or –TLR2−/− mice (B). These analyses were obtained by 16S rRNA sequencing.
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Figure S4 Bacterial phyla distribution in 16-wk-old-WT (A) or –TLR2−/− mice (B). These analyses were obtained by 16S rRNA sequencing.
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Figure S5 Bacterial phyla distribution in 1-y-old-WT (E) or –TLR2−/− mice (F). These analyses were obtained by 16S rRNA sequencing.
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Figure S6 Phosphorylation of the insulin receptor in muscle (A), liver (B), and white adipose tissue (WAT) (C). Tyrosine 172 phosphorylation of AMPK in muscle (D), liver (E), and WAT (F). PGC-1α expression in muscle (G), liver (H), and WAT (I). Insulin receptor and AMPK protein expression in muscle, liver, and WAT (lower panels). Equal protein loading in the gel was confirmed by reblotting the membrane with an anti-β-actin antibody (lower panels). Data are presented from six to eight mice per group, from experiments that were repeated at least three times. All evaluations were made with mice on standard chow. * p<0.05 between WT mice with or without insulin stimulus; ** p<0.05 between WT and TLR2−/− mice with insulin stimulus.
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Figure S7 Cecal samples of TLR2 knockout (TLR2−/−) mice were cultured in aerobic and anaerobic environments with or without the treatment with a mixture of antibiotics (AB) (0.5 g/kg neomycin, 1 g/kg metronidazole, and 1.0 g/kg ampicillin). Aerobic bacteria counts went below the detection limit in the groups treated with the AB. TLR2 knockout (TLR2−/−) mice exhibit taxonomical alterations in gut microbiota after the treatment with a mixture of AB (C), compared with controls, without antibiotics treatment (B). Mice stools were analyzed via 16S rRNA analysis. Data are presented from six to eight mice per group, from experiments that were repeated at least three times. All evaluations were made with mice on standard chow. * p<0.05 between aerobic bacteria of TLR2−/− mice with or without AB treatment; ** p<0.05 between anaerobic bacteria of TLR2−/− mice with or without AB treatment.
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Figure S8 Taxonomical characterization of WT gut microbiota after the treatment with a mixture of antibiotics (AB) (0.5 g/kg neomycin, 1 g/kg metronidazole, and 1.0 g/kg ampicillin). WT+AB stools were analyzed via 16S rRNA analysis.
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Click here for additional data file.
Figure S9 Taxonomical characterization of TLR2−/− gut microbiota after the treatment with a mixture of antibiotics (AB) (0.5 g/kg neomycin, 1 g/kg metronidazole, and 1.0 g/kg ampicillin). TLR2−/− + AB stools were analyzed via 16S rRNA analysis.
(TIF)
Click here for additional data file.
Figure S10 Taxonomical alteration obtained from the treatment with antibiotics individually (0.5 g/kg neomycin, 1 g/kg metronidazole, and 1.0 g/kg ampicillin). TLR2−/− mice treated only with metronidazole (A). TLR2−/− mice treated only with neomycin (B). TLR2−/− mice treated only with ampicillin (C).
(TIF)
Click here for additional data file.
We thank Mr. Luiz Janeri, Mr. Josimo Pinheiro, and Mrs. Dioze Guadagnini for the technical assistance and Nicola Conran for the English language editing.
The authors have declared that no competing interests exist.
This study was supported by grants from Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) and Conselho Nacional de desenvolvimento científico e tecnológico (CNPq). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Abbreviations
ASOantisense oligonucleotide
CFUcolony forming units
HFDhigh-fat diet
IRinsulin receptor
IRSinsulin receptor substrate
KOknockout
PBAphenyl butyric acid
PBSphosphate buffered saline
RERrespiratory exchange ratio
rRNAribosomal RNA
T2DMtype 2 diabetes mellitus
TJtight junction
TLRsToll-like receptors
TLR2Toll-like Receptor 2
ZOzonula occludens
==== Refs
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PLoS PathogPLoS PathogplosplospathPLoS Pathogens1553-73661553-7374Public Library of Science San Francisco, USA 22174681PPATHOGENS-D-11-0164110.1371/journal.ppat.1002418Research ArticleBiologyMicrobiologyVirologyEpstein-Barr Virus Nuclear Antigen 3C Stabilizes Gemin3 to Block p53-mediated Apoptosis EBNA3C Stabilizes Gemin3Cai Qiliang
1
Guo Yi
1
2
Xiao Bingyi
1
Banerjee Shuvomoy
1
Saha Abhik
1
Lu Jie
1
Glisovic Tina
3
Robertson Erle S.
1
*
1
Department of Microbiology and the Tumor Virology Program, Abramson Comprehensive Cancer Center, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, United States of America
2
Key Laboratory of AIDS Immunology, Ministry of Health, The First Affiliated Hospital, China Medical University, Shenyang, Liaoning, People's Republic of China
3
Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, United States of America
Raab-Traub Nancy EditorUniversity of North Carolina at Chapel Hill, United States of America* E-mail: [email protected] and designed the experiments: QC YG ESR. Performed the experiments: QC YG BX SB. Analyzed the data: QC YG ESR. Contributed reagents/materials/analysis tools: AS JL TG. Wrote the paper: QC YG ESR.
12 2011 8 12 2011 7 12 e100241822 7 2011 20 10 2011 Cai et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.The Epstein-Barr nuclear antigen 3C (EBNA3C), one of the essential latent antigens for Epstein-Barr virus (EBV)-induced immortalization of primary human B lymphocytes in vitro, has been implicated in regulating cell proliferation and anti-apoptosis via interaction with several cellular and viral factors. Gemin3 (also named DDX20 or DP103) is a member of DEAD RNA helicase family which exhibits diverse cellular functions including DNA transcription, recombination and repair, and RNA metabolism. Gemin3 was initially identified as a binding partner to EBNA2 and EBNA3C. However, the mechanism by which EBNA3C regulates Gemin3 function remains unclear. Here, we report that EBNA3C directly interacts with Gemin3 through its C-terminal domains. This interaction results in increased stability of Gemin3 and its accumulation in both B lymphoma cells and EBV transformed lymphoblastoid cell lines (LCLs). Moreover, EBNA3C promotes formation of a complex with p53 and Gemin3 which blocks the DNA-binding affinity of p53. Small hairpin RNA based knockdown of Gemin3 in B lymphoma or LCL cells remarkably attenuates the ability of EBNA3C to inhibit the transcription activity of p53 on its downstream genes p21 and Bax, as well as apoptosis. These findings provide the first evidence that Gemin3 may be a common target of oncogenic viruses for driving cell proliferation and anti-apoptotic activities.
Author Summary
Gemin3 (DDX20 or DP103) is a member of the DEAD-box family of RNA helicases involved in various cellular processes including DNA transcription and RNA processing. The Epstein-Barr virus (EBV) encoded nuclear antigen 3C (EBNA3C) is essential for EBV-induced immortalization of primary human B-lymphocytes in vitro. In this study, we discovered that Gemin3 directly binds to the tumor suppressor p53, and acts as a negative regulator blocking p53 functions. Importantly, EBNA3C induces Gemin3 accumulation and enhances the formation of the complex of Gemin3 and p53 in EBV- transformed primary human B lymphocytes. Remarkably, inhibition of Gemin3 production leads to cell death of B lymphoma cells, particularly EBNA3C positive cells. This is the first evidence which shows that Gemin3 directly impairs p53 function in EBV positive cells, and that Gemin3 could be a potential target for EBV-associated cancer therapy.
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Introduction
Epstein-Barr virus (EBV) is the first identified human tumor virus which causes infectious mononucleosis [1],[2], and is linked several lymphoproliferative diseases [3], including Burkitt’s lymphoma [4], nasopharyngeal carcinoma [5], and Hodgkin’s disease [6]. EBV, a ubiquitous human γ-herpesvirus, infects more than 90% of the worldwide adult population. Moreover, AIDS patients or post-transplant patients whose immune system is suppressed have a high probability of developing EBV-associated lymphomas. In vitro, EBV can transform normal resting human B-cells to continuous proliferation of latently infected B cells resulting in lymphoblastoid cell lines (LCLs). Like other herpesvirus, the EBV life cycle exhibits both latent (non-productive) and lytic (productive) phases. Four types of latency programs are classified: Type I latency is mainly represented by Burkitt lymphoma (BL) cells; there are two EBER genes expressed, the BART transcripts, and EBNA1 (EBV nuclear antigen 1) [7]. Type II latency is most frequently seen in Hodgkin's lymphoma and nasopharyngeal carcinoma with three additional latent-membrane proteins, LMP-1, LMP-2A and LMP-2B expressed [8]. Latency III is seen predominantly in lymphoproliferative diseases developed in immunocompromised individuals and EBV-transformed lymphoblastoid cell lines [3] . In this group all six EBNAs, three LMPs and the two EBERs are expressed [7],[9]. Type IV latency is less strictly defined and is associated with infectious mononucleosis patients [8],[9]. Some infected individuals is associated with the tightly latent program (latency 0), in which latent gene expression is almost undetectable [9]. The essential mediators for EBV transformation of B lymphocytes and establishment of latency in vitro include EBNA2, EBNA3A, 3C and LMP1 proteins. Importantly, EBNA3C plays a complex regulatory role in the transcription of several viral and cellular genes. For example, EBNA3C targets the cellular transcription factor RBP-Jκ to antagonize EBNA2 mediated transactivation [10],[11], and cooperates with EBNA2 to activate the major viral LMP1 promoter via interaction with the cellular transcription factor, Spi-1/Spi-B [12]. EBNA3C also regulates chromatin remodeling by recruiting both histone acetylase and deacetylase activities [13]–[15]. Furthermore, EBNA3C modulates the transcription of cellular genes involved in cell migration and invasion by targeting the metastasis suppressor Nm23-H1 [16]. In addition to its transcriptional functions, it has been reported that EBNA3C has cell-cycle regulatory functions, presumably mediated by direct protein–protein interactions [13],[17]–[19]. EBNA3C also stabilizes c-Myc and interacts with Mdm2 in modulating p53 transcription and apoptotic activities [20]–[22].
Gemin3 was discovered as a binding partner with EBV latent antigens (EBNA2 and EBNA3C), and the survival motor neuron (SMN) protein [23],[24]. It consists of 825 amino acids with 9 conserved motifs including the ASP-Glu-Ala-Asp motifs (or DEAD box motif), and belongs to DExD/H RNA helicase family, which plays many roles in RNA metabolism including pre-mRNA splicing, ribosome biogenesis, RNA transport, translation initiation, and RNA decay [24]–[28]. Both EBNA2 and EBNA3C combine with Gemin3 and SMN,while EBNA2 cooperate with SMN in transactivation of the viral LMP1 promoter [24]–[26]. It has been shown that Gemin3 also interacts with and modulates a variety of cellular transcription factors including steroidogenic factor 1(SF-1) [29],[30], early growth response protein 2 (Egr2) [31], forkhead transcription factor FOXL2 [32] , and mitogen Ets repressor METS [33]. Although Gemin3 was shown to play a role in gene transcription regulation, the function of Gemin3 in cell proliferation remains largely unclear. Here we show that Gemin3 is stabilized by EBNA3C via protein-protein interaction and can play a role in cell proliferation through formation of a complex with p53 which results in blocking p53-mediated transcriptional activity and apoptosis pathway. Knockdown of Gemin3 by RNA interference dramatically increases apoptosis of EBV-infected LCLs.
Results
EBNA3C forms a complex and co-localizes with Gemin3 in vivo
Similar to a screen by Grundoff, et al [24], a previous yeast two-hybrid study showed that C-terminal domain of EBNA3C interacts with Gemin3. To confirm that these two molecules do associate in vivo, we performed co-immunoprecipitation assays to determine if EBNA3C forms a complex with Gemin3 in EBV transformed B lymphoma cell lines (LCL1 and LCL2). The B lymphoma cell line Ramos which lacks EBNA3C expression because it is EBV negative was used as a control. The results showed that endogenous Gemin3 was dramatically immunoprecipitated by anti-EBNA3C antibody, but not by control mouse serum (Figure 1A). The reverse immunoprecipitation with anti-Gemin3 antibody further confirmed that EBNA3C does form a complex with Gemin3 in vivo (Figure 1B). Since Gemin3 is initially identified as a binding protein of EBNA2 and EBNA3C, we wanted to determine if the EBNA3C complex with Gemin3 was independent of EBNA2 and performed similar coimmunoprecipitation assays again by using the EBV negative B lymphoma BJAB derived cell lines BJAB7 and BJAB10 with EBNA3C stable expression alone. The results showed that EBNA3C alone was sufficient to form a complex with Gemin3 in vivo (Figure 1C).
10.1371/journal.ppat.1002418.g001Figure 1 EBNA3C forms a complex with Gemin3 in EBV transformed cells.
(A) and (B) Endogenous Gemin3 associates with EBNA3C. Twenty million of Ramos, LCL1, and LCL2 cells were individually lysed and subjected to immunoprecipitation (IP) with EBNA3C (A10) or Gemin3 (12H12) specific antibody. 5% of whole cell lysates (WCL) were loaded as input. Normal serum IgG was used for preclear (PC). Lysates and IP complexes were resolved by SDS-PAGE and subjected to western blotting (WB) with the indicated antibodies. (C) Exogenous EBNA3C associates with Gemin3. The EBNA3C stable expressing cells (BJAB7 and BJAB10) and parental BJAB cells were individually lysed and subjected to immunoprecipitation (IP) and western blotting as indicated.
To determine if the association of EBNA3C and Gemin3 occurs in the same cellular compartments in this scenario, we performed immunofluorescence assays by ectopically expressing FLAG-tagged EBNA3C and GFP-tagged Gemin3 in U2OS cells. The immunofluorescence results showed that although EBNA3C signals are shown as stippled, punctate dots with the exclusion of nuclei, and Gemin3 signal was displayed as intense staining of prominent but discrete foci in both nucleus and cytoplasm, and there were a number of positions where co-localization staining of EBNA3C with Gemin3 was evident (Figure 2A). To further visualize the interaction of these two proteins under physiological conditions, an EBV transformed cell line LCL1 was used. Endogenous EBNA3C and Gemin3 signals were evident with the co-localization pattern (Figure 2B), supporting the above data that these two proteins can associate in the same complex.
10.1371/journal.ppat.1002418.g002Figure 2 EBNA3C colocalizes with Gemin3.
(A) Colocalization of exogenous E3C and Gemin3 in U2OS cells. Cells transfected with E3C-FLAG and GFP-Gemin3 were stained by using M2 antibody, followed by goat anti-mouse antibody conjugated to Alexa Fluor 594 (red). (B) Endogenous Gemin3 colocalizes with E3C in the EBV-transformed cell line LCL1. Endogenous Gemin3 and E3C were individually detected by using mouse anti-Gemin3 (12H12) and rabbit anti-E3C antibodies, followed by Goat anti-mouse antibody conjugated to Alexa Fluor 594 (red) and goat anti-rabbit antibody conjugated to Alexa Fluor 488 (green). Nuclei were counterstained by using DAPI (blue). The images were sequentially captured using an Olympus confocal microscope. All panels are representative pictures from similar repeat experiments. Enlarged sections are shown at the bottom. Arrows show the colocalization of E3C and Gemin3.
EBNA3C interacts with Gemin3 through its C-terminal domains
To define which domain of EBNA3C interacts with Gemin3, we co-transfected HEK293 cells with expression constructs for GFP-tagged Gemin3 and FLAG-tagged full-length (residues 1 to 992) or a series of truncated mutant of EBNA3C (1–365, 366–620, or 621–992), and performed coimmunoprecipitation assays with GFP or FLAG antibodies. Consistently, the results showed that coimmunoprecipitation targeting Gemin3 or EBNA3C, always results in Gemin3 co-immunoprecipitating with both the full-length and C-terminal domain of EBNA3C with relatively high affinity (Figure 3A and B). To further determine if the C-terminal domain of EBNA3C alone is sufficient to bind with Gemin3, we performed GST pulldown assays by in vitro-translating the full length and truncated mutants of EBNA3C followed by co-incubation with bacterially expressed GST-Gemin3 protein. The results showed that the C-terminal domain (621–992) of EBNA3C directly interacted with Gemin3 (Figure 3C). Similarly, using in vitro-translated Gemin3 coincubated with different bacterially expressed GST-EBNA3C (1–365, 366–581, 582–792, and 900–992), we further found that the C-terminal amino acids 621 to 792, a smaller region than previously identified by yeast two-hybrid analysis (534–778aa, [24]) of EBNA3C was critical for binding to Gemin3 in vitro (Figure 3D and F). To determine the domain of Gemin3 responsible for interacting with EBNA3C, we generated three truncated mutants of GST-Gemin3 (residues 1–272, 307–547 and 546–825) and performed GST pulldown assays with in vitro-translated full-length EBNA3C. The result showed that Gemin3 bound with EBNA3C via its C-terminal domain (Figure 3E and F), supporting the previously identified data by yeast two-hybrid analysis [24], and further narrows the interacting domain.
10.1371/journal.ppat.1002418.g003Figure 3 EBNA3C and Gemin3 interact through their C-terminal domains.
(A) and (B) Gemin3 associates with the C-terminal domain of EBNA3C. Twenty million of HEK 293 cells were cotransfected with plasmids expressing GFP-tagged Gemin3 and Flag-tagged either full-length or the truncated mutants of EBNA3C. At 36 h post-transfection, cells were harvested and subjected to immunoprecipitation using anti-FLAG (M2) or GFP antibodies followed by western blotting with GFP or M2 antibody. 5% of whole cell lysates (WCL) were used as input. The membrane was stripped and reblotted with M2 or GFP antibody. (C) Gemin3 binds to C-terminal domain of E3C in vitro. The 35S-radiolabeled in vitro-translated proteins of E3C truncated mutants were precleared with GST bead, followed by incubation with GST or GST-Gemin3 beads. The bound protein mixtures were resolved by appropriate SDS-PAGE, and autography. 5% of in vitro translated protein is used as input. The quantification of relative amount of bound protein (RBU) was shown at the bottom. (D) The 35S-radiolabeled in vitro-translated protein full-length Gemin3 was pulled down by truncated mutants of EBNA3C fusion with GST (GST-E3C 1–365, 366–581,582–792, and 900–992). Coomassie blue staining of purified GST-EBNA3C truncated proteins is shown at the bottom panel. (E) E3C binds to C-terminal domain of Gemin3 in vitro. The 35S-radiolabeled in vitro-translated protein full-length EBNA3C was pulled down by truncated mutants of Gemin3 fusion with GST (GST-Gemin3 1–272, 307–547, and 546–825). Coomassie blue staining of purified GST-Gemin3 truncated proteins is shown at the bottom. (F) Schematics illustrate different structural domains of E3C and Gemin3. The respective binding domain(s) of two proteins are indicated by bold.
EBNA3C enhances the protein stability and production of Gemin3
To determine if EBNA3C plays a role on the production of Gemin3, we tested endogenous Gemin3 protein levels in EBNA3C stable expressing cell lines (BJAB7 and BJAB10) and parental BJAB cells, as well as EBV negative Ramos cells. Interestingly, the results showed that EBNA3C greatly induced the production of Gemin3 in BJAB cells (Figure 4A). To verify if whole EBV latent infection with EBNA3C expression also has the same effect on Gemin3 production, we did a western blot to detect Gemin3 protein levels in both PBMC and two EBV-infected derived LCL cell lines. Consistent with EBNA3C expression alone, the results showed that Gemin3 protein levels were significantly increased by EBV infection (Figure 4B). The results of Gemin3 protein levels in EBNA3C-knockdown BJAB10 and LCL cells were consistently much lower than in the control cells (Figure 4C), further suggesting that EBNA3C is required for maintenance of Gemin3 levels. To determine if the increased production of Gemin3 is due to post-translational regulation by EBNA3C, we co-expressed exogenous EBNA3C-FLAG with GFP-Gemin3 or GFP control vector into Saos-2 cells and determined the protein levels of GFP-Gemin3 and GFP. The results showed that the production of GFP-Gemin3 but not GFP was enhanced by EBNA3C in a dose-dependent manner (Figure 4D, left and middle panels), indicating that the interaction of EBNA3C with Gemin3 is important for Gemin3 accumulation. To this end, we carried out similar experiments by using an EBNA3C mutant lacking Gemin3-interacting domain, and found that deletion of the Gemin3-interacting domain efficiently abolished the effect of EBNA3C on Gemin3 production (Figure 4D, left and right panels). To further confirm that EBNA3C-induced Gemin3 levels were due to enhanced Gemin3 protein stability, we performed the protein stability assays for Gemin3 by treating BJAB (EBNA3C negative) and BJAB10 (EBNA3C positive) cells with cycloheximide (a protein translation inhibitor). The results showed that the stability of Gemin3 was dramatically enhanced in the presence of EBNA3C compared to cells without EBNA3C (Figure 4E).
10.1371/journal.ppat.1002418.g004Figure 4 EBNA3C enhances the protein stability of Gemin3.
(A) and (B) E3C increases the protein production of endogenous Gemin3. Ten million of EBV negative cells (Ramos, BJAB and PBMC), EBNA3C- expressing cells (BJAB7 and BJAB10), and EBV-transformed cells (LCL1 and LCL2) were lysed and subjected to western blot with indicated antibodies. The data show that the protein level of Gemin3 in the EBNA3C or EBV positive cells was significantly upregulated. (C) The protein level of Gemin3 is decreased in BJAB10 and LCL1 cells with EBNA3C stable knockdown. BJAB10 and LCL1 cells with Lentivirus-delivered GFP labeled shRNA against EBNA3C (shE3C) or scramble control (shCtrl) were showed on the left panel. The protein levels of E3C and Gemin3 are detected by western blotting with indicated antibodies. GAPDH immunoblotting was used as the loading control. The relative quantitation of Gemin3 and E3C protein levels are normalized by GAPDH. (D) E3C increases the protein level of exogenous Gemin3. Saos-2 cells were transfected GFP-Gemin3 or GFP with increasing amounts of full length (FL) EBNA3C-FLAG or its C-terminal deleted mutant (ΔC). The transiently transfected cells were harvested at 36 h post-transfection, the protein levels of exogenous GFP-Gemin3 and GFP were detected by western blotting with GFP antibody. The relative quantitation of GFP-Gemin3 or GFP is shown in the middle panel. (E) EBNA3C enhances the protein stability of Gemin3. The cell lysates of BJAB or BJAB10 cells treated with cycloheximide (CHX, 100 µg/ml) for 0, 2, 4 and 6 hours were subjected to immunoblotting as indicated. GAPDH immunoblotting was used as the loading control. The relative quantitation of Gemin3 is shown in the middle panel.
Gemin3 interacts with p53 and contributes to EBNA3C-mediated inhibition of p53 transcriptional activity
Our previous studies showed that EBNA3C blocks p53-mediated transcriptional as well as apoptotic activity [22],[34]. Gemin3 plays an essential role in cell survival and can function as a transcriptional repressor through interaction with a number of transcriptional factors [35],[36]. Therefore, we hypothesized that EBNA3C enhanced Gemin3 levels may be due to its interaction with p53 and in turn repress the transcriptional activity of p53. To this end, we first asked if Gemin3 can directly bind to p53 in vitro. Using in vitro-translated Gemin3, we incubated it with bacterially expressed GST or GST-p53 protein. We found that Gemin3 strongly bound to p53 but not the GST control (Figure 5A, lane 2 and 3). Using different truncation mutants of p53 fusion with GST, we found that the DNA-binding domain (amino acids 100–300) of p53 is critical for interaction with Gemin3 (Figure 5A, lane 4 to 6). To define the binding site of Gemin3 with p53, we utilized full length and truncated mutants of Gemin3 fusion with GST to incubate with in vitro-translated p53. The results showed that the C-terminal domain (amino acids 546–825) alone of Gemin3 presents similar strong binding with p53 as full length Gemin3 (Figure 5B). This further confirms that Gemin3 can directly interact with p53. To test if endogenous p53 interacts with Gemin3 in cells and whether this interaction is impaired by EBNA3C, we performed immunoprecipitation assays with anti-p53 antibody followed by western blotting with Gemin3 antibody individually from cell lysates of BJAB, BJAB10 and LCL1 cells. The results showed that Gemin3 and p53 do form a complex in EBV negative BJAB cells, and that the association of p53 with Gemin3 was greatly enhanced by 2.2 fold in EBNA3C-expressing BJAB10 cells and 5.2 fold in LCL1 cells when compared to that in the EBV negative BJAB cells (Figure 5C, bottom panel). Since Gemin3 bound to the DNA-binding domain of p53, we wanted to know if Gemin3 blocks sequence specific DNA-binding ability of p53, and so performed chromatin immunoprecipitation assays with exogenous p53 alone or p53 and Gemin3 in the presence and absence of p53-responsive DNA element (p53-RE). As shown in the Figure 5D, Gemin3 dramatically reduced the ability of p53 to bind to the p53-responsive DNA element. To further confirm the consequence of Gemin3 blocking DNA-binding ability of p53 and argue the possibility that Gemin3 can directly bind to DNA probe, we also performed reporter assays by co-expressing exogenous HA-p53 with increasing doses of GFP-tagged Gemin3 in the presence of 13 copies of p53-binding sites driven luciferase reporter into p53-null Saos-2 cells. The results showed that Gemin3 diminished p53-mediated transcriptional activity in a dose-dependent manner (Figure 5E). The expression levels of Gemin3, p53, and GAPDH as a loading control, were also analyzed by western blotting (Figure 5E, bottom panels). To answer if EBNA3C-mediated inhibition of p53 transcriptional activity is dependent on Gemin3 accumulation, we performed similar reporter assays by co-expressing p53 with or without EBNA3C in the presence of specific Gemin3 shRNA (shG3) or non-specific control shRNA (shCtrl). Strikingly, the results showed that EBNA3C-mediated inhibition of p53 transcriptional activity is dramatically reversed once Gemin3 is knocked down (Figure 5F, compare lane 3 with 6). This suggests that Gemin3 is important for EBNA3C-mediated inhibition of p53 function.
10.1371/journal.ppat.1002418.g005Figure 5 Gemin3 interacts with p53 and contributes to EBNA3C-mediated inhibition of p53 transcriptional activity.
(A) Gemin3 binds with DNA-binding domain of p53 in vitro. 35S-radiolabeled Gemin3 was in-vitro translated and incubated with bacterially purified GST, GST fusion with full length (1–393) or truncated mutants of p53 (1–83, 100–300, and 300–393). The pull-down complexes were resolved by appropriate SDS-PAGE and autography. The amount of bound protein was quantified with ImageQuant software. Coomassie blue staining of purified GST protein is shown in the middle panel. Schematic illustrates the binding domains of p53 with Gemin3 and EBNA3C. (B) C-terminal domain of Gemin3 binds with p53 in vitro. 35S-radiolabeled p53 was in vitro translated and incubated with bacterially purified GST control, full length GST-Gemin3 (1–825) or its truncations (1–272, 307–547, and 546–825). The GST pull down assay was analyzed as the described above. (C) EBNA3C increases the interaction of p53 with Gemin3. Twenty million of BJAB, BJAB10 and LCL1 cells were individually harvested, lysed and subjected to immunoprecipitation (IP) with p53 specific antibody and subsequently western blotted with Gemin3 or p53 antibody. The relative quantitation of Gemin3 complex with p53 was calculated by IP compared with input and shown in bottom panel. (D) Gemin3 reduces DNA-binding affinity of p53. Saos-2 cells were co-transfected HA-p53 with or without GFP-Gemin3 in the presence of p53-RE-Luc containing 13 copies of p53-binding sties or pGL3-basic vector alone. Chromatin immunoprecipitation assays were performed with anti-HA or normal lgG antibodies control. The DNA binding ability of p53 was detected by standard (upper) and quantitative real-time (lower) PCR with primers targeting ampicilin gene of pGL3 plasmid. (E) Gemin3 inhibits the transcriptional activity of p53 in the luciferase reporter assay. Saos-2 cells were co-transfected with 3 µg of the pGL3-p53-RE-Luc, 10 µg HA-p53 in the presence of either vector control or increasing amounts of GFP-Gemin3 (5 µg and 10 µg). Each transfection was performed in triplicate. Error bars indicated standard variations. Bottoms panels show the protein levels detected by western blotting analysis with the indicated antibodies. GAPDH was used as a control for equal loading. The relative luciferase unit (RLU) is showed by normalization with vector alone. (F) Gemin3 knockdown reduces E3C-mediated inhibition of p53 transcriptional activity in the luciferase reporter assays. Saos-2 cells with stable Gemin3 knockdown (shG3) or control (shCtrl) were individually co-transfected p53-RE-Luc with HA-p53, HA-p53 and E3C-FLAG or vector alone. The western blotting data of each protein is shown at the bottom panel. The relative luciferase unit (RLU) is showed by normalization with vector alone. Plasmid expressing RFP was used for normalized transfection efficiency.
Gemin3 knockdown attenuates EBNA3C-mediated inhibition of p53-induced apoptosis
Studies in our lab have shown that EBNA3C is able to inhibit p53-induced apoptosis in p53-null Saos2 cells [22],[34]. To further confirm the significance of Gemin3 on EBNA3C-mediated inhibition of p53 function, we verified whether or not Gemin3 knockdown affects EBNA3C-mediated inhibition of p53-induced apoptosis by using colony formation assays. The results showed that coexpression of EBNA3C with p53 markedly increased the colony formation of Saos-2 cell compared to that produced by p53 alone (Figure 6A, left panel). In contrast, although less colony formation was produced in the vector alone with the Gemin3 knockdown (shG3) compared to that with the control knockdown (shCtrl) (Figure 6A, top panel), the colony formation of p53 co-expressed with EBNA3C was significantly inhibited in Gemin3 knockdown cells (Figure 6A). Thus, these data revealed a role for EBNA3C in upregulation of Gemin3 for anti-apoptosis and promotes cell proliferation.
10.1371/journal.ppat.1002418.g006Figure 6 Gemin3 knockdown attenuates EBNA3C-mediated inhibition of p53-induced apoptosis.
(A) Saos-2 (p53−/−) cells were electroporated with different combinations of expression plasmids for HA-p53, EBNA3C-FLAG or vector alone in the presence of small hairpin RNA against Gemin3 (shG3) or control (shCtrl) as indicated. 2×103 transfected cells were cultured in the selection medium (DMEM supplemented with 100 mg/ml G418). After a 2-week selection, cells were fixed on the plates with 4% formaldehyde and stained with 0.1% crystal violet. The area of colonies (pixels) in each dish was calculated by LiCor Odyssey. The number represents the averages of data from two independent experiments. Plasmid expressing RFP was used for normalized transfection efficiency. (B) Western blots showing the protein level of Gemin3 in the lentivirus-mediated Gemin3 or control knockdown cell lines. GAPDH was used as the loading control. (C) Gemin3 knockdown increases apoptosis of EBV negative cells (Ramos and BJAB), EBNA3C positive cells (BJAB7 and BJAB10), and EBV transformed cells (LCL1). Cells were harvested after a 12-h serum starvation and fixed. Levels of cells undergoing apoptosis (sub-G1 phase) in individual PI-stained samples were detected by flow cytometry, and the data were analyzed by FlowJo software. The bar diagram shown at the bottom panel represents the mean of three independent experiments and the results of comparing with each control knockdown sample. (D) Quantitative real-time PCR analysis showed that p53, p21 and Bax genes were upregulated in the Gemin3 knockdown cells. Total RNA was individually isolated from the Gemin3 (shG3) or control (shCtrl) knockdown cells with 12-h serum starvation treatment. A 2 µg total RNA was used to synthesis cDNA. Error bars show standard deviations.
To further prove that Gemin3 plays an important role in cell proliferation, we performed apoptosis assays using B lymphoma cells or LCLs with or without lentivirus-mediated Gemin3 knockdown. Gemin3 expression was significantly knocked down in the EBV negative B lymphoma cell lines Ramos and BJAB, EBV transformed B cell line LCL1 as well as EBNA3C stably expressing BJAB cell lines BJAB7 and BJAB10 (Figure 6B). The results of apoptotic assays showed that Gemin3 knockdown cells had a dramatic increase in apoptosis compared to the control knockdown cells (Figure 6C). Furthermore, Gemin3 knockdown in EBV or EBNA3C positive cells showed significantly higher levels of apoptosis than that in EBV or EBNA3C negative cells (Figure 6C, bottom panel). Consistently, the quantitative PCR analysis showed that the transcriptional levels of p53 downstream genes including p21, Bax and p53 were enhanced when Gemin3 was knocked down in EBNA3C positive cells and were remarkably higher than that in EBNA3C negative cells (Figure 6D). Therefore, Gemin3 plays a critical role in cell proliferation in EBNA3C positive cells.
Discussion
Emerging evidence have indicated that Gemin3 is an essential gene for embryonic development and survival from mammal to Drosophila [23],[37]. Gemin3 (DP103/DDX20) was originally discovered in a screen for cellular factors that bind to EBNA2 and EBNA3C using the yeast two-hybrid system [24]. Gemin3 was shown to be involved in EBNA2-mediated transactivation and transformation, and a deletion mutant of EBNA2 lacking the Gemin3-binding site severely impeded LMP1 transactivation and viral transformation [26]. However, there was no previous report on the functional effect of EBNA3C on Gemin3. Our yeast two-hybrid screen showed that Gemin3 strongly interacted with EBNA3C and this was identified with high frequency in positive yeast clones when the C-terminal domain of EBNA3C was used as bait (data not shown). We now confirm that Gemin3 directly binds to EBNA3C in vitro and in vivo, and this interaction led to increased Gemin3 protein stability and its accumulation in B lymphoma cells and EBV-transformed lymphoblastoid cell lines. Furthermore, EBNA3C promoted the formation of a complex with p53 and Gemin3 in vivo which was important for blocking p53-mediated transcriptional activity and apoptosis. Inhibition of Gemin3 expression dramatically abolished EBNA3C-mediated inhibition of p53-induced apoptosis (Figure 7). The emerging evidence showing Gemin3 as a DEAD-box RNA helicase that plays a role in carcinogenesis, will inspire us to develop efficient helicase inhibitors as a potential target for anti-cancer in the future.
10.1371/journal.ppat.1002418.g007Figure 7 A schematic model depicting the Gemin3-mediated transcriptional regulation of p53 by EBNA3C in the EBV latently infected cells.
Left panel, in response to genotoxic stress, p53 achieves its anti-proliferative properties through its function as a DNA-binding transcriptional activator, and induce expression of downstream target genes (including p21 and BAX) for cell-cycle arrest and apoptosis. Right panel, in the EBV latency III cells, EBNA3C not only stabilizes Gemin3 through the interaction of their C-terminal domains and but also enhances a stable complex of Gemin3 with p53 for blocking p53-mediated transcriptional activity and cell apoptosis. Gemin3 knockdown (siRNA) increases the sensitivity of the EBNA3C-expressing cells to stress-induced apoptosis. p53-RE, p53 responsive element.
Gemin3 is also an integral component of the SMN complex, which plays an essential role in the production of spliceosomal small nuclear ribonucleoproteins (snRNPs) [38], regulation of DNA transcription [30], pre-mRNA splicing [37], and axonal RNA transport [39]. In addition to interaction with Gemin3, EBNA3C also has been shown to associate with SMN [25]. This further supports the notion that EBNA3C targets the SMN complex and can impair the function of each component including Gemin3 in the SMN complex. Here, we demonstrate a novel function of Gemin3 as a regulator that suppresses p53-mediated transcriptional activity and apoptosis. Interestingly, SMN was also shown to interact with p53 [40]. Therefore, our results showing that knockdown of Gemin3 sufficiently rescues p53, suggest that Gemin3 could be a critical member of the SMN complex to suppress p53 function. This provides an explanation as to why EBNA3C promotes complex formation of p53 and Gemin3 through enhancing Gemin3 protein stability.
The N-terminal domain of Gemin3 contains the conserved helicase motifs, while the non-conserved C-terminal domain can interact with a variety of cellular and viral factors. In this study, we found that Gemin3 binds with both EBNA3C and p53 through the same region (amino acid 548 to 825) of its C-terminal domain. Considering that EBNA3C individually binds to p53 and Gemin3 through its N-terminal and C-terminal domains. There is a strong possibility for EBNA3C to act as an adaptor to promote Gemin3 interaction with p53. This also may provide an explanation as to why the levels of p53 in complex with Gemin3 are dramatically enhanced in the presence of EBNA3C.
Although it has been shown that Gemin3 plays a diverse roles on repressing gene transcription [30], the mechanism of Gemin3-mediated transcriptional repression is not fully clear. One of the potential mechanisms could be through recruitment of specific transcription co-repressors. For examples, Gemin3 interacts with METS to repress Ets by assembling a complex of N-CoR, Sin3A, HDAC-2, and HDAC-5 [30],[33],[41]. Gemin3 was also found to form a complex with METS (mitogenic Ets transcriptional suppressor)/PE1 or ERF (Ets2 repressor factor) controlling cellular proliferation and differentiation through recruitment of HDAC2 and HDAC5 [42]. It also interacts with and represses the transcriptional activity of Egr2/Krox-20 which is dependent in part on HDAC recruitment [31]. Gemin3 also interacts with the transcriptional factor FOXL2 important for inducing apoptosis [32]. However, our study showed that Gemin3 reduces p53 transcriptional activity and now provides another potential mechanism through which Gemin3 can sequester a transactivation cofactor or may disrupt a transcriptionally active nucleic acid-protein complex.
Previous studies in our lab indicated that EBNA3C targets multiple factors to deregulate the p53 pathway and so drive cell proliferation. For example, EBNA3C has a unique activity of ubiquitylation and de-ubiquitylation [17],[18],[21], and also suppresses p53 function through formation of a complex with Mdm2 [21]. More recently, our lab also showed that EBNA3C directly competes with two p53-associated growth inhibitors ING4 and ING5 for attenuating p53 function [34]. Despite current studies which convincingly showed that EBNA3C inhibit p53 function, it is still unknown as to why the protein level of p53 is not affected in EBV-transformed LCLs or in B lymphoma expressing EBNA3C [43]–[46]. Here, our study further addresses a potential mechanism that EBNA3C can block p53 function through upregulating Gemin3 protein levels and so promoting the formation of a complex of p53 with Gemin3. This is distinctly different from the strategy used by KSHV encoded LANA where p53 ubiquitylation is induced and results in its degradation [47]. In summary, this study provides initial evidence that Gemin3 can directly interact with p53 through its DNA-binding domain and in turn inhibits its activities. This pathway is essentially targeted by EBNA3C in EBV-transformed lymphoblastoid cells.
Materials and Methods
DNA constructs and antibodies
pA3F-EBNA3C constructs expressing either full-length EBNA3C or different truncated versions of EBNA3C 1–365, 366–620, and 621–992 with a Flag tag at the carboxy-terminal end and Glutathione S-transferase (GST)-EBNA3C truncation mutation were described previously [20]. Constructs expressing green fluorescent protein (GFP)-tagged Gemin3 was prepared by cloning PCR-amplified fragments into pEGFP-C1 vector (BD Biosciences Clontech) at EcoRI and SalI restriction sites. The plasmid pGL3-p53-RE contains 13 copies of p53-binding site at the upstream of firefly luciferase gene was constructed by EcoRV insertion of p53-binding sequences into the pGL-3 luciferase reporter plasmid (Kindly provided by Wafik S. EI-Deiry at University of Pennsylvania, Philadelphia, Pennsylvania). pGEX-p53 expresses an N-terminal glutathione S-transferase (GST)-p53 fusion protein was derived from pGEX-2T (Amersham Pharmacia, Inc, Piscataway, NJ) by insertion of human p53 cDNA (A gift from Gary J Nabel, National Institutes of Health, Bethesda, MD) at the BamH1 and EcoR1 sites. pcDNA-HA-p53 was generated by cloning PCR-amplified p53 cDNA using pGEX-p53 as a template into the pcDNA3-HA vector at EcoR1 and Not1 sites. GST-p53 truncation mutants were constructed by insertion of PCR fragments into the pGEX-5x-1 backbone (gift from Shelley L Berger, The Wistar Institute, Philadelphia, PA). Plasmid expressing GST-Gemin3 was kindly provided by Dr. Gideon Dreyfuss (Howard Hughes Medical Institute Investigator at University of Pennsylvania Perelman School of Medicine) and described previously [23]. GST-Gemin3 truncation mutants GST-Gemin3 1–272 was constructed by insertion of PCR fragments into pGEX-2TK derivative vector at Kpn1 and EcoR1 sites, GST-Gemin3 307–547 and GST-Gemin3 546–825 into pGEX-2TK vector BamH1 and EcoR1 sites. All constructs and mutations were verified by DNA sequencing.
Mouse monoclonal anti-Gemin3 (12H12) was kindly provided by Dr. Gideon Dreyfuss (Howard Hughes Medical Institute Investigator at University of Pennsylvania School of Medicine). The monoclonal antibodies mouse anti-myc (9E10) and anti-EBNA3C (A10) were prepared from the respective hybridoma cultures. Mouse monoclonal antibody anti-FLAG epitope (M2) was purchased from Sigma-Aldrich Corp. (St. Louis, MO). Mouse monoclonal antibody reactive to p53 (DO-1) and GFP (F56-BA1) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Cell culture and transfection
Human embryonic kidney cells transformed with sheared adenovirus type 5 DNA HEK293 cell line and p53-null cell line SaoS-2 were obtained from Jon Aster (Brigham and Woman’s Hospital, Boston, MA) [48]. U2OS is also a human osteosarcoma cell line [49]. EBV negative Burkitt’s lymphoma cell lines BJAB and Ramos were provided by Elliott Kieff (Brigham and Woman’s Hospital, Boston, MA) [50]. LCL1 and LCL2 are in vitro-transformed EBV positive cell lines. BJAB cells expressing EBNA3C (BJAB7 and BJAB10) were generated previously by transfecting with pZipneo eukaryotic expression vector with EBNA3C cDNA followed by neomycin selection [51]. HEK 293, U2OS and Saos-2 cells were grown in Dulbecco's modified Eagle's medium (purchased from HyClone, Logan, UT) supplemented with 10% fetal bovine serum, 50 U/ml penicillin, 50 µg/ml streptomycin, and 2 mM L-glutamine. BJAB, Ramos and the EBV-positive cell lines were maintained in RPMI 1640 medium (HyClone, Logan, UT) supplemented as described for Dulbecco's modified Eagle's medium above. All cultures were incubated at 37°C in a humidified environment supplemented with 5% CO2. Cells were transfected by electroporation with a Bio-Rad Gene Pulser II electroporator. Briefly, 15×106 cells harvested in exponential phase were collected, washed in phosphate-buffered saline (PBS), and resuspended in 400 µl of the appropriate medium containing DNA for transfection [52]. Resuspended cells were transferred to a 0.4-cm-gap cuvette, and electroporation was performed at 975 µF, 210 V for HEK293 and Saos-2 cells. Transfected cells were transferred to a 100-mm petri dish containing 10 ml of complete medium and incubated at 37°C.
Immunoprecipitation and Western blotting
Transfected cells were harvested, washed with ice-cold PBS, and lysed in 0.5 ml ice-cold radioimmunoprecipitation (RIPA) buffer (1% Nonidet P-40 [NP-40], 10 mM Tris [pH 7.5], 2 mM EDTA, 150 mM NaCl), supplemented with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml pepstatin, and 1 µg/ml leupeptin). Cell debris was removed by centrifugation at 21,000×g (10 min and 4°C), and the supernatant was transferred to a fresh microcentrifuge tube. Lysates were then precleared by end-over-end rotation with normal mouse serum and 30 µl of a 1∶1 mixture of Protein A-Protein G-conjugated Sepharose beads (1 h, 4°C). Beads were spun out, and supernatant was transferred to a fresh microcentrifuge tube and approximately 5% of the lysate was saved for input control. The protein of interest was captured by rotating the remaining lysate with 1 µg of appropriate antibody overnight at 4°C. Immune complexes were captured with 30 µl of a 1∶1 mixture of Protein A and Protein G Sepharose beads, pelleted, and washed five times with ice-cold RIPA buffer. For Western blot assays, input lysates and immunoprecipitated (IP) complexes were boiled in Laemmli buffer [53], fractionated by SDS-PAGE, and transferred to a 0.45 µm nitrocellulose membrane. The membranes were then probed with appropriate antibodies followed by incubation with appropriate infrared-tagged secondary antibodies and viewed on an Odyssey imager (LiCor Inc., Lincoln, NE).
Purification of GST fusion proteins
Escherichia coli BL21 (DE3) cells were transformed with the plasmid constructs for each GST fusion protein. Single colonies were picked and grown overnight in 3 ml of Luria broth supplemented with 100 µg/ml ampicillin. One milliliter of the overnight culture was used to inoculate a 500 ml culture. The larger culture was incubated until the optical density at 600 nm was approximately 0.6, at which point it was induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 12 h at 30°C . The bacteria were pelleted, washed once with STE buffer (100 mM NaCl, 10 mM Tris, and 1 mM EDTA, pH 7.5), resuspended in 3 ml NETN buffer (0.5% NP-40, 100 mM NaCl, 20 mM Tris, 1 mM EDTA, pH 8.0), supplemented with protease inhibitors, and incubated on ice for 15 min. A volume of 150 µl of 1 M dithiothreitol (DTT) and 1.8 ml of a 10% solution of Sarkosyl in STE buffer was added, and the suspension was sonicated (for 3 min on ice) to solubilize the proteins. The lysates were centrifuged (12,000×g, 10 min, 4°C) to separate the unsolubilized fraction. The clear supernatant was transferred to a fresh tube, to which 3 ml of 10% Triton X-100 in STE buffer and 200 µl of Glutathione-Sepharose beads were added. The tube was rotated overnight at 4°C, after which the purified protein bound to Glutathione was collected by centrifugation (2 min, 600×g, 4°C) and washed five times with NETN buffer supplemented with protease inhibitors. The level of purification was determined by SDS-PAGE, and purified proteins were stored at 4°C.
GST pull-down assays
For pull-down assays from cell lysates, lysates were prepared in RIPA buffer (0.5% NP-40, 10 mM Tris [pH 7.5], 2 mM EDTA, 150 mM NaCl, supplemented with protease inhibitors). Lysates were precleared and then rotated with either GST control or the appropriate GST fusion protein bound to Glutathione-Sepharose beads. For in vitro binding experiments, GST fusion proteins were incubated with 35S-labeled in vitro-translated protein in binding buffer (1×PBS, 0.1% NP-40, 0.5 mM DTT, 10% glycerol, supplemented with protease inhibitors). In vitro translation was performed with the T7-TNT Quick Coupled transcription-translation system (Promega Inc., Madison, WI) according to the manufacturer's instructions.
Immunofluorescence
To check the co-localization of ectopically expressed GFP-Gemin3 and Flag-EBNA3C in the cells, we used Lipofectamine 2000 (Invitrogen, Carlsbad, CA) to transfect U2OS cells with the plasmids then cultured on coverslips. At 36 h posttransfection, cells were fixed using 3% paraformaldelhyde with 0.1% Triton X-100 for 20 min at room temperature. We used LCL1 cells to examine the co-localization of endogenous Gemin3 with EBNA3C, appropriate LCL1 cells were added onto slides and fixed using the same method after culture 5 h. Fixed cells were washed with PBS and subsequently blocked in 1% BSA for 10 min. Gemin3 was detected using mouse monoclonal antibody (12H12). Endogenous EBNA3C was detected using EBNA3C-reactive rabbit polyclonal antibody (1∶150 dilution); Flag-tagged EBNA3C was detected using M2 antibody (1∶1,000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Primary antibodies were incubated with the cells for 60 min at the room temperature. Cells were washed three times with PBS and exposed to secondary antibodies. Goat anti-mouse antibody conjugated to Alexa Fluor 594 to detect Gemin3 and goat anti-rabbit antibody conjugated to Alexa Fluor 488 to detect EBNA3C were respectively used for LCL1 cells, and Goat anti-mouse antibody conjugated to Alexa Fluor 594 to detect FLAG-EBNA3C was used for U2OS cells. Secondary antibodies were diluted in blocking buffer at 1∶1,000 and incubated for 1 h at RT, followed by three washes with blocking buffer. The last wash contained 4′, 6′-diamidino-2-phenylindole (DAPI; Promega, Madison, WI) to counterstain the nuclei. The slides were examined using Olympus confocal microscopy and the images were analyzed with a Fluoview FV300 (Olympus, Melville, NY) software.
Luciferase reporter assay
Twelve million Saos-2 cells were co-transfected by using a Bio-Rad electroporater (Bio-Rad Laboratories, Inc., Hercules, CA) with different combination of pGL3-p53-RE, pcDNA-HA-p53, pA3F-EBNA3C and GFP-Gemin3. At 24 h post-transfection, cells were harvested, washed in PBS, and lysed by cell lysis buffer (BioVision, Inc., Mountain View, CA). Forty microliters of cell lysate was used for the reporter assay, using an LMaxII384 luminometer (Molecular Devices, Inc., Sunnyvale, CA). A portion of the cell lysate was used for Western blotting. Transferred proteins were detected with Odyssey infrared scanning technology (LI-COR, Inc., Lincoln, NE), using Alexa Fluor 680 and Alexa Fluor 800 (Molecular Probes, Carlsbad, CA, and Rockland, Gilbertsville, PA, respectively). All the transfections were performed multiple times, and the results shown represent the means of the data from three independent experiments.
Lentiviral-mediated gene silencing
For the lentivirus-mediated knockdown of EBNA3C or Gemin3, the EBNA3C shRNA sequence (CCAUAUACCGCAAGGAAUA) or Gemin3 shRNA (ACUCCCCAGUGAGACCAUU) was respectively inserted into pGIPZ vector according to the manufacture’s instructions (Open Biosystem, Inc, Huntsville, AL), the vector expressing EBNA3C or Gemin3 small hairpin RNA is abbreviated as shE3C or shG3, respectively. A 21-mer oligonucleotide (UCUCGCUUGGGCGAGAGUAAG) that had no significant homology to any known human mRNA in the databases was cloned in the same vector and used as control. Control shRNA is hereinafter abbreviated as shCtrl.
Lentiviruses were produced by transient transfection into HEK 293T cells as previously described with the following modifications [54]. A total of 2×106 293T cells were seeded in 10-cm-diameter dishes in DMEM (HyClone, Logan, UT) supplemented with 10% FBS and cultured for 24 h prior to transfection. A total 20 µg of plasmid DNA was used for the transfection of each dish, including 1.5 µg of envelope plasmid pCMV-VSV-G (catalog no. 8454; Addgene, Inc., Cambridge, MA), 3 µg of packaging plasmid pRSV-REV (catalog no. 12251 Addgene, Inc., Cambridge, MA), 5 µg of packaging plasmid pMDLg/Prre (catalog no. 12251 Addgene, Inc., Cambridge, MA), and 10.5 µg of lentiviral vector plasmid. The precipitation was formed by adding the plasmids to a final volume of 438 µl of H20 and 62 µl of 2 M CaCl2, mixing well, adding 500 µl of 2×HEPES-buffered saline, and then incubating at room temperature for 30 min. Chloroquine was added to the 10 ml of plated media with a final concentration of 25 µM at 5 minutes prior to transfection. The medium was replaced after 12 h with DMEM supplemented with 10% FBS and 10 mM HEPES, and 10 mM sodium butyrate. The medium was replaced again 10 hours later using DMEM supplemented with 10% FBS and 10 mM HEPES. The conditioned medium was collected four times at 12 h interval, filtered through 0.45 µm pore-size cellulose acetate filters, and stored on ice. The virus was concentrated by spinning at 70,000×g for 2.5 h. The concentrated virus was resuspended in RPMI then used to infect 106 cells in the presence of 20 µM/ml Polybrene. After 72 h, add puromycin to final concentration of 2 µg/ml for selection. GFP immunofluorescence was assessed by using an Olympus IX71 microscope filtered with 560-nm excitation and 645-nm emission filters. Visible colonies were grown to 80% confluence in the presence of 2 µg/ml puromycin prior to western blot and apoptosis analysis.
Chromatin immunoprecipitation assay
The chromatin immunoprecipitation (ChIP) experiments were done essentially as previously described with some modifications [55],[56]. Saos-2 cells (10×106) transfected HA-p53 with or without GFP-Gemin3 in the presence of pGL-p53-RE or pGL3-basic vector alone were cross-linked with 1.1% (v/v) formaldehyde, 100 mM NaCl, 0.5 mM EGTA, and 50 mM Tris-HCl (pH 8.0) in growth medium at 37°C for 10 min, then at 4°C for 50 min. Formaldehyde was quenched by adding 0.05 vol 2.5 M glycine. Fixed cells were washed with PBS, incubated for 15 min in 15 ml of 10 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.5 mM EGTA, and 0.25% (v/v) Triton X-100, followed by 15 min in 15 ml of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5 mM EGTA, and 200 mM NaCl, and finally sonicated in 1 ml of 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5 mM EGTA, 1% (w/v) SDS plus 1 mM PMSF, 1 µg/ml aprotonin, leupettin, and pestatin to an average fragment size of 300–500 bp. 20% of solubilized chromatin extracts were saved for input followed with cross-link reverse step, and the remaining were clarified by centrifugation at 12,000 g, and diluted to 6 OD260 U/ml in IP buffer [140 mM NaCl, 1% (w/v) Triton X-100, 0.1% (w/v) sodium deoxycholate, 1 mM PMSF, 100 µg/ml salmon sperm DNA, and 100 µg/ml BSA]; preincubated for 1 h at 4°C with 10 µl/ml 50% (v/v) Protein A-agarose (Invitrogen Life Technologies, Camarillo, CA) with normal mouse/rabbit sera; reconstituted in PBS, and washed several times in IP buffer. Aliquots (600 µl) were incubated with 20 µg of each specific antibody for overnight at 4°C. Immune complexes were separated into bound and unbound complexes with protein A-agarose and cross-links were reversed by treatment at 65°C overnight. After treatment with RNase A and proteinase K, samples were extracted once with phenol/chloroform, and the DNA was precipitated with 2 volumes of ethanol. Precipitated DNA was pelleted, washed once with 70% ethanol, dried, and resuspended in 100 µl of water. The DNA was analyzed by quantitative PCR using Ampicilin primers (forward: 5’-CATCTTACGGATGGCATGAC-3’, reverse: 5’-CAACGATCAAGGCGAGTTAC-3’).
Colony formation assay
Ten million of Saos-2 cells were typically transfected using electroporation with different combinations of expression plasmids as shown in the text. Transfected cells were cultured in the selection medium (DMEM supplemented with 100 mg/ml G418). After 14 days, cells were fixed on the plates with formaldehyde and stained with 0.1% crystal violet. The amount of the colonies in each dish was scanned by Li-Cor Odyssey and counted. The data are presented as the average from two independent experiments.
Quantitative real-time PCR
Total RNA from cells was extracted using Trizol reagent and cDNA was made with a Superscript II reverse transcription kit (Invitrogen, Inc., Carlsbad, CA). The primers for real-time PCR were as followings: for p53: 5′-CCT GAGGTTGGCTCTGACTGTA-3′(sense) and 5′-TCCGTCCCAGTAGATTACCAC- 3′ (antisense), yielding a 136-bp product; for p21: 5′-GAGGGCAAGTACGAGTGG CAA-3′ (sense) and 5′- CTGCGCATTGCTCCGCTAACC-3′ (antisense), yielding a 170-bp product; for Bax: 5′- TGCTTCAGGGTTTCATCCAGGA-3′ (sense) and 5′- ACGGCGGCAATCATCCTCTG-3′ (antisense), yielding a 172-bp product; and for GAPDH (glyceraldehyde-3-phosphate dehydrogenase): 5′-CTCCTCTGACTTCAAC AGCG-3′ (sense) and 5′-GCCAAATTCGTTGTCATACCAG-3′ (antisense), yielding a 112-bp product. The cDNA was amplified by using 10 µl of Master Mix from the DyNAmo SYBR green quantitative real-time PCR kit (MJ Research, Inc.), 1 µM of each primer, and 2 µl of the cDNA product in a 20 µl total volume. Thirty cycles of 1 min at 94°C, 30 s at 55°C, and 40 s at 72°C were followed by 10 min at 72°C in an MJ Research Opticon II thermocycler (MJ Research, Inc., Waltham, MA). A melting curve analysis was performed to verify the specificities of the amplified products. The values for the relative levels of change were calculated by the “delta delta threshold cycle” (ΔΔCT) method and each sample were tested in triplicates.
Apoptosis assay
The apoptotic cells of stable Gemin3 and control knock down cells were analyzed by propidium iodide (PI) flow cytometric assay, which is based on the principle that DNA fragmentation and the consequent loss of nuclear DNA content occurs at the late phase of apoptosis. Briefly, 106 cells with serum starvation treatment of 0.1% serum for 12 h were collected and fixed with 100% ethanol for 2 h at 4°C, washed with 1x phosphate-buffered saline (PBS), and stained with 50 µg/ml propidium iodide (Sigma, St. Louis, MO) and 1 µg/ml RNase for 1 hour in the dark at 4°C. The stained cells were subseqeutly analyzed using FACSCalibur cytometer (Becton Dickinson, San Jose, CA) and FlowJo Software (Tree Star, Ashland, OR).
We are grateful to Gideon Dreyfuss, Gary J Nabel, Shelley L Berger, Jon Aster, Elliott Kieff, and Wafik S. EI-Deiry for generously providing reagents.
The authors have declared that no competing interests exist.
This study was supported by public health service grants: National Cancer Institute 5R01CA091792-08, 5R01CA108461-05, 1R01CA137894-01 and 1R01CA138434-01A209; National Institute of Allergy and Infectious Diseases 5R01AI067037-04 and National Institute of Dental and Craniofacial Research 5R01DE017338-03 (to ESR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22174839PONE-D-11-1355510.1371/journal.pone.0028563Research ArticleBiologyBiochemistryProteinsCytoskeletal ProteinsProtein InteractionsRecombinant ProteinsTransmembrane ProteinsImmunochemistryMolecular Cell BiologyCellular StructuresCytoskeletonCellular TypesMuscle CellsProteomicsProtein InteractionsDysferlin Interacts with Histone Deacetylase 6 and Increases alpha-Tubulin Acetylation Dysferlin Interacts with HDAC6Di Fulvio Sabrina
1
Azakir Bilal A.
1
Therrien Christian
2
Sinnreich Michael
1
2
*
1
Neuromuscular Research Group, Departments of Neurology and Biomedicine, University, Hospital, Basel, Switzerland
2
Neuromuscular Research Group, Montreal Neurological Institute and Hospital, McGill University, Montreal, Canada
Buratti Emanuele EditorInternational Centre for Genetic Engineering and Biotechnology, Italy* E-mail: [email protected] and designed the experiments: SDF BAA MS. Performed the experiments: SDF BAA. Analyzed the data: SDF BAA. Contributed reagents/materials/analysis tools: SDF BAA CT. Wrote the paper: SDF BAA CT MS.
2011 8 12 2011 6 12 e2856315 7 2011 10 11 2011 Di Fulvio et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Dysferlin is a multi-C2 domain transmembrane protein involved in a plethora of cellular functions, most notably in skeletal muscle membrane repair, but also in myogenesis, cellular adhesion and intercellular calcium signaling. We previously showed that dysferlin interacts with alpha-tubulin and microtubules in muscle cells. Microtubules are heavily reorganized during myogenesis to sustain growth and elongation of the nascent muscle fiber. Microtubule function is regulated by post-translational modifications, such as acetylation of its alpha-tubulin subunit, which is modulated by the histone deacetylase 6 (HDAC6) enzyme. In this study, we identified HDAC6 as a novel dysferlin-binding partner. Dysferlin prevents HDAC6 from deacetylating alpha-tubulin by physically binding to both the enzyme, via its C2D domain, and to the substrate, alpha-tubulin, via its C2A and C2B domains. We further show that dysferlin expression promotes alpha-tubulin acetylation, as well as increased microtubule resistance to, and recovery from, Nocodazole- and cold-induced depolymerization. By selectively inhibiting HDAC6 using Tubastatin A, we demonstrate that myotube formation was impaired when alpha-tubulin was hyperacetylated early in the myogenic process; however, myotube elongation occurred when alpha-tubulin was hyperacetylated in myotubes. This study suggests a novel role for dysferlin in myogenesis and identifies HDAC6 as a novel dysferlin-interacting protein.
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Introduction
Recessive mutations in the DYSF gene cause Limb girdle muscular dystrophy type 2B (LGMD2B) [1], Miyoshi Myopathy [1] and Distal anterior compartment myopathy [2]. Dysferlin is a large type II transmembrane protein composed of two DysF domains and seven C2 domains that mediate lipid [3], [4] and protein binding interactions [5], [6], [7], [8], [9]. Dysferlin is predominantly expressed in skeletal and cardiac muscle [10], and its expression is upregulated during myogenesis [11], [12]. The subcellular localization of dysferlin is at the sarcolemma, T-tubule membranes and in intracellular vesicular compartments of as yet unknown origin [13], [14]. Dysferlin is a critical component of the calcium-dependent sarcolemmal repair complex, but recent studies have proposed additional roles for dysferlin in myogenesis [15], [16], [17], intercellular calcium signaling [18] and cellular adhesion [19]. Our recent work identified alpha-tubulin and microtubules as novel binding partners of dysferlin [6], suggesting a possible role for dysferlin in microtubule dynamics or stability.
The upregulation of microtubule acetylation is essential for myogenesis [20]. Microtubule acetylation is regulated by alpha-tubulin acetyltransferases and deacetylases, the most notable one being histone deacetylase 6 (HDAC6) [21]. Unlike most classical HDACs which are located in the nucleus and deacetylate nuclear substrates such as histones, HDAC6 contains a nuclear exclusion signal and a cytoplasmic retention signal making it a cytoplasmic enzyme [21], [22]. HDAC6 has two catalytic hdac domains used to deacetylate alpha-tubulin [21], [23], [24], cortactin [23], [25], [26] and Hsp90 [27]. HDAC6-mediated microtubule deacetylation plays important regulatory roles in microtubule dynamics [28], [29], cellular motility [23], [26], [30], [31] and motor protein motility [32].
In this study, we identified HDAC6 as a novel dysferlin interacting protein. Our results revealed that dysferlin binds to HDAC6 and alpha-tubulin, and prevents HDAC6 from deacetylating its substrate, alpha-tubulin. We also demonstrated that inhibition of HDAC6 activity in the early stages of myoblast differentiation results in impaired myogenesis, whereas increased microtubule acetylation in myotubes results in myotube elongation. We suggest that the increasing dysferlin expression observed during myogenesis could be required to decrease HDAC6-mediated microtubule deacetylation.
Results
Dysferlin interacts with HDAC6 and prevents alpha-tubulin deacetylation
We had previously performed a mass spectrometric analysis of the dysferlin protein complex [6] and identified HDAC6 as a potential dysferlin interactor. This protein was also identified in another study [33]. To confirm this interaction, we performed binding assays using recombinant and native dysferlin and HDAC6 proteins. Recombinant dysferlin was able to bind either to recombinant FLAG-HDAC6 expressed in HEK293T cells (Figure 1A) or to native HDAC6 from homogenized murine testes (Figure 1B), which are a rich source of the enzyme. Co-immunoprecipitation assays performed in mouse skeletal muscle extracts showed that native dysferlin co-immunoprecipitated with native HDAC6 (Figure 1C).
10.1371/journal.pone.0028563.g001Figure 1 Dysferlin co-immunoprecipitates with HDAC6.
(A) HEK293T cells were transfected with GFP-myc-dysferlin (GFP-myc-Dysf) and FLAG-HDAC6, and recombinant dysferlin was immunoprecipitated (IP) with anti-myc antibodies. Immunoprecipitates were separated by SDS-PAGE and immunoblotted (IB) with the indicated antibodies. SM = starting material, 5% of total protein loaded. (B) GFP-myc-dysferlin (GFP-myc-Dysf) or GFP vector were transfected in HEK293T cells, immunoprecipitated with anti-GFP antibodies and incubated with testes homogenate from wildtype C57Bl/6 mice, which is a rich source of HDAC6. Immunoprecipitates were immunoblotted with the indicated antibodies. Alpha-tubulin was used as a loading control. (Right panel) HDAC6 protein levels in testes of wildtype C57Bl/6 mice (WT) versus HDAC6 knockout mice (KO), which were immunoblotted with anti-HDAC6 antibodies to demonstrate the specificity of the detected band. (C) Native dysferlin was immunoprecipitated with anti-dysferlin antibodies in mouse skeletal muscle extracts. Immunoprecipitates were separated by SDS-PAGE and immunoblotted with anti-dysferlin and anti-HDAC6 antibodies. (D) GFP-myc-dysferlin and FLAG-HDAC6 were overexpressed in Cos7 cells. Cells were fixed and immunostained with anti-GFP and anti-FLAG antibodies. (E) 134/04 cells were transfected with FLAG-HDAC6 and differentiated into myotubes. Cells were fixed and immunostained with anti-dysferlin and anti-FLAG antibodies. Scale bar: 20 µm.
To determine if dysferlin and HDAC6 co-localized in the same subcellular compartment, GFP-myc-dysferlin and FLAG-HDAC6 were transfected into Cos7 cells. Immunostaining showed partial co-localization between the two proteins in the cytoplasm and in the vicinity of the plasma membrane (Figure 1D). To determine if the proteins co-localized in muscle cells, FLAG-HDAC6 was transfected into a human myoblast cell line (134/04), which harbours two wildtype DYSF alleles, and cells were differentiated into myotubes. Immunostaining with anti-dysferlin and anti-FLAG antibodies demonstrated that the proteins partially co-localized in the cytoplasm and in the vicinity of the plasma membrane (Figure 1E).
To identify which of dysferlin's seven C2 domains could be involved in the interaction with HDAC6, we constructed a series of single C2 domain deletion mutants from full-length wildtype (WT) dysferlin, which harbours an N-terminal GFP tag and C-terminal myc-His tags (Figure 2A). Each mutant (ΔC2A to ΔC2G), or WT dysferlin or a GFP vector was expressed in HEK293T cells, immobilized on nickel affinity beads, and incubated either with recombinant FLAG-HDAC6 (Figure 2B) or with native HDAC6 from homogenized murine testes (Figure 2C). In both assays, dysferlin's C2D domain was required for the interaction with HDAC6.
10.1371/journal.pone.0028563.g002Figure 2 Dysferlin binds HDAC6 through its C2D domain and prevents alpha-tubulin deacetylation.
(A) Schematic of dysferlin C2 domain deletion constructs. (B) Wildtype dysferlin (WT), dysferlin deletion mutants (ΔC2A through ΔC2G) or GFP vector were transfected in HEK293T cells, pulled-down on Ni-NTA beads, and incubated with FLAG-HDAC6-transfected HEK293T cell lysates. Immunoprecipitates were immunoblotted with the indicated antibodies. (Right panel) FLAG-HDAC6 expression levels in transfected HEK293T cell lysates, immunoblotted with anti-FLAG antibody. (C) Wildtype dysferlin (WT), dysferlin deletion mutants (ΔC2A through ΔC2G) or GFP vector were transfected in HEK293T cells, pulled-down on Ni-NTA beads, and incubated with murine testes homogenate. Immunoprecipitates were immunoblotted with the indicated antibodies. (Right panel) This western blot is identical to the one displayed in Fig. 1B. (D) Cell lysates from (C) were immunoblotted for alpha-tubulin acetylation levels. Similar results observed with cell lysates from (B) (not shown).
Given that HDAC6 is a major alpha-tubulin deacetylase, we assayed for dysferlin's effect on alpha-tubulin acetylation. Alpha-tubulin acetylation levels increased in HEK293T cells expressing wildtype dysferlin (WT) or the following C2 domain deletion mutants: ΔC2B, ΔC2C, ΔC2E, ΔC2F and ΔC2G (Figure 2D). Cells expressing ΔC2A or ΔC2D showed no change in alpha-tubulin acetylation levels compared to GFP vector-expressing cells, indicating that dysferlin requires its C2A and C2D domains to prevent HDAC6 from deacetylating alpha-tubulin.
Dysferlin requires alpha-tubulin binding to prevent HDAC6 from deacetylating alpha-tubulin
We recently showed that dysferlin interacts with alpha-tubulin through its C2A and C2B domains, although this interaction is weaker with the C2B domain than with the C2A domain [6]. Figures 2B and 2C show that the ΔC2A deletion mutant interacted less strongly with alpha-tubulin when compared to wildtype dysferlin or the other six deletion mutants, which is in agreement with our previously published results. Notably, the interaction was not fully abolished since the ΔC2A deletion mutant retains its C2B domain, which also interacts with alpha-tubulin, albeit weakly. Theorizing that dysferlin requires both of its alpha-tubulin binding domains to interact with HDAC6, we used a truncated dysferlin mutant lacking its three N-terminal C2 domains, but retaining its DysF domains (DD) and the transmembrane domain (TM) (DD-DEFG-TM) (Figure 3A). As expected, this N-terminally truncated mutant did not pull down alpha-tubulin, and also showed weaker binding to HDAC6 (Figure 3B). This suggests that dysferlin also requires both of its alpha-tubulin binding domains (C2A and C2B) to fully interact with HDAC6.
10.1371/journal.pone.0028563.g003Figure 3 Dysferlin requires its alpha-tubulin binding domains to bind HDAC6 and prevent alpha-tubulin deacetylation.
(A) Schematic of dysferlin truncation construct (DD-DEFG-TM). (B) Wildtype dysferlin (WT), dysferlin deletion mutants ΔC2A and ΔC2D, or DD-DEFG-TM were transfected in HEK293T cells, pulled-down on Ni-NTA beads, incubated with murine testes homogenate and immunoblotted with the indicated antibodies. (C) Cell lysates from (B) were immunoblotted for alpha-tubulin acetylation levels. (D) FLAG-HDAC6 was co-transfected with wildtype dysferlin (WT), dysferlin deletion mutants (ΔC2A and ΔC2D), dysferlin truncation (DD-DEFG-TM) or GFP vector in HEK293T cells, immunoprecipitated with anti-alpha-tubulin antibodies, and immunoblotted with the indicated antibodies. As controls, cell lysates were immunoprecipitated without antibodies (CTL) or with anti-IgG antibodies (IgG).
We assessed whether the truncated mutant DD-DEFG-TM could affect alpha-tubulin acetylation levels in HEK293T cells. As shown in Figure 3C, DD-DEFG-TM did not alter the amount of acetylated alpha-tubulin, similarly to ΔC2A and ΔC2D deletion mutants. In agreement with Figure 2D, these results highlight the importance of dysferlin's C2A domain in preventing alpha-tubulin deacetylation.
Having shown that dysferlin's alpha-tubulin binding domains are important for impairing HDAC6-mediated deacetylation of alpha-tubulin, we theorized that dysferlin may be having this effect by affecting HDAC6's ability to interact with its substrate. To assess how dysferlin may affect HDAC6's interaction with alpha-tubulin, we performed an alpha-tubulin immunoprecipitation assay in HEK293T cells expressing FLAG-HDAC6 along with either wildtype dysferlin (WT), the ΔC2A or ΔC2D deletion mutants, or with the truncated mutant (DD-DEFG-TM). As shown in Figure 3D, in the absence of dysferlin, HDAC6 is able to bind to alpha-tubulin. However, in the presence of full-length dysferlin, alpha-tubulin no longer pulled down HDAC6, but only dysferlin. This effect was only observed if dysferlin retained its C2D domain as well as its alpha-tubulin binding C2A and C2B domains; if these domains were deleted, dysferlin did not prevent HDAC6 from interacting with alpha-tubulin. This is demonstrated by the ΔC2D and DD-DEFG-TM constructs, which showed an unimpaired HDAC6 interaction with alpha-tubulin (Figure 3D). Because the ΔC2A construct still had partial alpha-tubulin binding capabilities and an intact C2D domain, it was able to decrease HDAC6's interaction with alpha-tubulin, but not abolish it completely as did wildtype dysferlin. These results suggest that dysferlin prevents HDAC6 from interacting with its substrate, thus hindering alpha-tubulin deacetylation.
Dysferlin expression increases alpha-tubulin acetylation and resistance to microtubule depolymerization
Having demonstrated that recombinant dysferlin affects alpha-tubulin acetylation levels in HEK293T cells, we assessed whether native dysferlin expression also affected alpha-tubulin acetylation in muscle cells. We used three human myoblast cell lines: 134/04 cells harbouring two wildtype DYSF alleles, ULM1/01 cells harbouring two nonsense DYSF alleles, and 180/06 cells harbouring one missense DYSF allele and one nonsense DYSF allele. The cells were cultured, lysed and immunoblotted for acetylated-alpha-tubulin and alpha-tubulin levels. As shown in Figure 4A, wildtype cells (134/04) had higher levels of acetylated alpha-tubulin than dysferlin-deficient cells (180/06 and ULM1/01). To confirm that the effect was specific to dysferlin expression, wildtype dysferlin (WT) or the ΔC2A deletion mutant was overexpressed in 134/04 cells. In agreement with Figures 2D and 3C, dysferlin overexpression in muscle cells resulted in increased alpha-tubulin acetylation levels, whereas expression of the ΔC2A deletion mutant did not affect alpha-tubulin acetylation (Figure 4B).
10.1371/journal.pone.0028563.g004Figure 4 Dysferlin expression increases alpha-tubulin acetylation in muscle cells.
(A) 134/04, 180/06 and ULM1/01 cell lysates were immunoblotted with anti-dysferlin and anti-acetylated alpha-tubulin antibodies. Alpha-tubulin was used as a loading control. (B) GFP-dysferlin wildtype (WT) or GFP-dysferlinΔC2A (ΔC2A) were transfected into 134/04 myoblasts. Transfected and untransfected (CTL) cell lysates were separated by SDS-PAGE and immunoblotted with the indicated antibodies.
Microtubule post-translational modifications occur subsequent to microtubule stabilization; therefore alpha-tubulin acetylation can be considered as a marker of stabilized microtubules [34]. Stabilized microtubules are more resistant to microtubule depolymerization. We theorized that the increased alpha-tubulin acetylation levels observed in dysferlin-expressing cells were indicative of a pool of microtubules with increased resistance to depolymerization. To test this theory, we employed cold-induced and Nocodazole-induced microtubule depolymerization assays. In the cold-induced depolymerization assay, 134/04, 180/06 and ULM1/01 cells were incubated at 4°C for increasing lengths of time, and microtubule resistance was assessed by the amount of acetylated alpha-tubulin remaining post-treatment. As shown in Figures 5A and 5E, 134/04 cells retained significantly higher acetylated alpha-tubulin levels following cold treatments (30, 45 and 60 min) than dysferlin-deficient 180/06 and ULM1/01 cells. In the Nocodazole-induced depolymerization assay, 134/04, 180/06 and ULM1/01 cells were treated with increasing concentrations of Nocodazole, and microtubule resistance to depolymerization was assessed by the amount of acetylated alpha-tubulin remaining post-treatment. As shown in Figures 5B and 5F, 134/04 cells retained significantly higher acetylated alpha-tubulin levels following Nocodazole treatment (3 µg/ml and 9 µg/ml) than dysferlin-deficient 180/06 and ULM1/01 cells. To demonstrate that the effect was specific to dysferlin expression, wildtype dysferlin (WT) or ΔC2A were expressed in HEK293T cells, which were similarly treated with Nocodazole. As shown in Figures 5C and 5G, cells transfected with WT dysferlin showed significantly more acetylated alpha-tubulin levels post-Nocodazole treatment when compared to untransfected (CTL) cells. In agreement with our previous results, this effect required dysferlin's C2A domain, as cells transfected with ΔC2A showed acetylated alpha-tubulin levels that were comparable to untransfected (CTL) cells. These results suggest that dysferlin expression correlates with increased microtubule resistance to Nocodazole treatment, indicative of the presence of a larger pool of stable microtubules.
10.1371/journal.pone.0028563.g005Figure 5 Dysferlin expression increases resistance to microtubule depolymerization.
(A) 134/04, 180/06 and ULM1/01 cells were incubated at 4°C for increasing lengths of time. Cell lysates were immunoblotted with the indicated antibodies. To equalize the baseline (0 min) acetylated alpha-tubulin levels in the 180/06 and ULM1/01 cells with those of the 134/04 cells, the intensity of the bands was linearly increased post-acquisition. (B) 134/04, 180/06 and ULM1/01 cells were untreated (Unt), mock-treated (Mock) or treated with increasing concentrations of Nocodazole. Cell lysates were immunoblotted with the indicated antibodies. To equalize the baseline (Unt) acetylated alpha-tubulin levels in the 180/06 and ULM1/01 cells with those of the 134/04 cells, the intensity of the bands was linearly increased post-acquisition. (C) GFP-dysferlin wildtype (GFP-Dysf WT) or GFP-dysferlinΔC2A (GFP-DysfΔC2A) were transfected into HEK293T cells. Transfected and untransfected (CTL) cells were treated with increasing concentrations of Nocodazole. Cell lysates were immunoblotted with the indicated antibodies. (D) 134/04, 180/06 and ULM1/01 cells were untreated (Unt) or treated with 2.5 µg/ml Nocodazole, then the drug-containing media was replaced with fresh media and cells were allowed to recover for the indicated lengths of time. Cell lysates were immunoblotted with the indicated antibodies. To equalize the baseline (Unt) acetylated alpha-tubulin levels in the 180/06 and ULM1/01 cells with those of the 134/04 cells, the intensity of the bands was linearly increased post-acquisition. (E, F, G) The ratio of acetylated alpha-tubulin∶alpha-tubulin at each time point or Nocodazole concentration was normalized to 134/04 levels to equalize starting values. * indicates that 134/04 values or GFP-Dysf WT values were significantly greater (p<0.05) than 180/06 and ULM1/01 levels (E, F) or CTL and ΔC2A levels (G), at the indicated time point or concentration. (H) The ratio of acetylated alpha-tubulin: alpha-tubulin was calculated for each time point and normalized to 134/04 levels to equalize starting values. *, # and+indicate that the value at the indicated time point is significantly different (p<0.05) than the Unt value for 134/04, 180/06, ULM1/01, respectively.
Once the microtubule depolymerising agent is removed, microtubules will repolymerize during a recovery phase [35]. To study the effect of dysferlin expression on microtubule recovery from Nocodazole treatment, cells were treated with Nocodazole for 45 min and then the drug-containing media was removed and replaced with fresh media, thus allowing repolymerization of microtubules. After defined lengths of time, the alpha-tubulin acetylation levels were measured as a marker of microtubule repolymerization and stabilization. Figures 5D and 5H show that dysferlin expression (134/04 cells) resulted in faster recovery from Nocodazole treatment than was observed in dysferlin-deficient cells (180/06 or ULM1/01 cells). Taken together, these results show that dysferlin expression increases cellular alpha-tubulin acetylation levels, as well as promotes microtubule resistance to, and recovery from, induced depolymerization.
Hyperacetylation of alpha-tubulin impairs myogenesis
Microtubule acetylation and dysferlin expression are both upregulated during myogenesis [11], [20]. To demonstrate this in our cultured human myoblasts, 134/04 cells were cultured in differentiation media for up to four days to induce myotube formation. Lysates from these cells and from homogenized mouse skeletal muscle were immunoblotted for dysferlin and acetylated-alpha-tubulin levels. Results showed that both dysferlin and acetylated alpha-tubulin levels increased during myogenic differentiation (Figure 6A). Immunofluorescent staining for acetylated alpha-tubulin levels in 134/04 cells show significantly higher levels in differentiated myotubes than in undifferentiated myoblasts (Figure 6B). On the other hand, dysferlin-deficient myoblasts (180/06) do not differentiate into myotubes and the acetylated alpha-tubulin were unchanged even after four days in differentiation media (Figure 6B).
10.1371/journal.pone.0028563.g006Figure 6 Dysferlin and acetylated alpha-tubulin levels increase during differentiation.
(A) 134/04 cells were cultured in differentiation media for the indicated number of days. Cell lysates from these cells and mouse skeletal muscle extract were immunoblotted with the indicated antibodies. (B) 134/04 cells and 180/06 cells were cultured in differentiation media for 0 days or 4 days to induce myotube formation. Cells were fixed and immunostained with anti-acetylated alpha-tubulin antibodies and DAPI. Images were captured at the same fluorescence intensity and gain to compare alpha-tubulin acetylation levels between cells. Scale bar: 20 µm.
Our data suggests that upregulated dysferlin expression would increase microtubule acetylation via its interaction with HDAC6. Given that microtubule acetylation is a late-stage event in myogenesis, we theorized that early upregulation of dysferlin would result in prematurely increased acetylation levels, which could have detrimental effects on myotube formation. However, dysferlin has previously been shown to play a role in myogenesis [16], [17], for instance by affecting myogenin expression [16]. Therefore, it would not be possible to attribute any potential myogenic effect from overexpressing dysferlin in dysferlin-deficient myoblasts specifically to dysferlin's role on alpha-tubulin acetylation. Therefore, we used instead an HDAC6-specific inhibitor that causes alpha-tubulin hyperacetylation, to mimic the effect of dysferlin overexpression on alpha-tubulin acetylation specifically. Tubastatin A is a more selective derivative of the HDAC6 specific inhibitor Tubacin, which specifically inhibits alpha-tubulin deacetylation without affecting HDAC6's other substrates [24], [29], [30], [36], [37].
Myoblast differentiation assays were performed by treating 134/04 human myoblasts and C2C12 murine myoblasts continuously with Tubastatin A beginning at different stages of myoblast differentiation: at the stage when myoblasts were undifferentiated (Day 0), at the stage when myoblasts were beginning to form myotubes (Day 2) or at the stage when myotubes were terminally differentiated (Day 4) (Figure 7A). Acetylated alpha-tubulin levels in the cell lysates were assessed to confirm Tubastatin A efficacy (Figure 7B). The cells were immunostained for desmin and DAPI (Figure 7C). Desmin-stained myotubes were counted and categorized according to length (Figure 7D). The number of nuclei per myotube was also counted and the average number of nuclei in each category of myotube length was determined (Figure 7E).
10.1371/journal.pone.0028563.g007Figure 7 Effect of HDAC6 inhibition on myotube formation.
(A) C2C12 or 134/04 myoblasts were seeded in growth media on Day -1, then switched to differentiation media on Day 0. Cells were mock-treated (Mock) or treated with 7.5 µM Tubastatin A beginning on different days post-induction of myogenic differentiation (Day 0, Day 2, Day 4). On Day 5, cells were fixed and stained with an anti-desmin antibody and DAPI. (B) Alpha-tubulin acetylation levels were assayed to confirm Tubastatin A efficacy. (C) Representative immunofluorescence images of desmin-stained myotubes in each treatment regime. Scale bar: 60 µm. (D) Desmin-stained myotubes were categorized by their myotube length, and plotted against their relative number. (E) Desmin-stained myotubes were counted for their average number of nuclei and categorized by myotube length as in (D). * in (D) and (E) indicates p<0.05, significantly different from mock-treated myotubes in the same category. ** indicates that no myotubes were observed in the indicated category. (F) Absolute number of desmin-stained myotubes were counted and categorized by treatment regime. * indicates p<0.05, significantly different from Mock-treated. Shown here are results for C2C12 cells; similar results were obtained for 134/04 cells (not shown).
When treated early (Day 0, Day 2), myotube formation was significantly impaired (Figure 7C), resulting in fewer myotubes being formed (Figure 7F). The myotubes that did form were significantly shorter than mock-treated myotubes; additionally no long myotubes (>500 µm) were produced (Figure 7D). Conversely, when myotubes were hyperacetylated after terminal differentiation (Day 4), a significantly larger proportion of myotubes were longer than 600 µm when compared to mock-treated cells (Figure 7D), indicating that myotube length was increased. The average number of nuclei in this category of myotubes (>600 µm) was not significantly different from mock-treated cells (Figure 7E), indicating that the increased length was not due to increased myoblast fusion. These results suggest that microtubule hyperacetylation in the early-stages of myoblast differentiation is detrimental to myogenesis, whereas late-stage hyperacetylation can promote myotube elongation.
Discussion
Dysferlin is a multi-C2 domain transmembrane protein involved in skeletal muscle membrane repair, and has also been implicated in myogenesis, cellular adhesion and intercellular calcium signaling. In light of the growing evidence supporting dysferlin's multifunctionality, understanding dysferlin's biology will depend on identifying its interacting proteins. To this end, we previously undertook a proteomic search through the analysis of immunoprecipitated proteins from mouse skeletal muscle using liquid chromatography-tandem mass spectrometry [6]. In this study, we identified HDAC6 as a novel dysferlin binding partner, and present a new function for dysferlin in the modulation of alpha-tubulin acetylation via an interaction with this microtubule deacetylase.
We show that dysferlin prevents HDAC6 from deacetylating alpha-tubulin by physically binding both to the enzyme via its C2D domain and to the substrate, alpha-tubulin, via its alpha-tubulin binding C2A and C2B domains. Consequently, dysferlin expression increased the alpha-tubulin acetylation levels in muscle cells. The increased alpha-tubulin acetylation levels in dysferlin-expressing cells reflect a larger pool of stable microtubules, which we showed are more resistant to Nocodazole-induced depolymerization and cold-induced depolymerization. Dysferlin expression also promoted faster microtubule recovery from depolymerization, resulting in more microtubules being repolymerized, stabilized and consequently post-translationally modified by acetylation.
A question that emerges from this study is how dysferlin prevents HDAC6 from interacting with its substrate. One possibility is that dysferlin targets HDAC6 via its C2D domain and then reinforces the interaction by binding to alpha-tubulin. Dysferlin may then directly or indirectly block HDAC6 from interacting with alpha-tubulin, thus inhibiting its ability to deacetylate microtubules. This is demonstrated by the observation that (i) loss of dysferlin's C2A domain resulted in a decreased interaction between HDAC6 and alpha-tubulin (Figure 3D) and (ii) loss of both of dysferlin's C2A and C2B domains resulted in decreased binding between dysferlin and HDAC6 (Figure 3B). Another possibility is that dysferlin binds to alpha-tubulin via its C2A and C2B domains, and uses its HDAC6-binding C2D domain to block or dislodge the enzyme from alpha-tubulin. This is suggested by the alpha-tubulin co-immunoprecipitation experiment (Figure 3D), in which full-length dysferlin was pulled down by alpha-tubulin but HDAC6 was not. It is further possible that dysferlin may sequester HDAC6 away from its substrate, as Figure 2 demonstrated a strong interaction between full-length dysferlin and HDAC6 that may be indicative of a cytoplasmic subpopulation of the two proteins which would not be observed in the alpha-tubulin co-immunoprecipitation experiment. Further studies would be required to elucidate the mechanism involved in dysferlin's interaction with HDAC6.
To study the effect of alpha-tubulin hyperacetylation on myogenesis, we used Tubastatin A, a selective HDAC6 inhibitor. When HDAC6 was inhibited early during differentiation, myotube formation was impaired, whereas HDAC6 inhibition in differentiated myotubes promoted myotube elongation.
Impaired myogenesis arising from early microtubule hyperacetylation could be caused by disrupted microtubule dynamics and protein targeting. Dynamic microtubules are required to target the cell periphery and HDAC6 inhibition has been shown to decrease microtubule dynamics [28], [29]. Premature HDAC6 inhibition could promote a more stabilized microtubule pool, thus impairing the microtubule reorganization from a radial configuration in undifferentiated myoblasts to the organized longitudinal array observed in differentiated myotubes. Alpha-tubulin hyperacetylation would also compromise the microtubule tracks used for protein, vesicle and organelle delivery. Proteins, such as CLASPs, depend on spatial cues along microtubules to locally regulate microtubule dynamics, which are provided by discrete regions of microtubule acetylation [38]. Such signals would be disrupted by microtubule hyperacetylation.
In myotubes, on the contrary, increased microtubule acetylation could promote myotube elongation by affecting microtubule polarization and motor protein movement. It has been proposed that the linear microtubule array in differentiated myotubes directly promotes myotube elongation by providing polarization, which restricts myotube elongation to a single axis [39], [40]. Furthermore, microtubule acetylation enhances kinesin-1 recruitment to microtubules and anterograde movement along microtubules [32], [41], thus permitting the delivery of cytoskeletal remodelling factors, target site recognition molecules or adhesion molecules [42], [43]. Additionally, microtubule acetylation has been proposed to designate stabilized microtubules extending all the way to a destination [32], thus promoting the delivery of proteins necessary for myotube formation and elongation.
Dysferlin expression is upregulated during myogenesis, with higher levels being observed in differentiated myotubes [11], [12]. We showed that Tubastatin A treatment of undifferentiated myoblasts resulted in impaired myotube formation, such as is observed in dysferlin-deficient myoblasts [16], [17]. Thus, our study suggests that early expression of dysferlin would promote premature microtubule hyperacetylation by way of inhibiting HDAC6, which would impair myotube formation. On the other hand, later expression of dysferlin would promote myotube formation and elongation through increased microtubule acetylation. This could explain the temporal expression pattern of dysferlin during myogenesis. The observed reduction in the number of long myotubes, and concomitant increase in short myotubes, in dysferlin-deficient cell cultures [15], [17] is in keeping with our explanation for the temporally-regulated dysferlin expression during myogenesis. We theorize that the impaired myotube elongation in these cells might in part be explained by active HDAC6, which would maintain low levels of acetylated microtubules.
For the design of dysferlin gene therapies, our study would caution against the use of ubiquitous promoters or promoters expressed early in muscle development, such as CMV and CAG [44]. Instead our data would support the use of promoters that are expressed at later stages of muscle differentiation, such as C5–12 [45] and the human α-skeletal actin promoter [44].
In summary, we have identified HDAC6 as a novel dysferlin-interacting protein, and demonstrated that the interaction between these two proteins is mediated by dysferlin's C2D domain and that dysferlin prevents HDAC6 from deacetylating alpha-tubulin by physically binding to both the enzyme and to the substrate, alpha-tubulin. Finally, we demonstrated the importance of late alpha-tubulin hyperacetylation during the myogenic process, and propose that dysferlin may act as an inhibitor of HDAC6-mediated microtubule deacetylation during myogenesis.
Materials and Methods
Ethics Statement
All animals were handled in strict accordance with good animal practice as defined by the relevant national and/or local animal welfare bodies, and all animal work was approved by the appropriate committee: Cantonal Veterinary Office of Basel, Switzerland (Approval IDs: 2391 and 51). Primary human myoblasts (134/04, 180/06 and ULM1/01) were obtained from EuroBioBank (www.eurobiobank.org) along with the required regulatory permissions (Approval ID: LMU 107/01).
Cell Cultures
C2C12 murine myoblasts and Human embryonic kidney derived cells (HEK293T) were purchased from ATCC (Burlington, Ontario) (ATCC number CRL-1573 and CRL-1772 respectively) [6]. 134/04 cells contain two wildtype DYSF alleles; 180/06 cells harbour one DYSF allele containing the missense mutation C1663T (Arg555Trp) and an additional null allele 3708delA (D1237TfsX24). Myoblast culture ULM1/01 harbours two null alleles: a C4819T (R1607X) substitution and a 5085delT (F1695LfsX48) deletion. Myoblast cultures were immortalized with a retrovirus carrying the E6E7 early region from human papillomavirus type 16, as previously described [46]. Cells were maintained in growth media (10% FBS (Gibco) in DMEM (Sigma)).
Plasmids, Constructs, Antibodies
FLAG-tagged HDAC6 was purchased from AddGene (Plasmid number 13823). The GFP-myc-His-Dysferlin was a kind gift from Dr. K. Bushby, Newcastle [6], [47]. GFP-tagged dysferlin C2-domain deletion mutants were cloned from the full-length dysferlin construct, each having a single C2 domain deleted. Deletion of the C2 domains coding region was performed by PCR mutagenesis using domain spanning oligonucleotides and the QuikChange Site-Directed Mutagenesis Kit (Stratagene), using either the single [48] or double mutagenic primers approach (Supplementary Table S1). The procedure was performed according to the manufacturer's protocols. Dysferlin truncation construct (DD-DEFG-TM), retaining its DysF domains (DD) and C2D-E-F-G domains (DEFG) and transmembrane domain (TM), was cloned from the full-length dysferlin construct. The C-terminal DD-DEFG-TM portion was first cloned into the pcDNA/TO/myc-His vector (Invitrogen), and then eGFP was added N-terminally in a second cloning step (Supplementary Table S1). All clones were sequenced in both DNA strands to confirm the deletions and the conservation of the reading frame.
Mouse monoclonal antibodies against dysferlin, FLAG and acetylated alpha-tubulin were purchased from Vector Laboratories, Sigma and Santa Cruz, respectively. Rabbit polyclonal antibodies against HDAC6 and alpha-tubulin were purchased from Abcam. Rabbit polyclonal GFP antibody was purchased from Invitrogen. Secondary antibodies, Alexa Fluor 680 goat anti-mouse IgG and InfraRed Dye 800 goat anti-rabbit IgG, were purchased from Invitrogen and Rockland, respectively.
Co-immunoprecipitation assays, pulldown assays and Western blot
Cells were transfected using Lipofectamine 2000 (Invitrogen) in OptiMEM (Gibco) for 48 hours. Cells were lysed and immunoprecipitated (IP) as previously described [6]. For pulldown assays, dysferlin-transfected cells were similarly lysed, and supernatants were incubated with His-Select Nickel Affinity (Ni-NTA) Gel (Sigma) in the presence of 20 mM Imidazole (Sigma) overnight at 4°C, washed with 50 mM Imidazole, and incubated with FLAG-HDAC6-expressing cell extracts (similarly prepared), or with wildtype murine testes homogenates (prepared as described [6]), overnight at 4°C in IP buffer. Beads were washed with 50 mM Imidazole and separated by SDS-PAGE. Proteins were transferred onto PVDF membranes, blocked in Blocking buffer (3% Top-Block (LubioScience) with 0.05% sodium azide (Sigma)), incubated overnight with the indicated antibodies in Blocking buffer plus 0.05% Tween-20 (Merck) and detected by Fluorimetric analysis (Odyssey version 2.1.12). All experiments were performed in triplicates. Densiometric analysis was performed using ImageJ 1.43 u (NIH, USA). Statistical analysis was performed using the Student's T-test with a significance level of at least 0.05.
Microtubule depolymerization and recovery assays
For microtubule depolymerization assays, cells were grown to confluency and treated for 45 min with the indicated concentration of Nocodazole (Sigma) in growth media, or alternatively incubated at 4°C for the indicated lengths of time. Cells were washed, lysed and western blotted as described above. For recovery experiments, cells were treated similarly, then Nocodazole was replaced with fresh media and cells were incubated at 37°C, 5% CO2 for the indicated lengths of time. Cells were washed, lysed and western blotted as described above. All experiments were performed in triplicates. Densiometric analysis was performed using ImageJ 1.43 u (NIH, USA). Statistical analysis was performed using the Student's T-test with a significance level of at least 0.05.
Immunofluorescence assays
Cells were grown on matrigel-coated coverslips in growth media or in differentiation media for four days to induce myotube formation. Cells were fixed with 4% paraformadehyde (PFA) for 20 min, blocked 15 min in 2% fish skin gelatin, 1% normal goat serum, 0.15% Triton X-100 in PBS and incubated for 1 hour at room temperature with the indicated antibodies. Incubation with anti-FLAG antibodies was performed at 37°C for 2 hours. Cells were captured on a LSM 710 inverted confocal microscope (Zeiss) and analyzed using Zen 2009 LE software (Zeiss). All experiments were performed in triplicates.
Myoblast differentiation assays
Cells were grown to 80% confluency then switched to differentiation media (2% horse serum (Sigma) in DMEM). Cells were treated with 7.5 uM Tubastatin A (BioVision, LubioScience) either at the same time at media switch (Day 0 or D0), after 48 hours in differentiation media (Day 2 or D2) or after 72 hours (Day 4 or D4). 24 hours later (Day 5), cells were lysed and western blotted as described above, or fixed with 4% PFA for 20 min, blocked 15 min in 2% fish skin gelatin, 1% normal goat serum, 0.15% Triton X-100 in PBS, then stained for desmin and DAPI. Myotubes were imaged with a Leica DMI6000B fluorescence microscope with the Volocity 5.2.0 software (Improvision Ltd) and analyzed with AnalySISD 5.0 (Soft-imaging). All experiments were performed in triplicates. Statistical analysis was performed using the Student's T-test with a significance level of at least 0.05.
Supporting Information
Table S1
Primers used for dysferlin C2 domain deletion constructs. List of primers used to clone dysferlin C2 deletion constructs from full-length dysferlin. Residues that were deleted from each construct are indicated. Deletion of the C2 domains coding region was performed by PCR mutagenesis using either the single or double mutagenic primers approach (see Materials and Methods).
(DOC)
Click here for additional data file.
We thank Muscle Tissue Culture Collection (MTCC) and Dr. Schneiderat from EuroBioBank for providing the human myoblast cultures used in this work. The Muscle Tissue Culture Collection is part of the German network on muscular dystrophies (MD-NET, service structure S1, 01GM0601) funded by the German ministry of education and research (BMBF, Bonn, Germany). The Muscle Tissue Culture Collection is a partner of Eurobiobank (www.eurobiobank.org) and TREAT-NMD (www.treat-nmd.eu). We thank Dr. Bushby (Newcastle, UK) for the GFP-myc-His-dysferlin cDNA. We thank Dr. Matthias and Gabriele Matthias (Basel, Switzerland) for the HDAC6-knockout mouse testes. We thank Dr. Spiess for helpful discussions. We thank Beat Erne (Basel, Switzerland) and Steven Salomon (Montreal, Canada) for technical assistance.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This study was supported by Myosuisse, l'Association française contre les myopathies (AFM) (grant number 14994), MDAC-ALS-CIHR Partnership and the Swiss National Science Foundation (SNF) (grant number 31003A_130286). The first author is funded by l’Association française contre les myopathies (AFM) (grant number 15111). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22174910PONE-D-11-0942610.1371/journal.pone.0028841Research ArticleBiologyMolecular cell biologySignal transductionSignaling cascadesMAPK signaling cascadesMedicineOncologyCancer TreatmentAntiangiogenesis TherapyCancers and NeoplasmsGastrointestinal TumorsColon AdenocarcinomaLung and Intrathoracic TumorsNon-Small Cell Lung CancerAntitumor Activity of Sorafenib in Human Cancer Cell Lines with Acquired Resistance to EGFR and VEGFR Tyrosine Kinase Inhibitors Sorafenib in Resistant Cell LinesMorgillo Floriana
*
Martinelli Erika Troiani Teresa Orditura Michele De Vita Ferdinando Ciardiello Fortunato
Division of Medical Oncology, Department of Clinical and Experimental Medicine and Surgery "F. Magrassi e A. Lanzara" Second University of Naples, Naples, Italy
Fusco Alfredo EditorConsiglio Nazionale delle Ricerche (CNR), Italy* E-mail: [email protected] and designed the experiments: FM EM TT MO FDV FC. Performed the experiments: FM. Analyzed the data: FM. Contributed reagents/materials/analysis tools: FC. Wrote the paper: FM.
2011 9 12 2011 6 12 e2884125 5 2011 16 11 2011 Morgillo et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Treatment of non small cell lung cancer (NSCLC) and colorectal cancer (CRC) have substantially changed in the last years with the introduction of epidermal growth factor receptor (EGFR) inhibitors in the clinical practice. The understanding of mechanisms which regulate cells sensitivity to these drugs is necessary for their optimal use.
An in vitro model of acquired resistance to two tyrosine kinase inhibitors (TKI) targeting the EGFR, erlotinib and gefitinib, and to a TKI targeting EGFR and VEGFR, vandetanib, was developed by continuously treating the human NSCLC cell line CALU-3 and the human CRC cell line HCT116 with escalating doses of each drug. MTT, western blot analysis, migration, invasion and anchorage-independent colony forming assays were conducted in vitro and experiments with established xenografts in athymic nude mice were performed in vivo in sensitive, wild type (WT) and TKI-resistant CALU-3 and HCT116 cell lines.
As compared to WT CALU-3 and HCT116 human cancer cells, TKI-resistant cell lines showed a significant increase in the levels of activated, phosphorylated AKT, MAPK, and of survivin. Considering the role of RAS and RAF as downstream signals of both the EGFR and VEGFR pathways, we treated resistant cells with sorafenib, an inhibitor of C-RAF, B-RAF, c-KIT, FLT-3, RET, VEGFR-2, VEGFR-3, and PDGFR-β. Sorafenib reduced the activation of MEK and MAPK and caused an inhibition of cell proliferation, invasion, migration, anchorage-independent growth in vitro and of tumor growth in vivo of all TKI-resistant CALU-3 and HCT116 cell lines.
These data suggest that resistance to EGFR inhibitors is predominantly driven by the RAS/RAF/MAPK pathway and can be overcame by treatment with sorafenib.
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Introduction
The epidermal growth factor receptor (EGFR) is a central regulator of cancer cell proliferation and progression in several human cancer types. The clinical efficacy of EGFR inhibitors (cetuximab, panitumumab, erlotinib, gefitinib and vandetanib) introduced in the clinical practice for the treatment of metastatic cancers is limited to a subgroup of patients with the majority of cancer patients showing either intrinsic or acquired resistance to these drugs [1].
The recent progresses in the knowledge of cancer biology and drug-resistance mechanisms have identified, among the intracellular signalling pathways, that act as down-stream to the EGFR, the AKT and RAS/RAF/ mitogen-activated protein kinase (MAPK) pathways as major responsible for the development of cancer cell resistance to EGFR inhibitors [2]–[4].
However, we recently demonstrated that, in our in vitro non small cell lung cancer (NSCLC) model of acquired resistance to erlotinib and gefitinib, treatment with several agents known to target directly or indirectly the AKT signalling pathway, such ad LY294002, deguelin and everolimus, was not efficacious in inhibiting erlotinib- (ERL-) and gefitinib- (GEF-) resistant cancer cell proliferation [5].
On the other side, mutations of the K-RAS gene has been described both in NSCLC and colorectal cancer (CRC) patients as responsible for a poor prognosis and poor response to EGFR inhibitors [6]. These mutations cause KRAS proteins to accumulate in the GTP-bound, active form leading to constitutive, growth-factor-receptor independent activation of KRAS downstream signaling in tumor cells [7]. The development of therapeutic strategies for patients with KRAS mutations is thus an important clinical goal. RAF serine-threonine kinases are the principal effectors of RAS in the MAPK signaling pathway and is therefore a potential target for cancer therapy. To date, the most successful clinical inhibitor of RAF activity is sorafenib (Nexavar, BAY 43-9006) [8]–[10], an orally available multi-targeted kinase inhibitor, that blocks the activation of C-RAF, B-RAF (both the wild-type and the activated V600E mutant), c-KIT, FLT-3, RET, vascular endothelial growth factor receptor 2 (VEGFR-2), VEGFR-3, and platelet-derived growth factor receptor β (PDGFR-β) [8]–[10], currently approved for the treatment of metastatic renal cell carcinoma (RCC) and for advanced hepatocellular carcinoma (HCC), and under investigation in other malignancies. Sorafenib affects tumor growth by directly inhibiting tumor cell proliferation and promoting apoptosis in a variety of tumor types as well as by inhibiting tumor-induced neoangiogenesis.
Our laboratory has recently provided evidence of a synergistic interaction between sorafenib and erlotinib or between sorafenib and cetuximab, a monoclonal antibody targeting the extracellular domain of the EGF receptor, in a panel of NSCLC and colorectal cancer (CRC) cell lines, in vitro and in vivo, which is accompanied by a marked and sustained inhibition of the MAPK- and AKT-dependent intracellular signals [11].
We hypothesized that treatment with sorafenib could overcome the induced EGFR TKI-resistance by its ability to block several growth factor receptor-driven signals. Moreover, because sorafenib blocks B-RAF, and it could be effective in cancer cell lines expressing activating K-RAS mutations.
In the present study, we report on the development and on the characterization of human NSCLC and CRC cell lines with acquired resistance to two tyrosine kinase inhibitors (TKI) targeting the EGFR, erlotinib and gefitinib, and a TKI targeting EGFR, VEGFR and RET, vandetanib, and on the antitumor effects of sorafenib in these resistant cancer cell lines.
Results
Development and characterization of TKI-resistant CALU-3 and HCT116 cancer cells
The human NSCLC CALU-3 cell line and the human CRC HCT116 cell line harbour the wild type EGFR gene and an activating K-RAS (KRASp.G13D) gene mutation. In contrast to the other K-RAS mutations, this mutation has been described as not influencing the sensitivity to anti-EGFR treatment, in particular cetuximab [11]. These cancer cell lines has been previously characterized by our group for the expression of the four EGF-related growth factor receptors (EGFR, ERBB2, ERBB3, and ERBB4) and of three VEGF receptors (VEGFR-1,VEGFR-2, VEGFR-3), as well as for the expression of three EGFR ligands (amphiregulin, EGF, and TGFα) and of three VEGFR ligands (VEGF-A, VEGF-B, VEGF-C), by using quantitative RT-PCR (qRT-PCR) [12]. All tested ligand mRNAs were expressed in CALU-3 and HCT116 cell lines. CALU-3 cells also expressed EGFR mRNA, whereas low levels of ERBB2 and ERBB3 mRNAs were measurable. VEGFR-2 and VEGFR-3 mRNA expression was detected in CALU-3 cell line. Expression of EGFR and its specific ligands suggests that in these human cancer cell lines an EGFR-driven autocrine pathway is relevant for cancer cell proliferation. In fact, CALU-3 and HCT116 cells are growth-inhibited by treatment with selective EGFR TKIs, such as gefitinib or erlotinib [13]. Furthermore, these cancer cells express both VEGF ligands and VEGFRs and are growth inhibited by treatment with anti-angiogenic TKIs [13].
Therefore, CALU-3 and HCT116 cells were selected as a model for exploring the acquired resistance mechanisms to treatment with the EGFR TKIs erlotinib and gefitinib, or with the dual EGFR/VEGFR tyrosine kinase inhibitor vandetanib.
The gefitinib- (GEF-R), erlotinib- (ERL-R) and vadetanib- (VAN-R) resistant cell lines were obtained by continuously culturing CALU-3 and HCT116 cells in the presence of increasing doses of each drug for 12 months. After the establishment of three different TKI-resistant CALU-3 and three different TKI-resistant CALU-3 HCT116 cell lines, we characterized their resistant phenotype by doing cell proliferation assays in the presence of each of these inhibitors. As illustrated in Table 1, an approximately 10-fold increase in the IC50 for each TKI-resistant cell line as compared with parental cells was observed. ERL-R, GEF-R and VAN-R CALU-3 and HCT116 human cancer cell lines were cross-resistant to either gefitinib, erlotinib or vandetanib treatment. We next confirmed the establishment of stable TKI-resistant CALU-3 and HCT116 cancer cells in a drug-free culture medium. In fact, all six TKI-resistant cell lines could grow in the absence of each drug for long periods of time (three to six months) and maintain their TKI-resistant phenotype (data not shown).
10.1371/journal.pone.0028841.t001Table 1 IC50 for treatment with erlotinib, gefitinib or vandetanib in parental CALU-3 and HCT116 cell lines (WT) and their TKI-resistant derivatives (ERL-R, GEF-R, VAN-R).
CALU-3 HCT116
WT ERL-R GEF-R VAN-R WT ERL-R GEF-R VAN-R
ERLOTINIB 3 µM ≥25 µM ≥25 µM ≥25 µM 7 µM ≥25 µM ≥25 µM ≥25 µM
GEFITINIB 6 µM ≥25 µM ≥25 µM ≥25 µM 10 µM ≥25 µM ≥25 µM ≥25 µM
VANDETANIB 5 µM ≥25 µM ≥25 µM ≥25 µM 6 µM ≥25 µM ≥25 µM ≥25 µM
To further characterize the TKI-resistant CALU-3 and HCT116 cell lines, we examined differential protein expression among wild type, sensitive CALU-3 and HCT116 cells and their TKI-resistant derivatives.
Activation of the EGFR leads to a complex intracellular signalling which includes the activation of the pro-survival PI3K/AKT pathway and the mitogenic RAS/RAF/MEK/MAPK pathway [13], [14]. We, therefore, investigated by immunoblotting analysis these molecular pathways. As illustrated in Figure 1, EGF-stimulated activation of the EGFR was efficiently blocked in WT and ERL-R, GEF-R and VAN-R cells as demonstrated by the inhibition of EGFR auto-phosporylation (P-EGFR).
10.1371/journal.pone.0028841.g001Figure 1 Analysis of EGFR downstream pathways in parental CALU-3 and HCT116 cells (WT) and in their TKI-resistant derivatives 8ERL-R, GEF-R, VAN-R).
Western blotting analysis of EGFR and of down-stream signalling pathways in parental human CALU-3 and HCT116 cells (WT) and in their TKI-resistant derivatives (ERL-R, GEF-R, VAN-R). β-actin was included as a loading control.
Since the activated, phosphorylated forms of AKT and MAPK are key intracellular mediators of growth factor-activated cell survival and proliferation signals [13], [14] investigating the activation state of these molecular pathways may be of interest in the understanding the resistance mechanisms. Activation of MAPK and AKT with an increase in their phosphorylated forms (P-MAPK and P-AKT) as well as an increase in survivin protein levels were observed in all three TKI-resistant CALU-3 cell lines and in all three TKI-resistant HCT116 cell lines as compared to their parental counterpart (Figure 1).
Taken together, these results suggest that in this cancer cell model of acquired resistance to three different TKIs, activation of AKT- and MAPK-driven intracellular signals may be responsible for cancer cell growth in the presence of either selective anti-EGFR TKIs, such as gefitinib or erlotinib, or in the presence of broad spectrum TKI, such as vandetanib.
Effects of sorafenib on TKI-resistant CALU-3 and HCT116 cancer cell growth
In light of the role of RAS and RAF as downstream mediators of both the EGFR and VEGFR signals from cell surface, we tested the anti-proliferative effect of sorafenib, a multi-targeted kinase inhibitor, blocking the activation of C-RAF, B-RAF, c-KIT, FLT-3, RET, VEGFR-2, VEGFR-3, and PDGFR-β [8] on the parental WT and TKI-resistant CALU-3 and HCT116 cancer cells. A significant inhibition of cell growth was observed in both WT CALU-3 cells and WT HCT116 cells and their corresponding three TKI-resistant derivatives following sorafenib treatment, with an IC50 ranging from 0. 1 to 0.5 µM, (Figure 2).We further characterized the effects of sorafenib treatment on intracellular signalling by Western blotting. As illustrated in Figure 3, treatment of ERL-R, GEF-R and VAN-R CALU-3 and HCT116 cells with sorafenib for 48 hours did not affect total MEK and MAPK protein levels, while it caused a marked decrease of the phosphorylated, activated forms of MEK (P-MEK) and of MAPK (P-MAPK). Moreover, we investigated the activation status of all molecular targets of sorafenib by studing the protein expression levels of C-RAF, B-RAF, c-Kit, FLT-3, RET, VEGFR-2, VEGFR-3 and PDGFRβ, and their phosphoryation status by western blotting analysis. Among all the targets of sorafenib activity, only C-RAF and B-RAF resulted activated in resistant CALU-3 and HCT116 cancer cell lines, and therefore strongly inhibited by sorafenib treatment (Figure 3).
10.1371/journal.pone.0028841.g002Figure 2 Growth inhibitory effects of treatment with sorafenib in parental and TKI-resistant CALU-3 and HCT116 cancer cells.
MTT cell proliferation assays were performed in parental lung adenocarcinoma CALU-3 and colorectal cancer HCT116 cells. (WT) and in their TKI-resistant derivatives (ERL-R, GEF-R, VAN-R ), treated for three days with the indicated concentrations of sorafenib. Results represent the average (±SD) of three separate experiments, each performed in quadruplicate.
10.1371/journal.pone.0028841.g003Figure 3 Western blotting analysis of CALU-3 and HCT116 cells TKI-resistant derivatives (ERL-R, GEF-R, VAN-R) following treatment with sorafenib.
Western blotting analysis of C-RAF, B-RAF, MEK and MAPK activation following treatment with the indicated concentration of sorafenib of lung adenocarcinoma CALU-3 and colorectal cancer HCT116 cells TKI-resistant derivatives (ERL-R, GEF-R, VAN-R ). β-actin was included as a loading control.
Effects of sorafenib treatment on the invasion, migration and anchorage-independent growth of TKI-resistant CALU-3 and HCT116 cancer cells
It has been suggested that cancer cells undergoing resistant to anti-EGFR drugs could gain a more aggressive and metastatic phenotype with increased ability to invade, migrate and to form colonies in semisolid medium [15]. Therefore, we evaluated these properties in TKI-sensitive WT CALU-3 and HCT116 cancer cells and in their TKI-resistant derivatives. As illustrated in Figure 4, WT CALU-3 and HCT116 cells demonstrated little or no ability in invasion and migration. On the contrary, all TKI-resistant CALU-3 and HCT116 cell lines exhibited significant invasive and migratory abilities. Moreover, their anchorage-independent colony growth was increased of approximately 3-fold as compared to WT cells (Figure 4). Collectively, these results suggest that cancer cell lines with acquired resistance to erlotinib, gefitinib and vandetanib have acquired a more invasive and, potentially, more metastatic behaviour.
10.1371/journal.pone.0028841.g004Figure 4 Effects of treatment with sorafenib on the invasive, migratory and anchorage-independent colony forming capabilities of TKI-resistant CALU-3 and HCT116 cancer cells.
Anchorage-independent growth (A), migration (B) and invasion (C), were evaluated in TKI-resistant CALU-3 and HCT116 derivatives (ERL-R, GEF-R, VAN-R) after treatment with the indicated concentrations of sorafenib. The results are the average ± SD of three independent experiments, each done in triplicate. Representative pictures are shown for the migration and invasion abilities of CALU-3 WT and Resistant cell lines.
We next evaluated the effects of sorafenib on the invasive and migratory capabilities of the TKI-resistant CALU-3 and HCT116 cell lines. We did not tested the effect of sorafenib treatment on TKI-sensitive WT cancer cell lines in consideration of the absence of migratory and invasive ability. A significant dose-dependent inhibition of invasion and migration was observed in all TKI-resistant cell lines following treatment with sorafenib (Figure 4).
Effects of sorafenib on TKI-resistant CALU-3 and HCT116 tumor xenografts
We finally investigated the in vivo antitumor activity of sorafenib in nude mice bearing WT CALU-3 and HCT116 or TKI-resistant CALU-3 and HCT116 cell lines which were grown subcutaneously as xenografts. In WT CALU-3 and HCT116 tumor xenografts, treatment with sorafenib caused a significant decrease in tumor size as compared to control untreated mice. For example, at day 35 from the starting of treatment, the mean tumor volume in mice bearing WT tumor xenografts and treated with sorafenib was respectively 38% and 31% in CALU-3 and HCT116 as compared to control untreated mice (Figure 5, 6). Also in mice bearing ERL-R, GEF-R or VAN-R CALU-3 and HCT116 tumor xenografts, treatment with sorafenib induced a significant reduction in tumor growth (Figure 5, 6). In this respect, at day 35 from the starting of treatment, the mean tumor volumes in the sorafenib-treated mice ranged between 27% and 40%, as compared to control untreated mice.
10.1371/journal.pone.0028841.g005Figure 5 Antitumor activity of the sorafenib in parental and TKI-resistant CALU-3 xenografts.
A, Parental (WT) CALU-3 cancer cells; B, GEF-R CALU-3 cancer cells; C, VAN-R CALU-3cancer cells; D, ERL-R CALU-3 cancer cells. Athymic nude mice were injected subcutaneously into the dorsal flank with 107 cancer cells. After 7 to 10 days (average tumor size, 75 mm3), mice were treated as indicated in Materials and Methods for 5 weeks. Each treatment group consisted of 8 mice. Data represent the average (±SD). Student's t test was used to compare tumor sizes among different treatment groups at day 35 following the start of treatment. A, CALU-3 WT: sorafenib versus control (two-sided p<0.001); B, CALU-3 GEF-R: sorafenib versus control (two-sided p<0.001). C, CALU-3 VAN-R: sorafenib versus control (two-sided p<0.001); D, CALU-3 ERL-R: sorafenib versus control (two-sided p<0.001).
10.1371/journal.pone.0028841.g006Figure 6 Antitumor activity of the sorafenib in parental and TKI-resistant HCT116 xenografts.
A, Parental (WT) HCT116 cancer cells; B, GEF-R HCT116 cancer cells; C, VAN-R HCT116 cancer cells; D, HCT116 cancer cells. Athymic nude mice were injected subcutaneously into the dorsal flank with 107 cancer cells. After 7 to 10 days (average tumor size, 75 mm3), mice were treated as indicated in Materials and Methods for 5 weeks. Each treatment group consisted of 8 mice. Data represent the average (±SD). Student's t test was used to compare tumor sizes among different treatment groups at day 35 following the start of treatment. A, HCT116 WT: sorafenib versus control (two-sided p<0.001); B, HCT116: sorafenib versus control (two-sided p<0.001). C, HCT116 VAN-R: sorafenib versus control (two-sided p<0.001); D, HCT116 ERL-R: sorafenib versus control (two-sided p<0.001).
Discussion
Activation of the EGFR and the VEGFR pathways play a key role in the development and progression of the majority of epithelial cancers including NSCLC and CRC. However, only a subgroup of patients benefits of treatments with drugs targeting the EGFR or the VEGFR pathways [16]. Indeed, even in initially responding patients secondary or acquired resistance occurs causing cancer progression and treatment failure. Several molecular mechanisms have been suggested to explain the acquisition of cancer cell resistance to molecularly targeted anti-cancer drugs [16].
Cancer cell resistance to EGFR antagonists could be due to several reasons. Host related mechanisms, such as defective immune-system activity, rapid metabolism or poor absorption, are responsible for intrinsic or primary resistance. Moreover, the genetic instability of these cells generates cancer cell clones with an acquired resistance following prolonged exposure to EGFR inhibitors. Since EGFR antagonists interfere with the activation of several intracellular pathways that control cell proliferation, survival, apoptosis, metastatic capability, invasion and tumor-induced angiogenesis, it is clear that several different molecular changes could be responsible for the development of resistance to these inhibitors [16].
In the present study, we found that cancer cells which develop resistance to the EGFR tyrosine kinase inhibitors following long time treatment (acquired resistance) exhibit activated RAS/RAF/MAPK and AKT pathways. The EGFR-independent activation of these downstream pathways makes cancer cells insensitive to the EGFR inhibition and represents one of the most common reported causes of resistance to EGFR-targeted therapy in solid tumors.
The constitutive activity of at least one of these two pathways has been demonstrated to be able to defines a resistant phenotype unaffected by the treatment with gefitinib and cetuximab [2], [17].
Persistent phosphorylation of the RAS/RAF/MAPK and/or AKT pathways can be explained with the activation of cell surface receptors other than EGFR such as insulin-like growth factor-1 receptor (IGF-1R) and MET [18]–[21], which are known to be iperexpressed or activated in the presence of persistent inhibition of the EGFR.
Moreover, an increased receptor-independent activity of the RAS/RAF/MAPK and/or AKT pathways could results from direct gene amplification, activating/inactivating mutations or loss of molecular regulator [22], [23].
However, whereas inhibition of the AKT pathway does not interfere with the proliferation of resistant cells, the inhibition of the RAF/MEK/MAPK pathway by sorafenib treatment strongly reduces cell growth and survival. Indeed, among all the molecular targets of sorafenib, only C-RAF and B-RAF resulted activated in resistant cell lines probably by upstream receptors not yet tested in this work; however this information suggests that the activation of RAF-dependent intracellular signals could be an important mechanism in the acquisition of resistance to anti-EGFR therapy.
The RAS/RAF/MAPK signaling pathway is a promising therapeutic target given its central role in regulation of mammalian cell proliferation, relaying extracellular signals from ligand-bound receptor tyrosine kinases at the cell surface to the nucleus via a cascade of specific phosphorylation events. The mutational status of RAS and B-RAF genes has been demonstrated to affect the sensitivity of tumor cell lines to inhibitors of the EGFR [24]. However, therapeutic targeting of the RAS pathway has so far been unsuccessful. RAF serine-threonine kinases are the principal effectors of RAS and are considered an important target for cancer therapy. Agents such as sorafenib that bypass RAS and inhibit effector molecules downstream of the mutant GTPase (e.g. RAF) are being evaluated. Preclinical data have suggested that sorafenib inhibits cell growth by inducing G1 arrest in NSCLC cell lines independent of KRAS genotype [8].
Another possible mechanism of resistance to the EGFR inhibition may be an increased angiogenic potential through enhanced endothelial cell proliferation and permeabilization. On the basis of this informations, several preclinical studies have been realized with the intent to discover the effects of a combined targeting of the erbB and VEGF pathways by using different approaches [25]–[29]. In addition to the option of using anti- EGFR therapies in combination with anti-VEGF drugs, a series of tyrosine kinases that block both the EGFR and the VEGF receptor TK were developed, such as vandetanib, which has demonstrated significant activity as single agent and in combination with traditional chemotherapeutics in several human tumor types [27]–[29].
Often, EGFR inhibitor-resistant human cancer cell lines exhibit, as common feature, VEGFR overexpression, increased secretion of VEGF and placental growth factor, and augmented migration capabilities. Sorafenib inhibits several RTKs that participate in neovascularization, including vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 [30]. Inhibition of angiogenesis might thus be expected to contribute to the inhibition of tumor growth by this drug in addition to its effects on RAF signaling. Although sorafenib was previously shown to inhibit the growth of a variety of human tumor xenografts in mice [8], it has been difficult to measure the relative contributions of its antiangiogenic activity and its direct antitumor activity mediated by RAF inhibition. Moreover, in our work, the development of human cancer cells resistant to vandetanib, excluded the possibility that sorafenib'efficacy may depend on the inhibition of the VEGFR.
Evidence of a positive interaction between sorafenib and anti-EGFR drugs have recently been provided by our group [12]. Preclinical evidences supported a strong anti-proliferative and anti-migratory effects in NSCLC and CRC cancer cell lines following the combination with sorafenib plus ant-EGFR drugs [12]. Moreover, a recent phase II clinical study supported the combination of erlotinib and sorafenib in elderly patients with advanced NSCLC in light of the higher 1-year survival rate [30].
In the present study, we have provided evidences that sorafenib is active in inhibiting tumor cell growth in vitro and in vivo of human cancer cells resistant to inhibitor of the EGFR and/or VEGFR. The activity of sorafenib is strictly linked to its ability to block RAF signaling through the RAS/RAF/MEK/MAPK pathway.
Methods
Cell lines, drugs and chemicals
The human NSCLC CALU-3 and the human CRC HCT116 cell lines were provided by the American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS; Life Technologies, Gaithersburg, MD) in a humidified atmosphere with 5% CO2. Gefitinib and vandetanib were provided by AstraZeneca, Macclesfield, UK; erlotinib was provided by Roche, Basel, Switzerland; sorafenib was provided by Bayer Schering Pharma, Leverkusen, Germany. Primary antibodies against P-EGFR (Tyr1173), EGFR, P-MAPK44/42 (Thr202/Tyr204), MAPK44/42, P-AKT (Ser473), AKT, P-MEK (Ser217/221), MEK, P-B-RAF (ser 445), P- C-RAF (ser 338), survivin were obtained from Cell Signaling Technology, Danvers, MA, USA. Cell invasion and migration assay kits were obtained by Chemicon, Millipore, CA, USA. All other chemicals were purchased from Sigma Aldrich, MO, USA.
Establishment of CALU-3 and HCT116 cancer cell lines with acquired resistance to different TKIs
Over a period of 12 months, human CALU-3 lung adenocarcinoma cells and human HCT116 colorectal carcinoma cells were continuously exposed to increasing concentrations of either gefitinib, erlotinib or vandetanib, as previously described (12). The starting dose was the dose causing the inhibition of 50% of cancer cell growth (IC50) for each EGFR inhibitor (i.e.: erlotinib, 3 µM; gefitinib, 6 µM; vandetanib, 1 µM). The drug dose was progressively increased to 15 µM in approximately two months, to 20 µM after other two months, to 25 µM after additional two months, and, finally, to 30 µM for a total of 12 months. The established resistant cancer cell lines were then maintained in continuous culture with the maximally achieved dose of each TKI that allowed cellular proliferation (30 µM for each drug).
Cell proliferation assay
Cancer cells were seeded in 96-well plates and were treated with different drugs, such as erlotinib, gefitinib, vandetanib or sorafenib for 72 hours. Cell proliferation was measured with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The IC50 were determined by interpolation from the dose-response curves. Results represents the median of three separate experiments each performed in quadruplicate.
Western blotting analysis
Following treatment, cancer cells were lysed with Tween-20 lysis buffer (50 mmol/L HEPES, pH 7.4, 150 mmol/L NaCl, 0.1% Tween-20, 10% glycerol, 2.5 mmol/L EGTA, 1 mmol/L EDTA, 1 mmol/L DTT, 1 mmol/L phenylmethylsulfonylfluoride, and 10 µg/mL of leupeptin and aprotinin) and sonicated. Equal amounts of protein were analyzed by SDS-PAGE. Thereafter, proteins were transferred to nitrocellulose membranes and analyzed by specific primary antibodies, as indicated in the experiment. Proteins were detected via incubation with horseradish peroxidase–conjugated secondary antibodies and ECL chemiluminescence detection system.
Invasion assay
The invasive ability in vitro was measured by using transwell chambers, according to the manufacturer's protocol. Briefly, cells were seeded onto the membrane of the upper chamber of the transwell at a concentration of 2×105/ml in 500 µl of RPMI medium and were left untreated or treated with the indicated doses of sorafenib for 24 hours. The medium in the upper chamber was serum-free. The medium at the lower chamber contained 10% FBS as a source of chemo-attractants. Cells that passed through the Matrigel coated membrane were stained with Cell Stain Solution containing crystal violet supplied in the transwell invasion assay (Chemicon, Millipore, CA) and photographed after 20 hours of incubation. Absorbance was measured at 562 nm by an ELISA reader after dissolving of stained cells in 10% acetic acid. Assays were performed in triplicate.
Migration assay
Cell migration was assessed using a commercially available chemotaxis assay. Briefly, cells were incubated in RPMI serum-free medium for 24 hand were left untreated or treated with the indicated doses of sorafenib, following which they were detached from flasks, suspended in quenching medium (serum-free medium containing 5% bovine serum albumin) and EDTA, and seeded into Boyden migration chamber inserts placed in a 24-well plate. The inserts contain a microporous membrane with an 8-µm pore size. Inserts were placed over wells containing serum-free media plus chemo-attractant (10% FBS). After a 48-h treatment period, cells/media were discarded from the top side of the migration chamber insert and the chamber was placed in the wells of a new 24-well plate containing cell detachment solution. Following incubation for 30 min at 37°C, the insert was discarded, and a solution of lysis buffer and CyQuant GR dye was added to each well. CyQuant is a green fluorescent dye that exhibits strong enhancement of fluorescence when bound to cellular nucleic acids released by the lysis buffer, enabling assessment of the relative number of migrated cells. Fluorescence was determined with a fluorimeter at 480/520 nm. Assays were performed in triplicate.
Growth in soft agar
Cells (104 cells/well) were suspended in 0.5 mL 0.3% Difco Noble agar (Difco, Detroit, MI) supplemented with complete culture medium. This suspension was layered over 0.5 mL 0.8% agar-medium base layer in 24 multiwell cluster dishes (Becton Dickinson, Lincoln Park, NJ) and treated with different concentrations of sorafenib. After 14 days, cells were stained with nitro blue tetrazolium (Sigma, St. Louis, MO) and colonies larger than 0.05 mm were counted. Assays were performed in triplicate.
Tumor xenografts in nude mice
Four- to six-week old female balb/c athymic (nu+/nu+) mice were purchased from Charles River Laboratories (Milan, Italy). The research protocol was approved by the Second University of Naples Animal Care and Use Committee (IT2010/20299). Mice were maintained in accordance with the institutional guidelines of the Second University of Naples Animal Care and Use Committee. Mice were acclimatized for one week prior to being injected with cancer cells and injected subcutaneously with 107 CALU-3 (WT, ERL-R, GEF-R, or VAN-R) cells or with 107 HCT116 (WT, ERL-R, GEF-R, or VAN-R) cells, that had been resuspended in 200 µL of Matrigel (Becton Dickinson). When established tumors of approximately 75 mm3 in diameter were detected, mice were treated with oral administrations of sorafenib (50 mg/kg/day), for the indicated time periods. Each treatment group consisted of 8 mice. Tumor volume was measured using the formula π/6 x larger diameter x (smaller diameter)2.
Statistical analysis
The Student's t test was used to evaluate the statistical significance of the results. All P values represent two-sided tests of statistical significance. All analyses were performed with the BMDP New System statistical package version 1.0for Microsoft Windows (BMDP Statistical Software, Los Angeles, CA).
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by a grant from the Associazione Italiana per la Ricerca sul Cancro (AIRC), Milan, Italy. Floriana Morgillo is the recipient of an European Society of Medical Oncology (ESMO) translational research fellowship. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Int J DentInt J DentIJDInternational Journal of Dentistry1687-87281687-8736Hindawi Publishing Corporation 2219093210.1155/2011/548068Research ArticleEvaluation by an Aeronautic Dentist on the Adverse Effects of a Six-Week Period of Microgravity on the Oral Cavity Rai Balwant
1, 2, 3
*Kaur Jasdeep
1, 4
Foing Bernard H.
5
1JBR Institute of Health Education Research & Technology, Utrechtseweg 64, 3704 HE Zeist, The Netherlands2Kepler Space University, Bluffton, SC 29909, USA3VU Amsterdam, de Boelelaan 1105, 1081 HV Amsterdam, The Netherlands4Catholic University, Leuven, Belgium5Faculty of Earth & Life Sciences, Free University Amsterdam, ILEWG & ESTEC Noordwijk, 1081 HV Amesterdam, The Netherlands*Balwant Rai: [email protected] Editor: Francesco Carinci
2011 10 12 2011 2011 5480682 7 2011 7 10 2011 8 10 2011 Copyright © 2011 Balwant Rai et al.2011This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Objective. HDT bed rest condition is a simulated microgravity condition in which subject lies on bed inclined −6 degree feet up. To determine the influence of a simulated microgravity (HDT bed rest) on oral cavity, 10 healthy male volunteers were studied before, during, just after, and after 6 weeks of the simulated microgravity condition of −6° head-down-tilt (HDT) bed rest. Materials and Methods. Facial nerve function, facial sensation, chemosensory system, salivary biomarkers were measured. Results. Lactate dehydrogenase, MIP 1 alpha, malonaldehyde, 8-hydroxydeoxyguanosine, and thiocyanate were found to increase significantly, while flow rate, sodium, potassium, calcium, phosphate, protein, amylase activity, vitamin E and C, and mouth opening were decreased in simulation environments in contradiction to normal. The threshold for monosodium glutamate (MSG) and capsaicin increased during microgravity as compared to normal conditions. Moderate pain of teeth, facial oedema, mild pain, loss of sensation of pain and temperature, decreased tongue, and mandibular movement in simulation microgravity environments were observed. Conclusions. These results suggest that reversible effect of microgravity is oedema of face, change in taste, abnormal expression of face, teeth pain, and xerostomia. Further study will be required on large scale on long-term effects of microgravity on oral cavity to prevent the adverse effects.
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1. Introduction
For many years, the prevailing concept in space human factors research has been that microgravity has an impact on human physiology and astronauts are faced with several health risks during both short- and long-duration spaceflights. Some of these health problems include bone loss, muscle atrophy, cardiac dysrhythmias, and altered orientation [1, 2]. It has been reported that saliva composition is changed and oral health compromised during simulated skylab mission [3, 4]. Previous studies observed some adverse effects of simulated microgravity (HDT bed rest condition) on oral cavity [5–12]. HDT bed rest condition is a simulated microgravity condition in which subject lies on bed inclined −6 degree feet up [2].This study is extension of our previous studies [6]. To the best of our knowledge, no long-term study has been conducted on effect of oral cavity such as facial nerve functions, prevalence and pattern of oral disease, bone loss, tooth pain, salivary flow rate, and different salivary biomarkers. The prevalence of oral diseases such as dental caries, periodontal disease and cancer, and bone loss was estimated by salivary biomarkers, which is noninvasive. Hence, this study was planned to evaluate the effect of simulated microgravity on oral cavity.
2. Materials and Methods
The subjects of this investigation were 10 male volunteers (aged 22–30 years, body mass index 18.7 (2.6), participated in a 6-week 6° HDT bed-rest position). We chose 6-week duration of study taken into account for three continues ISS mission for astronaut. Females were not selected in this study because females were not willing to take part in this study. Each subject was given a detailed explanation of the experimental protocol and provided written and verbal consent. The average energy, calcium, and vitamin D expended by the subjects during the simulation was 2300 kcal/day (range 2080–3010 kcal/day), 1200 mg/day, and 10 μg, respectively. Each subject completed a questionnaire on their medical and dental history to determine the status of systemic diseases, smoking, and history of alcohol and drug use. They also underwent a clinical examination for systemic diseases, chronic diseases such as autoimmune diseases, cancer, cardiovascular diseases such as cerebrovascular diseases, heart failure, ischemic cardiopathy, chronic fatigue syndrome, chronic graft-versus-host disease (GVHD), chronic hepatitis, chronic pain syndromes, chronic osteoarticular diseases, chronic renal failure, chronic respiratory diseases, and diabetes mellitus, and oral and dental diseases. Patients were excluded if they had a systemic or chronic disease, an oral or dental disease, if they were smokers, or if they had a history of alcohol or drug abuse. Subjects with caries, cavities, sealants, nontreated cavities, mal-adjusted crowns, and previous channel treatments were excluded. All parameters and samples were taken just before HDT and last day of HDT. Facial function tests, mouth opening, jaw movements, tongue movements, facial sensation (touch, pressure, temperature), taste, odor, perception of food, salivary vitamins E and C, lactate dehygrogenase isoenzyme, MIP 1 alpha, glucosyltransfer B, malonaldehyde, 8-hydroxydeoxyguanosine, thiocyanate, salivary contents, and salivary flow rate were measured as in our previous studies [5–12]. To measure pain, we used a visual scale analog ranging from 0.5 (very mild pain) to 5 (severe pain). Change in mouth opening was measured. Salivary vitamin C and vitamin E were estimated by HPLC. HPLC separations were accomplished at room temperature (approximately 370°C) with a cecil liquid chromatography system (series, 1100, USA) consisting of a sample injection valve with a 30 u and sample loop, an ultraviolet (mv) spectrophotometric detector, a integrator, and techsphere ODS-2 packed (4 um particle and 80.4 pore size) column (250 × 4.6 ID) with a methanol: acetonitrile: chloroform (45 : 41 : 10 V/V) as mobile phase at 1 mL/min flow rate. All procedures were performed under light protected conditions [6, 13]. MIP 1 alpha, lactate dehygrogenase isoenzyme, and amylase levels were measured by using ELISA kit (MIP 1 alpha, R&D Systems, Inc._Minn, USA; L type LDH J; Wako Chemical Industry, Osaka, Japan; Yanaihara Institute, Shizuoka, Japan). We determined glucosyltransfer B present in the salivary samples using an ELISA assay by employing a kit assay obtained from Kirkegaard and Perry Laboratories, Gaithersburg, Md, USA [6]. Salivary 8-OHdG levels were measured by a competitive ELISA kit (Japan Institute for control of Aging, Shizuoka, Japan) [6]. Lipid peroxidation product MDA was analyzed by thiobarbituric acid (TBA) reaction [6, 14]. Saliva was assessed colorimetrically by a spectrophotometer and using affiliated kits for analysis of saliva sodium, potassium, calcium, and phosphate. The total protein concentration was measured by the biuret method [6]. Salivary 8-OHdG levels were measured by using (ELISA kit-competitive method, Cayman Chemical, USA). The data were analyzed using SPSS version 11 and applied the student t-test.
3. Results
Lactate dehygrogenase, MIP 1 alpha, malonaldehyde, 8-hydroxydeoxyguanosine, and thiocyanate were significantly higher; while flow rate, sodium, potassium, calcium, phosphate, protein, amylase activity, and vitamins E and C were decreased in HDT as compared to normal (Table 1, P < 0.05). The threshold for MSG and capsaicin was increased about 1.5 dilution step, while sodium chloride was decreased about 2 dilution during microgravity as compared to normal (Table 2). Pain scores were increased in teeth, mandibular angle regions, sublingual and submandibular opening duct regions HDT as compared to normal (Table 3). Face, submandibular and sublingual duct opening area showed swelling in HDT as compared to normal (Table 3). Face showed abnormal expression during HDT. Mouth opening, tongue, and mandibular movement were decreased in HDT as compared to normal, although levels were not statically significant (Tables 2 and 3).
4. Discussion
Flow rate, sodium, potassium, calcium, phosphate, and protein levels were increased in simulation environments as compared to normal, while same findings were observed in urine [6, 15]. Increased bone resorptions contribute significantly to raise the salivary state of saturation with respect to the calcium salts, namely, calcium oxalate and calcium phosphate [6]. In addition, other environmental and dietary factors may adversely affect salivary composition and increase stone formation risk during space flight [15, 16]. Although observations to date have suggested that there could actually be a reduced food intake during the early phase of flight, crew members on longer-duration flights could also increase food intake and be at increased risk for salivary stone formation [6, 17]. The most important effect of restricting energy intake is on calcium and bone metabolism. The MIP 1 alpha level was decreased in microgravity which is potential markers of bone loss [9, 16]. In agreement with earlier reports by Parazynski et al. [17], an increased fluid excretion was observed in simulated microgravity, which leads to dehydration and finally to a reduction of plasma volume and an increase in the hematocrit. A reduction of plasma volume may result in increase in serum electrolyte levels, and therefore serum osmolality and urine osmolality increase too. The plasma volume decreases together with increases in serum and urine osmolality and electrolyte levels, influences body fluid regulation by activating hormonal regulatory factors, that is, vasopressin, rennin, and aldosterone [17]. In agreement with earlier reports by Kirsch et al. [18], plasma volume contraction occurs quickly in microgravity. This hemoconcentration probably results from increased upperbody vascular pressures in microgravity [16] and perhaps reduced interstitial pressures; both factors would encourage transcapillary fluid filtration into upperbody interstitial spaces, and substantial filtration can occur in minutes [19].
The levels of calcium were increased in microgravity as compared to control as reported in our previous study [6]. MIP 1 alpha was elevated during HDT condition as biomarker of bone loss as reported in previous studies [6, 16]. Loss of bone calcium during bed rest is the result of increased bone resorption by osteoclasts and it is not due to endocrine changes. Parathyroid hormone (PTH) promotes the release of calcium from bone and stimulates the kidneys to release the active form of Vitamin D, 1,25-dihydroxyvitamin D, which increases intestinal calcium absorption [18–23]. Insufficient calcium consumption leads to a reduction in serum calcium levels and thereby to a secretion of parathyroid hormone (PTH) and calcitriol synthesis. Both rises in PTH and calcitriol induce an increase in calcium retention either from the intestine or from bone. Based on that, a long-lasting insufficient calcium intake and insufficient vitamin D are the main factors leading to a decrease in bone mineral density [20, 21]. It might lead to periodontitis and facture of facial bones.
The decreased levels of vitamins E and C and increased malonaldehyde levels denoted increase in free radical activity as in microgravity environments [21]. So the free radical activity increased in microgravity as compared to normal gravity as reported in previous studies [21]. The 8-hydroxy deoxyguanosine levels were increased in saliva in microgravity environments as compared to normal, it may be due to increase in oxidative stress [6, 17, 21]. The threshold for MSG and capsaicin increased about 1.5 dilution step, while sodium chloride decreased about 2 dilution during microgravity as compared to normal. It might be due to fluid shift mechanism. It could be due to physiological changes including an upward shift of body fluids toward the head, which may lead to an attenuation of the olfactory component in the flavour of foods, pressing the nerve regions or dysfunction of nerve as well as increased activity of b-AR agonists [21]. Lactate dehygrogenase isoenzyme levels increased during HDT, this implied the development of muscular atrophy as reported in previous studies [23]. Salivary glucosyltransferase B levels were increased in HDT as compared to normal as reported in previous study [6] and indicated that caries prevalence is more during microgravity. Thiocyanate levels were significantly increased in HDT as compared to normal as reported in previous study [6]. Thiocyanates (SCN–) are ubiquitous in nature. There are indispensable parts of host defense system that act as a substrate for lactoperoxidase (LPO). LPO oxidizes saliva SCN–thereby generating antimicrobial agent hypothiocyanite (OSCN–) [6]. Pain scores increased in teeth and mandibular angle regions in HDT as compared to normal. It might be due to the pain mechanism appears somatic due to excessive expansion. Sublingual and submandibular opening duct regions, abnormal facial expression, loss of sensation of pain and temperature, decreased tongue, and mandibular movements in simulation microgravity environment were observed due to fluid shift mechanism.
The limitations of this study are short duration, small sample size, female subjects who were not included, no followup of volunteers, and other factors. These results suggest that reversible effect of microgravity is oedema of face, change in taste, abnormal expression of face, teeth pain, and xerostomia. The nonreversible effects of microgravity such as periodontal diseases and dental caries occur in different pattern than normal, and stone formation in salivary duct, precancer or cancer, fracture of maxillary and mandibular bone, and xerostomia are more prevalent in astronauts as compared to normal persons. Further study will be required on large-scale and on long-term effects of microgravity on oral cavity to prevent the adverse effects.
Table 1 The median (range) unstimulated salivary whole flow rate, compositions, biomarkers (sodium, potassium, calcium, phosphate, protein, lactate dehygrogenase, MIP 1 alpha, malonaldehyde, 8-hydroxydeoxyguanosine, thiocyanate, amylase activity, vitamins E, C), and mouth opening before and during last day of HDT in 10 healthy persons.
Parameters Before HDT Last day of HDT
P value
Flow rate (mL/min) 0.026 (0.01–0.03) 0.009 (0.008–0.02) <0.01
Na (mM) 13 (8.0–15.0) 12 (9.0–18.2) <0.01
K (mM) 23.6 (6.7–34.8) 22.2 (7.3–36.9) <0.05
Total calcium (mM) 3.4 (1.4–4.6) 3.9 (2.4–4.8) <0.01
Total phosphate (mM) 7.4 (1.3–11.3) 7.8 (2.1–12.1) <0.01
Total protein (mg/mL) 4.4 (1.6–13.6) 4.5 (1.7–14.2) <0.05
Cl (Mm) 22.3 (14.9–42.1) 22.0 (14.7–43.2) <0.05
Total protein output (mg/min) 0.44 (0.23–0.96) 0.42 (0.26–0.85) <0.001
Amylase activity (micro kat/L) 324 (145–567) 262 (112–345) <0.001
Vitamins E (mg/mL) 0.56 (0.32–0.76) 0.51 (0.31–0.73) <0.005
Vitamins C (mg/mL) 0.35 (0.12–54) 0.32 (0.09–0.52) <0.05
Lactate dehygrogenase (IU/L) 2.4 (1.2–3.4) 2.3 (1.6–3.7) <0.05
MIP 1 alpha (pg/mL) 17.6 (12.6–21.5) 18.1 (13.2–22.4) <0.01
Malonaldehyde (ng/mL) 2.46 (1.45–4.34) 2.65 (1.67–4.89) <0.05
8-hydroxydeoxyguanosine (ng/mL) 0.66 (0.45–1.34) 0.78 (0.51–1.43) <0.001
Thiocyanate (U/L) 34.3 (21.7–56.9) 39.8 (22.7–57) <0.001
Mouth opening (In cm) 45.4 (34.6–53.2) 42.3 (31.5–49.6) <0.005
Table 2 The square means of the thresholds of all persons before and on last day of HDT in 10 healthy persons. The thresholds are reported as the dilution series values (dilution 10 is most conc., dilution 1 is least conc.).
Parameters Before HDT Last day of HDT
P value
Sucrose 3.8 3.3 <0.05
Citric acid 3.1 2.4 <0.05
Sodium chloride 3.4 2.2 <0.05
Quinine 4.6 4.1 <0.05
MSG 6.2 6.9 <0.05
Capsaicin 6.4 7.1 <0.05
Amyl butyrate 3.2 2.8 <0.05
Methone 3.1 2.5 <0.05
Table 3 The symptoms of subjects before and on last day of HDT in 10 healthy persons.
Parameters Before HDT Last day of HDT
Teeth pain while articulation of teeth 0 1.0
Facial swelling No swelling Moderated oedema
Pain while closing or opening the mouth 0 0.5
Salivary gland Normal Moderated pain in submandibular and sublingual duct opening area and swelling
Pain while moderate pressing the facial region 0 0.5
Facial expression Normal Abnormal
Sensation tests Normal Pain and temperature sensation are not present
Movements of tongue Normal Decreased in all direction
Jaw movements Normal Decreased in all direction
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22174954PONE-D-11-2133510.1371/journal.pone.0029192Research ArticleAgricultureCropsCerealsRiceCrop DiseasesBiologyImmunologyMicrobiologyBacterial PathogensGram NegativeImmunityInnate ImmunityHost-Pathogen InteractionPlant MicrobiologyPlant SciencePlant PathologyPlant PathogensPlant MicrobiologySmall Protein-Mediated Quorum Sensing in a Gram-Negative Bacterium Protein-Mediated QSHan Sang-Wook
1
Sriariyanun Malinee
1
¤
Lee Sang-Won
1
2
Sharma Manoj
1
Bahar Ofir
1
Bower Zachary
1
Ronald Pamela C.
1
2
*
1
Department of Plant Pathology and the Genome Center, University of California Davis, Davis, California, United States of America
2
The Department of Plant Molecular System Biotechnology and Crop Biotech Institute, Kyung Hee University, Yongin, South Korea
Chakravortty Dipshikha EditorIndian Institute of Science, India* E-mail: [email protected] and designed the experiments: SWH M. Sriariyanun SWL PCR. Performed the experiments: SWH M. Sriariyanun M. Sharma OB ZB. Analyzed the data: SWH M. Sriariyanun PCR. Contributed reagents/materials/analysis tools: SWH M. Sriariyanun. Wrote the paper: SWH M. Sriariyanun PCR.
¤ Current address: Department of Chemical and Process Engineering, Thai-German Graduate School of Engineering, King Mongkut's University of Technology North Bangkok, Bangkok, Thailand
2011 12 12 2011 6 12 e2919228 10 2011 22 11 2011 Han et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.The rice XA21 pattern recognition receptor binds a type I secreted sulfated peptide, called axYS22, derived from the Ax21 (activator of XA21-mediated immunity) protein. The conservation of Ax21 in all sequenced Xanthomonas spp. and closely related genera suggests that Ax21 serves a key biological function. Here we show that the predicted N-terminal sequence of Ax21 is cleaved prior to secretion outside the cell and that mature Ax21 serves as a quorum sensing (QS) factor in Xanthomonas oryzae pv. oryzae. Ax21-mediated QS controls motility, biofilm formation and virulence. We provide genetic evidence that the Xoo RaxH histidine kinase serves as the bacterial receptor for Ax21. This work establishes a critical role for small protein-mediated QS in a Gram-negative bacterium.
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Introduction
Given the demonstrated importance of plant and animal receptors [also called pattern recognition receptors (PRRs)] of conserved microbial signatures [also called pathogen associated molecular patterns (PAMPs)], there is great interest in elucidating the biological function of these ligands [1]. We have recently shown that the rice XA21 receptor binds a sulfated peptide, called axYS22, derived from the Ax21 (activator of XA21-mediated immunity) protein from the Gram-negative bacterium, Xanthomonas oryzae pv. oryzae (Xoo). XA21/axYS22 binding triggers XA21-mediated innate immunity [2], [3].
The conservation of Ax21 in all sequenced Xanthomonas spp., Xylella fastidiosa and the human pathogen Stenotrophomonas maltophilia suggests that Ax21 serves a key biological function. To elucidate this function, we previously isolated and characterized eight genes required for Ax21 activity (rax genes). raxA, raxB and raxC encode components of a predicted type I secretion system (TOSS). Ax21 requires this RaxABC TOSS for activity and secretion [3], [4]. The RaxB protein carries two highly conserved N terminal proteolytic subdomains characteristic of transporters in Gram-positive bacteria that cleave N-terminal peptides prior to substrate secretion [4]. These data, together with the presence of a predicted N-terminal signal sequence in Ax21, suggest that Ax21 is cleaved by the RaxB transporter prior to secretion. raxST, raxP and raxQ encode enzymes involved in sulfation; and raxH and raxR encode a predicted histidine kinase and cognate response regulator, respectively [4], [5], [6]. The expression of the eight rax genes is density-dependent [7]. Their expression at low densities can be rescued by the addition of high-performance liquid chromatography (HPLC)-fractionated Xoo PXO99 supernatants. Fractions from Xoo strains lacking Ax21 activity cannot induce density dependent expression. We therefore, hypothesized that Ax21 serves as a quorum sensing (QS) factor.
QS is a process where small molecules serve as signals to recognize cell population size, leading to changes in expression of specific genes when the QS factor has accumulated to a certain threshold concentration [8]. In Gram-positive bacteria, QS is controlled by oligopeptides, whereas Gram-negative bacteria generally use acylated homoserine lactones (AHLs) or diffusible signal factors (DSF) for QS [9]. One instance of peptide-mediated QS in Gram-negative bacteria has been reported [10]. Although QS factors are abundant in the host vicinity, none have previously been shown to bind host receptors of conserved microbial signatures.
Results and Discussion
To determine if Ax21 can serve as a QS factor to regulate density-dependent expression of rax genes, we monitored rax gene expression in PXO99 and in a mutant strain lacking Ax21 (PXO99Δax21). We found that the six rax genes were highly expressed in PXO99 cultures grown to high population densities [108 colony forming unit (CFU)/ml], but not in PXO99Δax21 cultures (Table. S1). These experiments indicate that Ax21 regulates density-dependent expression of rax genes.
We next purified Ax21 using gel filtration and immobilized metal ion affinity chromatography from culture supernatants of an Xoo strain expressing biologically active mature 6x-His-tagged Ax21 (rAx21) (without the N-terminal signal sequence) (Figure S1 and S2). A 7 kDa cut-off spin column was used to remove small peptides and other small molecules from the supernatants (Figure S2A). Elution was carried out using elution buffers containing various concentrations of imidazole (Figure S2B). Western blot analysis using an anti-Ax21 antibody revealed that the 150 mM imidazole buffer-eluted fraction contains highly purified rAX21 (Figure S2B).
To test whether the mature Ax21 protein itself could restore rax gene expression to the PXO99Δax21 strain, we added rAx21 to this strain. We found that addition of the 150 mM imidazole-eluted fraction carrying rAx21, complemented rax gene expression in PXO99Δax21 whereas addition of flow-through or 250 mM imidazole buffer-eluted fractions lacking rAx21 did not (Figure 1). Furthermore, the peptides, axYS22 and axM178, derived from Ax21 that were previously identified in HPLC-fractionated Xoo PXO99 supernatants [3], did not restore rax gene expression to the PXO99Δax21 strain (Figure S4 and S5). These results conclusively demonstrate that the mature rAx21 protein serves as the QS factor and that the activity is not due to small peptides or other molecules present in the active fraction.
10.1371/journal.pone.0029192.g001Figure 1 Purified recombinant Ax21 complements density-dependent expression of raxST, raxB, and raxR in PXO99Δax21.
Expression of the raxST, raxB, and raxR genes in PXO99 (106 and 108 CFU/ml, grey), PXO99Δax21 (108 CFU/ml, black), and PXO99Δax21 (108 CFU/ml) supplemented with exogenous addition of the flow-through (FT), 150 mM (150), or 250 mM (250) imidazole buffer-eluted fractions (figure S2) was assessed. Levels of gene expression were calculated relative to 16sRNA expression. Primers specific to each gene are as listed in table S8. Data are mean of four replicates ± standard deviation (SD.).
As an additional test to investigate the nature of Ax21, we carried out liquid chromatography–tandem mass spectrometry of supernatants from PXO99Δax21(rAx21). Nine peptides spanning nearly entire Ax21 protein, except for the predicted N-terminal signal sequence were identified. These results demonstrate that the entire, mature Ax21 protein is secreted and that the predicted N-terminal signal sequence is cleaved before secretion (Figure S3).
To test if the biological activity of rAx21 is dependent on the predicted tyrosine sulfotransferase, RaxST, we isolated rAx21 from the PXO99ΔraxST strain [4] expressing rAx21. rAx21 purified from this strain, displayed significantly less activity compared with rAx21 purified from the PXO99Δax21 strain (Figure S6). These results indicate that RaxST is required for full Ax21 biological activity.
Bacteria use QS communication to regulate diverse biological processes, including motility, virulence and transition from a planktonic (free swimming) state to a sessile state, called a biofilm. To elucidate the biological function of Ax21, we compared expression profiles of PXO99 and PXO99Δax21 at three different population densities. PXO99 and PXO99Δax21 were diluted to 105 CFU/ml and continuously grown until population densities reached 106, 107 and 108 CFU/ml (representing early log, middle log, and late log phases of bacterial growth, respectively). RNA was then isolated and subjected to whole genome expression profiling. We found that 489 genes (approximately 10% of Xoo genome) are significantly differentially regulated by Ax21 (Figures S7, S8, S9 and Tables S1, S2, S3, S4, S5, S6, S7).
Ten of these genes encode proteins containing the amino acid domains GGDEF, EAL, and HD-GYP (Figure 2A). Such proteins have previously been shown to control cyclic diguanylate (c-di-GMP) turnover, a nucleotide-based secondary messenger that regulates diverse microbial phenotypes including growth, motility, virulence, and biofilm formation. In Xanthomonas spp., the RpfC/G sensor kinase and response regulator are required for DSF perception and signal transduction leading to c-di-GMP degradation through a protein containing an HD-GYP domain [11]. In the opportunistic pathogen Pseudomonas aeruginosa, AHL-mediated c-di-GMP production is regulated by a tyrosine phosphatase (TpbA) [12]. Thus, three distinct QS systems (AHL-, DSF- and Ax21-mediated) control expression of genes encoding proteins that regulate c-di-GMP turnover. Bacterial c-di-GMP has also recently been shown to trigger the innate immune response of mouse and human cells [13], [14].
10.1371/journal.pone.0029192.g002Figure 2 Whole genome transcriptomics of PXO99 vs. PXO99Δax21 at different population densities identifies Ax21-regulated genes.
A heat map represents the ratio of gene expression levels in a log2-based pseudocolor scale (red, positive; green, negative). (A) Expression pattern of genes encoding proteins predicted to regulate c-di-GMP turnover in PXO99 (Left) and PXO99Δax21 (Right). (B) Ax21 up-regulated genes that are highly expressed in early log phase including bacterial motility genes and xanthan gum biosynthesis. The functions of the genes marked by a star were assessed for phenotypes in Xoo knockout strains (Figure S13).
Our expression analysis also identified a set of genes that are up-regulated by Ax21 during early log phase (Figure 2B). These include the gumE, gumJ, and gumK genes, which encode proteins required for biosynthesis of xanthan gum, an important component of the Xanthomonas extracellular polymeric substance (EPS) [15] (up-regulated by Ax21 2.0, 1.8, and 1.8 fold, respectively in PXO99 vs. PXO99Δax21). EPS enables bacteria to adhere to each other or to a solid surface, a key component of biofilms.
To assess if Ax21 is required for biofilm formation, we examined biofilm formation in the PXO99, PXO99Δax21, and PXO99ΔraxST strains using a plate adherence assay. The PXO99Δax21 strain formed significantly less biofilms as compared with the PXO99 strain. Exogenous addition of purified rAx21 restored biofilm formation in PXO99Δax21 (Figure 3A). Aggregation assays comparing PXO99Δax21 and PXO99 revealed that Ax21 is also required for in vivo aggregation of Xoo (Figure 3B). Similar results were obtained using confocal microscopy (Figure S10). These experiments demonstrate that Ax21-mediated QS controls biofilm formation in Xoo.
10.1371/journal.pone.0029192.g003Figure 3 Ax21 regulates Xoo biofilm formation, cell aggregation, motility, and virulence.
(A) Biofilm formation of PXO99, PXO99Δax21, PXO99Δax21 supplemented with exogenous addition of the flow-through (FT), 150 mM (150), or 250 mM (250) imidazole buffer-eluted fractions (figure S2) were measured according to absorbance at A590 using the polyvinyl chloride plate assay and normalized with the value of PXO99. Bars represent the mean of ten biological replicates ± SD. (B) Aggregation assays were performed using PXO99 and PXO99Δax21 strains carrying a green fluorescent protein. Xoo strains were observed with a microscope equipped with a fluorescein isothiocyanate filter (excitation filter, 450 to 490 nm; emission filter, 520 nm; dichroic mirror, 510 nm). The bars indicate 10 µm. (C) Swimming motility of PXO99 and PXO99Δax21 strains were quantified by measuring the diameter of colony expansion in swimming motility assays. Bars represent the mean of four biological replicates ± SD. (D) Virulence of PXO99 and PXO99Δax21 was monitored at low population densities. Rice leaves (TP309 cultivar) were inoculated with PXO99, PXO99ΔraxST, and PXO99Δax21 strains using the soaking method (103 CFU/ml) for two days. Bacterial populations were determined two days after inoculation. Bars represent the mean from at least seven leaves ± SD. The experiment was repeated four times with similar results.
Our microarray data also revealed that, at early log phase, Ax21 up-regulates expression of genes involved in bacterial motility (Figure 2B). For example, fliC, fliD (flagella biosynthesis genes C and D) and the chemotaxis gene PXO_04752, are up-regulated 3.3-, 3.3-, and 16.4- fold, respectively in the PXO99 vs. PXO99Δax21 strains. To test whether Ax21 controls Xoo motility, we assayed the phenotype of Xoo PXO99 and PXO99Δax21 strains using a swimming motility plate assay. We found that the motility of PXO99 was two-fold higher than that of PXO99Δax21 (Figure 3C) indicating that Ax21 regulates Xoo swimming motility on semi-solid media.
We have previously shown that the predicted histidine kinases PhoQ and RaxH are required for Ax21-mediated activities [5], [16]. We therefore hypothesized that one of these proteins was the bacterial receptor for Ax21. In support of this hypothesis, we observed that biofilm formation in both the PXO99ΔraxH and PXO99ΔphoQ strains is reduced compared to the PXO99 strain (Figure S11). We next tested whether biofilm activity could be rescued by addition of purified rAx21 protein to these mutant strains. We found that PXO99ΔphoQ but not PXO99ΔraxH could form biofilms after complementation with rAx21 (Figure S11). These results indicate that the defect in biofilm formation in PXO99ΔphoQ is not due to perception of Ax21. In contrast, the Xoo strain carrying a mutation in raxH could no longer respond to rAx21, supporting the hypothesis that RaxH is the Ax21 receptor. How can RaxH, a predicted inner membrane bound kinase with a periplasmic sensor domain, detect the Ax21 protein? Ax21 may be imported into the periplasm via a channel located in the outer membrane. For example, the E. coli extracellular small protein, colicin M (29 kDa), is localized to the periplasm via the FhuA porin located in the outer membrane [17]. Alternatively, Ax21 may form a structure that allows it to directly integrate into the membrane.
The observation that Ax21 is a QS factor that controls density-dependent expression of genes involved in motility, c-di-GMP turnover, and biofilm formation, suggests that PXO99Δax21 strains would be impaired in virulence. However, earlier experiments indicated no significant changes in virulence phenotypes when PXO99Δax21 infection was tested by clipping rice leaves with bacteria dipped in high-density cultures (108 CFU/ml) [3], [18]. Because under field conditions, Xoo infection through hydathodes or wounded sites requires only a low inoculation density (104 CFU/ml) to initiate infection [19], we hypothesized that an effect of Ax21 on virulence has been masked by the high-density inoculation approach.
To test this hypothesis, we established a new inoculation method. Xoo strains PXO99, PXO99ΔraxST, and PXO99Δax21 strains were cultured in PSA (peptone sucrose media) plates and then diluted with water to 103 CFU/ml. Unclipped rice leaves were then soaked in bacterial suspensions for two days, and bacterial populations assessed two days following inoculation. We found that the population of the wild-type PXO99 strain is two fold higher than that of the PXO99ΔraxST and PXO99Δax21 strains using the low density soaking method (Figure 3D). In contrast, the populations of all three strains are similar two days after inoculation using the high-density scissor clipping method (Figure S12). These results indicate that ax21 and raxST are required for full virulence during early stages of infection that mimic field conditions.
To investigate the mechanism with which Ax21 regulates motility, virulence, and biofilm formation, we generated Xoo strains mutated for twelve genes that are regulated by Ax21 (table S9 with the primers listed in table S10). Virulence of five strains (PXO99Δ01391, PXO99Δ01395, PXO99Δ02671, PXO99Δ04882, and PXO99Δ06202) is partially or completely lost in the knockout mutants. Six strains (PXO99Δ00678, PXO99Δ01395, PXO99Δ02637, PXO99Δ02671, PXO99Δ04882, and PXO99Δ06202) displayed a reduction in biofilm formation and eleven strains partially lost swimming motility (Figure S13). These analyses indicate that Ax21 exerts its complex control through regulation of target genes.
The discovery that a small protein from a Gram-negative bacterium has a dual role in QS and in activation of the host innate immune response has not previously been demonstrated. We do not, however, believe this is an anomaly or that the biological importance of Ax21 is restricted to plant pathogens. For example, we previously reported that Ax21 is also conserved in the nosocomial pathogen Stenotrophomonas maltophilia and proposed a similar role for Ax21 in this species [3]. Consistent with our hypothesis, a synthetic Ax21 protein has been shown to regulate gene expression, motility, and biofilm formation in S. maltophilia, extending our findings to an animal pathogen [20]. Furthermore, analysis of the genome sequences of other Gram-negative bacteria reveals an abundance of TOSS predicted to cleave N-terminal leader sequences and secrete mature proteins [21].
These results suggest that not only do these other Gram-negative bacteria use N-terminal processed small proteins for QS, but that some of the hundreds of the predicted receptors in rice and other species, for which no corresponding conserved microbial signature has yet been identified, detect such molecules [22]. Such knowledge can be used to develop reagents to immunize hosts against infection or antagonists to disrupt QS-mediated virulence activities and biofilm formation [23], a process thought to be involved in 65-80% of bacterial infections of plants and animals [24].
Materials and Methods
Bacterial strains and growth conditions
The Xanthomonas oryzae pv. oryzae (Xoo), Escherichia coli strains and plasmids used are listed in Table S11. Peptone sucrose media (PSM) [25] and nutrient broth (NB) (Difco Laboratories), containing 20 µg/ml of cephalexin (MP Biomedicals), and/or other antibiotics as appropriate were used for growing cultures of Xoo at 28°C. E. coli strains were cultured in Luria-Bertani (LB) medium at 37°C. For E. coli, kanamycin at 50 µg/ml, ampicillin at 100 µg/ml, cephalexin at 15 µg/ml and gentamycin at 25 µg/ml (10 µg/ml for Xoo) were used for selection of transformants. Rice varieties Taipei 309 (TP309, a rice line susceptible to PXO99 and a TP309 transgenic line (106-17-3-37) carrying the Xa21 gene (TP309-XA21, resistant to Xoo strain PXO99) [2] were used to assess biological activity of the Xoo strains.
Construction of Xoo knockout mutants by marker exchange mutagenesis
Xoo knockout mutants were generated using marker exchange mutagenesis [26]. For homologous recombination in PXO99, DNA fragments were synthesized using the polymerase chain reaction (PCR) method. Primer sequences used for each gene are shown in Table S8. PCR was carried out using Programmable Thermal Controller (MJ Research). The amplified DNA fragments were cloned into the pGEM®-T-easy vector. A kanamycin-resistant cassette was inserted into the appropriate restriction enzyme cleavage site. Sequencing of the The pGEM®-T-easy constructs carrying the mutagenized genes was performed using the dideoxy chain termination method and an automated sequencer (Model 400 I, Li-Cor) with M13 forward and reverse primers or specific primers designed based on sequencing data. Confirmed constructs were then introduced into competent PXO99 wild type cells. After electroporation, the cells were incubated for 3 h at 28°C with PS broth media, and then spread on PS agar plates containing 50 µg/ml of kanamycin. Colonies that grew on those plates were duplicated onto PS agar plates containing 50 µg/ml of kanamycin as well as plates containing 50 µg/ml of kanamycin/ampicillin in order to select for double cross-over events. Colonies that only grew on the kanamycin plates were collected and confirmed as insertional mutants using PCR. The primers that were used for gene cloning were used also used to confirm that each gene had been knocked out in these strains.
Construction of recombinant Ax21 and generation of Xoo mutants expressing the recombinant protein
To generate recombinant ax21, a 21-bp 6x-His tag was added to the C-terminal region as follows: The ax21 gene was amplified from the genome of PXO99 with the following primers: 5′- GTCGACGATGCAGCTCCATCCGTGTG-3′ and 5′- GTCGACTTAATGATGATGATGATGATGCCAGCTGAAGCGCGGGCCGA-3′ using the PCR method with Taq polymerase in a Programmable Thermal Controller (MJ Research Inc.). The amplified fragment was then inserted into pGem®-T Easy (Promega). The pGem®-T Easy vector containing the ax21 gene (pGem-Ax21) was extracted and treated with the restriction enzymes SalI. This fragment was then inserted into the pML122 vector [26], [27] to promote expression (pML122-rAx21) in Xoo and introduced into the PXO99Δax21
[3] by electroporation. After incubation in PS broth for 3 hours, the cells were spread onto PS agar plates containing 10 µg/ml of gentamycin and 50 µg/ml of kanamycin [PXO99Δax21(rAx21)]. The electroporated strain was confirmed to carry the ax21 gene by PCR using primer combinations specific for raxST and pML122 vector sequences. Expression of the recombinant Ax21 protein was verified by western blot analysis using a His-tag antibody and Ax21-specific antibody. For the anti-Ax21 antibody, synthetic peptides and monospecific antibodies were generated by Pacific Immunology. Detailed information about their methods can be obtained at Pacific Immunology (http://www.pacificimmunology.com/). Ax21 activity of the PXO99Δax21(rAx21) strain was confirmed using the scissors clipping method [3] (Figure S1).
Purification of the recombinant Ax21 protein
The procedure for purification of recombinant Ax21 from strain PXO99Δax21 expressing rAx21 is shown in fig. S2A. PXO99Δax21(rAx21) was incubated in 6 liters of nutrient broth for 3 days, harvested, and washed twice with sterilized water. The washed cells were incubated in 20 ml of modified M9 minimal media [6] for 3 days. The supernatants were collected and filtered with a 0.22 µm syringe filter to remove bacteria. Small molecules [(<7 kDa) including peptides and ions] were removed using a Zeba spin desalting column (Thermo scientific). Desalted samples were fractionated on a Superdex 75 (GE Health care) in 50 mM Na2HPO4, pH 8.0, 10 mM EDTA, 150 mM NaCl, and 0.1% Triton X-100. The flow rate of the column was set at 1 ml/min and the eluate was fractionated into 1 ml fractions. The fractions containing rAx21 were desalted again using the Zeba spin desalting column. One ml of the 50% Ni-NTA slurry was added to desalted samples and the Ni-NTA mixture incubated in 50 mM NaH2PO4, pH 8.0, 300 mM NaCl, and 10 mM imidazole for 30 minutes at room temperature. The Ni-NTA mixture was loaded onto a Poly-Prep chromatography column (BIO-RAD) and eluted with 700 µl of 50 mM Na2HPO4, pH 8.0, 300 mM NaCl containing 50, 100, 150, 200, 250, or 500 mM imidazole. All fractions were confirmed by western blot analysis using anti-Ax21 and anti-His-tag antibodies. After desalting using the Zeba spin desalting column, purified rAx21 was used for the complementation experiments.
Silver staining
After rAx21 was purified from PXO99Δax21(pML122-rAx21) as described above, the fractions were separated using a 12% Sodium dodecyl sulfate polyacrylamide gel. The gel was stained with Silver stain plus (Bio-Rad) as previously described [3] (Figure S3). The fraction eluted with 150 mM imidazole buffer contained highly purified rAx21 (Figure S3, lane 6). This fraction as well as control fractions, the flow-through fraction (Figure S3, lane 3) and 250 mM imidazole buffer-eluted fraction (Figure S3 lane 8) were desalted and the used for the biological assays.
Western blot analysis
Samples were fractionated in a 12% polyacrylamide gel. The proteins were then transferred onto Hybond ECL nitrocellulose membranes (Amersham Pharmacia Biotech) using standard procedures. The membranes were incubated with blocking solution consisting of 5% (w/v) skimmed milk in T-TBS buffer (10 mM Tris-HCl, pH 8.0, containing 150 mM NaCl and 0.1% [v/v] Tween 20) for 1 h. The membranes were then incubated in the presence of anti-Ax21 or anti-His antibody (Sigma) at a dilution of 1∶3,000 in T-TBS buffer for 1 h, and then washed with the T-TBS buffer for 10 min, three times. The membranes were then incubated for 1 h with T-TBS buffer containing a 1∶6,000 dilution of anti-rabbit IgG for anti-Ax21 and anti-mouse IgG for anti-His conjugated with horseradish peroxidase (Jackson ImmunoResearch Laboratories). After three washes with T-TBS buffer (of 15, 5, and 5 min, respectively), the membranes were incubated for 5 min with the Super Signal West Pico chemiluminescent substrate (Pierce) and then exposed to X-ray film (Fujifilm Medical Systems). Films were developed following standard autoradiographical practices.
RNA preparation
RNA from Xoo PXO99 and PXO99Δax21 harvested cells were isolated using TRIzol® reagent (Invitrogen) following the manufacturer's protocol. The RNA samples were treated with 10 units of RNase-free DNaseI (Invitrogen) for 30 min at room temperature, followed by column purification using RNeasy mini kits (Qiagen). The quality of RNA was determined by subjecting samples to gel electrophoresis on 1% agarose gels and by measuring the absorbance at 260 nm and 280 nm. Protein content in the RNA samples was assessed using A260/A280.
cDNA generation and labeling
For Q-RT-PCR analysis, cDNA was generated by using SuperScript™III First-Strand kit (Invitrogen). One microgram of RNA was mixed with 50 ng random hexamers and 10 nMol dNTP mix. RNA mixture was incubated at 70°C for 5 min, then placed on ice for 2 min. Then cDNA synthesis mix (2 µl of 10X RT buffer, 4 µl of 25 mM MgCl2, 2 µl of 0.1 M DTT, 1 µl of 40U/µl RNaseOUT™, 1 µl of 200U/µl SuperScript ™III Reverse Transcriptase) was added into RNA mixture and incubated at 25°C for 10 min, followed by 50°C for 1 hour. The reactions were terminated at 85°C for 5 min, then chilled on ice. 2 U of RNase H was added and incubated at 37°C for 20 min. Synthesized cDNA was kept at −20°C until it was used as template for Q-RT-PCR analysis.
For microarray analysis, SuperScript™III Indirect cDNA Labelling System (Invitrogen) was used to synthesize label probe for microarray. Twenty microgram RNA was mixed with 3 µg random hexamers and incubated at 70°C for 5 min, then placed on ice for 2 min. Then cDNA synthesis mix (6 µl of 5X First-Strand buffer, 1.5 µl of 0.1 M DTT, 1.5 µl dNTP mix including amino-modified nucleotides, 1 µl of 40U/µl RNaseOUT™, 2 µl of 400U/µl SuperScript ™III Reverse Transcriptase) was added into RNA mixture and incubated at 25°C for 10 min, followed by 46°C for 3 hours. After cDNA synthesis, alkaline hydrolysis reaction was performed immediately to degrade the original RNA by adding 15 µl of 1 N NaOH and then incubated mixture at 70°C for 10 min. To neutralize the pH, 15 µl of 1 N HCl was mixed gently. Synthesized first strand cDNA was purified with Purification Module provided with the kit and proceeded to coupling reaction with fluorescent dye. Purified cDNA was mixed with 5 µl of 2X coupling buffer and 5 µl of DMSO in the vial of Amersham CyDye™ reactive dye (GE Healthcare Biosciences). Then labelled cDNA was purified with Purification Module and subjected to hybridization process.
Hybridization and scanning
Labeled cDNA probes were evaporated in a vacuum centrifuge setting at 60° C to a volume of approximately 2–3 µl. Evaporated probes were then resuspended in 100 µl of a salt based hybridization solution (Ocimum Biosolutions) at room temperature. All hybridization and scanning steps were performed in a hepa and carbon filtered clean room. Hybridization was carried out using a Tecan HS 4800 hybridization station. To block nonspecific hybridization, a pre-hyridization buffer (5X SSPE, 6M Urea, 0.5% Tween-20, 10X Denhardt's solution) was applied to the slides at 50°C and agitated for 15 min on the medium setting. Labeled probes were denatured by heating the mixture at 95°C for 3 min and then cooling snapped on ice for 30 second. Probes were applied into the injector to hybridize with printed slides. Samples were hybridized for 16 hour at 42°C, then following hybridization, the slides were consecutively washed at 37°C with three salt based buffers of increasing stringency (2X SSC, 0.1% SDS, 1.0X SSC, and 0.5X SSC). Each buffer wash step was repeated twice, with a soak time of one minute followed by a one minute wash. A final wash step with water was performed. Following the final wash, slides were dried under a constant stream of N2 at 30°C. Slides were kept under N2 until scanning. Hybridized microarray slides were imaged using a GenePix 4000B dual laser microarray scanner (Axon Instruments) at 5 µm resolution. Slides were imaged using 100% laser power for both lasers (532 nm and 635 nm) and scanned twice using the high PMT and low PMT settings. All images were processed using GenePix software (Axon Instruments) for element identification and quantification. The metadata associated with the hybridizations, along with the “raw” intensities obtained from the GenePix quantitation.
Array analysis and functional classification of differentially expressed genes
To identified differentially expressed genes from metadata, the LMGene package for R language statistic analysis that computes the p-values per gene via gene by gene ANOVA method developed by Rocke was used [28]. Genes that were differentially expressed more than 1.75 fold (log2ratio >0.8 or <–0.8) and have an FDR less than 5% were selected as described in our previous work [29]. To annotate the functions of the Ax21-regulated genes, COG terms (Clusters of Orthologous Groups of proteins: http://www.ncbi.nlm.nih.gov/COG/) of Xoo PXO99 were assigned. The TIGR Multiexperiment Viewer (MeV, http://www.tm4.org/mev.html) software was used to cluster and view expression patterns of differentially expressed genes.
Validation of expression patterns of candidate genes using quantitative RT-PCR
To validate expression pattern from microarray analysis, the synthesized cDNA samples were diluted by adding 100 µl DEPC water. 1 µl was used as template for each reaction (10 µl). cDNA template were mixed with SYBR® Green PCR Master Mix kit (Applied Biosystems) and specific primers as listed in Table S8. Each reaction included an initial heat for 5 min at 95°C, followed by 40 cycles of PCR (95°C, 10 sec; 60°C, 20 sec). The level of gene expression of each samples were relatively calculated comparing to 16sRNA amount.
Exogenous addition of recombinant Ax21 and quantitation of rax gene expression
To test whether rAx21 can restore rax gene expression in the PXO99Δax21 mutant strain, we supplemented the strain exogenously with the flow-through fraction, the 150 mM imidazole-eluted fraction containing purified recombinant Ax21 (rAx21), the 250 mM imidazole buffer-eluted fraction, axYS22, or axM178. Xoo PXO99 and PXO99Δax21 were cultured in PS broth media overnight and diluted to 105 CFU/ml. rAx21, control fractions or peptides were then added exogenously and the culture continued until the cell density reached 108 CFU/ml. Cultured cells were harvested and RNA isolated as described above (see RNA isolation and cDNA generation methods). The final concentration of exogenous addition to the bacterial culture was 1 µM for axYS22, and 5 nM for rAx21. All experiments were carried out with at least three biological replicates including four technical replicates in each experiment. All biological replicates gave similar results.
Swimming motility assays
The impact of Ax21 regulation on bacteria swimming motility was evaluated as described previously [30]. All tested Xoo strains were cultured in PSB media until cell densities reached to 108 CFU/ml. Cells were pelleted, washed with sterilized water twice, and resuspended to 109 CFU/ml. Then 3 µl of concentrated cultures were dropped in the middle of swimming assay plate (modified minimal media containing 0.15% agar), and incubated at 28°C for three days. The diameters of expanding colonies were measured and mean values were calculated from four biological replicates.
Virulence assays
In this study, two inoculation methods, the standard clipping [18] and the soaking method, were used for evaluation of virulence of the Xoo strains. For the standard clipping method, Xoo strains were cultured in PSB media until cell densities reached 5×108 CFU/ml. Six week-old rice cultivar TP309 (susceptible to PXO99 strain) was inoculated by clipping the leaf tip with scissor that dipped into bacterial culture. The leaf lesion lengths were measured two weeks after inoculation using our standard procedures [2]. For the soaking method, rice leaves were soaked for two days in Xoo cultures of 103 CFU/ml. Bacterial populations were determined as previously described [31] at two days after inoculation.
Biofilm formation assays
To quantitate biofilm formation in Xoo, we used the polyvinyl chloride microplate method [32]. Xoo strains were cultured in PS broth media overnight and then diluted to concentration 5×105 CFU/ml in minimal media. For complementation experiments, we supplemented the PXO99Δax21 mutant strain exogenously with the flow-through fraction, the 150 mM imidazole-eluted fraction containing purified recombinant Ax21 (rAx21), or the 250 mM imidazole buffer-eluted fraction and then continued cultured for seven days. After seven days, the cultures remaining in the plate were removed with pipettes. The remaining cells were stained with a 0.1% crystal violet solution for 30 min and excess dye was washed twice with distilled water. Dye that stained on the adhesive cells was resuspended with 95% EtOH, the optical density was measured at 590 nm and average values from four biological replicates were calculated. The final concentration of exogenous addition to the bacterial culture is 5 nM for rAx21.
Confocal microscopy
PXO99 and PXO99Δax21 strains carrying a green fluorescent protein (GFP) (Han et al., 2008) were cultured in M9 minimal media with glass slides (105 CFU/ml). After 4 days incubation, the glass slides with attached cells were washed nine times with sterilized water, and then air-dried. Biofilm formation was observed with a Leica True Confocal Scanner (TCS) SPE confocal microscope. The GFP was imaged under the following conditions: excitation: 488 nm; dichroic mirror: 405/488/543; emission: 495–530 nm. Images were analyzed using the program ImageJA (Ver 1.45b). After imaging, the attached bacterial cells were immediately recovered from the glass slide with 2 ml of sterilized water by extensively pipetting. The population of the recovered cells was determined using a colony counting method on PSA plates.
Aggregation assays
PXO99 and PXO99Δax21 strains carrying the green fluorescent protein were generated as described previously [31]. Xoo strains were grown on PSA plates, harvested, and washed with sterilized distilled water. The washed cells were resuspended to 106 CFU/ml. The diluted cells were dropped into an 8-chamber slide and incubated for three days at room temperature. Aggregated cells were observed using a Zeiss Axiophot fluorescence microscope (Jena) equipped with a fluorescein isothiocyanate filter (excitation filter, 450 to 490 nm; emission filter, 520 nm; dichroic mirror, 510 nm).
Supporting Information
Figure S1 Recombinant Ax21 protein is biologically active. After culturing PXO99 (blue), PXO99ΔraxST (red), PXO99Δax21 (yellow), or PXO99Δax21(rAx21) (purple) strains on PS agar plates containing cephalexin, kanamycin, and/or gentamycin, the cells were diluted to 5×108 CFU/ml and inoculated onto TP309 rice leaves (lacking XA21) and TP309-XA21 (carrying XA21) by the scissor clipping method. Each bar represents the average ± standard deviation (SD) of six sampled leaves per treatment. The experiment was repeated two times.
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Figure S2 Silver staining and western blot analyses reveal that recombinant Ax21 is highly purified. (A) Scheme for the purification of recombinant Ax21 (rAx21). rAx21 was purified from supernatants from the PXO99Δax21(rAx21) strain using Superdex 75 for a gel filtration and an Ni-NTA for his-tag purification. Samples were run in an 12% acrylamide gel (B) Silver staining and (C) Western blot analysis using anti-Ax21 antibody. Lanes are designated as follows: M, Marker; 1, Desalted total supernatants; 2, after gel filtration chromatography; 3, flow through; 4, elution with 50 mM imidazole buffer; 5, 100 mM imidazole buffer; 6, 150 mM imidazole buffer; 7, 200 mM imidazole buffer; 8, 250 mM imidazole buffer; 9, 500 mM imidazole buffer. The arrow indicates the rAx21 protein. A band corresponding the full-length N-terminal processed rAx21 reacted with the anti-His tag antibody indicating that the mature protein is secreted.
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Figure S3 The mature, processed rAx21 lacking the N-terminal signal peptide is secreted into supernatants. Desalted supernatants from the PXO99Δax21(rAx21) strain were digested by trypsin, and LC-MS/MS analysis carried out as described previously [2]. (A) Deduced amino acid sequence of rAx21. Yellow indicates amino acid sequence from the nine unique peptides obtained from LC-MS/MS analysis (B to J). The red box designates the predicted N-terminal signal peptide. The spectra of the nine peptides, which covered nearly the entire region of rAx21 except for the predicted N-terminal signal peptide, obtained from LC-MS/MS analysis are as follows: (B) AENLSYNFVEGDYVR, (C) TPTDGRDADGWGVK, (D) ASYAVAPNFHVFGEYSK, (E) NTNSDFQQWGVGVGFNHEIATSTDFVAR, (F) RLDLDSPNINFDGYSVEAGLR, (G) NAFGEHFEVYALAGYEDYSK, (H) GIDAGNDFYGR, (I) MDGDGNKEWSVGPR, and (J) FSWHHHHHH (include the 6x His tag).
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Figure S4 The AxYS22 peptide is stable and biologically active after two days incubation with Xoo. Two µM of AxYS22 were incubated in (1) water, (2) PXO99, and (3) PXO99ΔraxST for 2 days and then supernatants were collected. As a control, supernatants from (4) PXO99ΔraxST culture without AxYS22 peptide were included. These samples were used to pretreat TP309-XA21 rice leaves as described previously [2]. After pretreatment, the rice leaves were inoculated with PXO99ΔraxST. Lesion lengths were measured two weeks after PXO99ΔraxST inoculation. Each bar indicates the average lesion length ± SD from 5 or 7 leaves.
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Figure S5 The axYS22 and axM178 peptides derived from the Ax21 protein do not confer QS activity. Expression of the raxST (left), raxB (middle), and raxR (right) genes from PXO99 at 108 CFU/ml, PXO99 at 106 CFU/ml, and PXO99 at 106 CFU/ml supplemented with (A) axYS22 or (B) axM178 peptides, which were previously identified in biologically active fractions [3], was assessed using Q-RT-PCR with primers specific to each gene. Primers are as listed in table S8. Levels of gene expression were calculated relative to 16sRNA expression. Data are the mean of three replicates ± standard deviation (SD).
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Figure S6 Lack of the Xoo raxST significantly reduces Ax21-mediated QS. Density dependent expression of the genes from PXO99 at 108 CFU/ml, PXO99 at 106 CFU/ml, and PXO99 at 106 CFU/ml with exogenous addition of rAx21 isolated from 1, PXO99Δax21(rAx21) or 2, PXO99ΔraxST was assessed as described in Materials and Methods. Levels of gene expression were calculated relative to 16sRNA expression. Primers specific to raxST are as listed in table S8. Data are the mean of three replicates ± standard deviation (SD).
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Figure S7 Expression of ten Ax21 regulated genes is validated by Q-RT-PCR analysis. To validate the microarray results, we assessed expression levels of ten Ax21-regulated genes (from the 108 CFU/ml dataset) using Q-RT- PCR with specific primers as listed in Table S8. The relative fold change of transcriptional levels in PXO99 and PXO99Δax21 strains were determined by microarray analysis (grey) and Q-RT-PCR (black) (+ = expression level of PXO99 is higher than of PXO99Δax21, - = expression level of PXO99 is lower than of PXO99Δax21). Although the amplitude of the observed gene expression fold changes differ between the two techniques, as might be expected due to their sensitivity, the general trend of gene expression is similar.
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Figure S8 COG enrichment analysis indicates that Ax21 controls density-dependent expression of genes controlling diverse bacterial processes. To predict putative biological functions of Ax21 we applied COG (Cluster of Orthologous Groups of proteins) enrichment analysis. Each Ax21-regulated gene was assigned a COG term and grouped according to its biological function (http://www.ncbi.nlm.nih.gov/COG/). The numbers of Ax21-regulated genes in each functional category were calculated as a percentage of the total number of Ax21-regulated genes in each dataset (early log, mid log, and late log). Then the percentage of Ax21-regulated genes of each functional category was divided by the original percentage of each functional category present in the array platform (total numbers of genes in one functional categories/total numbers of genes on the array platform). The ratios between percentage of Ax21-regulated gene number and arrays in each category are presented in a pseudocolor numeric scale (Yellow, Ax21 up-regulated; Blue, Ax21 down-regulated). This analysis indicates that when cell population density is low, Ax21 controls up-regulation of genes (red box) involved in cell motility, cell cycle, inorganic ion metabolism, defense mechanism, coenzyme metabolism, and intracellular trafficking, but down-regulation of genes (green box) involved in transcription and translation. In contrast, when cell population density is high, Ax21 up-regulates genes (green box) involved in transcription and translation. The pattern of COG enrichment, created by MeV (www.tm4.org/mev, Multi experiment Viewer), of the microarray data supports our finding that Ax21 is a quorum sensing factor that controls diverse gene functions.
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Figure S9 Whole genome transcriptomics of PXO99 vs. PXO99Δax21 at three different population densities. (A) A heat map represents the ratio of gene expression levels in a log2-based pseudocolor scale in (i) late log phase (108 CFU/ml), (ii) early log phase (106 CFU/ml) (red, Ax21-up-regulated; green, Ax21-down-regulated). (B) Expression profiles of selected Ax21-regulated genes that are highly expressed at high population densities reveal an enrichment for putative transcriptional regulators. Of these, only the colR response regulator has been characterized (up-regulated 2 fold in PXO99 vs. PXO99Δax21). colR is critical for host colonization and infection in P. flurorescens and X. campestris pv. campestris, respectively. Our analysis also revealed that a gene encoding a (ppGpp)ase (ppGpp, guanosine-3,5-bispyrophosphate) is up-regulated by Ax21 suggesting that Ax21 controls ppGpp turnover.
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Figure S10 PXO99Δax21 shows significantly reduced biofilm formation compared with PXO99. (A) Biofilm formation assay on glass slides was performed using PXO99 and PXO99Δax21 strains carrying a green fluorescent protein as described in Materials and Methods. GFP was imaged under the following conditions: excitation: 488 nm; dichroic mirror: 405/488/543; emission: 495–530 nm. Width (X) x Height (Y) x Depth (Z): 1.1 × 1.1 × 0.141 mm. (B) Images were analyzed using the program ImageJA (Ver 1.45b). After imaging, the attached bacterial cells were immediately recovered from the glass slides with 2 ml of sterilized water by extensively pipetting. The population of the recovered cells was determined using a colony counting method. Bars indicate average of three replicates ±SD.
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Figure S11 PXO99ΔraxH does not respond to exogenous addition of Ax21. PXO99, PXO99ΔphoQ, PXO99ΔraxH, and PXO99Δax21 strains (5×105 CFU/ml), with or without purified rAx21, were tested for biofilm formation as described in Materials and Methods. The higher OD reflects more biofilms formed. Bars indicate average of four biological replicates ±SD.
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Figure S12 Growth of PX099, PXO99ΔraxST and PXO99Δax21 strains using the scissors clipping method. TP309 rice leaves were inoculated by clipping the leaf tip with scissors dipped into Xoo cultures of 5×108 CFU/ml. PXO99, PXO99ΔraxST, and PXO99Δax21 strains were extracted from rice leaves two days after inoculation and populations quantified using established procedures [3]. Each bar indicates the average of nine leaves ± SD. Four replicate experiments gave similar results.
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Figure S13 Phenotypic validation of twelve selected Ax21-regulated genes. Twelve of the Ax21-regulated genes listed in Table S9 were selected for knockout analysis using the marker exchange mutagenesis method (primers listed in Table S10). All Xoo mutant strains, generated in this study, were tested for (A) Swimming motility, (B) virulence (by standard clipping method), and (C) biofilm formation. Each bar represents the average ± standard deviation (SD). Mutations in genes that are regulated by Ax21 show phenotypic defects in the PXO99Δax21 strain compared to PXO99. These results indicate Ax21 controls the observed phenotypes through regulation of the Ax21-regulated genes. Nine of the twelve gene tested have been previously characterized in Xanthomonas spp or other bacteria. Three genes encode hypothetical proteins containing putative GGDEF and EAL domains. Strains carrying knockouts in these three genes also displayed reduced biofilm, swimming motility and virulence phenotypes.
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Table S1 Density-dependent expression of rax genes is controlled by Ax21.
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Table S2 Genes up-regulated in early log phase by Ax21.
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Table S3 Genes up-regulated in mid log phase by Ax21.
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Table S4 Genes up-regulated in late log phase by Ax21.
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Table S5 Genes down-regulated in early log phase by Ax21.
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Table S6 Genes down-regulated in mid log phase by Ax21.
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Table S7 Genes down-regulated in late log phase by Ax21.
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Table S8 List of primers used for quantitative realtime PCR.
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Table S9 Expression profile of Xoo genes that were selected to generate knockout strains for functional analysis.
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Table S10 List of primers used to generate Xoo knockout strains.
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Table S11 Bacterial strains and plasmids used in this study.
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We thank Dr. Benjamin Schwessinger for helpful discussions and critical reading of the manuscript. The microarray data have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO series accession number GSE24989.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by NIH grant GM55962 to PCR. S-WL's work, in part, was supported by the Next-Generation BioGreen 21 Program (PJ0080982011) and the Mid-Career Researcher Program (2010-0026679). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Arthritis Res TherArthritis Research & Therapy1478-63541478-6362BioMed Central ar34412183500110.1186/ar3441Research ArticleFibroblast growth factor receptor 1 is principally responsible for fibroblast growth factor 2-induced catabolic activities in human articular chondrocytes Yan Dongyao [email protected] Di [email protected] Simon M [email protected] Wijnen Andre J [email protected] Katalin [email protected] Gillian [email protected] Hee-Jeong [email protected] Department of Biochemistry, Rush University Medical Center, 1735 W Harrison Street, Chicago, IL 60612, USA2 Department of Internal Medicine, Section of Rheumatology, Rush University Medical Center, 1735 W Harrison Street, Chicago, IL 60612, USA3 Orthopedic Surgery, Rush University Medical Center, 1735 W Harrison Street, Chicago, IL 60612, USA4 Department of Bioengineering, University of Illinois, 1304 West Springfield Avenue, Chicago, IL 60612, USA5 Department of Stem Cells and Tissue Repair, Institute of Medical Biology, A*STAR, 8A Biomedical Grove, #06-06 Immunos, 138648 Singapore6 Division of Musculoskeletal Oncology, Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, 5 Lower Kent Ridge Road, 119074 Singapore7 Department of Cell Biology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA8 Department of Oncology, Cambridge University, Cancer Research Institute, Li Ka Shing Center, Robinson Way, Cambridge, CB2 ORE, UK2011 11 8 2011 13 4 R130 R130 22 12 2010 6 6 2011 11 8 2011 Copyright ©2011 Yan et al.; licensee BioMed Central Ltd.2011Yan et al.; licensee BioMed Central Ltd.This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Introduction
Cartilage degeneration driven by catabolic stimuli is a critical pathophysiological process in osteoarthritis (OA). We have defined fibroblast growth factor 2 (FGF-2) as a degenerative mediator in adult human articular chondrocytes. Biological effects mediated by FGF-2 include inhibition of proteoglycan production, up-regulation of matrix metalloproteinase-13 (MMP-13), and stimulation of other catabolic factors. In this study, we identified the specific receptor responsible for the catabolic functions of FGF-2, and established a pathophysiological connection between the FGF-2 receptor and OA.
Methods
Primary human articular chondrocytes were cultured in monolayer (24 hours) or alginate beads (21 days), and stimulated with FGF-2 or FGF18, in the presence or absence of FGFR1 (FGF receptor 1) inhibitor. Proteoglycan accumulation and chondrocyte proliferation were assessed by dimethylmethylene blue (DMMB) assay and DNA assay, respectively. Expression of FGFRs (FGFR1 to FGFR4) was assessed by flow cytometry, immunoblotting, and quantitative real-time PCR (qPCR). The distinctive roles of FGFR1 and FGFR3 after stimulation with FGF-2 were evaluated using either pharmacological inhibitors or FGFR small interfering RNA (siRNA). Luciferase reporter gene assays were used to quantify the effects of FGF-2 and FGFR1 inhibitor on MMP-13 promoter activity.
Results
Chondrocyte proliferation was significantly enhanced in the presence of FGF-2 stimulation, which was inhibited by the pharmacological inhibitor of FGFR1. Proteoglycan accumulation was reduced by 50% in the presence of FGF-2, and this reduction was successfully rescued by FGFR1 inhibitor. FGFR1 inhibitors also fully reversed the up-regulation of MMP-13 expression and promoter activity stimulated by FGF-2. Blockade of FGFR1 signaling by either chemical inhibitors or siRNA targeting FGFR1 rather than FGFR3 abrogated the up-regulation of matrix metalloproteinases 13 (MMP-13) and a disintegrin and metalloproteinase with a thrombospondin type 1 motif 5 (ADAMTS5), as well as down-regulation of aggrecan after FGF-2 stimulation. Flow cytometry, qPCR and immunoblotting analyses suggested that FGFR1 and FGFR3 were the major FGFR isoforms expressed in human articular chondrocytes. FGFR1 was activated more potently than FGFR3 upon FGF-2 stimulation. In osteoarthritic chondrocytes, FGFR3 was significantly down regulated (P < 0.05) with a concomitant increase in the FGFR1 to FGFR3 expression ratio (P < 0.05), compared to normal chondrocytes. Our results also demonstrate that FGFR3 was negatively regulated by FGF-2 at the transcriptional level through the FGFR1-ERK (extracellular signal-regulated kinase) signaling pathway in human articular chondrocytes.
Conclusions
FGFR1 is the major mediator with the degenerative potential in the presence of FGF-2 in human adult articular chondrocytes. FGFR1 activation by FGF-2 promotes catabolism and impedes anabolism. Disruption of the balance between FGFR1 and FGFR3 signaling ratio may contribute to the pathophysiology of OA.
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Introduction
Osteoarthritis (OA) is a debilitating disease afflicting millions of people worldwide, which imposes a tremendous burden upon society. OA is a multifactorial heterogeneous disease that is influenced by both genetic and environmental factors [1]. A wide array of enzymes, such as matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with a thrombospondin type 1 motif (ADAMTS), and pro-inflammatory cytokines, have been implicated in pathological processes associated with OA, such as cartilage degradation, synovial inflammation and bone abnormalities [2]. Notably, the products of cartilage degeneration not only further promote matrix degradation, but also stimulate the synovium to overproduce inflammatory mediators and degrading proteases, which, in turn, exacerbate cartilage matrix loss [2]. Such autocrine and paracrine loops perpetuate joint destruction, frequently resulting in irreversible disease progression.
Progressive damage to articular cartilage is a hallmark of OA, and a principal cause of tissue break-down is the destruction rather than formation of the cartilage extracellular matrix by chondrocytes. Thus, metabolic homeostasis is perturbed at the cellular level in OA because chondrocyte catabolism predominates over anabolism resulting in net cartilage degeneration. Elevated levels of pro-inflammatory cytokines, inflammatory mediators and certain growth factors potently heighten the expression of matrix-degrading enzymes. Destructive proteases such as MMP-13 and ADAMTS-5 are able to cleave major components in the extracellular matrix of chondrocytes, including type II collagen and aggrecan [3,4]. In response to tissue damage, chondrocytes make attempts at matrix repair, but they often fail to restore the eroded cartilage to its original pristine hyaline state, due to multiple impairing mechanisms [5-8].
FGF-2 participates in the regulation of cartilage homeostasis in addition to its well-established mitogenic role [9]. Released from the extracellular matrix upon tissue injury [10], FGF-2 stimulates MMP-13 expression, which may accelerate cartilage degradation [11]. In both articular chondrocytes and meniscal chondrocytes, FGF-2 alters the ratio between type II and type I collagen, thus possibly resulting in the formation of fibrocartilage, a defective substitute for healthy hyaline cartilage [12,13]. In porcine articular chondrocytes, FGF-2 antagonizes IGF-1/TGF-β-mediated type II collagen and decorin production [14]. Moreover, FGF-2 potently inhibits IGF-1/BMP-7-enhanced proteoglycan accumulation and synthesis in human articular chondrocytes, even though it stimulates proliferation, and markedly affects physical properties of normal cartilage [5,15]. Recent studies by others, suggest a chondroprotective role of FGF-2 in cartilage biology, which merits additional studies to resolve the physiological complexities linked to the opposing biological functions of FGF-2 in human articular cartilage [16,17].
Our group has clearly established that FGF-2 exerts catabolic effects in primary human articular chondrocytes cultured ex vivo, thus mechanistically predicting cartilage degradation in human patients. Previously, we showed that FGF-2 inhibits the synergistic anabolic effects of IGF-1 and BMP-7, and also stimulates MMP-13 expression via protein kinase C δ (PKCδ)-mediated activation of multiple MAP kinases (ERK1/2, p38 and JNK) [5,18]. We also showed that FGF-2 activates the NFκB pathway, which converges with the MAP kinase pathway on the activation of transcription factor Elk-1 to stimulate MMP-13 transcription [19].
There are four different isoforms of FGF receptors (FGFR1 to FGFR4) that are responsible for the biological impact of FGF-2 through the developmental stages [20]. It is still not clear which receptor(s) mediate the catabolic and/or anti-anabolic signaling by FGF-2 as we previously observed, and what other target genes than MMP-13 are regulated by FGF-2 in human adult articular cartilage [5,18,19]. In this study, we examined which of the main FGFR isoforms mediate the biological effects of FGF-2, characterized critical FGF-2-regulated genes that depend on FGF-2/receptor signaling. We also determined the potential pathological alterations in the expression profiles of FGFR isoforms by comparing cartilage from healthy (Collin' grade 0 or 1) and age- and gender-matched osteoarthritic knee joints (surgically removed).
Materials and methods
Materials
Human recombinant FGF-2 was purchased from the National Cancer Institute (Bethesda, MD, USA). Human recombinant FGF18 was purchased from PeproTech (Rocky Hill, NJ, USA). Antibodies against human Flg (FGFR1), Bek (FGFR2), FGFR3, FGFR4, and phospho-Tyrosine (PY99) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibody against human β-actin was purchased from Abcam (Cambridge, MA, USA). The antibody against human ADAMTS5 was purchased from Millipore (Billerica, MA, USA) and the antibody against MMP-13 and FGFR1 neutralizing antibody were provided by courtesy of Dr. Gillian Murphy and Dr. Simon Cool, respectively. The MMP-13 antibody was described previously [21]. A full characterization of the neutralizing antibody against FGFR1 is provided elsewhere [22]. The titer of the latter antibody is equal or higher than 1:200,000 as determined by standard enzyme-linked immunosorbent assay (ELISA). Pharmacological inhibitor SU5402 (FGFR1i) and PD98059 (ERKi) were purchased from Calbiochem/EMD Chemicals (Gibbstown, NJ, USA). The SU5402 concentrations used in this study (5 μM and 2 μM) did not lead to significant inhibition of FGFR3 phosphorylation, as determined by immunoprecipitation and immunoblotting (data not shown). Stealth small interfering RNA (siRNA) targeting FGFR1 and FGFR3 were purchased from Invitrogen (Carlsbad, CA, USA).
Chondrocyte isolation and culture
Normal human knee cartilage tissues were obtained within 24 hours of death of donors (age ranging from 40 to 65) from the Gift of Hope Organ and Tissue Donor Network (Elmhurst, IL, USA) with approval by the local ethics committee and consent from the families. Prior to dissection, each specimen was graded for overall degenerative changes based on the modified 5-point scale of Collins [23]. Surgically removed cartilage from OA patients (age ranging from 40 to 65) were obtained from the Orthopedic Tissue and Implant Repository Study with consent from the patients. Human tissues were handled according to the guidelines of the Human Investigation Committee of Rush University Medical Center.
Chondrocytes were isolated by enzymatic digestion of cartilage using Pronase for one hour, followed by overnight digestion with collagenase-P as described previously [5,24]. For monolayer cultures, isolated cells were washed and suspended in culture media at 3 × 106 cells/mL, and seeded onto 12-well plates using 1 mL media/well. Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM)/F-12 (1:1) containing 10% fetal bovine serum and antibiotics (complete media) for three days before the treatments. For alginate bead culture, cells were suspended in alginate (2 × 106 cells/mL) immediately after enzymatic digestion and washing steps, and beads were formed in CaCl2 solution, as described previously [25,26]. Beads were cultured in DMEM/F-12 medium (1:1), supplemented with 1% mini-ITS+ premix and 0.1% ascorbic acid, at eight beads per well in 24-well plates. Chondrocytes used for profiling FGFR isoform expression were processed immediately after cell isolation from cartilage.
Chondrocyte stimulation and immunoblotting
Prior to treatments, chondrocytes were growth factor deprived in serum-free DMEM/F-12 (1:1) for 24 hours. Media were replaced again with fresh serum-free DMEM/F-12 (1:1) two hours before stimulation. When inhibitors were applied, cells were pre-incubated with individual pathway-specific inhibitors for one hour before stimulation with FGF-2 (100 ng/mL). After terminating the experiments, conditioned media and whole cell lysates were collected. Media were stored at 4°C with 0.1% NaN3, and used within five days. Cell lysates were prepared using modified cell lysis RIPA buffer: 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, 0.25% deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM glycerol phosphate, 1 mM NaVO4, and 2 mM phenylmethylsulfonyl fluoride (Sigma, St Louis, MO, USA). Total protein concentrations of the cell lysates were determined using the bicinchoninic acid (BCA) assay (Thermo Scientific, Rockford, IL, USA). Equal amounts of protein were resolved in 10% SDS-polyacrylamide gels and transferred to nitrocellulose membrane for immunoblotting analyses as described previously [24]. Immunoreactivity was visualized using an ECL system (Pierce, Rockford, IL, USA).
Immunoprecipitation
Whole cell lysates were prepared as described above and centrifuged at 12,500 rpm for 20 minutes. Supernatants were transferred and incubated with antibody against FGFR1 or FGFR3 immobilized to Protein A agarose beads (Thermo Scientific, Rockford, IL, USA). Beads were maintained in homogenous suspension overnight at 4°C using a rotary wheel that supports 'end-over-end' mixing. Beads were then washed three times with binding buffer (0.14 M NaCl, 0.008 M sodium phosphate, 0.002 M potassium phosphate, 0.01 M KCl, pH 7.4). The samples were eluted and subjected to SDS-PAGE.
Total RNA extraction, cDNA synthesis, and quantitative real-time PCR
Total RNA from normal and osteoarthritic human articular chondrocytes was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the instructions provided by the manufacturer. Reverse transcription (RT) was carried out with 1 μg total RNA using ThermoScript TM RT-PCR system (Invitrogen) for first strand cDNA synthesis. For real-time PCR, cDNA was amplified using MyiQ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Relative gene expression was determined using the ΔΔCT method, using detailed guidelines provided by the manufacturer (Bio-Rad). 18S rRNA and GAPDH were used as internal controls for normalization. The standard deviations in samples were calculated using data from at least five different donors in independent experiments. The primer sequences are summarized in Table 1.
Table 1 Real-time PCR primer sequences
Gene Primer Sequence (5'→3') NCBI Gene Number Anneal Tm
MMP13 F: ACCCTGGAGCACTCATGTTTCCTA
R: TGGCATCAAGGGATAAGGAAGGGT NM_002427.3 60°C
ADAMTS5 F: CTGTGACGGCATCATTGGCTCAAA
R: TTCAGGAATCCTCACCACGTCAGT NM_007038.3 60°C
ACAN F: TCTTGGAGAAGGGAGTCCAACTCT
R: ACAGCTGCAGTGATGACCCTCAGA NM_001135.3 60°C
FGFR1 F: AACCTGACCACAGAATTGGAGGCT
R: ATGCTGCCGTACTCATTCTCCACA NM_023110.2 60°C
FGFR2 F: TGATGGACTTCCTTATGTCCGCGT
R: AGCGTCCTCTTCTGTGACATTGGT NM_000141.4 60°C
FGFR3 F: ACCAATGTGTCTTTCGAGGATGCG
R: AGAGCACGCAGCTTGTCACATAGA NM_000142.4 60°C
FGFR4 F: ATGGAACTGGTGTGCTCAAGAAGC
R: TTCACATGTCCTCCGACCAACACA NM_002011.3 60°C
18S rRNA F: CGGCTACCACATCCAAGGAA
R: GCTGGAATTACCGCGGCT NR_003286.2 60°C
GAPDH F: TCGACAGTCAGCCGCATCTTCTTT
R: GCCCAATACGACCAAATCCGTTGA NM_002046.3 60°C
ACAN, aggrecan core protein; ADAMTS5, a disintegrin and metalloproteinase with a thrombospondin type 1 motif 5; FGFR, fibroblast growth factor receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MMP13, matrix metalloproteinase-13.
Dimethylmethylene blue (DMMB) assay and DNA assay
Cultured cells on alginate beads were collected and processed for quantitative assays using the DMMB binding method, as previously described [26,27]. The pH of DMMB solution used in this study was 1.5, in order to minimize the interfering effect of alginate, as previously demonstrated [28]. The proteoglycan levels in the cell-associated matrix were measured. Cell viability and cell numbers were determined using PicoGreen (Invitrogen), as previously described [26].
Transient transfection
Nucleofection was optimized for human articular chondrocytes based on the manual of the Nucleofector™ kit (Lonza, Walkersville, MD, USA) as described previously [29,30]. Chondrocytes were cultivated for three days before transfection. For FGFR knockdown experiments, siRNA at a concentration of 200 nM (20 pmol/sample) was used during transfection. After 48 hours, cell lysates were subjected to SDS-PAGE and immunoblotting for validation of successful knockdown. In parallel, stimulations were performed 48 hours after the transfection. In promoter activity assays, as internal control for transfection efficiency, the Renilla Luciferase vector (pRL-TK) was co-transfected with the MMP-13 promoter/firefly luciferase constructs as we described previously [18]. Both Renilla and firefly luciferase activity were measured simultaneously using a dual-luciferase reporter assay system (Promega, Madison, WI, USA) and a luminometer (Berthold, Huntsville, AL, USA).
Flow cytometry analysis
Immunofluorescence labeling of FGFRs was performed as previously described [31]. Human articular chondrocytes were incubated with anti-CD32/CD16 monoclonal antibody to block Fc receptor-mediated nonspecific antibody binding. Primary antibodies against FGFR1, FGFR2, FGFR3, and FGFR4 were incubated with cells, followed by addition of secondary antibody, goat-anti-rabbit Alexa Fluor 488 (Invitrogen). Cells were also incubated with goat-anti-rabbit Alexa Fluor 488, or non-immune rabbit serum plus goat-anti-rabbit Alexa Fluor 488 as controls. FGFRs present on the plasma membrane of chondrocytes were analyzed using a FACS Calibur instrument and CellQuest software (BD Flow Cytometry Systems, San Jose, CA, USA).
Statistical analysis
Statistical significance was determined by Student's t-test, or one-way repeated measures ANOVA followed by Sidak post-hoc test using the SPSS 17 program. P-values lower than 0.05 were considered to be statistically significant in each test.
Results
FGF-2-mediated cellular proliferation and proteoglycan loss is via FGFR1 in human articular chondrocytes
Previously, we reported FGF-2-mediated cell proliferation and significant proteoglycan loss in human articular cartilage using in vitro and ex vivo explant culture systems [5]. Dynamic interactions of FGF-2 with its cognate receptors, FGFR1 and FGFR3 were shown to elicit distinctive biological responses. FGF-2 binds to all FGFR isoforms, yet with higher affinity to FGFR1 and FGFR3 [32]. We investigated which specific FGFR isoforms are responsible for the FGF-2-mediated cellular proliferation and proteoglycan loss.
Human articular chondrocytes in alginate beads were incubated with FGF-2 for 21 days in the presence or absence of a pharmacological inhibitor that blocks the tyrosine kinase activity of FGFR1 (SU5402, 2 μM) or a neutralizing antibody directed against FGFR1 antibody [22]. Treatment with FGF18 was included in parallel, because its pro-anabolic activity for matrix formation is exclusively mediated through activation of FGFR3 in human articular cartilage [33]. Accumulated cell-associated proteoglycan and cellular proliferation were assessed by DMMB assay. Our results show that FGF-2 significantly reduced proteoglycan production, whereas FGF18 was pro-anabolic (Figure 1A), consistent with previous findings [5,33,34]. In parallel, we found that FGF-2 but not FGF18 significantly induced cell proliferation (up to two-fold) (Figure 1B). This reduction of proteoglycan biosynthesis and stimulation of proliferation by FGF-2 were each completely abolished by co-incubation with either the pharmacological inhibitor of FGFR1 (2 μM) or the neutralizing anti-FGFR1 antibody, while neither agent affected FGF18-mediated proteoglycan production (Figure 1A, B). Consistent with our previous report, FGF-2 did not alter cell survival, suggesting FGF-2 does not impact cell death (data not shown) [5]. These data indicate that the biological impact of FGF-2 is achieved via the activation of FGFR1 but not FGFR3. Our data corroborate the current concept that FGFR1 and FGFR3 have distinct biological roles in articular chondrocytes, and that activation of FGFR3 by FGF18 is anabolic for human articular cartilage homeostasis [33,34].
Figure 1 The effect of FGF-2 on proteoglycan accumulation and cell proliferation. Human articular chondrocytes were cultured in alginate beads with indicated treatments for 21 days before assaying for proteoglycan and DNA content. (A) Proteoglycan accumulation was assessed by dimethylmethylene blue (DMMB) assay, normalized by DNA content. (B) Cellular proliferation was quantified by total DNA content in beads from each treatment group. * P < 0.05; ** P < 0.01. FGF, fibroblast growth factor.
FGFR1 is responsible for the up-regulation of MMP-13 and ADAMTS5, as well as down-regulation of aggrecan by FGF-2 in human articular chondrocytes
We examined whether FGFR1 is the major receptor responsible for FGF-2-dependent regulation of genes encoding cartilage-degrading enzymes (for example, MMP-13 and ADAMTS5), or major cartilage-related proteoglycans (for example, aggrecan). Because we were interested in investigating the FGFR1-altered transcriptional regulation of the target gene that requires 24-hour incubation, we utilized a monolayer culture system instead of a long-term alginate bead culture system. Human chondrocytes in monolayer were pre-incubated with either SU5402 (5 µM), a pharmacological inhibitor that blocks the tyrosine kinase activity of FGFR1, or a neutralizing antibody directed against FGFR1 (lane 3 and 4) followed by stimulation with FGF-2 (100 ng/mL) for 24 hours. Based on a previous report, the SU5402 inhibitor has an IC50 of 10 to 20 μM for FGFR1 inhibition [35]. Our empirically determined SU5402 concentration (5 μM) differs from the reported IC50, which may be attributed to differences in biological context, experimental conditions and readouts. Conditioned media were collected for analyses of MMP-13 and ADAMTS5 mRNA levels using RT-qPCR with human-specific primer sets for MMP13 and ADAMTS5 that had been validated in our laboratory [36]. Stimulation of cells with FGF-2 alone significantly induced MMP-13 (Figure 2A; P < 0.05) and ADAMTS5 (Figure 2B; P < 0.05) at both mRNA and protein levels. The biological modulations of FGF-2 on MMP13 and ADAMTS5 were abolished in the presence of either the FGFR1 inhibitor (P < 0.05) or the FGFR1 neutralizing antibody (P < 0.05). By clear contrast, FGF18 (100 ng/mL), which specifically activates FGFR3, failed to stimulate catabolic enzyme production, and its action was not altered by SU5402 or the neutralizing FGFR1 antibody (data not shown).
Figure 2 Effects mediated by FGFR1 upon FGF-2 binding. (A, B, E) Human articular chondrocytes in monolayer were treated with FGF-2 (100 ng/mL), in the absence or presence of FGFR1 neutralization antibody (1:1,000, 1:200) or SU5402 (5 μM), for 24 hours. In parallel, chondrocytes were transfected with siRNA targeting FGFR1 or FGFR3, and subjected to 24-hour FGF-2 (100 ng/mL) stimulation. Conditioned media were collected for immunoblotting analyses of matrix metalloproteinase-13 (MMP-13) and a disintegrin and metalloproteinase with a thrombospondin type 1 motif 5 (ADAMTS5). Total RNA was extracted for cDNA synthesis and qPCR quantification of gene expression. * P < 0.05. (C) Chondrocyte lysates were prepared 48 hours after siRNA transfection. The samples were resolved by SDS-PAGE, and immunoblotted for FGFR1 and FGFR3 protein expression. GAPDH was used as a loading control. (D) -1600MMP-13 Luciferase-promoter construct was transiently transfected into human articular chondrocytes. The transfected cells were pre-incubated with neutralizing antibody (1:1,000, 1:200) or 5 μM SU5402 for 1 hour, and then stimulated with FGF-2 (100 ng/mL) for 24 hours. The luciferase activity representing MMP-13 promoter activity was measured. A Renilla vector was co-transfected as an internal control for normalization. * P < 0.05. ADAMTS, a disintegrin and metalloproteinase with a thrombospondin type 1 motif; FGF, fibroblast growth factor; FGFR, FGF receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MMP, matrix metalloproteinase; qPCR, quantative polymerase chain reaction; siRNA, small interfering RNA.
To substantiate our findings, human articular chondrocytes in monolayer were transfected with either FGFR1-specific or FGFR3-specific siRNA. Cells were then stimulated with FGF-2 (100 ng/mL), followed by immunoblotting and qPCR to assess MMP-13 and ADAMTS5 expression. Our results indicate that both FGFR1 and FGFR3 were significantly knocked down on their protein levels (Figure 2C). We did not observe any off-target effects of these siRNA on the other FGFR isoforms (data not shown). Consistent with the results acquired using FGFR inhibitors, FGFR1 knockdown resulted in reversal of FGF-2-mediated up-regulation of MMP-13 and ADAMTS5 (Figure 2A, B; P < 0.05). By stark contrast, FGFR3 knockdown did not significantly modulate FGF-2-mediated MMP-13 or ADAMTS5 induction (Figure 2A, B), suggesting FGFR1 is the FGFR isoform responsible for the catabolic actions of FGF-2 in articular chondrocytes.
Coherent results were obtained in transient transfection studies that monitored integration of upstream metabolic signals on the promoter of the MMP13 gene. In previous studies, we showed that the first 1.6 kb of the MMP-13 promoter (-1600 MMP-13 Luc reporter construct) responded to FGF-2 stimulation by increasing MMP-13 gene transcription [18]. Here we found that FGF-2-induction of MMP-13 promoter-driven luciferase activity was completely inhibited by SU5402 or neutralizing FGFR1 antibody, with luciferase activity levels returning to the control level (no treatment) (Figure 2D). These results further support our hypothesis that FGFR1 is responsible for the FGF-2-induced MMP-13 expression in human articular chondrocytes.
Administration of FGF-2 significantly reduced the expression of aggrecan (Figure 2E; P < 0.05), a major component of proteoglycan in human articular chondrocytes. The inhibition of aggrecan expression by FGF-2 was completely reversed in the presence of either SU5402 (5 µM), or neutralizing FGFR1 antibody in a dose-dependent manner. Likewise, FGFR1 knockdown abolished aggrecan down-regulation by FGF-2, whereas FGFR3 knockdown failed to exert such an effect (Figure 2E; P < 0.05). Taken together, our results suggest that pro-catabolic and anti-anabolic biological signals mediated by FGF-2 are relayed through FGFR1 but not FGFR3 in human adult articular chondrocytes.
FGFR1 and FGFR3 are predominantly expressed in normal human adult articular chondrocytes
We examined the basal expression levels of FGFR family members (FGFR1-4) at the cell surface to understand which receptors mechanistically influence the biological response to FGF-2 in human adult articular cartilage. To determine expression levels of FGFRs, normal knee chondrocytes (Collin's grade 0 or 1, age group from 40 to 65) were subjected to flow cytometric analyses, using anti-FGFR1 to four antibodies. Our results indicate that FGFR3 and FGFR1 were the two most prominent receptors at the cell surface of normal human articular chondrocytes (n = 3) (Figure 3A). Similarly, cellular mRNA and protein levels of FGFR3 and FGFR1 were predominantly higher than the levels of FGFR2 and FGFR4 based on RT-qPCR data (Figure 3B) and immunoblotting analyses (Figure 3C). Compared with healthy chondrocytes, osteoarthritic chondrocytes did not up-regulate FGFR2 or FGFR4 expression level (data not shown). We also performed FGFR isoform expression analyses using mRNA and protein acquired via different procedures (that is, direct extraction from cartilage explants, extraction from cells immediately after enzymatic digestion, and extraction from cells after up to five-day culture), and we did not observe notable differences between these methods regarding FGFR expression levels (data not shown). These results collectively suggest that FGFR3 and FGFR1 are the principal sensors of FGF ligands in articular chondrocytes.
Figure 3 Abundant FGFR1 and FGFR3 expression in articular chondrocytes. (A) Human articular chondrocytes were incubated with anti-CD32/CD16 monoclonal antibody to block nonspecific antibody binding. FGFR1, FGFR2, FGFR3, and FGFR4 antibodies were incubated with cells, followed by incubation with a secondary antibody, goat-anti-rabbit Alexa Fluor 488. Cells were also incubated with goat-anti-rabbit Alexa 488, or non-immune rabbit serum plus goat-anti-rabbit Alexa Fluor 488 as controls. FGFRs on the plasma membrane of chondrocytes were analyzed with a FACS Calibur instrument and CellQuest software. (B) Human articular chondrocytes in monolayer were subjected to RNA extraction, cDNA synthesis, and qPCR quantification of FGFR isoform expression. * P < 0.05. (C) Human articular chondrocytes in monolayer were directly lysed. Equal amounts of total protein were resolved on a SDS-PAGE gel, followed by transfer and immunoblotting of FGFR isoforms. FGF, fibroblast growth factor; FGFR, FGF receptor; qPCR, quantative polymerase chain reaction.
FGF-2 preferentially activates FGFR1, and to a lesser extent, FGFR3 in human articular chondrocytes
FGF-2 signaling is initiated by ligand binding and subsequent activation of specific FGFRs. Because we observed that FGFR1 and FGFR3 were predominantly expressed in articular chondrocytes, we next asked a question: Which type of FGFR(s) is activated in response to FGF-2? We included FGF18 treatments in the experiments, in parallel for comparison, as FGF18 exclusively activates FGFR3, in human adult articular chondrocytes.
Either FGF-2 or FGF18 (100 ng/mL) was administered in serum-free media to human knee articular chondrocytes in monolayer. Immunoprecipitation (IP) analyses were performed using either FGFR1 or FGFR3 antibody, followed by immunoblotting with a phospho-Tyr (PY99) antibody. In our initial time-course studies, we observed the strongest receptor activation reflected by increased phospho-tyrosine levels within 5 minutes, followed by a rapid decrease within 30 minutes after treatment with FGF-2 or FGF18 (data not shown). Therefore, we selected the five-minute time point to determine specific FGFR activation in response to FGF-2. Our IP results suggest that FGF-2 activated both FGFR1 and FGFR3 with a modest preference towards FGFR1 over FGFR3 in knee articular chondrocytes (Figure 4A). However, FGF18 exclusively activated FGFR3 without detectable activation of FGFR1 (Figure 4B). These results suggest that selective FGFR-ligand interactions drive distinctive signaling pathways and result in differential biological outcomes in human adult articular chondrocytes.
Figure 4 Activation of FGFR1 and FGFR3 by FGF-2. (A) Human articular chondrocytes in monolayer were stimulated with FGF-2 (100 ng/mL) for five minutes before whole cell lysates were prepared. The lysates were incubated with antibody against FGFR1 or FGFR3 in separate tubes. Equal amounts of total protein after immunoprecipitation (IP) were resolved on SDS-PAGE gel, followed by immunoblotting of phospho-tyrosine. Antibody against total FGFR1 or FGFR3 was used to evaluate loading precision. (B) Human articular chondrocytes in monolayer were stimulated with FGF18 (100 ng/mL) for five minutes before whole cell lysates were prepared. The following steps were performed as described above. FGF, fibroblast growth factor; FGFR, FGF receptor.
FGFR3 is down-regulated in OA cells, and is regulated by growth factors
FGFR3 has been shown to elicit anabolic responses in cartilage (for example, FGF18), and Fgfr3-knockout mice exhibit premature cartilage degradation and arthritis [34,37]. Previously, we reported hyper-sensitization of osteoarthritic cells to FGF-2 compared to normal chondrocytes [18]. We postulated that the functional differences in the FGF-2 response that are evident between normal and OA cells, or between knee and ankle (unpublished data) is related to altered levels of FGFR subtypes in osteoarthritic chondrocytes. We investigated this hypothesis by comparing FGFR1 and FGFR3 expression at both mRNA and protein levels in normal and osteoarthritic chondrocytes. To minimize biological variability in each group, the specimens were carefully matched for age and gender. Results from RT-qPCR and immunoblot analyses reveal a significant down-regulation of FGFR3 in osteoarthritic cells at both mRNA (Figure 5A; P < 0.05) and protein levels (Figure 5B) when compared to normal cells. To understand changes in the relative abundance of FGFR1 and FGFR3 in normal and osteoarthritic specimens, we simultaneously quantified the mRNA levels of FGFR1 and FGFR3 in each donor, and calculated the expression ratios of FGFR1 to FGFR3. The data show that expression ratio of FGFR1 to FGFR3 was significantly increased in OA compared to normal chondrocytes (P < 0.05) (Figure 5C). In addition, no apparent correlation between donor age and FGFR expression level was observed (data not shown). Thus, imbalanced FGFR signaling may account for the altered cellular responses to FGF-2 and FGF18 in the osteoarthritic state.
Figure 5 FGFR expression profiles and FGFR3 regulation in articular chondrocytes. (A) Age-matched, normal and osteoarthritic human articular chondrocytes in monolayer were subjected to RNA extraction, cDNA synthesis, and qPCR quantification of FGFR3 expression. (B) Age-matched, normal and osteoarthritic human articular chondrocytes were directly lysed for immunoblotting analyses of FGFR3. (C) Age-matched, normal and osteoarthritic chondrocytes were subjected to RNA extraction, cDNA synthesis, and qPCR quantification of FGFR1 and FGFR3 expression. The FGFR1 to FGFR3 expression ratio was calculated in each donor. (D) Human articular chondrocytes in monolayer were pre-incubated with SU5402 (5 μM) or PD98059 (ERK inhibitor, 50 μM) for 1 hour, and then treated with FGF-2 (100 ng/mL) for 24 hours. In parallel, chondrocytes were transfected with siRNA targeting FGFR1 or FGFR3, and then stimulated by FGF-2 (100 ng/mL) for 24 hours. Total RNA was extracted afterwards for cDNA synthesis and qPCR analyses of FGFR3 expression. * P < 0.05. (E) Human articular chondrocytes in monolayer were administered with BMP7 (100 ng/mL) and cultured for 24 hours. Total RNA was extracted afterwards for cDNA synthesis and qPCR analyses of FGFR1 and FGFR3 expression. ERK, extracellular signal-regulated kinase; FGF, fibroblast growth factor; FGFR, FGF receptor; qPCR, quantative polymerase chain reaction.
Because progression of OA depends on external signaling ligands (for example, FGFs and BMPs), we investigated whether FGFR3 expression is down-regulated by such factors in chondrocytes. Importantly, administration of FGF-2 significantly decreased the expression level of FGFR3 (Figure 5D; P < 0.05). By contrast, BMP-7, a well-known anabolic growth factor in cartilage, markedly increased expression of FGFR3 (Figure 5E; P < 0.001) whereas the level of FGFR1 was not significantly modulated in the presence of BMP-7 in human articular chondrocytes. The suppression of FGFR3 expression by FGF-2 was completely abolished in the presence of either FGFR1 inhibitor or FGFR1 neutralizing antibody. Similarly, knockdown of FGFR1 by siRNA rescued FGFR3 suppression in the presence of FGF-2 (Figure 5D; P < 0.05). Furthermore, the inhibitor of the ERK MAPK pathway significantly reversed FGF-2-mediated reduction of FGFR3, suggesting that FGF-2-suppression of FGFR3 is via FGFR1-ERK/MAPK axis in human articular chondrocytes (Figure 5D).
Discussion
The balance between FGFR1 and FGFR3 signaling, and the cognate ligands FGF-2 and FGF18, appears to be vital for normal cartilage homeostasis [9]. While FGF-2 binds to all FGFR isoforms in vitro, it has greater affinity for FGFR1 and FGFR3 [32]. The anabolic growth factor FGF18 appears to act selectively through FGFR3 to activate distinct downstream pathways in human articular chondrocytes. Of the four receptors for FGFs, we found that FGFR1 and FGFR3 were predominantly expressed in human adult articular chondrocytes. To assess the specific roles of FGFR1 versus FGFR3, we used several different experimental criteria. We applied multiple approaches: two inhibitors with distinct modes of action (that is, a chemical inhibitor that blocks the tyrosine kinase activity of FGFR1 and an antibody directed against FGFR1), specific siRNAs that target FGFR1 or FGFR3, as well as comparisons between FGF-2 versus FGF-18 treatments. Published data and our own empirical findings indicate that the chemical inhibitor, the antibody, and the siRNA for FGFR1 each selectively target FGFR1, while FGF-2 and FGF-18 have different downstream effects. These criteria together permit interpretations that the primary functions of FGFR1 and FGFR3 signaling by FGF-2 are distinctive in human adult articular chondrocytes. While contributing functions of FGFR3 in mediating FGF-2 signaling cannot be ruled out categorically, our biological results favor the interpretation that sustained FGF-2/FGFR1 signaling, but not FGFR3 signaling, is primarily responsible for proliferation, pro-catabolism as well as anti-anabolism in adult human articular chondrocytes.
Absence of signaling from FGFR3 was demonstrated to result in increased MMP-13 expression and cartilage degradation, which resembles the osteoarthritic features observed in mice overexpressing MMP-13 [37,38]. This increase in MMP-13 may be due to elevated FGF-2 signaling through FGFR1, which is a dominant, major FGFR subtype in the absence of FGFR3 [33]. The orchestrated and fine-tuned activities of FGFR1 and FGFR3 seem to be essential to extracellular matrix turnover under normal condition [39]. The results presented in this study collectively suggest that FGFR1 and FGFR3 promote catabolism and anabolism, respectively.
Arthritic tissues from OA patients exhibited substantially decreased expression of FGFR3, thus possibly intensifying FGFR1 signaling in human primary knee joint articular chondrocytes. We also found that FGF-2/FGFR1 signaling down-regulated FGFR3 expression in articular chondrocytes. The observation that FGF-2 levels are abnormally elevated in synovial fluid of OA patients fits a molecular model [18], in which FGF-2 initiates a self-reinforcing feedback loop that perpetuates the characteristic degeneration of cartilage in OA, via promoting FGF-2/FGFR1 signaling and simultaneously, suppressing FGFR3-related pathways (for example, FGFR3/FGF18 signaling). We observed distinct cellular responses to FGF-2 in different biological contexts (for example, between knee and ankle; normal and OA; young and old, and so on). For example, we consistently observed a more adverse effect of FGF-2 in aged tissue donors ( > 45 years old) with damaged femoral regions (Collin's grade > 2) or clinical OA as previously published [19]. Nevertheless, we do not always observe the same biological effects by FGF-2 using donor tissues from young and normal knee cartilage with Collin's grade 0 or ankle tissues. In particular, although our IP results suggest that FGF-2 more potently activates FGFR1 than FGFR3 in knee articular chondrocytes, we observed differential activities of FGFR1 and FGFR3 in ankle chondrocytes after FGF-2 stimulation, in which FGF-2-induced activation of FGFR3 was as potent as FGFR1 (data not shown). We found these results very interesting as it may provide a molecular mechanistic understanding explaining, in part, why knee joints are more vulnerable to OA compared with ankles. Our data remain more consistent with the initial publications that FGF-2 stimulates catabolism and/or anti-anabolism by inducing cartilage degrading enzymes (for example, MMP1, MMP13) and proteoglycan loss in articular cartilage in vitro and ex vivo [11,18,19]. However, we are aware of some animal studies that demonstrated that FGF-2-mediates anabolism in knee joints [40]. Although these apparent discrepancies in biological effects are not clearly understood yet, it may result from: age- and grade-dependent differences (correlates with degree of damage) between animal (young and healthy grade 0) and human tissues ( > 45 years old, grade 0/1) or perhaps, disease stage-specific expression/activation pattern of FGF receptors (for example, FGFR1 versus FGFR3) as we have shown in this study.
The findings presented here indicate that FGF-2 signaling via FGFR1 is required for expression and/or transcriptional regulation of the collagenase MMP-13 and aggrecanase ADAMTS5, as well as suppression of the key anabolic gene aggrecan. Cartilage degradation is linked to ADAMTS5, and specific inhibition of ADAMTS5 is chondroprotective for articular joints [41,42]. Interestingly, we did not observe a significant induction of ADAMTS4 upon FGF-2 stimulation. This finding suggests that ADAMTS5 is preferentially modulated by FGF-2 and may have a selective patho-physiological function that complements MMP-13 and other proteolytic enzymes in OA. Aggrecan turnover occurs in healthy cartilage, and the imbalance between its production and degradation results in defective extracellular matrix [39]. The repression of aggrecan by FGF-2-FGFR1 possibly disrupts normal extracellular matrix metabolism and facilitates further pathogenic progression.
The activation of chondrocyte proliferation by FGF-2/FGFR1 activation observed in this study is consistent with studies in various cell types, including chondrocytic cells [43]. The effect of FGF-2 on stimulating chondrocyte proliferation and proteoglycan-degrading enzymes, and reducing proteoglycan production, may compromise the integrity of the extracellular matrix surrounding newly divided chondrocytes. We previously reported that the endogenous level of FGF-2 is highly increased in osteoarthritic synovial fluids [18]. Increased chondrocyte proliferation was also observed in certain osteoarthritic populations [44]. Therefore, pathologically elevated levels of FGF-2 in osteoarthritic synovial fluid may influence not only cartilage but also the "whole joint organ", including synovial lining and subchondral bone, which may promote fibroblastic proliferation of chondrocytes, resulting in the formation of fibrocartilage with altered biological and biomechanical properties. In addition, neural ingrowth and angiogenesis in synovium, which have been shown in the OA animal model and patients with painful knee joint OA, may be directly and/or indirectly promoted by FGF-2 [45].
Previously, we have shown that FGF-2 potently abrogated BMP-7-mediated proteoglycan synthesis and accumulation [5,46]. Our current study demonstrates that administration of FGF-2 suppressed the FGFR3 gene via the activation of ERK/MAPK in human articular chondrocytes. Interestingly, we found that BMP-7 markedly up-regulated FGFR3 expression, and this induction was effectively blocked by the FGF-2-ERK/MAPK axis (unpublished data). It is possible that BMP-7 augments its anabolic activity, in part, via induction of FGFR3, thus indirectly potentiating endogenous FGF18-FGFR3 signaling. This may explain the additive effect (if not synergistic) of BMP-7 plus FGF18 on proteoglycan production observed in another set of our studies in human articular chondrocytes (unpublished data). One plausible mechanism is that FGF-2 overrides stimulatory effects of BMP-7 on FGFR3 expression, which negates the responsiveness to FGF18 and diminishes proteoglycan production. These speculations need to be confirmed by a set of experiments in future studies.
Conclusions
In conclusion, we have provided evidence that FGFR1 primarily transmits detrimental signals in adult human articular chondrocytes upon FGF-2 stimulation, as opposed to FGFR3. FGFR1 signaling leads to inhibition of proteoglycan accumulation, increased catabolic gene expression, and decreased anabolic gene expression. FGFR1 and FGFR3, which represent receptors with the highest affinity for FGF-2, are dominantly expressed in articular chondrocytes. FGFR1 is preferentially activated by FGF-2 over FGFR3, which corroborates the catabolic role of FGF-2. FGFR3 is significantly down regulated in osteoarthritic chondrocytes, and the FGFR1 to FGFR3 expression ratio is elevated in OA. In addition, FGFR3 is down-regulated by FGF-2 signaling through FGFR1-ERK axis. Our findings suggest that FGFR1 specifically has a predominant function in FGF-2-promoted cartilage degeneration and OA pathophysiology.
Abbreviations
ADAMTS: a disintegrin and metalloproteinase with a thrombospondin type 1 motif; BCA: bicinchoninic acid; DMEM: Dulbecco's modified Eagle's medium; DMMB: dimethylmethylene blue; ERK: extracellular signal-regulated kinase; FGF-2: fibroblast growth factor 2; FGFR: FGF receptor; IP: immunoprecipitation; MMP: matrix metalloproteinase; OA: osteoarthritis; qPCR: quantitative real-time polymerase chain reaction; RT: reverse transcription; siRNA: small interfering RNA.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
DY designed the experiments for this study, acquired the data, interpreted the data, carried out the flow cytometry analysis and drafted the manuscript. DC and H-JI designed the experiments for this study, interpreted the data and drafted the manuscript. SC and GM interpreted the data. KM interpreted the data and carried out the flow cytometry analysis. AW interpreted the data and drafted the manuscript. All authors read, edited and approved the final manuscript.
Acknowledgements
We would like to thank the tissue donors, Drs. Gabriella Cs-Szabo, Arkady Margulis, and the Gift of Hope Organ and Tissue Donor Network for normal and OA human joint tissue samples. We also thank Dr. Prasuna Muddasani for her excellent technical assistance, and Dr. Richard Loeser (Wake Forest University School of Medicine) for the MMP13/Luc reporter gene construct. FGF-2 was kindly provided by NCI. This work was supported by grants (to H-JI) from NIH R01AR053220, the Arthritis Foundation, and the National Arthritis Research Foundation.
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Arthritis Res TherArthritis Res. TherArthritis Research & Therapy1478-63541478-6362BioMed Central ar32572133851510.1186/ar3257Research ArticleReduced immunomodulation potential of bone marrow-derived mesenchymal stem cells induced CCR4+CCR6+ Th/Treg cell subset imbalance in ankylosing spondylitis Wu Yanfeng [email protected] Mingliang [email protected] Rui [email protected] Xinjun [email protected] Yuanchen [email protected] Yong [email protected] Lin [email protected] Jichao [email protected] Keng [email protected] Peng [email protected] Huiyong [email protected] Department of Othopaedics, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, 107# Yanjiangxi Road, Guangzhou 510120, PR China2 Biotherapy Centre, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, 107# Yanjiangxi Road, Guangzhou 510120, PR China3 Department of Othopaedics, Guangdong Provincial People Hospital, 106# Zhongshan Road 2, Guangzhou 510080, PR China2011 21 2 2011 13 1 R29 R29 12 10 2010 16 1 2011 21 2 2011 Copyright ©2011 Wu et al.; licensee BioMed Central Ltd.2011Wu et al.; licensee BioMed Central Ltd.This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Introduction
Ankylosing spondylitis (AS) is a chronic autoimmune disease, and the precise pathogenesis is largely unknown at present. Bone marrow-derived mesenchymal stem cells (BMSCs) with immunosuppressive and anti-inflammatory potential and Th17/Treg cells with a reciprocal relationship regulated by BMSCs have been reported to be involved in some autoimmune disorders. Here we studied the biological and immunological characteristics of BMSCs, the frequency and phenotype of CCR4+CCR6+ Th/Treg cells and their interaction in vitro in AS.
Methods
The biological and immunomodulation characteristics of BMSCs were examined by induced multiple-differentiation and two-way mixed peripheral blood mononuclear cell (PBMC) reactions or after stimulation with phytohemagglutinin, respectively. The interactions of BMSCs and PBMCs were detected with a direct-contact co-culturing system. CCR4+CCR6+ Th/Treg cells and surface markers of BMSCs were assayed using flow cytometry.
Results
The AS-BMSCs at active stage showed normal proliferation, cell viability, surface markers and multiple differentiation characteristics, but significantly reduced immunomodulation potential (decreased 68 ± 14%); the frequencies of Treg and Fox-P3+ cells in AS-PBMCs decreased, while CCR4+CCR6+ Th cells increased, compared with healthy donors. Moreover, the AS-BMSCs induced imbalance in the ratio of CCR4+CCR6+ Th/Treg cells by reducing Treg/PBMCs and increasing CCR4+CCR6+ Th/PBMCs, and also reduced Fox-P3+ cells when co-cultured with PBMCs. Correlation analysis showed that the immunomodulation potential of BMSCs has significant negative correlations with the ratio of CCR4+CCR6+ Th to Treg cells in peripheral blood.
Conclusions
The immunomodulation potential of BMSCs is reduced and the ratio of CCR4+CCR6+ Th/Treg cells is imbalanced in AS. The BMSCs with reduced immunomodulation potential may play a novel role in AS pathogenesis by inducing CCR4+CCR6+ Th/Treg cell imbalance.
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Introduction
Ankylosing spondylitis (AS) is a chronic autoimmune inflammatory disease, the prototypic seronegative spondylarthritis that primarily affects the sacroiliac joints and the axial skeleton, which was characterized by inflammatory back pain, enthesitis, and specific organ involvement [1]. AS is a complex multifactorial disease; several pathogenetic factors, including infection [1,2], environmental triggers [1], genetic susceptibility such as HLA-B27 positivity [3,4] and HLA-E gene polymorphism [5], and in particular, autoimmune disorders [1] have been reported to potentially trigger the onset or maintain the pathogenesis progress of AS. Additionally, the genome-wide association study of AS identifies non-MHC susceptibility loci [6], such as IL-23R (rs11209026) and ERAP1 (rs27434). There were also, however, some controversies; for example, no candidate bacteria were detected by PCR in biopsies from sacroiliac joints [7] and most HLA B27-positive individuals remain healthy [1]. The precise pathogenesis of AS is therefore largely unknown at present. Nowadays, more and more studies have focused on the immunological factors for AS.
Mesenchymal stromal cells (MSCs) isolated from a variety of adult tissues, including the bone marrow, have multiple differentiation potentials in different cell types, and also display immunosuppressive (in vitro [8,9], in vivo [10-12]) and anti-inflammatory properties [13], so their putative therapeutic role in a variety of inflammatory autoimmune diseases is currently under investigation. Recently, many findings indicate that MSC immunomodulation potential plays a critical role in severe aplastic anemia [14]. Simultaneously, substantial disorders and abnormalities of MSCs exist in many autoimmune diseases [15]. Few studies, however, have so far focused on whether there were some abnormalities in bone marrow-derived mesenchymal stem cells (BMSCs) of patients with ankylosing spondylitis (ASp) with regard to the biological and immunological properties.
More recently, two additional subsets, the forkhead box P3 (Fox-P3)-positive regulatory subset (Treg) and the IL-17-producing subset (Th17) [16-19], have emerged and together with Th1 and Th2 cells, formed a functional quartet of CD4+ T cells that provides a closer insight into the mechanisms of immune-mediated diseases such as AS. Autoimmune diseases are thought to arise from a breakdown of immunological self-tolerance leading to aberrant immune responses to self-antigen. Ordinarily, regulatory T (Treg) cells - including both natural and induced Treg cells - control these self-reactive cells [20]. Several studies of patients with connective tissue diseases found reduced [21] or functionally impaired [22] Treg cells, and Treg cells of autoimmune hepatitis patients have reduced expression of Fox-P3 and CTLA-4, which may lead to impaired suppressor activity [23]. On the contrary, these proinflammatory Th17 cells are implicated in different autoimmune disease models [24-26]. Furthermore, these cells typically express IL-23R on their membrane [27], and recent studies in AS [28-30] show an important genetic contribution for polymorphisms in the gene that codes for this IL-23R. The active polymorphisms in the IL-23R gene could thus indicate an important role for this pathogenic T-cell subset (Th17) in the development and maintenance of AS. The involvement of Treg and Th17 cells in AS, however, has not yet been clearly established.
As previously described, skewing of responses towards Th17 and away from BMSCs or Treg cells may be responsible for the development and/or progression of AS [31]. Furthermore, CCR6 and CCR4 identified true Th17 memory cells producing IL-17 [32] and the majority of Th17 cells were CCR6+CCR4+ [33]. Aimed at investigating the puzzling issues above, the present study was designed to examine the biological and immunological properties of BMSCs, to examine the frequencies and phenotypes of Treg cells and proinflammatory CCR4+CCR6+ Th memory cells, and to study the interactions between BMSCs and CCR4+CCR6+ Th/Treg cells in peripheral blood mononuclear cells (PBMCs) for AS.
Materials and methods
Patients and controls
The present study was approved by the ethics committee of the Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University, Guangzhou, China. In addition, informed consent was obtained from all patients and all healthy donors (HDs). Fifty-one ASp (eight women and 43 men) with an average age of 29.4 years (17 to 45 years) and 49 HDs (eight women and 41 men) with an average age of 27.3 years (18 to 39 years) were included in the study. All of the AS patients were diagnosed according to the New York modified criteria [34] and were HLA-B27-positive; conversely, 37 healthy donors were HLA-B27-negative (HD1) and 12 healthy donors were HLA-B27-positive (HD2). Sixteen patients were diagnosed for the first time, and the research samples from all ASp were taken at the active stage (all Bath Ankylosing Spondylitis Disease Activity Index ≥4) and without taking any medicine for at least 2 weeks.
Bone marrow aspiration, human BMSCs and PBMCs
After being informed regarding the scientific contributions, possible risks and complications and the corresponding prevention and treating measures for bone marrow aspirations, all of the healthy controls and ASp expressed approval and signed the informed consent. The bone marrow aspirations were all performed by skilled allied health professionals strictly according to the international standardized procedure for bone marrow aspirations. The bone marrow samples of AS patients and HDs were diluted with DMEM (low-glucose DMEM) containing 10% FBS. The mononuclear cells were prepared by gradient centrifugation at 900 × g for 30 minutes on Percoll (Pharmacia Biotech, Uppsala, Sweden) of density 1.073 g/ml. The cells were washed, counted, seeded at 2 × 106 cells/cm2 in 25-cm2 flasks containing low-glucose DMEM supplemented with 10% FBS and cultured at 37°C, 5% carbon dioxide. Medium was replaced and the cells in suspension were removed at 48 hours and every 3 or 4 days thereafter. BMSCs were recovered using 0.25% Trypsin-ethylenediamine tetraacetic acid and replated at a density of 5 × 103 to 6 × 103 cells/cm2 surface area as passage 1 cells when the culture reached 90% confluency. BMSCs after the third subculture were used for described experiments. PBMCs were obtained by the Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden) gradient separation of the buffy coat of ASp and HDs.
Cell viability and proliferation test for BMSCs
BMSCs were seeded in 96-well plates at a concentration of 1 × 104/ml, in a final volume of 100 μl fresh medium (10% FBS + low-glucose DMEM), and three wells of each sample were digested using 0.25% Trypsin-ethylenediamine tetraacetic acid for cell counting per day up to 12 days. The BMSC growth curves were made using the data for cell proliferation obtained above. Using MTT (5 mg/ml; Sigma-Aldrich Co., St. Louis, MO, USA), dimethyl sulphoxide (Sigma) and an EL800 microplate reader (BioTek Instruments, Winooski, VT, USA) that was to determine absorbance at 490 nm, the cell viability curves for BMSCs were acquired in the same way according to the day and the absorbance. The BMSC proliferation ability was also examined by 3H-TdR assay. Fresh medium was used as a negative control.
In vitro differentiation potential assay of BMSCs
To induce osteogenic differentiation, BMSCs were initially seeded in six-well plates at a concentration of 104/cm2. After preculturing for 24 hours, the BMSCs were allowed to grow in osteogenic medium (high-glucose DMEM supplemented with 10% FBS, 50 mg/l ascorbic acid, 10 mM β-glycerolphosphate and 10 nM dexamethasone; all these inducing reagents from Sigma). The BMSCs were then incubated in 5% carbon dioxide at 37°C, according to the experimental requirements for up to 14 days, and the medium was replaced every 3 days before harvest. The alkaline phosphatase (ALP) and mineralization of BMSCs were assayed using Cell Alkaline Phosphatase Staining assay (Sigma) and Alizarin Red staining (AR-S, 40 mmol/l, pH 4.2; Sigma) on the 14th day, respectively.
To induce adipogenic differentiation, the BMSCs were seeded in six-well plates at a concentration of 104/cm2. After preculturing for 24 hours, the BMSCs were shifted to adipogenic medium (low-glucose DMEM supplemented with 10% FBS, 1 μM dexamethasone, 10 μg/ml insulin, 0.5 mM 3-isobutyl-1-methylxanthine and 0.2 mM indomethacin; all these inducing reagents from Sigma). The BMSCs were then incubated in 5% carbon dioxide at 37°C, and the medium was replaced every 3 days before harvest. The intracellular lipid accumulation as an indicator was visualized on the 14th day by Oil Red O staining after fixed with 4% cold paraformaldehyde in PBS (pH 7.4) and washed with distilled water.
To induce chondrogenic differentiation, aliquots of 2.5 × 105 BMSCs were centrifuged at 1,000 rpm for 5 minutes in 15-ml polypropylene conical tubes to form pellets, which were then cultured in high-glucose DMEM supplemented with 1% ITS-Premix (Becton-Dickinson, Mountain View, CA, USA), 50 mg/ml ascorbic acid (Sigma), 10-3 M sodium pyruvate (Sigma), 10-7 M dexametazone (Sigma), and 10 ng/ml transforming growth factor-β3 (R&D Systems, Minneapolis, MN, USA) for 28 days. The pellets were then fixed with 4% paraformaldehyde, embedded in paraffin, and subjected to Alcian blue staining to confirm chondrogenic differentiation.
The BMSCs in fresh medium (high-glucose DMEM supplemented with 10% FBS) without these differentiation-inducing factors were used as the experimental control, and fresh medium without any cells was used as a negative control. All measurements were performed in triplicate. The images were visualized using an inverted phase contrast microscope (Nikon Eclipse Ti-S, Nikon Corporation, Tokyo Prefecture, Japan).
Alkaline phosphatase measurement
On the 14th day the osteogenic medium was removed, and then 1.0 ml Triton X-100 (Sigma) was added to each well. A cell scraper was used to remove the BMSCs from the well bottom, and then the 1.0 ml cell lysate were placed in a 1.5-ml centrifuge tube. The samples were then processed through two freeze-thaw cycles (-70°C and room temperature, 45 minutes each) to rupture the cell membrane and extract the proteins and DNA from the cells. A p-nitrophenyl phosphate liquid substrate system (Stanbio, Boerne, TX, USA) was used to analyze the ALP concentration from the cells of each group. Then 10 μl each cell lysate solution was added to 190 μl p-nitrophenyl phosphate substrate and incubated in the dark at room temperature for 1 minute. The absorbance was read using a plate reader (M5 SpectraMax; Molecular Devices, Sunnyvale, CA, USA) at 405 nm and normalized to the PicoGreen assay [35]. DNA was quantified using the Quant-iT PicoGreen Kit (Invitrogen, Carlsbad, CA, USA) following standard protocols. Briefly, 100 μl each cell lysate solution was added to 100 μl PicoGreen reagent and incubated in the dark at room temperature for 5 minutes. The absorbance was read at an excitation/emission of 480 to 520 nm on the plate reader.
Immunomodulation potential of BMSCs
The inhibitory effects of BMSCs on mixed PBMC reaction (MLR) and PBMC proliferation stimulated by phytohemagglutinin (PHA) (4 μg/ml; Roche, Mannheim, Germany) were measured using the MTT assay [36] and the 3H-TdR assay [10] as described previously. Briefly, BMSCs were seeded in V-bottomed, 96-well culture plates for 4 hours for adherence, and then irradiated (30 Gy) with Co60 before being cultured with the mixed PBMCs or the PBMCs stimulated by PHA.
Two-way mixed PBMC reaction
For the two-way MLR, allogeneic PBMCs (15 × 104 cells/cm2) from a healthy volunteer were mixed in a 1:1 ratio with PBMCs from another unrelated healthy volunteer (third-party setting). The mixed PBMCs were then mixed with different amounts (15 × 103 cells/cm2 = 1:20 BMSC:PBMC ratio, 3 × 104 cells/cm2 = 1:10, 6 × 104 cells/cm2 = 1:5, 15 × 104 cells/cm2 = 1:2, 3 × 105 cells/cm2 = 1:1) of BMSCs (experiment wells) or without BMSCs (blank wells) in V-bottomed, 96-well culture plates to ensure efficient cell-cell contact for 5 days in 0.2 ml modified RPMI-1640 medium (Gibco, BRL, Grand Island, NY, USA) supplemented with 10% FBS.
Allogeneic PBMC proliferation assay
Compared with the MLR, the allogeneic PBMC proliferation assay only uses one allogeneic PBMC reaction (30 × 104 cells/cm2) from a healthy volunteer stimulated with PHA, instead of two PBMC reactions. Inhibitory effects were measured on the 5th day using the MTT assay with an EL800 microplate reader at 570 nm and the 3H-TdR assay with a microplate scintillation and luminescence counter (Packard NXT, Meriden, CT, USA). Results were expressed as mean absorbance (optical density (OD)) ± standard deviation (SD) and as mean counts per minute (CPM) ± SD, respectively. All measurements were performed in triplicate.
The data are presented as percentage inhibition values calculated using the following formulae (Table 1):
Table 1 Details regarding the formula for percentage inhibition in the present study
Experiment wells Adjusted wells Blank wells
Two-way MLR BMSCs + 2 HD-PBMCs BMSCs 2 HD-PBMCs
Allogeneic PBMC proliferation assay BMSCs + 1 HD-PBMC + PHA BMSCs 1 HD-PBMC + PHA
BMSC, bone marrow-derived mensenchymal stem cell; PBMC, peripheral blood mononuclear cell; HD-PBMC, peripheral blood mononuclear cell of healthy donor; PHA, phytohemagglutinin; MLR, mixed PBMC reaction.
OD(exp), OD(adj) and OD(bla) represent the mean absorbance of experiment wells, adjusted wells (only BMSCs) and blank wells, respectively, and CPM(exp), CPM(adj) and CPM(bla) represent the mean counts per minute of the corresponding wells. Depending on the experimental design, there were some wells used for controlling. Results were expressed as the mean (% inhibition) ± SD.
Direct contact co-culture of BMSCs and PBMCs
BMSCs were trypsinized and then irradiated (30 Gy) with Co60 before being co-cultured with PBMCs from a healthy volunteer in the presence of PHA (4 μg/ml; Roche) in 24-well plates (Nunclon, Roskilde, Denmark) and plated at a ratio of 1:10 in a total volume of 2 ml/well in triplicate for 72 hours. The cell density was 5 × 104/cm2 BMSCs and 5 × 105/cm2 PBMCs in a mix. Phorbol myristate acetate (50 ng/ml; Sigma, St Louis, MO, USA) and calcium ionomycin (1 μg/ml; Sigma) were added 6 hours prior to the end of the 72-hour co-culture. All of the PBMCs were then collected to be assayed by flow cytometry for the CCR4+CCR6+ Th and Treg cells. PBMCs were also grown alone in BMSC-free medium and used as control.
Antibodies and flow cytometry
To detect the surface markers [37] of BMSCs and the frequency of CCR4+CCR6+ Th and Treg cells in PBMCs, the antibodies (Table 2) - including CD105(FITC), CD73(FITC), CD90(FITC), CD34(FITC), CD45(FITC), CD14(PE) and HLA-DR(FITC) for BMSCs; CCR4(PE-Cy7), CD196(CCR6)(PE) and CD4(FITC) for CCR4+CCR6+ Th cells [29]; and CD4(FITC), CD25(APC) and Fox-P3(PE) antibodies for Treg cells - were used according to the manufacturers' recommendations. BMSCs and PBMCs marked with appropriate antibodies were measured with a FACScan laser flow cytometry system (Becton Dickinson) immediately. In each experiment, control staining with the appropriate isotype monoclonal antibodies was included. Results were expressed as the mean (frequency, %) ± SD.
Table 2 Antibodies used to detect CCR4+CCR6+ Th and Treg cells in PBMCs and phenotype BMSCs by flow cytometry
Antibody Isotype Clone/fluorochrome Concentration (μg/ml) Source
CD105 (ENG)a Mouse IgG1 266/FITC 10 Becton Dickinson (Bedford, MA, USA)
CD73 (NT5E)a Mouse IgG1 AD2/FITC 20 Becton Dickinson (Bedford, MA, USA)
CD90 (THY1)a Mouse IgG1 5E10/FITC 1 Becton Dickinson (Bedford, MA, USA)
CD34a Mouse IgG1 581/FITC 50 Southern Biotech (Birmingham, AL, USA)
CD45 (PTPRC)a Mouse IgG1 HI30/FITC 10 Caltag (Burlingame, CA, USA)
CD14a Mouse IgG1 61D3/PE 200 Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA)
HLA-DRa Mouse IgG2a L243/FITC 200 Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA)
CD4b Mouse IgG2b OKT4/FITC 12 eBioscience (San Diego, CA, USA)
CD25b Mouse IgG1,κ BC96/APC 25 eBioscience (San Diego, CA, USA)
FOX-P3b Mouse IgG1 236A/E7/PE 50 eBioscience (San Diego, CA, USA)
CCR4b Mouse IgG1,κ 1G1/PE-Cy7 200 BD Pharmingen™ (Basel, Switzerland)
CCR6 (CD196)b Mouse IgG1,κ 11A9/PE 200 BD Pharmingen™ (Basel, Switzerland)
aAntibodies used to phenotype bone marrow-derived mensenchymal stem cells (BMSCs) by flow cytometry. bAntibodies used to detect CCR4+CCR6+ CD4+ T-helper (Th) cells and forkhead box P3-positive regulatory T (Treg) cells by flow cytometry. PBMC, peripheral blood mononuclear cell.
Statistical analysis
Data are expressed as the mean ± SD, and the significance of the results was determined using the unpaired Student's t test. The product-moment correlation coefficient was used to test the correlations between the suppression ratios of BMSCs and the ratio of CCR4+CCR6+ Th cells to Treg cells in peripheral blood. Statistical analysis was performed using the SPSS computer program (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered statistically significant.
Results
Growth characteristics and cell viability of AS-BMSCs are normal
To evaluate the biological properties of AS-BMSCs, compared with those of HD-BMSCs, the studies for growth characteristics, cell viability and multiple differentiation potentials in vitro were performed. The AS-BMSC growth curves have the same tendency as those for HD-BMSCs. The BMSC proliferation data of these two groups at each day (1 to 12 days) were tested by unpaired Student's t test, and the statistical result indicates that there was no statistically significant difference in BMSC growth characteristics between ASp and HDs (HD1 and HD2) (P > 0.05, Student's t test for independent samples). Established cultures (12 days) of BMSCs exhibited close, even equivalent, cell viability at each point of time from 1 to 12 days, as determined by cellular viability assays, and the difference of OD at 490 nm between ASp and HDs (HD1 and HD2) at each day (1 to 12 days) was not also statistically significant (P > 0.05, Student's t test for independent samples). The cultures have similar purities: (QL [the lower point of interquartile range], QU [the upper point of interquartile range]) = (95%, 99%) for AS-BMSCs, (QL, QU) = (96%, 98%) for HD1-BMSCs and (QL, QU) = (96%, 99%) for HD2-BMSCs.
Triple differentiation potentials of AS-BMSCs in vitro were not changed
To explore whether the multiple differentiation potentials of BMSCs in AS were abnormal, we investigate the osteogenic, adipogenic and chondrogenic differentiation potentials of AS-BMSCs and HD-BMSCs in the present study. Obvious differentiated osteocytes and adipocytes were detected as early as the 7th day after being induced for osteogenic and adipogenic differentiation, and obvious differentiated chondrocytes were seen at about 14 days since induction (Figure 1A to 1C).
Figure 1 Bone marrow-derived mensenchymal stem cell triple differentiation potentials from ankylosing spondylitis patients and healthy donors. (A), (B), (C) Morphological characteristics of bone marrow-derived mensenchymal stem cells (BMSCs) for osteogenic, adipogenic and chondrogenic differentiation evaluated by the inverted phase contrast microscope; ankylosing spondylitis (AS)-BMSCs have the same morphological properties as the HLA-B27-negative healthy donor (HD1)-BMSCs and HLA-B27-positive healthy donor (HD2)-BMSCs. Osteocytes were stained for calcium deposition using Alizarin Red-S (A1, B1, C1: x400) and for alkaline phosphatase (ALP) with the Cell Alkaline Phosphatase-S assay (A2, B2, C2: x200). Adipocytes were filled with many fat vacuoles, and Red Oil O was used to stain the fat vacuoles of adipocytes (A3, B3, C3: x200). Chondroblast differentiation from BMSCs was identified with Alcian blue staining (A4, B4, C4: x200). (D) General photographs of BMSCs for osteogenic differentiation, stained with Alizarin Red-S. (E) ALP activities of AS-BMSCs, HD1-BMSCs and HD2-BMSCs were 644 ± 45, 655 ± 49 and 646 ± 51, respectively; differences not statistically significant (P > 0.05). Data presented as mean ± standard deviation. MLR, mixed peripheral blood mononuclear cell reaction; PHA, phytohemagglutinin.
There appeared to be two stages in the BMSC differentiation process for both ASp and HDs. In the early stage, only a few osteocytes, adipocytes and chondrocytes were found within the undifferentiated BMSCs. Gradually, these three kinds of cells increased; simultaneously, the cell's body got bigger and cytoplasm became more abundant because, for example, osteocytes made closer contact, fat vacuoles of adipocytes multiplied and grew bigger, and chondrocytes began to gain many collagen fibers. In the later stage, these three kinds of cells increased rapidly and nearly predominated. For the adipocytes, osteocytes and chondrocytes derived from BMSCs, the purities were (QL, QU) = (90%, 97%), (QL, QU) = (91%, 96%) and (QL, QU) = (88%, 95%) for ASp, (QL, QU) = (88%, 98%), (QL, QU) = (90%, 97%) and (QL, QU) = (90%, 96%) for HD1, and (QL, QU) = (86%, 95%), (QL, QU) = (89%, 98%) and (QL, QU) = (92%, 97%) for HD2, respectively.
The calcium nodules were stained to present a red color (Figure 1A1 to 1D1), after Alizarin Red staining for calcium deposits of osteocytes was performed to determine the mineralization of BMSCs. For the adipogenic differentiation, the mass fat vacuoles of adipocytes were also stained to present a red color by Oil Red O staining (Figure 1A3 to 1C3). The well-differentiated chondrocytes were Alcian Blue-positive, and presented a bright blue color after staining (Figure 1A4 to 1C4).
The ALP activity, normalized to DNA concentration, is plotted in Figure 1E. The ALP activity (mean ± SD) was 644 ± 45 (mM p-nitrophenyl phosphate/minute per mg DNA) for AS-BMSCs (n = 51), which is lower than the 655 ± 49 for HD1-BMSCs (n = 37) (P > 0.05) and the 646 ± 51 for HD2-BMSCs (n = 12) (P > 0.05). All three values were much higher than those of the baseline ALP for BMSCs of ASp, HD1 and HD2 (85 ± 40, 88 ± 48 and 82 ± 13, respectively) (P < 0.001) in control medium without the osteogenic factors. ALP staining was performed on the 14th day to investigate the maturity degree of osteocytes in the groups of Asp, HD1 and HD2 (Figure 1A2 to 1C2).
Phenotype of bone marrow-derived mesenchymal stem cells
The AS-BMSCs and HD-BMSCs were then examined for typical MSC phenotypic surface markers. Flow cytometric analysis showed that the AS-BMSCs and HD-BMSCs (HD1-BMSCs and HD2-BMSCs) have the same phenotypic surface markers, just as the typical MSCs did. The samples all express high levels of the surface markers CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 and HLA-DR surface molecules (Figure 2).
Figure 2 Phenotyping of bone marrow-derived mensenchymal stem cells for typical mensenchymal stromal cell surface markers. Single-parameter histograms for (A1) to (A3), (B1) to (B3), (C1) to (C3) individual mensenchymal stromal cell (MSC) markers and (A4) to (A7), (B4) to (B7), (C4) to (C7) MSC exclusion markers, representative of samples from patients with ankylosing spondylitis (AS) and from healthy donors (blue lines). Red lines indicate background fluorescence obtained with isotype control IgG. x axis, fluorescence intensity; y axis, cell counts. BMSC, bone marrow-derived mensenchymal stem cell; HD1, HLA-B27-negative healthy donors; HD2, HLA-B27-positive healthy donors.
Decreased suppressive potential of AS-BMSCs on either two-way MLR or PBMC proliferation stimulated with PHA
Under the condition that the proliferation characteristics, cell viability, multiple-differentiation potentials and surface markers of AS-BMSCs were normal, compared with HD-BMSCs, the immunomodulation potential of AS-BMSCs was evaluated in the present study. The effects of BMSCs from ASp (n = 51), HD1 (n = 37) and HD2 (n = 12) on two-way MLR or PBMC proliferation in the presence of PHA were evaluated by mixing BMSCs and mixed PBMCs for two-way MLR, or by PBMCs from a third healthy volunteer in the presence of PHA for PBMC proliferation assay at five BMSC:PBMC ratios of 1:20, 1:10, 1:5, 1:2 and 1:1, respectively.
Two-way mixed PBMC reaction
The differences of absorbance between 0 days and 5 days were not statistically significant for the allogeneic PBMCs from a healthy volunteer (P = 0.351) and the PBMCs from another unrelated healthy volunteer (P = 0.418) (Figure 3A1). For the mixed PBMCs, however, the absorbance at 5 days was significantly higher than the value at 0 days (P < 0.001, Figure 3A1). As shown in Figure 3A2,A3, there was a statistically significant reduction in suppressive potential (% inhibition) of BMSCs from ASp on two-way MLR at all five ratios, compared with the percentage inhibition of HD-BMSCs (P < 0.001, Figure 3A2,A3).
Figure 3 Reduced suppressive potential of bone marrow-derived mensenchymal stem cells of patients with ankylosing spondylitis. (A1) Absorbance of mixed peripheral blood mononuclear cells (PBMCs) at 5 days (0.194 ± 0.038) was significantly higher than the value at 0 days (0.104 ± 0.023) (*P < 0.001), showing the significant mixed PBMC reaction. (A2), (A3) Compared with healthy donor (HD)-bone marrow-derived mensenchymal stem cells (BMSCs), the decreased percentage inhibition of ankylosing spondylitis (AS)-BMSCs on two-way mixed peripheral blood mononuclear cell reaction (MLR) at different ratios showed that suppressive potentials of AS-BMSCs were reduced (% inhibition reduced, *P < 0.001). (B1) PBMCs derived from a healthy volunteer could proliferate significantly the presence of phytohemagglutinin (PHA) in vitro (P < 0.001). (B2), (B3) Percentage inhibition of AS-BMSCs on PBMC proliferation induced by PHA was significantly lower than the values of HD-BMSCs at varied BMSC:PBMC ratios of 1:20, 1:10, 1:5, 1:2 and 1:1 (% inhibition reduced, *P < 0.001). Data expressed as mean ± standard deviation of triplicates of three separate experiments. (A2), (B2) and (A3), (B3) were performed by MTT assay and 3H-TdR assay respectively. There were no statistically significant differences of suppressive potentials between (A2), (A3) HLA-B27-negative healthy donor (HD1)-BMSCs and (B2), (B3) HLA-B27-positive healthy donor (HD2)-BMSCs. OD, optical density.
Allogeneic PBMC proliferation assay
Similarly, when PBMC proliferation was elicited by means of PHA, the addition of BMSCs from ASp also produced a statistically significant decreased inhibitory effect on PBMC proliferation (Figure 3B2,B3; P < 0.001, Student's t test for independent data), ranging from a BMSC:PBMC ratio of 1:20 to 1:1. Additionally, the differences of absorbance between 0 days and 5 days without PHA were not significant (P = 0.223), while the value for 5 days with PHA was significantly higher than the value at 0 days (P < 0.001, Figure 3B1).
Furthermore, in either the MLR (Figure 3A3) or the PBMC proliferation assay stimulated with PHA (Figure 3B3), the 3H-TdR assay data suggested a significant relationship between dose and suppression of immunoreactivity of BMSCs for ASp, HD1 and HD2. The MTT assay also presented this phenomenon basically, but not clearly.
Increased CCR4+CCR6+ Th and decreased Treg populations in peripheral blood of patients with AS
Recent studies have independently revealed enhanced Th17 response and weakened Treg response in some autoimmune diseases [38,39], so we also examined the frequencies of CCR4+CCR6+ Th and Treg cells in PBMCs of ASp and HDs (Figure 4). The PBMCs from ASp and HDs were examined for the subset populations using flow cytometry, defined as the percentages of CCR4+CCR6+ Th cells (CCR4/CCR6 double-positive) [32] and Treg cells (CD4/CD25/Fox-P3 triple-positive) accounting for the total CD4-positive Th cells (CCR4+CCR6+ Th/Th, Treg/Th), CD3-positive T cells (CCR4+CCR6+ Th/T, Treg/T), lymphocytes (CCR4+CCR6+ Th/L, Treg/L), and peripheral blood mononuclear cells (CCR4+CCR6+ Th/PBMCs, Treg/PBMCs) respectively. The proportions of Fox-P3-positive occupying CD4/CD25 double-positive cells (Fox-P3+/CD4+CD25+) and PBMCs (Fox-P3+/PBMCs) were also tested. Compared with healthy donors (HD1 and HD2), the CCR4+CCR6+ Th population of ASp was significantly increased (P < 0.001, Table 3 and Figure 5), whereas Treg cells and Fox-P3-positive cells were found to be significantly decreased (P < 0.001, Student's t test for independent data, Table 3 and Figure 5). There were no significant differences between HD1 and HD2.
Figure 4 Representative plots of CCR4+CCR6+ T-helper and regulatory T cells. Representative plots of peripheral circulating populations of (A1) to (A3) CCR4+CCR6+ T-helper (Th) cells (green) and (B1) to (B3) regulatory T (Treg) cells (red) in peripheral blood mononuclear cells (PBMCs) of patients with ankylosing spondylitis (ASp) (A1, B1), HLA-B27-negative healthy donors (HD1) (A2, B2) and HLA-B27-positive healthy donors (HD2) (A3, B3). FOX-P3, forkhead box P3; FSH, forward scatter-height; SSH, side scatter-height.
Table 3 Percentages of CCR4+/6+ Th and Treg cells in appropriate cell subsets
CCR4+/CCR6+ Th/Th CCR4+/CCR6+ Th/T CCR4+/CCR6+ Th/L Treg/Th Treg/T Treg/L Fox-P3+/CD4+CD25+
ASp 9.81 ± 0.62 7.69 ± 0.63 4.24 ± 0.56 2.82 ± 0.24 2.12 ± 0.23 1.23 ± 0.13 22.23 ± 5.13
HD1 5.51 ± 0.59 3.53 ± 0.56 2.24 ± 0.48 5.27 ± 0.28 4.13 ± 0.26 2.54 ± 0.15 32.54 ± 7.05
HD2 5.34 ± 0.52 3.33 ± 0.45 2.17 ± 0.37 5.31 ± 0.23 4.21 ± 0.19 2.64 ± 0.18 34.92 ± 6.71
Data presented as the percentage mean ± standard deviation. The differences for all percentages between patients with ankylosing spondylitis (ASp) and either HLA-B27-negative healthy donors (HD1) or HLA-B27-positive healthy donors (HD2) were significant (P < 0.001, according to a two-tailed significant level of 0.05). There were no significant differences between HD1 and HD2 (P > 0.05). Th, CD4+ T-helper cells; T, T lymphocytes; L, lymphocytes; Treg, forkhead box P3-positive regulatory T cells.
Figure 5 Percentages of CCR4+CCR6+ T-helper, regulatory T and Fox-P3-positive cells in peripheral blood mononuclear cells. Statistical multiple bar graphs showing percentages of increased CCR4+CCR6+ T-helper (Th) cells, reduced regulatory T (Treg) cells and reduced forkhead box P3 (Fox-P3)-positive cells in the peripheral blood mononuclear cells (PBMCs) of patients with ankylosing spondylitis (ASp), compared with values for healthy donors (HLA-B27-negative healthy donors (HD1) and HLA-B27-positive healthy donors (HD2)). *P < 0.001, Student's t test for independent data.
BMSCs of patients with AS-induced CCR4+CCR6+ Th/Treg imbalance
We performed the direct contact co-culture of BMSCs and PBMCs to explore whether the reduced immunomodulation potential of AS-BMSCs altered the balance of CCR4+CCR6+ Th/Treg cells. The PBMCs were collected to be assayed by flow cytometry for the CCR4+CCR6+ Th and Treg cells after co-culture with BMSCs of ASp and HDs (HD1 and HD2) for 3 days. The percentages of Treg cells (0.63 ± 0.23%) and Fox-P3-positive cells (0.74 ± 0.11%) in PBMCs after co-culture with AS-BMSCs for 3 days reduced significantly, whereas the percentages of CCR4+CCR6+ Th cells (1.87 ± 0.29%) in PBMCs after co-culture with AS-BMSCs for 3 days increased significantly, compared with these values of groups, including 3-day HD1, 3-day HD2, 3-day control, and 0 days (P < 0.001, Figure 6). Impressively, the ratio of CCR4+CCR6+ Th cells to Treg cells (CCR4+CCR6+ Th/Treg) in PBMCs after co-culture with AS-BMSCs for 3 days increased significantly (P < 0.001, Figure 6).
Figure 6 Ratios of Fox-P3/PBMCs, CCR4+CCR6+ Th/PBMCs, Treg/PBMCs and CCR4+CCR6+ Th/Treg. Peripheral blood mononuclear cells (PBMCs) were collected to be assayed by flow cytometry for the CCR4+CCR6+ T-helper (Th) cells, regulatory T (Treg) cells and forkhead box P3 (Fox-P3)-positive cells after co-culture with or without (3 day-control) ankylosing spondylitis (AS)-bone marrow-derived mensenchymal stem cells (BMSCs) (3 day-ASp), HLA-B27-negative healthy donors (HD1)-BMSCs (3 day-HD1) or HLA-B27-positive healthy donors (HD2)-BMSCs (3 day-HD2) for 72 hours. Statistical multiple bar graphs show that AS-BMSCs induced the ratio of CCR4+CCR6+ Th/Treg imbalance via reducing Treg/PBMCs but increasing CCR4+CCR6+ Th/PBMCs; it also produced a significant reduction of Fox-P3-positive cells in PBMCs (*P < 0.001, Student's t test for independent data).
Negative correlations between percentages of CCR4+CCR6+ Th/Treg cells and the suppressive ratio of BMSCs
When examining data from all subjects tested, we observed positive correlations between the percentage inhibition of BMSCs (MLR) and the percentage inhibition of BMSCs (PHA) for all of the 51 ASp, 37 HD1 and 12 HD2. Interestingly, however, for all of the ASp (Figure 7A), HD1 (Figure 7B) and HD2 (Figure 7C) there were significantly negative correlations between the ratios of CCR4+CCR6+ Th cells to Treg cells in the peripheral blood and either percentage inhibition of BMSCs (MLR) or percentage inhibition of BMSCs (PHA) at all five ratios (P < 0.01, respectively).
Figure 7 Correlation analysis between CCR4+CCR6+ Th/Treg ratio and immunomodulation potential of bone marrow-derived mensenchymal stem cells. (A) All patients with ankylosing spondylitis (ASp), (B) HLA-B27-negative healthy donors (HD1) and (C) HLA-B27-positive healthy donors (HD2) presented significantly negative correlations between the ratio of CCR4+CCR6+ T-helper (Th) cells to regulatory T (Treg) cells in the peripheral blood mononuclear cells (PBMCs) and percentage inhibition either from two-way mixed PBMC reaction (MLR) (upper panel) or PBMC proliferation induced by phytohemagglutinin (PHA) (lower panel) at a bone marrow-derived mensenchymal stem cell (BMSC):PBMC ratio of 1:10 (P < 0.01, respectively).
Discussion
In the present study, we found that AS-BMSCs showed normal proliferation, cell viability, surface markers and multiple differentiations characteristics, but significantly reduced immunomodulation potential; also, the frequencies of Treg and Fox-P3+ cells in AS-PBMCs decreased, but CCR4+CCR6+ Th cells increased. Moreover, the AS-BMSCs induced the ratio of CCR4+CCR6+ Th/Treg cell imbalance when co-cultured with PBMCs. Additionally, no differences were found between HD1 and HD2. Impressively, the immunomodulation potential of BMSCs has negative correlation with the ratios of CCR4+CCR6+ Th to Treg cells in peripheral blood.
Characteristic symptoms of AS are spinal stiffness, ankylosis and syndesmophytes [1], which are explained by spinal inflammation, structural damage, or both [40]. As the ankylosis of the spine or even spinal stiffness was probably initiated by the heterotopic ossification of osteoblasts, and most of these osteoblasts derived from BMSCs [41,42], and, simultaneously, there were some abnormalities with the biological properties, including the multiple differentiation potentials in some autoimmune disorders, such as severe aplastic anemia [14], we performed research to examine the biology properties of AS-BMSCs. We did not, however, detect any abnormality about the biological characteristics of AS-BMSCs in vitro, including the proliferation ability, cell viability, morphological features, differentiation potentials and surface markers. Especially, the activity of osteogenic differentiation and mineralization capacity are totally normal. In addition, Braun and colleagues reported that immunohistological studies on sacroiliac joint biopsies have shown cellular infiltrates, including T cells and macrophages, and that TNFα is overexpressed in sacroiliac joints [43]. These events indicated that the endogenous osteogenic differentiation potential of BMSCs may be not the real murderer, which was thought to induce MSCs to produce heterotopic ossification; the appropriate cell activity and cytokine function [44] existing in the internal environments, which maintain BMSCs in vivo, may play a critical role in the process of BMSC heterotopic ossification.
There appears to be a reciprocal relationship between the development of Treg cells and Th17 cells. Recent studies have independently revealed enhanced Th17 response and weakened Treg response in some autoimmune diseases [38,39], indicating an important role for Th17/Treg imbalance in the pathogenesis of autoimmunity. The present study revealed that the Th17/Treg imbalance existed in the peripheral blood of ASp, suggesting its potential role in the breakdown of immune self-tolerance in AS. Moreover, the physiological frequency of Fox-P3+ and Treg cells can suppress autoimmune disorders, but the reduction or even depletion of Fox-P3+ cells could lead to induction of autoimmunity by specific ablation of Treg cells in genetically targeted mice [45], these results indicated that the reduction of Treg cells probably enhanced the pathological process of AS. The balance between Treg and Th17 cells is dependent on the localized cytokine milieu including levels of IL-2, IL-6 [46] and transforming growth factor beta (TGFβ) [47], and the differentiation of both Treg and Th17 cells required TGFβ, but depends on opposing activities: at low concentrations, TGFβ synergizes with IL-6 and IL-21 to promote IL-23R expression, favoring Th17 cell differentiation, while high concentrations of TGFβ repress IL-23R expression and favor Fox-P3+ Treg cells [48].
Rivino and colleagues reported that the combination of CCR4 and CCR6 does not uniquely define Th17 cells; it also demarcated an IL-10-producing population of T cells [49]. There are several reasons why the Th17 cells were defined with the combination of CCR4 and CCR6 in this study. At first, CCR4 had been shown to mark skin-homing T cells [50], expression of which has been associated with the ability of cells to traffic into peripheral tissues [51]; in addition, the percentage of CD4+/CCR4+ T cells showed significant positive correlations with the Bath Ankylosing Spondylitis Disease Activity Index in AS [52]. Furthermore, the findings of Napolitani and colleagues provide a functional link between CCR6 and IL-17 [32], which have been independently associated with tissue pathology. CCR6 has been shown to be involved in the recruitment of pathogenic T cells in rheumatoid arthritis [53], experimental autoimmune encephalitis [54] and psoriasis [55,56], and Th17 cells are increasingly recognized as essential mediators of those diseases [25,57-61]. Besides, just like CCR4, CCR6 also mediates T-cell homing to skin and mucosal tissues [62], and its expression facilitates the recruitment of both dendritic cells and T cells in different diseases [60]. These findings illustrated that the Th17 cells CCR4+CCR6+ were the most active and aggressive pathogenic ones. Second, only a fraction of IL-10-producing CCR6+ T cells co-expresses CCR4 [49]. Finally, Hill Gaston and colleagues reported increased frequency of IL-17-producing T cells in AS [33]; these findings were consistent with our study's results. Moreover, they did not detect any differences in the frequency of IL-10-positive CD4+ T cells between patients with arthritis and control subjects, and none of the IL-17-positive cells co-expressed IL-10. This means that the increased frequency of IL-17-producing T cells in AS was not compensated for by an increased frequency of IL-10-producing cells.
In the present study, we failed to find any significant differences between HLA-B27-negative and HLA-B27-positive healthy donors, which were essentially the same in all respects we had studied. These findings indicated that HLA-B27-positivity may be not responsible for those abnormalities of ASp. Inflammation is one important link within the pathogenesis of AS [1], while few studies reported whether inflammation be responsible for the altered properties of AS. Rheumatoid arthritis is a typical inflammatory disease, One study reported that the number of IL-17+ Th cells and CD4+CD25+ Treg cells in the peripheral blood of patients with rheumatoid arthritis is elevated compared with that of healthy individuals [33,63], whereas other studies suggest no differences between these two groups [64,65]. Two groups have reported that peripheral blood Treg cells isolated from patients with rheumatoid arthritis and from control individuals showed no difference in their ability to suppress effector T-cell proliferation [38]. Another group, however, reported a striking defect in the capacity of Treg cells from patients with rheumatoid arthritis to suppress effector T-cell proliferation [66]. These divergent results could reflect differences in the populations of patients, the methods used to purify Treg and Th17 cells, or how the suppression assays were performed.
Recently, a study indicated that an alteration in the balance of Th1, Th2, Th17, and Treg cells contributes to the development of experimental autoimmune myastheia gravis, and that the administration of BMSCs can ameliorate the severity and, in a process dependent on the secretion of TGF-β, presenting to inhibit the proliferation of antigen-specific T cells, normalize the distribution of the four T-helper subsets and their corresponding cytokines [67]. In vitro experiments have shown that human MSCs can induce the generation of CD4+ T-cell subsets displaying a regulatory phenotype (Treg) [8,68]. These results demonstrated that administration of BMSCs from healthy donors to ASp may be a novelty therapeutic strategy for AS.
The imbalance of Th17/Treg cell subsets may contribute to the inflammatory responses [69] and heterotopic ossification of MSCs [44] in AS by secreting the proinflammatory T-cell cytokines. There is therefore a potential mechanism of AS that reduced the immunomodulation potential of BMSC induced CCR4+CCR6+ Th/Treg imbalance and led to excessive activation of T cells, and then to the increased proinflammatory CCR4+CCR6+ Th cells and reduced Treg cells. Fox-P3+ cells compounded by the synergistic actions of activated T-cell cytokines drive the local BMSCs into both osteoblasts and osteoclasts at localized sites of inflammation, and then the induced BMSCs result in syndesmophytes, fusion of the sacroiliac joint and even spinal stiffness via heterotopic ossification. Finally, AS occurs.
Increasing evidence suggests that MSCs might be a suitable cell population for immunosuppressive therapy in solid organ transplantation and may be strong candidates for cell therapy against human autoimmune diseases [70-72]. The advantages of MSCs are obvious: they can be easily harvested from a multitude of tissues, can be cultured to nearly unlimited extent, and have very promising immunomodulation effects [73]. The immunoregulatory function of BMSCs thus appears to represent a promising strategy for cytotherapy of autoimmune diseases, such as AS, which is central to human health and disease, and provides novel insights into new therapeutic interventions. The retinoic acid receptor-alpha [74] could be another considered candidate for the treatment of autoimmunity, because signaling through a specific nuclear retinoic acid receptor can favor the decision to adopt the Treg cell fate at the expense of the Th17 cell fate. The further elucidation of the precise mechanism may aid in the identification of targets for future immunomodulatory therapy of AS.
Conclusions
The reduced immunomodulation potential of BMSCs may be an initiating factor for AS pathogenesis, and may play a novelty role in triggering the onset of AS via inducing the CCR4+CCR6+ Th/Treg cell subset imbalance. BMSCs may therefore be an interesting therapeutic target in AS, suggesting the use of BMSCs from HDs in the disease.
Abbreviations
ALP: alkaline phosphatase; AS: ankylosing spondylitis; AS-BMSC: bone marrow-derived mensenchymal stem cell of patient with AS; ASp: patients with ankylosing spondylitis; AS-PBMC: peripheral blood mononuclear cell of patient with AS; BMSC: bone marrow-derived mensenchymal stem cell; CPM: counts per minute; DMEM: Dulbecco's modified Eagle's medium; FBS: fetal bovine serum; Fox-P3: forkhead box P3; 3H-TdR: 3H-thymidine; HD: healthy donor; HD1: HLA-B27-negative healthy donors; HD2: HLA-B27-positive healthy donors; HD-BMSC: bone marrow-derived mensenchymal stem cell of healthy donor; HD-PBMC: peripheral blood mononuclear cell of healthy donor; IL: interleukin; MLR: mixed peripheral blood mononuclear cell reaction; MSC: mensenchymal stromal cell; MTT: methyl thiazolyl tetrazolium; OD: optical density; PBMC: peripheral blood mononuclear cell; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PHA: phytohemagglutinin; QL: the lower point of interquartile range; QU: the upper point of interquartile range; SD: standard deviation; TGFβ: transforming growth factor beta; Th: T-helper; TNF: tumor necrosis factor; Treg: regulatory T.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
RY, XL, YM and YT carried out the experimental work and the data collection and interpretation. LH, KC and JY participated in the design and coordination of experimental work, and the acquisition of data. MR and YW participated in the study design, data collection, analysis of data and preparation of the manuscript. PW and HS carried out the study design, the analysis and interpretation of data and drafted the manuscript. All authors read and approved the final manuscript.
Acknowledgements
The authors thank Dr Xiaoping Wang at Sun Yat-sen University for assistance with cell culturing, including BMSCs and PBMCs. They also thank Dr Jing Wei for flow cytometry, and Wenfeng Xie and Hua Zeng for help with the samples from ASp. The study was financially supported by the National Natural Science Foundation of China (30973033), the Yat-sen Innovative Talents Cultivation Program for Excellent Tutors (81000-3126203), the Guangzhou Science and Technology Project (2008A1-E4011-9), the Natural Science Foundation of Guangdong Province (9151008002000015), and the Guangdong Provincial Science and Technology Project (2009B060300023).
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22216167PONE-D-11-0742010.1371/journal.pone.0029058Research ArticleBiologyBiochemistryEnzymesMolecular Cell BiologyCell DeathGene ExpressionMedicineObstetrics and GynecologyBreast CancerA Short Caspase-3 Isoform Inhibits Chemotherapy-Induced Apoptosis by Blocking Apoptosome Assembly Caspase-3s Inhibits Apoptosome AssemblyVégran Frédérique
1
2
3
Boidot Romain
1
2
Solary Eric
2
3
Lizard-Nacol Sarab
1
2
*
1
Unit of Molecular Biology - Centre Georges François Leclerc, Dijon, France
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Federative Institute of Research IFR “Santé-STIC” - University of Burgundy, Dijon, France
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UMR-INSERM U-866, Dijon, France
Wong Nai Sum EditorUniversity of Hong Kong, Hong Kong* E-mail: [email protected] and designed the experiments: SL-N. Performed the experiments: FV RB. Analyzed the data: FV RB ES SL-N. Contributed reagents/materials/analysis tools: FV RB. Wrote the paper: ES SL-N.
2011 22 12 2011 6 12 e2905827 4 2011 20 11 2011 Végran et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Alternative splicing of caspase-3 produces a short isoform caspase-3s that antagonizes caspase-3 apoptotic activity. However, the mechanism of apoptosis inhibition by caspase-3s remains unknown. Here we show that exogenous caspase-3 sensitizes MCF-7 and HBL100 breast cancers cells to chemotherapeutic treatments such as etoposide and methotrexate whereas co-transfection with caspase-3s strongly inhibits etoposide and methotrexate-induced apoptosis underlying thus the anti-apoptotic role of caspase-3s. In caspase-3 transfected cells, lamin-A and α-fodrin were cleaved when caspase-3 was activated by etoposide or methotrexate. When caspase-3s was co-transfected, this cleavage was strongly reduced. Depletion of caspase-3 by RNA interference in HBL100 containing endogenous caspase-3s caused reduction in etoposide and methotrexate-induced apoptosis, whereas the depletion of caspase-3s sensitized cells to chemotherapy. In the presence of caspase-3s, a lack of interaction between caspase-3 and caspase-9 was observed. Immunoprecipitation assays showed that caspase-3s binds the pro-forms of caspase-3. This result suggested that the absence of interaction with caspase-9 when both variants of caspase-3 are present contribute to block the apoptosome assembly and inhibit apoptosis. These data support that caspases-3s negatively interferes with caspase-3 activation and apoptosis in breast cancer, and that it can play key roles in the modulation of response to chemotherapeutic treatments.
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Introduction
Caspases are a family of evolutionary conserved cysteine proteases that play a central role in a majority of apoptotic cell death pathways. Death signals activate the proteolytic cascade of caspases through two main pathways, i.e. an extrinsic pathway that starts at the level of plasma membrane death receptors and an intrinsic pathway that activation is a response to irreversible cellular damage [1]. Both pathways converge to the activation of caspase-3, the closer homolog of the in Caenorhabditis elegans CED-3 [2]. Procaspase-3 is a 32 kDa caspase-3 zymogen (also known as CPP32). CPP32 exists in the cells as inactive dimmers. Its proteolytic cleavage on C-terminal side of aspartate residues eliminates the pro-domain, separates the remaining protein into a large and a small subunit and generates an active tetramer constituted by two large and two small subunits. In turn, caspase-3 activates downstream enzymes of the caspase family and contributes with them to generate the characteristic apoptotic cell death phenotype.
Activation of caspase-3 is required for membrane bleeding and internucleosomal DNA fragmentation that occur during apoptosis. Caspase activity is regulated at several levels, including gene transcription and post-translational modifications. The alternative splicing of caspase genes generates full-length and truncated proteins whose functions can be antagonistic [3]. This characteristic maintains the threshold of response to certain levels of stimuli [4].
The human Caspase-3 gene is located on 4q33-q35.1 and possesses 2635 base pairs leading to 7 exons. Its alternative splicing generates two transcripts, caspase-3 and caspase-3s that were detected in all the studied tissues [5] and may have different apoptotic activities [5], [6]. The principal mRNA variant, caspase-3, is 834 base-long and the short transcript, caspase-3s, has lost the sixth exon leading to a loss of 122 bases (representing 95 amino-acids initially present in the procaspase-3 protein). The lost sequence includes the short subunit and the C-terminal part of the long subunit in which is located the QACRG motif that participates in the formation of the catalytic site. The caspase-3s protein has around a 20 KDa molecular weight and expression of the short isoform of caspase-3 in 293T cells prevents DNA fragmentation and poly(ADP-ribose) polymerase 1 (PARP1) cleavage in response to an apoptotic stimulus [5].
The role of caspase-3 in the response of breast cancer cells to chemotherapeutic drugs remains a controversial issue. The lost of caspase-3 expression as well as defaults in cytochrome c release from the mitochondria, which is requested in most apoptotic pathways to activate caspase-3 through caspase-9 activation, are associated with multidrug resistance. Accordingly, expression of caspase-3 in the human MCF-7 breast tumor cell line (which is deficient for caspase-3) restores the apoptotic response to the topoisomerase II inhibitor, etoposide [7]–[9]. Caspase-3 was also involved in breast cancer cell apoptosis upon exposure to anthracyclines [9]–[12] and cisplatin [13]–[16]. Its role in tumor cell response to paclitaxel has been challenged [17]–[24].
Currently, the role of caspase-3s in chemotherapy response is unexplored. We also previously studied the impact of caspase-3s expression in a population of breast carcinomas treated with neoadjuvant cyclophosphamide-based chemotherapy and observed an inverse relationship between caspase-3s/caspase-3 ratio level expression and pathological response [6]. Therefore, the present study was performed to precise the molecular mechanism in apoptosis inhibition of caspase-3s by using breast tumor cell lines MCF-7 treated by various chemotherapeutic agents known to induce an apoptotic mode of cell death.
Materials and Methods
Cell lines
Human breast cancer cell lines MCF-7 (deficient for caspase-3), HBL-100 and MDA-MB-231 (proficient for caspase-3) were purchased from the ATCC (American Type Culture Collection, Manassas, VA, USA). The cell lines were cultured according to the manufacturer's instructions.
Full-length cDNA synthesis and cloning
The full-length of caspase-3 and caspase-3s coding sequences were obtained using SuperScript™ One-Step long templates RT-PCR (Invitrogen, Carlsbad, CA, USA) with 1.25 µg of total RNA from the UACC3199 cell line (Arizona Cancer Center Tissue Culture Shared Resource) containing high levels of the two transcripts. Specific primers used were reported in Table S1. PCR program was performed by one cycle at 45°C for 30 min, 94°C for 2 min followed by 35 cycles of 15 s at 94°C, 30 s at 50°C, 1 min at 68°C, and one final cycle for 5 min at 72°C (Abi Prism 9700 thermocycler, Applied Biosystems, Foster City, CA, USA). The two transcripts were separated by a 3% agarose gel electrophoresis and purified by specific extraction with QIAquick Gel Extraction Kit (Qiagen, Courtaboeuf, France).
The inserts were cloned into pcDNA3.1/CT-GFP-TOPO or pcDNA3.1/CT-YFP-TOPO and amplified in One Shot® TOP10 Chemically Competent E. Coli with Fusion TOPO® TA Expression Kits (Invitrogen). The plasmids from a few randomly picked colonies were isolated. The orientations of the caspase-3 or caspase-3s fragments were tested by PCR and automatic sequencing as described below.
Stable transfection
For stable transfection, 5×105 MCF-7 and HBL100 cells were grown in a medium without antibiotics in 12-well plates. Two days later, cells were transfected with 0.5 µg of plasmid containing either caspase-3 or caspase-3s insert and with GFP or YFP control vector. Stable transfections were performed by LipofectAMINE 2000 (Invitrogen) according to manufacturer's instructions. The stable colonies were selected by 1000 µg/ml Geneticin® or 10 µg/ml Blasticidin (Invitrogen). To ensure that the inserts were sufficiently expressed, the transfection efficiency was controlled by quantitative real-time RT-PCR amplification, the presence of GFP and YFP was controlled by microscopy and the presence of extrinsic caspase-3 and caspase-3s was determined by Western Blot. Relative change expressions were calculated between control and transfected cells.
Caspase-3 and caspase-3s extinction by siRNA
HBL100 or MDA-MB-231 cells (3×104 per wells), which are proficient for caspase-3, were grown in a medium without antibiotics in 6-well plates during 2 days. Transfections (procaspase-3 siRNA: CGACUUCUUGUAUGCAUACUCCACA, caspase-3s siRNA: GGGTTATTATTCTTGGCGAA) were performed by LipofectAMINE 2000. Twenty four, 48, 72 and 96 hours of extinction were tested by Western Blot (Figure S2A) or quantitative PCR (Figure S2B). Cytotoxic treatments were performed during the best extinction time lapse (Figure S2A and B). For treatment after caspase-3s down-regulation, chemotherapeutic drug yields were 10 fold inferior to the IC50.
Sequencing of PCR products
The specificity of all PCR amplifications was verified by sequencing of PCR-products. Briefly, products were excised from 3% agarose gels and isolated as described above. The purified PCR products were sequenced using the Abi-Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) with the respective primers used in the initial PCR according to the manufacturer's protocol.
RNA extraction, cDNA synthesis, and Quantitative real-time PCR amplification
Total RNA was extracted with Trizol reagent (Invitrogen) and its quality was checked by 28S/18S ratio on agarose gel. One microgram of total RNA was reverse transcribed as previously described [6].
For real-time PCR, amplification was performed in a total volume of 25 µL in the presence of 600 nM of each primers, 200 nM of probe, 12.5 µL of Universal Master Mix (Applied Biosystems), and 12 ng of cDNA (or water as negative control). PCR was performed with an initial denaturation step of 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. All samples were amplified in duplicate and results were analyzed at the CT level. Control 18S reactions (Applied Biosystems) were used to normalize ΔCT values.
Total protein extractions
Total protein extraction was performed by addition of lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Sodium pyrophosphate, 1 mM β-Glycerophosphate, 1 mM Na3VO4, 1 µg/ml Leupeptin, 1 mM PMSF added immediately prior to use) on cell monolayer for 5 min on ice. Afterwards, the lysate was harvested and sonicated.
Western Blot analysis
30 µg of protein extracts were separated SDS-PAGE 10% acrylamide and transferred to PVDF or nitrocellulose membranes (according to the molecular weight of the protein) with an electrophoretic transfer apparatus. After blocking with buffer containing 3% of ECL Advance™ blocking agent (GE Healthcare, Buckinghamshire UK) in PBS/Tween 0.3% for 1.5 hour at room temperature, the membrane was probed with specific polyclonal antibodies (Abcam, Cambridge, UK) diluted in blocking solution (β-actin 1/25000, caspase-3 1/7500, GFP 1/50000, caspase-9 1/1000, APAF-1 1/1000, lamin A 1/4000, or α-fodrin 1/2000) for 1 hour at room temperature. The antibody used for procaspase-3 and caspase-3 detection was generated with the epitope residues between amino acids 161 and 175. This part of the protein is common to procaspase-3 and caspase-3 as it is located in the large subunit.
The GFP antibody recognizes YFP in YFP transfected cells and thus the caspase-3s YFP tag. Next, the membrane was incubated with biotinylated anti-rabbit IgG (1∶50000 in blocking solution) secondary antibody (Invitrogen) 30 min at room temperature. Then, the membrane was probed with streptavidin-HRP (1/50000) for 15 min at room temperature in PBS/Tween 0.3%. Signals were detected using a chemiluminescent detection system (ECL Advance™ Western Blotting Detection Kit; GE Healthcare) and Chemidoc XSR device (Bio-Rad).
Flow cytometric assessment of apoptosis after dual staining with Annexin-V-PE and 7AAD
Apoptosis was induced by a large scale of chemotherapeutic drugs in the two cell lines (MCF-7 and HBL100) (Table S2). Assessment of apoptosis was accomplished by measuring Annexin-V-PE and 7AAD staining (Apoptosis Detection Kit I BD Pharmingen™, Franklin Lakes (NJ), USA) according to manufacturer's instructions. Cells (30 000) were counted by flow cytometry using Becton Dickinson LSRII and the experiments were performed in triplicate in two different clones.
Immunoprecipitation assays (IP)
IP assays were carried out on total protein extract of HBL100 cell line with Exacta Cruz matrix (Tebu-bio, Le Perray En Yvelines, France) according to manufacturer's instructions and analyzed by western blot.
Fluorescence microscopy
Wild-type, non tagged procaspase-3 or caspase-3s stably transfected MCF-7 cells were transiently transfected with procaspase-3-GFP or caspase-3s-YFP. Cells were plated onto 6-well plates for 24 hours, then treated or not with anticancer drugs for indicated times before replacing the culture medium with phenol red-free medium. Cells were scanned in phase contrast, and with a GFP or YFP filter under UV light.
Statistical analysis
Data were analyzed using the student t test on the Statview 5.0 software.
Results
Caspase-3s inhibits drug-induced apoptosis in breast cancer cells
We previously showed that in breast carcinoma, increase in caspase-3s/caspase3 ratio expression was significantly associated with chemoresistance to cyclophosphamide-based neoadjuvant treatment [6]. To go further, procaspase-3 and caspase-3s were stably transfected, either alone or in combination in MCF-7 cells. The constructs were fused to GFP (pro-caspase-3) or YFP (caspase-3s) and the GFP vector was used as a control (Figure S1). Transfected MCF-7 cells were exposed to the topoisomerase II inhibitor etoposide, the tubulin poison vinblastine and the folate metabolism inhibitor methotrexate for 48 hours before analyzing apoptosis induction by the use of a flow cytometry assay. Expression of procaspase-3 cDNA strongly increased the ability of MCF-7 to undergo apoptosis in response to etoposide and methotrexate but did not demonstrate any significant effect on vinblastine-induced cell death in the tested conditions or slightly enhanced their apoptotic response to some of the drugs used (Figure 1A, light grey bars). Expression of the caspase-3s either did not sensitize the cells to drug-induced apoptosis (Figure 1A, black bars). Remarkably, co-expression of both procaspase-3 and caspase-3s completely suppressed the ability of procaspase-3 expression to sensitize MCF-7 cells to etoposide and methotrexate-induced cell death (Figure 1A, dark grey bars). These results were further completed by studying the consequences of caspase-3s and/or procaspase-3 expression on apoptosis induced by a series of other cytotoxic agents in MCF-7 and HBL100 cells. Both cell lines demonstrated specific profiles of sensitivity to drug-induced apoptosis. While procaspase-3 expression (in MCF-7) or overexpression (in HBL100) increased their apoptotic response to cytotoxic agents (5FU, Bleomycin, Cisplatine, Epirubicin, Vincristine or Staurosporine), expression of caspase-3s always counteracted this sensitization (Table S3). Moreover, siRNA experiments targeting a sequence located in the caspase-3 specific exon 6 or a specific sequence of caspase-3s, to down-regulate the expression of procaspase-3 or caspase-3s respectively, in HBL100 cells were performed to determine the effect of endogenous caspase-3s after apoptosis induction. Cells were treated with etoposide, vinblastine and methotrexate for 48 hours. In absence of procaspase-3 (Figure 1B), HBL100 cells were less sensitive to apoptosis induced by etoposide and methotrexate. As expected, in absence of caspase-3s (Figure 1C), both HBL100 and MDA-MB-231 cells were more sensitive to etoposide and methotrexate induced apoptosis, confirming that endogenous caspase-3s plays an important role in caspase-3 dependant apoptosis regulation.
10.1371/journal.pone.0029058.g001Figure 1 Caspase-3s inhibits drug-induced apoptosis.
A. MCF-7 cells were stably transfected with GFP vector (white), procaspase-3-GFP (light grey), caspase-3s-YFP (black) or with C3-GFP and C3s-YFP (dark grey) and treated with etoposide, vinblastine or methotrexate for 48 H. B. HBL100 WT cells (white) were transfected either with scRNA (grey) or with siRNA against procaspase-3 (black). The efficiency of procaspase-3 extinction is shown in the small inset. Apoptosis was next induced by etoposide, vinblastine or methotrexate and detected by flow cytometry. Asterisks correspond to p<0.01. C. HBL100 and MDA-MB-231 cells (white) were transfected either with scRNA (white) or with siRNA against caspase-3s (black). The efficiency of caspase-3s extinction is shown in the small inset. Apoptosis was next induced by etoposide, vinblastine or methotrexate and detected by flow cytometry. The drug used yields in panel C were 10 fold inferior than the IC50 used in panel B, explaining the differences between controls of panels B and C. Asterisks correspond to p<0.01.
Caspase-3s prevents procaspase-3 activation
We first analyzed procaspase-3 activation in treated MCF-7 cells transfected with pro-caspase-3-GFP or with caspase-3s-YFP. Whereas vinblastine did not demonstrate any effect on procaspase-3 activation (Figure 2A, fifth and sixth tracks), exposure to etoposide and methotrexate induced the proteolytic cleavage of the procaspase-3-GFP into a p40 fragment indicating the activation of caspase-3-GFP (Figure 2A, third and seventh tracks). The expression of caspase-3s-YFP with caspase-3-GFP prevented the ability of etoposide and methotrexate to trigger the proteolytic cleavage of the proenzyme (Figure 2A, fourth and eighth tracks). In procaspase-3 proficient cells, etoposide and methotrexate induced the cleavage of caspase-3 specific substrates, the 70 KDa Lamin-A into a 28 KDa fragment and the cleavage of the 250 kDa α-Fodrin into a 160 kDa fragment [25], [26]. These cleavages were not observed in MCF-7 cells expressing only caspase-3s and in those expressing both procaspase-3 and caspase-3s in response to etoposide (Figure 2B) and methotrexate (Figure 2D). Vinblastine treatment had not effect on Lamin-A and α-Fodrin cleavage (Figure 2C) whatever the pro-caspase-3 or caspase-3s expression.
10.1371/journal.pone.0029058.g002Figure 2 Caspase-3s prevents procaspase-3 activation.
A. MCF-7 cells stably transfected with caspase-3 were treated with etoposide, vinblastine or methotrexate (lanes 1, 3, 5 and 7). Caspase-3 was cleaved in procaspase-3 transfected cells after 48 hour treatment with etoposide, or methotrexate but not after vinblastine treatment. In procaspase-3-GFP/C3s-YFP co-transfected treated MCF-7 cells (lanes 2, 4, 6, and 8), procaspase-3 cleavage was completely abolished. β-actin was used as loading control. B–D. Analysis of lamin-A and α-fodrin cleavage by Western Blot. GFP MCF-7 cells, procaspase-3-GFP (ProC3-GFP) or caspase-3s-YFP (C3s-YFP) transfected cells were treated with etoposide (B), vinblastine (C) or Methotrexate (D) during 48 hours. Lamin-A and α-fodrin were cleaved only in single procaspase-3-GFP transfected cells treated with etoposide or Methotrexate. ProC3-GFP/C3s-YFP co-transfected cells treated with etoposide or methotrexate did not show cleavage of neither lamin-A nor α-fodrin. In accordance with panel A, vinblastine did not induce cleavage of neither lamin-A nor α-fodrin β-actin was used as loading control.
Caspase-3s influences procaspase-3 location
Fluorescent microscopy analyses of MCF-7 cells indicated that, in the absence of any treatment, procaspase-3-GFP was located mainly in the cytoplasm (Figure 3A, first line), whereas caspase-3s-YFP was in both nucleus and cytoplasm (Figure 3B, first line). Upon exposure to etoposide or methotrexate for 10 hours, part of the GFP-tagged caspase-3 was translocated into the nucleus (Figure 3A, second and fourth lines). After vinblastine exposure, GFP-tagged procaspase-3 remained in the cytoplasm (Figure 3A, third line). Caspase-3s location was not influenced by any treatment (Figure 3B, second, third and fourth lines). When non tagged caspase-3s was stably expressed in MCF-7 cells before transient expression of GFP-tagged procaspase-3, the fluorescent procaspase-3 was located in both nucleus and cytoplasm and exposure to the tested drugs did not change this subcellular repartition (Figure 3C). As expected, when non tagged procaspase-3 was stably expressed in MCF-7 cells before transient expression of YFP-tagged caspase-3s, the fluorescent caspase-3s was located in both nucleus and cytoplasm and again, exposure to the tested drugs did not change significantly this subcellular repartition (Figure 3D). The fluorescent nuclear staining quantification is shown in Table S4.
10.1371/journal.pone.0029058.g003Figure 3 Caspase-3s influences the localization of procaspase-3.
Twenty-four hours after GFP-coupled procaspase-3 (C3-GFP) or YFP-coupled caspase-3s (C3s-YFP) transfection in parental MCF-7 cells (A and B respectively) or in non tagged caspase-3s (C3s) or procaspase-3 (C3) stably transfected MCF-7 cells (C and D respectively) MCF-7 cells were treated or not with etoposide, vinblastine or methotrexate for 10 hours. Cells were scanned in phase contrast and with a GFP or YFP filter under UV light. Nuclei were delimited by dotted lines. The nulear staining quantification is presented in Table S4.
Caspase-3s structure predicts an absence of catalytic activity
To understand why caspase-3s has no catalytic activity, we studied its protein sequence and structure. As shown in Figure 4A, caspase-3s has no catalytic site compared with procaspase-3, and especially the cysteine residue that is crucial for the catalytic activity of caspases. Moreover, caspase-3s does not possess the cleavage site between the small and the large subunit, resulting in an absence of cleavage into two subunits. Finally, in the caspase-3s, there are no sites for binding of caspase inhibitors such as Ac-DEVD-CHO [27], XIAP [28]… However, caspase-3s possesses the same pro-domain as procaspase-3, but, contrary to procaspase-3, caspase-3s pro-domain is not cleaved. Indeed, the activation of procaspase-3 is sequential. The first step is the cleavage between the large and the small subunits. Once this cleavage is done, the large+small subunit complex induces the cleavage of the pro-domain, involving the activation of caspase-3. Thus, if the protein is not divided in two subunits, the pro-domain can not be cleaved. Therefore, the absence of caspase-3s pro-domain cleavage may be explained by the absence of caspase-3s cleavage in two subunits.
10.1371/journal.pone.0029058.g004Figure 4 Protein sequence and 3D structure of procaspase-3 and caspase-3s.
A. Alignment of procaspase-3 (C3) and caspase-3s (C3s) amino acid sequences. Prodomain is framed, amino acids forming the catalytic site are in bold blue, interaction sites with caspase-3 inhibitors are in bold green [27]–[28], cleavage site between small and large subunits are in bold and underlined [28], aspartate residue cleaved during activation are in bold red [29]. B. 3D structure of procaspase-3 and caspase-3s. Bold arrows represent the end of common sequence between both proteins and pink asterisks show the β-sheets and α-helixes present in procaspase-3 and absent in caspase-3s.
The difference between pro-caspase-3 and caspase-3s sequences has a strong impact on the 3D structure. In fact, when the pro-caspase-3 3D structure was compared with the caspase-3s one, it appeared that caspase-3s lacked 2 β-sheets and 2 α-helixes compared with procaspase-3 (Figure 4B). These structures are necessary for the catalytic activity of caspase-3.
Caspase-3s interacts with procaspase-3 and prevents its interaction with apoptosome
To understand the apoptosis inhibition mechanism of caspase-3s, co-immunoprecipitation experiments were performed in HBL100 cells stably transfected with YFP-coupled caspase-3s. Using an anti-caspase-3 monoclonal antibody that targets an epitope located in the short subunit of caspase-3, detecting only procaspase-3 and active caspase-3 but not caspase-3s, we observed, upon exposure to etoposide and methotrexate, caspase-3s interacted with procaspase-3 (Figure 5A). Moreover, co-immunoprecipitation experiments with an anti-GFP antibody (that recognizes caspase-3s tag) demonstrated that, in cells exposed to etoposide or methotrexate, caspase-3s-YFP could pull down procaspase-3 (Figure 5B).
10.1371/journal.pone.0029058.g005Figure 5 Caspase-3s interacts with procaspase-3 and inhibits the apoptosome assembly.
A–B. This experiment was carried out in HBL100 cells stably transfected with caspase-3s-YFP or YFP alone and treated with etoposide, vinblastine or methotrexate for 48 hours. A. In YFP transfected cells, immunoprecipitations were performed with anti-caspase-3 antibody and western blot with anti-GFP (that recognizes YFP in YFP transfected cells and the caspase-3s YFP tag) and anti-caspase-3 antibodies. The results revealed no interaction between caspase-3 and YFP with or without treatment. In HBL100 transfected with caspase-3s-YFP, an interaction between procaspase-3 and caspase-3s after etoposide or methotrexate treatments was observed. B. In YFP transfected cells, immunoprecipitations were performed with anti-GFP antibody and western blot with anti-caspase-3 and anti-GFP antibodies. The results revealed no interaction between YFP and procaspase-3 whatever the conditions. In caspase-3s-YFP transfected HBL100, an interaction between caspase-3s and procaspase-3 was observed after etoposide or methotrexate treatment. C–F. MCF-7 cells were transfected with empty-vector (YFP), procaspase-3 (ProC3) or caspase-3s-YFP (C3s-YFP) or both caspase-3s-YFP and procaspase-3. The cells were treated or not with etoposide. C. Immunoprecipitations were performed with anti-active caspase-9 antibody and western blot with anti-active caspase-9, anti-Apaf-1, anti-caspase3 and anti-GFP (that recognizes caspase-3s tag) antibodies. The results showed that procaspase-3 and caspase-3s interacted, in treated cells, with active caspase-9 and anti-Apaf-1 only when they are alone. As soon as caspase-3s and procaspase-3 are present at the same time, the interaction with active caspase-9 and anti-Apaf-1 was abolished. D. Immunoprecipitations were performed with anti-caspase-3 antibody and western blot with anti-caspase-3, anti-Apaf-1, and anti-active caspase-9 antibodies. The results showed that procaspase-3 interacted with active caspase-9 and anti-Apaf-1 in treated cells only in absence of caspase-3s. E. Immunoprecipitations were performed with anti-GFP antibody and western blot with anti-GFP, anti-Apaf-1 and anti-active caspase-9 antibodies. The results showed that YFP did not interact with active caspase-9 and anti-Apaf-1 whereas caspase-3s interacted with active caspase-9 and anti-Apaf-1 in treated cells only in absence of procaspase-3. F. Immunoprecipitations were performed with anti-Apaf-1 antibody and western blot with anti-Apaf-1, anti-active caspase-9, anti-caspase-3 and anti-GFP antibodies. The results showed that procaspase-3 and caspase-3s interacted, in treated cells, with active Apaf-1 and caspase-9 only when they are alone. As soon as caspase-3s and procaspase-3 are present at the same time, the interaction with Apaf-1 and active caspase-9 was abrogated.
Then, we wondered whether the interaction of caspase-3s with procaspase-3 prevents pro-caspase-3 activation by inhibiting apoptosome assembly. For that, MCF-7 cells were transfected with a vector encoding either YFP alone or the YFP-tagged caspase-3s or procaspase-3 without any tag. These cells were left untreated or treated with etoposide for 48 hours. Afterwards, cell lysates were immunoprecipitated with an anti-active caspase-9 antibody and Apaf-1 antibody representing the Apaf-1/cytochrome c/caspase-9 complex, precursor of apoptosome. The active form of caspase-9 was detected by immunoblot analysis in etoposide treated cells (Figure 5C, Active caspase-9 panel) and its interaction with Apaf-1 was confirmed in etoposide treated samples (Figure 5C, APAF-1 panel). The results showed an interaction between active caspase-9/Apaf-1 and procaspase-3 (as detected with an anti-procaspase-3 antibody) or caspase-3s (as detected with an anti-GFP antibody, recognizing caspase-3s tag) when these proteins were expressed alone in etoposide-treated cells. These interactions were no more detected when both procaspase-3 and caspase-3s were expressed simultaneously (Figure 5C, Procaspase-3 and C3s-YFP panels). The results were confirmed by inverse immunoprecipitation with an anti-procaspase-3 antibody (Figure 5D), an anti-GFP antibody (Figure 5E), and an anti-Apaf-1 antibody (Figure 5F). These results suggested that caspase-3s prevent the apoptosome assembly by sequestrating procaspase-3.
Discussion
A variety of mechanisms has been described to account for the resistance of breast cancer cells as well as other tumor types to anticancer agents. Indeed, patients often develop drug resistance towards drugs of unrelated structures and activities. To fight against chemotherapy, cancer cells are able to develop various mechanisms of resistance such as activation of survival pathways or apoptosis resistance. Apoptosis resistance can reflect resistance to chemotherapeutic treatments [1]. Caspase-3 is a major pro-apoptotic protein and is a key enzyme in drug-induced apoptosis of tumor cells [9]. In contrast, caspase-3s overexpression in breast carcinomas might be indicative of chemoresistance to neoadjuvant cyclophosphamide-containing treatment [6]. Thus, it was suggested that caspase-3s might be an apoptosis protagonist which could be a new predictive marker of apoptosis resistance. To evaluate this hypothesis, the ability of caspase-3s to counteract apoptosis induced by various pro-apoptotic chemotherapeutic drugs was evaluated in this study with the use of breast tumor cell lines which are deficient or proficient for caspase-3.
The present study demonstrates that caspase-3s, generated by alternative splicing of caspase-3 pre-mRNA, negatively interferes with caspase-3 activation and apoptosis in breast cancer cells exposed to cytotoxic drugs. This effect appears to involve an interaction of caspase-3s with procaspase-3 that prevents its recruitment in the apoptosome and its activation.
Our results confirm and extend the observation that procaspase-3 expression in MCF-7 cells, which do not express spontaneously this protease, enhances the cell ability to undergo cell death in response to etoposide, methotrexate and other cytotoxic drugs [30]. Caspase-3 activation was confirmed by immunoblot showing the proteolytic cleavage of the protein. The cleavage of α-fodrin and lamin A, which was described as caspase-3 targets, also argues for a role of caspase-3 in these apoptotic pathways. In addition, siRNA-mediated down-regulation of caspase-3 in HBL100 decreased the apoptotic response to the drugs used, whereas caspase-3s expression inhibition increases cell death induced by the treatment in HBL100 and MDA-MB-231. It is important to note that there are no differences between HBL100 and MDA-MB-231 cells in expression of other apoptotic factor analyzed in this study. In MCF-7 cells, caspase-3 is absent and caspases-4 and -10 are downregulated. Nevertheless, as caspase-4 is an inflammatory caspase and caspase-10 is only involved in extrinsic apoptosis pathway, both these proteins have no influence on the studied apoptosis pathway.
In apoptosis involving the mitochondrial pathway, procaspase-3 is activated by caspase-9, subsequently to apoptosome formation [31]. Thus, it was of interest to precise the interaction between caspase-3s, pro-caspase-3 and/or caspase-9. Indeed, the ability of caspase-3s to interact with pro-caspase-3 and/or caspase-9 might, at least in part, contribute to explain its anti-apoptotic activity. We observed that procaspase-3 or caspase-3s expressed in MCF-7 cells interacted with active caspase-9 when these cells were exposed to apoptotic stimuli. This interaction was abrogated when caspase-3s and procaspase-3 are expressed simultaneously. These observations could suggest that interaction of active caspase-9 with a catalytically inactive caspase-3s could stimulate caspase-9 activity or activation at the apoptosome level explaining why, in MCF-7, the overexpression of caspase-3s alone increased slightly apoptosis rate. However this situation was observed only in MCF-7, which is deficient for caspase-3, and not in HBL100 as caspase-3s is expressed with procaspase-3 (Table S3). Nevertheless, in human tissues, caspase-3s is always expressed with procaspase-3. Taken together, these results strongly suggested that caspase-3s prevent the apoptosome assembly by sequestrating procaspase-3. These data evocate the mechanism of caspase-9 gene which also possesses a splice variant with antagonist apoptotic properties to the principal transcript. Indeed,, casp9-γ, an alternative splice variant of caspase-9, functions as an endogenous apoptotic inhibitor by interfering with the Apaf-1/procaspase-9 interaction [32]. Similarly, forced expression of the caspase-8 alternative splice variant, caspase-8L, protected hematopoietic cells from extrinsic apoptosis by preventing CD95 to connect to the caspase cascade [33].
In conclusion, our study indicates that caspase-3s could have its anti-apoptotic function by sequestration of procaspase-3, inhibiting the interaction of procasase-3 with caspase-9 and apoptosome, thus counteracting the activation of the apoptotic machinery (Figure 6). Our data support that the investigation of caspase-3s in breast tumors might contribute to explain certain forms of drug resistance, and to develop more adapted and efficient treatments.
10.1371/journal.pone.0029058.g006Figure 6 Model of apoptosis inhibition by caspase-3s.
After apoptotic signal, cytochrome c is released from mitochondria to the cytoplasm. Then, Apaf-1/cytochrome c oligomers are formed and interact with caspase-9. In presence of procaspase-3 only, the complex Apaf-1/cytochrome c/active caspase-9 interacts with procaspase-3 to form the complete apoptosome. Then, active caspase-3 is release from the complex and apoptosis occurs. In presence of caspase-3s, the complex Apaf-1/cytochrome c/active caspase-9 interacts with caspase-3s to form a “short” apoptosome having not the ability to induce apoptosis. Finally, in presence of both procaspase-3 and caspase-3s, caspase3s interacts with procaspase-3. This interaction induces the sequestration of procaspase-3, inhibiting the complete apoptosome assembly.
Supporting Information
Figure S1
Control of transfection by Western Blot assay with anti-GFP antibody in MCF-7 cell line. Procaspase-3-GFP (ProC3-GFP) and caspase-3s-YFP (C3s-YFP) were stably overexpressed in single transfection and in co-transfection. GFP vector (GFP) was used as control. β-actin was used as loading control.
(TIF)
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Figure S2
Control of procaspase-3 and capsase-3s extinction after siRNA transfection.
A. Endogenous procaspase-3 was inactivated in HBL100 cells by siRNA transfection and the expression was monitored every 24 hours by western blot analysis from 24 to 96 hours after transfection. The transfection of siRNA induced the complete extinction of procaspase-3 from 48 to 96 hours after transfection. Caspase-3s expression was not affected by procaspase-3 siRNA transfection. B. Endogenous caspase-3s was inactivated in HBL100 and MDA-MB-231 cells by siRNA transfection and the expression was monitored every 24 hours up to 96 hours by real time quantitative PCR. The transfection of siRNA induced the complete extinction of caspase-3s from 48 to 96 hours after transfection. Nonetheless, procaspase-3 expression was not down-regulated by caspase-3s siRNA transfection, but its expression was strongly increased. C. The extinction of endogenous caspase-3s induced a strong increase of the endogenous procaspase-3/caspase-3s ratio. Double arrows indicate the optimal time lapse for cytotoxicity assay.
(TIF)
Click here for additional data file.
Table S1 Primers and probes sequences used in this study.
(DOC)
Click here for additional data file.
Table S2 Chemotherapeutic agents and doses used in this study to induce apoptosis.
(DOC)
Click here for additional data file.
Table S3 The cells (MCF7: deficient for caspase-3; HBL100: proficient for caspase-3) were incubated for 48 H in drug-containing medium and apoptosis was detected by flow cytometry. *Data represent the percentage (± Standard Deviation) of apoptotic cells in each treatment conditions (mean of 3 experiments). CT: control, 5FU: 5- Fluorouracil, BLM: Bleomycine, CDDP: Cisplatin, EPI: Epirubicin, DOC: Docetaxol, VIN: Vincristine, STAU: Staurosporine. For the different drugs, the concentrations used to induce apoptosis are different in MCF-7 and HBL100, and are thus indicated in Table S2.
(DOC)
Click here for additional data file.
Table S4 Numbers indicate the mean fluorescence intensity of nuclei for each condition.
(DOC)
Click here for additional data file.
Competing Interests: The authors have declared that no competing interests exist.
Funding: The authors have no support or funding to report.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22216200PONE-D-11-1475010.1371/journal.pone.0029170Research ArticleBiologyDevelopmental BiologyStem CellsStem Cell NicheMolecular Cell BiologyCellular TypesStem CellsStem Cell NicheMedicineOncologyCancers and NeoplasmsGenitourinary Tract TumorsProstate CancerUrologyProstate DiseasesProstate CancerProstate Cancer Cell Lines under Hypoxia Exhibit Greater Stem-Like Properties Stem-Like Properties of Prostate CancerMa Yuanyuan
1
5
Liang Dongming
1
Liu Jian
1
Axcrona Karol
2
Kvalheim Gunnar
3
Stokke Trond
4
Nesland Jahn M.
1
5
Suo Zhenhe
1
5
*
1
Department of Pathology, University of Oslo, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
2
Department of Urology, University of Oslo, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
3
Department of Cell Therapy, University of Oslo, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
4
Department of Radiation Biology, Institute for Cancer Research, University of Oslo, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway
5
Department of Pathology, Faculty of Medicine, Institute of Clinical Medicine, University of Oslo, Oslo, Norway
Tang Dean G. EditorThe University of Texas M.D Anderson Cancer Center, United States of America* E-mail: [email protected] and designed the experiments: YM DL ZS. Performed the experiments: YM DL JL. Analyzed the data: YM DL JL KA GK TS JMN ZS. Contributed reagents/materials/analysis tools: ZS. Wrote the paper: YM DL JL KA GK TS JMN ZS.
2011 28 12 2011 6 12 e291701 8 2011 22 11 2011 Ma et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Hypoxia is an important environmental change in many cancers. Hypoxic niches can be occupied by cancer stem/progenitor-like cells that are associated with tumor progression and resistance to radiotherapy and chemotherapy. However, it has not yet been fully elucidated how hypoxia influences the stem-like properties of prostate cancer cells. In this report, we investigated the effects of hypoxia on human prostate cancer cell lines, PC-3 and DU145. In comparison to normoxia (20% O2), 7% O2 induced higher expressions of HIF-1α and HIF-2α, which were associated with upregulation of Oct3/4 and Nanog; 1% O2 induced even greater levels of these factors. The upregulated NANOG mRNA expression in hypoxia was confirmed to be predominantly retrogene NANOGP8. Similar growth rates were observed for cells cultivated under hypoxic and normoxic conditions for 48 hours; however, the colony formation assay revealed that 48 hours of hypoxic pretreatment resulted in the formation of more colonies. Treatment with 1% O2 also extended the G0/G1 stage, resulting in more side population cells, and induced CD44 and ABCG2 expressions. Hypoxia also increased the number of cells positive for ABCG2 expression, which were predominantly found to be CD44bright cells. Correspondingly, the sorted CD44bright cells expressed higher levels of ABCG2, Oct3/4, and Nanog than CD44dim cells, and hypoxic pretreatment significantly increased the expressions of these factors. CD44bright cells under normoxia formed significantly more colonies and spheres compared with the CD44dim cells, and hypoxic pretreatment even increased this effect. Our data indicate that prostate cancer cells under hypoxia possess greater stem-like properties.
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Introduction
Somatic tumors, including prostate cancer, contain a small subset of stem-like cells, called cancer stem cells, with capacities for self-renewal, differentiation, and initiation of new tumors. It has been demonstrated that cancer stem cells can escape from radiotherapy and chemotherapy, and are able to form metastatic tumors in other organs [1], [2]. Cancer stem cells preferentially reside in specific hypoxic microenvironment-niches, often existing inside tumors [3], [4].
The hypoxia inducible factors (HIFs) are key regulators found in mammalian cells under lower oxygen tension; they are involved with multiple functions, such as cell survival, angiogenesis, and stem cell maintenance, and play essential roles in cellular and systemic homeostasis in response to hypoxia [5]. HIFs are heterodimers; HIF1A (HIF-1α) and EPAS1 (HIF-2α) are the two major isoforms of the α-subunit, and share a high degree of sequence homology. Oct3/4 (also called POU5F1) and Nanog are embryonic stem cell markers that are important for transcription and in maintaining self-renewal of embryonic stem cells and primordial germ cells. They have also been identified in different somatic tumors, including head and neck, lung, colorectal, ovarian, and prostate cancers [6]–[11]. Comparatively, the expressions of these genes are down-regulated in all differentiated somatic cell types, in vitro as well as in vivo
[12], [13]. NANOG, also called NANOG1, has several pseudogenes, and among them NANOGP8 has later been confirmed to be a retrogene. Both NANOG1 and NANOGP8 expressions have been identified in different cancer cell types [14], [15], including prostate cancer cells [16].
A number of surface markers have been used to isolate putative cancer stem/progenitor cells. In prostate cancer, the early progenitor cells are associated with several specific surface markers, such as CD44, CD133, and CXCR4 [17]–[19]. Side population technology has also been used to isolate cancer stem cells with the ability to pump out Hoechst 33342 [20]. Efflux of the dye is attributed to members of the ATP-binding cassette family, such as ABCG2 (breast cancer resistance protein, BCRP). The upregulation of ABCG2 is also responsible for chemotherapeutic resistance in certain cancer cells [21]. In breast and prostate cancers, previous studies have identified CD44+ or CD117+/ABCG2+ cells with stem-like characteristics, such as increased clonogenic/tumorigenic properties, higher expressions of stemness genes, and stronger ability to form tumors in animal models [19], [22], [23].
Hypoxia helps embryonic stem cells to maintain stemness and higher oxygen tensions drive cells into proliferation and differentiation, which are more susceptible to conventional treatment modalities [24], [25]. Similar results have been observed in adult cells, like adipocytes, fibroblasts, and several types of cancer cells [26]–[28]. However, the effects of hypoxia on prostate cancer cells have not been fully elucidated. Therefore, in this study we examined the prostate cancer cell lines PC-3 and DU145 at different oxygen tensions in order to better understand the effect of hypoxia on the stem-like properties of the cells. Stemness factors, Oct3/4 and Nanog, were expressed at higher levels in the cells under hypoxic cultivation and these cells exhibited elevated colony formation potential compared to the cells under normoxic condition. Furthermore, the upregulated NANOG mRNA expression under hypoxia was confirmed mainly derived from the retrogene NANOGP8. In these prostate cancer cell lines, hypoxia also increased the fraction of side population cells, extended the G0/G1 stage and resulted in higher levels ABCG2 and CD44 expressions. Additional experiments demonstrated that CD44bright cells exhibited significantly greater stemness, as verified by colony formation assay, sphere growth assay, and stemness factor expression analyses.
Materials and Methods
Cell culture
Human prostate cancer cell lines, PC-3 and DU145, were purchased from ATCC (American Type Culture Collection, USA) and maintained in our lab for this study. For conventional cell culture, cells were seeded in culture flasks with RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cultures were maintained at 37°C in a humidified incubator in an atmosphere of 20% O2, 5% CO2, and 75% N2.
The Xvivo Closed Incubation System (Xvivo system 300 C, BioSpherix, New York, USA) was used in this study in order to accurately maintain different oxygen tensions in different chambers. After 24 hours of cultivation in conventional cell culture (allowing cells to attach onto the flasks), the cells were transferred into different chambers with 1% O2, 5% CO2, and 94% N2; 7% O2, 5% CO2, and 88% N2; or 20% O2, 5% CO2 and 75% N2 for variable periods of time before being harvested for additional analysis.
Semiquantitative reverse transcription-PCR (RT-PCR)
Total RNA was extracted from the cultivated cells using the RNeasy Kit (Qiagen, CA, USA) according to the manufacturer's instruction. To eliminate any DNA, DNase I was used in the RNA isolation procedure. RNA sample concentrations were quantified using a spectrophotometer (Nanodrop ND-1000, USA) at OD260/280.
Complementary DNA (cDNA) was subsequently synthesized from 5 µg total RNA using the Multiscribe reverse transcriptase (Applied Biosystems, Foster City, CA). The conditions for reverse transcription were: 25°C for 10 minutes, 37°C for 12 minutes, 85°C for 5 minutes, followed by holding at 4°C. cDNA was stored at −70°C for later PCR analyses.
PCR amplification of cDNA was performed using a PCR system (DOPPIO VWR, UK) and the following program: initial denaturation at 95°C for 10 minutes; followed by 30 cycles (28 cycles for GAPDH) of annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds; followed by termination with a 10 minute elongation step at 72°C. The primers used for RT-PCR were: for Nanog, F 5′-ATGCCTCACACGGAGACTGT-3′ and R 5′-AGGGCTGTCCTGAATAAGCA-3′, amplifying a 66-bp fragment; for Oct3/4, F 5′-ACATGTGTAAGCTGCGGCC-3′ and R 5′-GTTGTGCATAGTCGCTGCTTG-3′, amplifying a 297-bp fragment; for HIF-1α, F 5′-AGTGTACCCTAACTAGCCGAGGAA-3′ and R 5′-CTGAGGTTGGTTACTGTTGGTATCA-3′, amplifying a 113-bp fragment; for HIF-2α, F 5′-GACCAGCAGATGGACAACTTGTAC-3′ and R 5′-CAGAAAGATCATGTCGCCATCTT-3′, amplifying a 84-bp fragment; and for GAPDH, F 5′-CCTCAAGATCATCAGCAATGC-3′ and R 5′-TGGTCATGAGTCCTTCCACG-3′, amplifying a 101-bp product. The experiments were repeated at least three times.
The amplified PCR products were separated by 7.5% polyacrylamide gel electrophoresis, stained with GelRed (Molecular Probes, Invitrogen), and visualized with a syngene image system (G: BOX Syngene, USA).
The mRNA expressions of NANOG1 and NANOGP8 were measured by quantitative PCR using a Taqman ABI 7900 Sequence Detector System (Applied Biosystems). The published specific primers and probes [29] were used in this study . For NANOG1: forward primer was 5′-CGCCCTGCCTAGAAAAGACATTT -3′, reverse primer was 5′-AGAAGCCGTCTCTGGCTATAGATAA -3′, and the probe was CTGCTAAGGACAACATTGAT; for NANOGP8: forward primer was 5′-CGCCCTGCCTAGAAAAGACATTT-3′ , reverse primer was 5′-ACGAGTTTGGATATCTTTAGGGTTTAGAATC-3′, and the probe was CCTTGGCTGCCGTCTCTG. All the primers and probes labeled with FAM-MGB were obtained from Applied Biosystems. The GAPDH was used as an internal control and the Ct values at 20% O2 were used as calibrators for evaluating NANOG1 and NANOGP8 expression levels in response to hypoxia. The experiments were performed in duplicate. The expression levels of the NANOG1 and NANOGP8 were analyzed through the ΔΔCt method [29].
Immunoblotting
Cells were quickly rinsed with ice-cold phosphate-buffered saline (PBS) and scraped into RIPA buffer (25 mM Tris HCl pH 7.6, 100 mM NaCl, 1% NP40, 1% sodium deoxycholate, 0.1% SDS; Thermo Scientific Pierce, Germany), with protease inhibitors (0.1 µM aprotinin, 1.0 mM PMSF, 1 µM leupeptin, 1 µM pepstatin) added immediately before use. The samples were centrifuged at 15,000 rpm for 15 minutes at 4°C and the supernatants were transferred to new tubes. The protein concentrations were measured with a Bio-Rad protein assay according to the manufacturer's instruction. The samples were heated with a benchtop heater (Model 111002, Boekel Scientific, USA) at 100°C for 5 minutes in SDS-loading buffer (500 mM Tris-HCl pH 6.8; 10% Glycerol, 2% SDS, 0.6 M DTT, 0.05% bromophenol blue), and then an equal amount of protein (50 µg) per sample was subjected to 10% SDS-PAGE and transferred to polyvinylidene difluoride transfer membrane (BIO-RAD, USA). Membranes were blocked with 5% non-fat dry milk in TBS-Tween for 60 minutes at room temperature and then incubated with the primary antibodies at optimal dilution in TBST/5% milk overnight at 4°C. The optimized antibodies used in this study included: HIF-1α (1 µg/ml MAB1536, R&D), HIF-2α (1 µg/ml MAB2886, R&D), GAPDH (0.2 µg/ml AF5718, R&D), Oct3/4 (1 µg/ml MAB1759, R&D), Nanog (1 µg/ml AF1997, R&D), and ABCG2 (0.5 µg/ml B7059, Sigma-Aldrich). The membranes were then incubated with secondary HRP-conjugated antibodies and immunocomplexes were visualized by enhanced chemiluminescence (GE Healthcare, UK). Western blotting experiments were repeated at least three times.
Cell block preparation
Cells were grown to 80% confluence and then were digested with 0.25% trypsin and EDTA (Invitrogen, USA), harvested, and centrifuged at 2000 rpm for 10 minutes. Supernatants were discarded, 3 drops of plasma and 2 drops of thrombin were added to the sedimentation, and the contents were carefully mixed by tube rotation. One minute later, the mixture was coagulated and 4% buffered formalin was added to the tube for fixation. The coagulated mass was then placed in linen paper and used to construct a paraffin block by the conventional process.
Immunocytochemistry
Cell blocks were sliced into 4-µm paraffin sections that were then deparaffinized using PT-Link apparatus. Next, the sections were rinsed with DAKO wash buffer, incubated with hydrogen peroxide for 5 minutes, and then incubated with primary antibody for 30 minutes at room temperature. The antibodies used and their concentrations were: HIF-1α (mouse, 1∶200; catalog number: NB100-150, Novus), HIF-2α (rabbit, 1∶100; catalog number: NB100-122, Novus), Oct3/4, (goat, 10 µg/ml; catalog number: AF1759, R&D) and Nanog (goat, 5 µg/ml; catalog number: AF1997, R&D).
After another rinse with DAKO wash buffer, mouse/rabbit EnVision FLEX+Linker reagent was added and samples were incubated for 15 minutes at room temperature, followed by incubation with EnVision FLEX+HRP for 30 minutes at room temperature. Samples with primary antibodies from goat were incubated for 30 minutes at room temperature with mouse anti-goat IgG before the addition of mouse EnVision FLEX+Linker reagent and EnVision FLEX+HRP as described above. The sections were rinsed, color reaction developed with DAB reagent, counterstained in hematoxylin for 20 seconds, dehydrated, and mounted under glass cover slips in preparation for evaluation by microscopy.
Cell counting (cell proliferation rate)
Cell proliferation was evaluated by counting cell numbers using the Electronics Countess Automated Cell Counter (Invitrogen, USA). After trypsinization, the floating cells were collected to create a cell suspension that contained no obvious cell clusters. In each preparation, 10 µl of cell suspension was mixed with 0.4% trypan blue dye (1∶1) before being loaded onto a cell counting chamber slide for cell counting. The number of viable cells that were able to exclude the dye was counted for each experimental condition. For each sample, the cell number was counted at least three times.
Colony formation assay
Using six-well plates, 500 cells per well were maintained in oxygen tensions of 1%, 7%, and 20% for 48 hours. All plates were then placed in an incubator with 20% O2 for 2 weeks for observation of colony formation. Colonies were fixed with 4% buffered formalin for 15 minutes, and then stained with 1% crystal violet for 30 minutes. The plates were gently washed with PBS and dried before microscopic colony evaluation. Colonies that contained more than 30 cells were counted. Colony formation efficiency was calculated as follows: colony formation efficiency = colonies/input cells×100%. Data are representative of three independent experiments.
Cell cycle analysis
After incubation under different oxygen tensions, including 1% or 20% O2, the PC-3 and DU145 cells were harvested and fixed with methanol at −20°C overnight. These cells were used to prepare a single cell suspension to which was added 1.5 µg/ml of Hoechst 33258 before the cells were kept on ice for 30 minutes. After that, the samples were analyzed with an LSRII flow cytometer (Becton Dickinson, San Jose, CA, USA).
Side population assay
Cells grown to 80% confluence (about 1×106 cells) were harvested and suspended in prewarmed RPMI 1640 medium containing 2% fetal bovine serum and 2 mM HEPES buffer. Hoechst 33342 dye (stock solution of 1 mg/ml; Sigma) was then added to a final concentration of 5 µg/ml, and the mixture was incubated with intermittent shaking for 90 minutes at 37°C, in the presence or absence of verapamil (50 µM; Sigma). Then, the cells were washed with ice-cold HBSS with 2% FBS, centrifuged at 4°C, and resuspended in ice-cold HBSS containing 2% FBS. Propidium iodide was added to the suspended cells to a final concentration of 2 µg/ml, in order to reveal viable cells. Before analysis, the cells were filtered through a 70-µm cell strainer to obtain a single cell suspension. The cell aggregates were discarded from the analysis by doublet discrimination and single cells were analyzed on a LSRII flow cytometer (BD Biosciences). Hoechst 33342 dye was excited at 350 nm and the side-population cells were visualized by the use of red (red, 660/10 nm) vs. blue (blue, 424/44 nm) detection.
ABCG2/CD44 phenotype and Fluorescence-activated cell sorting (FACS)
After 48 hours of incubation at oxygen tensions of 1% and 20%, the PC-3 and DU145 cells were trypsinized, counted, washed with cold FACS buffer (PBS+BSA 0.03%), and resuspended to a final concentration of 106 cells/ml. The cells were pre-blocked with 5% BSA for 30 minutes on ice before they were stained with primary antibodies (anti-CD44 monoclonal antibody directly conjugated with APC (allophycoyanin) and anti-ABCG2 monoclonal antibody directly conjugated with FE (phycoerythin); BD Pharmingen Company) on ice, in the dark, for 30 minutes. Cell suspensions were washed twice, resuspended in 400 µl FACS buffer, and filtered through a 70-µm nylon mesh. Samples were analyzed on a flow cytometer (Becton Dickinson, San Jose, CA, USA) for detection of ABCG2 and CD44, and a FACS DiVa cell sorter was used for cell sorting. After cultivation at 1% or 20% O2 for 48 hours, the PC-3 and DU145 cells were sorted based on CD44 expression. For the CD44 positive cells, only the top 10% expressing cells were selected (called CD44bright); for the CD44 negative cells, the bottom 10% expressing cells were isolated (called CD44dim). For each sample, viable and single cells were gated; APC Mouse IgG2b (BD Pharmingen, USA) and FE Mouse IgG2b (BD Pharmingen, USA) isotype controls were used as negative controls.
Sphere formation assay
The assay used was based on previously described methods [30]. After the CD44bright cells were sorted with the method as described above, single CD44bright and CD44dim cells were plated at a density of 300 cells per well, in ultralow attachment six-well plates (Ultra low cluster plates, Life sciences). These cells were cultivated in serum free DMEM/F12 medium (Invitrogen) supplemented with 20 ng/ml EGF and 20 ng/ml bFGF for ten days under normoxia conditions before the spheres were evaluated under inverse miscopy and counted (more than 30 cells within a sphere was considered to be a full sphere). Data are representative of three independent experiments.
Statistical analyses
For each experiment, data are shown as mean ± SEM of at least three independent experiments; SPSS software (version 16.0) was used for data analysis. Statistical analyses were performed using the one-way ANOVA test and Student's t-test (P<0.05 was considered statistical significance).
Results
Hypoxia induces expression of HIF-1α, HIF-2α, Oct3/4 and Nanog
HIF-1α and HIF-2α, the major transcriptional factors responding to hypoxia, were examined in human prostate cancer cells PC-3 and DU145 that were exposed to different oxygen tensions for variable periods of time. At 20% O2 tension, HIF-1α and HIF-2α were weakly expressed at both the mRNA and protein levels; comparatively higher levels of HIF-1α and HIF-2α expression were observed at 7% O2 tension, and the highest levels were seen at 1% O2 tension (Figure 1A). At reduced oxygen tension levels, these two factors were already upregulated after 6 hours of cultivation. Their expressions reached the highest levels at 12 hours of cultivation at 7% O2 tension, but reached highest levels at only 6 hours of cultivation at 1% O2. Protein expression after 48 hours of cultivation at different oxygen tensions was also studied by immunocytochemistry (Figure 1B). The expressions of HIF-1α and HIF-2α were consistently higher in the hypoxia-treated cells, in agreement with the findings obtained by RT-PCR and immunoblotting.
10.1371/journal.pone.0029170.g001Figure 1 Hypoxia increases the expressions of HIF-1α and HIF-2α.
(A) In comparison to the cells cultivated at 20% O2, the cells cultivated at 7% O2 show higher levels of HIF-1α and HIF-2α, and the cells cultivated at 1% O2 show the highest levels of HIF-1α and HIF-2α by both RT-PCR and immunoblotting. (B) Immunocytochemistry reveals higher levels of HIF-1α and HIF-2α expressions in the cells cultivated under 7% O2 and the highest levels of HIF-1α and HIF-2α expressions in the cells cultivated under 1% O2 for both cell lines. The breast carcinoma sections were used as positive controls for both HIF-1α and HIF-2α. All photos were originally taken at 200× and all the insets were taken at 400×.
Oct3/4 and Nanog are frequently used as markers for the undifferentiated cells and play an essential role in sustaining capacity of self-renewal in adult stem cells [31], [32]. The expressions of these transcription factors were also examined in PC-3 and DU145 cells that were cultivated at different oxygen tensions. At both the mRNA and protein levels (Figure 2A), weak expressions of Oct3/4 and Nanog were revealed in these two cell lines cultivated at 20% O2 tension, while their expressions were upregulated in cells cultivated under 7% and 1% O2 conditions. In both cell lines, the expressions of these two factors were higher at 1% O2 than at 7% O2. As was also seen in immunoblotting analyses (Figure 2A), Oct3/4 and Nanog began to substantially increase as early as 6 hours after the cells were transferred to 1% O2, and reached maximum levels at 12 hours for both cell lines. When the cells were placed in 7% O2, the expressions of these two factors were also substantially elevated after 6 hours, and reached the highest levels at 12 hours cultivation; however, these expression levels were weaker than those observed in cells exposed to 1% O2. Similar results were also obtained by immunocytochemistry (Figure 2B). Following exposure to hypoxic condition, enhanced Oct3/4 and Nanog expressions were seen in both PC-3 and DU145 cells.
10.1371/journal.pone.0029170.g002Figure 2 Hypoxia increases the expressions of Oct3/4 and Nanog.
(A) In comparison to the cells cultivated at 20% O2, the cells cultivated at 7% O2 show higher levels of Oct3/4 and Nanog expressions, and the cells cultivated at 1% O2 show the highest levels of Oct3/4 and Nanog expressions in PC-3 and DU145 cell lines by both RT-PCR and immunoblotting. (B) Immunocytochemistry reveals corresponding higher levels of Oct3/4 and Nanog expressions at 7% O2 and the highest levels of Oct3/4 and Nanog expressions at 1% O2, in comparison to the cells cultivated at 20% O2 for both cell lines. Human seminoma tissue sections were used as positive controls for these two antibodies. All photos were originally taken at 200× and all the insets were taken at 400×.
To determine whether the elevated NANOG expression was derived from NANOG1 or NANOGP8, quantitative RT-PCR analyses were further performed, with the corresponding cells cultivated under normoxia as calibrators. It was repeatedly verified that the NANOG1 expression in the cells under normoxia was at very low level, with average Ct values 37.24 and 37.37 for the PC-3 cell and DU145 cells, respectively. However, the NANOGP8 expression in the cells under normoxia was relatively high, with average Ct values 33.56 and 33.51 for the PC-3 cell and DU145 cells, respectively. Although higher levels NANOG1 and NANOGP8 expressions could be observed in both cell lines under hypoxia, the elevated NANOG expression was confirmed predominantly NANOGP8, with up to 6-fold and 10-fold increase in expression in the PC-3 and DU145 cells under 1% O2, respectively (Figure 3). Since the NANOG1 expression in the cells under normoxia was extremely low, the 2.6-fold and 3.1-fold increase in expression of this gene in the PC-3 and DU145 cells under 1% O2 represented still quite low expression level.
10.1371/journal.pone.0029170.g003Figure 3 Quantitative PCR results of NANOG1 and NONOGP8.
Compared to the cells under normoxia, there are elevated NANOG1 and NONOGP8 expressions in the cells under 7% O2 for both cell lines, with up to 1.8-fold increase NANOG1 expression and up to 2.5-fold NONOGP8 increase in both cell lines; the cells under 1% O2 express even higher levels of NANOG1 and NONOGP8, with 2.6-fold and 3.1-fold increase in NANOG1 expression in the PC-3 and DU145 cell lines, respectively, and with 6-fold and 10-fold increase in NONOGP8 expressions in the PC-3 and DU145 cell lines, respectively.
Hypoxia increases colony formation capability and extends G0/G1 stage
Since hypoxia increased the expression of stemness factors, we next investigated whether hypoxia influenced the proliferation of these cells. The PC-3 and DU145 cells were cultivated under normoxia (20% O2) or hypoxia conditions (1% O2 and 7% O2) for 48 hours for the proliferation assay. As shown in Figure 4A, for each cell line there were no statistically significant differences in proliferation of the cells cultivated at different oxygen tensions (P>0.05), although the cells grew somewhat slower under hypoxia than normoxia. Next, we asked whether hypoxia-pretreatment could influence clonogenicty in these cells. The cells were initially exposed to different oxygen tensions for 48 hours, followed by transfer to a normoxic chamber (20% O2) for 14 days for the colony formation assay. Compared to the cells that were steadily cultivated at 20% O2, more colonies were observed in the cells that were pretreated at 7% O2, and even more colonies were seen in the cells pretreated at 1% O2 (Figure 4B). Compared to the cells always cultivated under normoxia, statistically significantly higher colony formation efficiency was identified in the hypoxia-pretreated PC-3 and DU145 ells (Figure 4C). Cell cycle analyses demonstrated an extended G0/G1 stage in the cells that were exposed to 1% O2 for 48 hours in comparison with the cells cultivated at 20% O2 as controls (P<0.05) (Figure 4D and E), indicating more cells in a quiescent status under hypoxia.
10.1371/journal.pone.0029170.g004Figure 4 Hypoxic effects on cell proliferation, colony formation and cell cycle.
(A) Cell proliferation curves show no statistical difference for cell growth under different oxygen tensions (P>0.05). (B) Colony formation assay for both cell lines shows more colonies in the cells pre-treated at 7% O2 for 48 hours and even more colonies in the cells pre-treated under 1% O2 (C) Histograms for the colony formation efficiency shows statistically higher efficiency in the cells pre-treated under hypoxia (7% or 1% O2) for both cell lines (P<0.0001). (D) Flow cytometry shows extended G0/G1 stage for both cell lines which have been cultivated under 1% O2 for 48 hours, in comparison to the cells always kept under normoxia. (E) Statistical analyses reveal significantly extended G0/G1 stage in the cells cultivated under hypoxia for both cell lines (P<0.05).
Hypoxia increases the fraction of cells with stem-like phenotype
Side population cells, assumed to contain putative prostate cancer stem cells, are known to pump out the dye Hoechst 33342 [20], [33]. Cells exhibiting this activity were further assessed during cultivation at different oxygen tensions. Higher fractions of these side population cells were observed in cultures kept at 1% O2 tension for 48 hours in comparison to the cells cultivated at 20% O2 (Figure 5A and B).
10.1371/journal.pone.0029170.g005Figure 5 Stem-like phenotype analyses by flow cytometry.
PC-3 and DU145 cells were cultivated in 1% O2 and 20% O2 coditions for 48 hours to analyze stem-like phenotype through flow cytometry assay. (A) The representative images show that side population cells were induced in both cell lines after hypoxic treatment. (B) Statistic analyses show significant difference in side population. (C) Flow cytometry analyses show higher levels of ABCG2 expression intensity in both cell lines under hypoxia. (D) Histogram shows statistically significant difference in ABCG2 expression. (E) Flow cytometry analyses show higher CD44 expression intensity in both cell lines under hypoxia. (F) Histogram shows statistically significant difference in CD44 expression.
ABCG2 and CD44 have been described as prostate cancer stem-like markers based on clinical investigations and studies in prostate cancer cell lines [19], [33]. Therefore, ABCG2 and CD44 expressions in the PC-3 and DU145 cells that were incubated at 1% or 20% O2 tensions for 48 hours were examined with flow cytometry. As shown in Figures 5C and D, threw were 1.20-fold and 1.42-fold increase in ABCG2 expression in the PC-3 and DU145 cells under 1% O2, respectively, in comparison to the cells always cultivated under normoxia. Similarly, there were 1.50-fold and 1.45-fold increases in CD44 expression in the PC-3 and DU145 cells under 1% O2, respectively, in comparison to the cells always cultivated under normoxia (Figures 5E and F).
Since hypoxia could induce both ABCG2 and CD44 expression in both cell lines, we further investigated the relationship between CD44 and ABCG2. Double staining of these two surface markers revealed that the hypoxia-induced ABCG2+ cells were primarily CD44bright cells, and the CD44dim cells under the same culture condition were mostly negative for ABCG2 expression (Figure 6A).
10.1371/journal.pone.0029170.g006Figure 6 CD44bright cells are mainly positive for ABCG2, Oct3/4 and Nanog.
(A) Double staining of CD44 and ABCG2 surface markers with flow cytometry assay shows higher levels expressions of these factors in both cell lines under hypoxia for 48 hours. (B) The CD44bright cells under normoxia express higher levels of ABCG2, Oct3/4 and Nanog, but the CD44dim cells under the same normoxia condition express very low levels of these factors. The CD44bright cells pretreated under 1% O2 for 48 hours show even higher levels of these factors compared to the CD44bright cells cultivated under normoxia.
CD44bright cells show stem-like properties
The sorted CD44bright and CD44dim cells were further examined by immunoblotting. As shown in Figure 6B, in both PC-3 and DU145 cell lines grown under normoxia, the CD44bright cells expressed higher levels of ABCG2, Oct3/4, and Nanog than the CD44dim cells. Hypoxic pretreatment of these cell lines for 48 hours resulted in even greater levels of expression of these factors in the CD44bright cells.
After verifying the higher stemness factor and ABCG2 expressions in the CD44bright cells, both under hypoxia and normoxia, we examined whether the CD44bright cells had greater colony formation capability (a property of stem-like cells). As can be seen in Figures 7A and 7B, the CD44bright cells formed significantly more colonies compared to the CD44dim cells. Next, we tested how the CD44bright and CD44dim cells responded to hypoxic pretreatment (1% O2), in comparison to the cells consistently cultivated at normoxia. The CD44bright cells which were hypoxic-pretreated for 48 hours could form significantly more colonies than the CD44bright cells without hypoxic pretreatment.
10.1371/journal.pone.0029170.g007Figure 7 CD44bright cells show stem-like properties.
(A) For both PC-3 and DU145 cell lines, more colonies are shown in the CD44bright cells than the CD44dim cells under nomoxia condition, while even more colonies can be seen in the CD44bright cells pretreated under 1% O2 for 48 hours. (B) Histograms for the colony formation efficiency show statistically significantly higher efficiency in the CD44bright cells than the corresponding CD44dim cells (P<0.0001 for both cell lines), and even higher level efficiency in the hypoxia- pretreated CD44bright cells in comparisons with the CD44bright cells without hypoxic pretreatment (P<0.05 for both cell lines). (C) Sphere formation assay shows spheres in the CD44bright cells in both normoxia and hypoxic pretreated cells while there is no qualified sphere in the corresponding CD44dim cells for both cell lines. (D) Histograms show sphere formation efficiency in the CD44bright and CD44dim cells with and without hypoxia pretreatment.
The sorted subpopulations of CD44bright and CD44dim cells were additionally examined by sphere growth assays since sphere growth is common in stem-like cells. As shown in Figure 7C, the CD44dim cells hardly formed any sphere, no matter the cells were hypoxia pre-treated or not. However, there was a great number of spheres in the CD44bright cells from both cell lines, either from the nomoxia or from the hypoxia pretreatment. Although there was no significant sphere formation efficiency difference in the hypoxia pre-treated CD44bright cells compared to the nomoxia pre-treated CD44bright cells in both cell lines, the hypoxia pre-treated CD44bright cells in the PC-3 cell line demonstrated a 1.21-fold increase in sphere formation efficiency, and the hypoxia pre-treated CD44bright cells in the DU145 cells revealed a 1.14-fold increase in sphere formation efficiency, in comparison with their control cells.
Discussion
Hypoxia often occurs in the inner part of solid tumors, creating an environment where undifferentiated tumor cells can exist. For prostate cancer, hypoxia is commonly associated with poor prognosis [34]. In this study, we have demonstrated that hypoxia can upregulate stem-like properties of the prostate cancer cell lines PC-3 and DU145. It has been hypothesized that cancer stem-like cells may persist as a distinct population within tumors and cause relapse and metastasis following general cancer therapies like radiotherapy and chemotherapy. Therefore, it is of great value to identify factors that affect the stem-like properties of cancer cells and to determine what conditions influence the differentiation of stem cells.
The hypoxia inducible factors HIF-1α and HIF-2α are reportedly activated in aggressive tumor cells [35], [36]. HIF-1α expression is increased in primary prostate cancers, prostate cancer bone marrow metastasis of PC-3 and brain metastasis of DU145, compared to in normal prostate epithelium [37], [38]. In this study, HIF-1α was weakly expressed under normoxia and their expressions were increased under hypoxic conditions. This is in agreement with earlier reports of higher expressions in response to hypoxia [39]–[41]. In our study, HIF-2α was also upregulated in response to hypoxia treatment in consistent with increased expression of HIF-1α. Moreover, in a clinical observation, HIF-1α expression was elevated in high-grade prostatic intraepithelial neoplastic lesions (the precursor of a majority of invasive prostate adenocarcinoma), relative to expression levels in normal epithelium, stromal cells, and benign prostatic hyperplasia [42].
The biological impact of hypoxia is exerted through transcriptional factors, such as Oct3/4 and Nanog [43]. Oct3/4 and Nanog are key players in a transcriptional network for maintenance of embryonic stem cell and primordial germ cells self-renewal [6], [11]. Our present study revealed that Oct3/4 and Nanog were expressed in the prostate cancer cell lines PC-3 and DU145, albeit in a lower level. Invasive tumor cells are shown with a stem-like genomic signature expressing a number of stem cell genes, including Oct3/4 and Nanog and these cells are more tumorigenic compared to their non-invasive counterpart [44]. In our present study, we found that the expressions of these genes were upregulated upon hypoxia exposure, at both the mRNA and protein levels, in parallel with the increasing expressions of HIF-1α and HIF-2α. The upregulation of Oct3/4 expression could be correlated with the enhanced HIF-2α expression under hypoxia and this observation is consistent with previous findings that HIF-2α binds to the promoter of Oct3/4 and induces its expression and activity directly [45], supporting a potential role for the interaction of HIF-2α and Oct3/4 in prostate cancer cells response to hypoxia.
In order to discriminate whether the upregulated NANOG mRNA expression was derived from NANOG1 and/or NANOGP8 genes in response to hypoxia, specific primers and probes were used to detect the expressions of these two genes in PC-3 and DU145 cell lines under different oxygen tensions by quantitative RT-PCR. In line with previous reports, there was extremely low level NANOG1 expression in these cells [16], [29]. Although hypoxia could upregulate the expression of this gene in both cell lines, the expressions of the gene in these cells were still very low even in the cells under hypoxia. However, the NANOGP8 was expressed in higher levels in both cell lines under normoxia, and its expression was greatly upregulated by hypoxia, with up to 6-fold and 10-fold increase in the PC-3 and DU145 cell lines, respectively, indicating that NANOGP8 expression was predominantly influenced by hypoxia. Since Nanog protein expression was upregulated under hypoxia, and the cells under hypoxia revealed greater stem-like features, our results may support the notion that NANOGP8 plays an important role on the cancer stem-like properties in prostate cancer cells [16], [29].
It is well documented that oxygen plays an important role in the development of tissues and cells. Hypoxia often occurs in pathophysiological conditions, especially when growth exceeds the blood supply in the tumor. It has been speculated that the formation of a hypoxic niche may drive selection of cancer stem cells that can maintain tumors by regulating specific developmental programs. Based on these reports, we wanted to know whether hypoxia influenced cell proliferation. Although there was no statistically significant difference between cells' proliferation under hypoxia compared to that under normoxia, the cells grew slowly under hypoxia with extended G0/G1, indicating higher quiescent status of these cells in hypoxia.
To study the effect of hypoxia on stem-like cell phenotypes, we assessed the fraction of side population cells. The side population assay has been successfully used for identification of cancer stem-like cells in studies of common urological malignancies, including prostate, bladder, and renal cancers [47]. Our experiments revealed a significantly higher number of side population cells in both the PC-3 and DU145 cells that were treated with 48 hours in 1% O2, strongly indicating that stem-like properties of these cancer cells could be greatly upregulate in vitro. Side population cells contain active ATP-cassette family members (e.g., ABCG2) that can pump Hoechst 33342 dye out of the cells and ABCG2 is associated with multi-drug resistance. Importantly, ABCG2 is a key molecular determinant for the side population cells and has been reviewed as a universal stem cell marker [33]. Consistent with the elevated fraction of side population cells, the expression of ABCG2 was correspondingly increased in the cells treated at 1% O2 tension, in comparison with the cells cultivated at 20% O2 for both cell lines. Since CD44 is considered a putative surface marker for cancer stem/progenitor cells in breast and prostate cancers [19], [22], [48], [49], we analyzed CD44 expressions in these cells as well. Our study showed that CD44 expression was significantly induced under hypoxia. The putative prostate cancer stem-like cell marker CD44 has been associated with prostate cancer stem like feature [46], and has been indicated to play an essential role in the quiescence of hematopoietic stem cells in the osteoblastic niche [50]. The extended G0/G1 stage and induced CD44 and ABCG2 expressions in our present study may suggest more slow-cycling cancer stem-like cells under hypoxia, which is in line with the study reported by Ishimoto et al [51].
Both CD44 and ABCG2 have been strongly indicated as stem cell markers in previous studies [19], [52], [53]. In the present study, we examined these two factors in the same cell populations, in order to explore whether their responses to hypoxic treatments were related. Using flow cytometry, we found that most of the PC-3 and DU145 cells were CD44 positive, but only a small portion of cells in these two cell lines were ABCG2 positive. The ABCG2 positive cells were mostly CD44bright cells, and hypoxia induced expressions of CD44 and ABCG2 in both cell lines, indicating that ABCG2 expression may be associated with CD44bright cells. Next, we sorted the CD44bright cells and verified that they were primarily strongly positivity for ABCG2. On the contrary, all the CD44dim cells were almost ABCG2 negative. Furthermore, there were higher levels ABCG2 expressions in the hypoxia pretreated CD44bright cells than the CD44bright cells without hypoxia-pretreatment in both cell lines. Collectively, our results suggest a strong association between ABCG2+ and CD44bright cells.
In consistent with previous report that both NANOGP8 mRNA and Nanog protein are enriched in the CD44bright human prostate cancer cells [16], we also found that higher protein expressions of Oct3/4 and Nanog in the CD44bright cells compared to the corresponding CD44dim cells. Moreover, we examined additional stemness features of these cells using colony formation and sphere growth assays. These experiments showed that the CD44bright cells formed more colonies than the CD44dim cells. The sphere growth assay also showed significantly higher sphere formation efficiency for the CD44bright cells, and this finding is supported by the former report that CD44bright prostate cancer cells display stem –like characteristics such as more proliferative, clonogenic, tumorigenic and metastatic than the corresponding CD44dim cells [54]. Furthermore, the colony efficiency of the CD44bright cells was significantly improved following hypoxic pretreatment, whereas the CD44dim cells did not respond to such hypoxic exposure. Since colony formation and sphere formation capabilities are confined to stem cells [46], [55], [56], these results may support the hypothesis that CD44bright cells possess stem-like properties in the prostate cancer cells [54].
In summary, hypoxia enhanced the stem-like properties of the human prostate cancer cell lines PC-3 and DU145. Hypoxic treatment also induced growth capability of CD44 positive cells in these two cell lines, and CD44bright cells possessed greater stem cell-like features, as verified by higher expressions of stemness factors and ABCG2, and significantly higher colony and sphere formation efficiency. Furthermore, the stem-like properties of the CD44bright cells were significantly increased upon hypoxic pretreatment.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by The Norwegian Radium Hospital Research Foundation with grant number 333005 to ZS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Sensors (Basel)Sensors (Basel, Switzerland)1424-8220Molecular Diversity Preservation International (MDPI) 10.3390/s100606128sensors-10-06128ArticleAn Emergency-Adaptive Routing Scheme for Wireless Sensor Networks for Building Fire Hazard Monitoring Zeng Yuanyuan 14*Xiong Naixue 2Park Jong Hyuk 3*Zheng Guilin 41 School of Electronic Information, Wuhan University, China2 Department of Computer Science, Georgia State University, USA; E-Mail: [email protected] Department of Computer Science and Engineering, Seoul National University of Technology, Korea4 School of Power and Mechanical Engineering, Wuhan University, China; E-Mail: [email protected]* Author to whom correspondence should be addressed; E-Mails: [email protected] (Y.Z.); [email protected] (J.H.P.); Tel.: +86-27-6877-2272, +82-2-970-6702; Fax: +86-27-6877-2272, +82-2-977-9441.2010 21 6 2010 10 6 6128 6148 8 5 2010 4 6 2010 7 6 2010 © 2010 by the authors; licensee MDPI, Basel, Switzerland.2010This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).Fire hazard monitoring and evacuation for building environments is a novel application area for the deployment of wireless sensor networks. In this context, adaptive routing is essential in order to ensure safe and timely data delivery in building evacuation and fire fighting resource applications. Existing routing mechanisms for wireless sensor networks are not well suited for building fires, especially as they do not consider critical and dynamic network scenarios. In this paper, an emergency-adaptive, real-time and robust routing protocol is presented for emergency situations such as building fire hazard applications. The protocol adapts to handle dynamic emergency scenarios and works well with the routing hole problem. Theoretical analysis and simulation results indicate that our protocol provides a real-time routing mechanism that is well suited for dynamic emergency scenarios in building fires when compared with other related work.
wireless sensor networksbuilding firesreal-timepower adaptation
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1. Introduction
In the near future, it can be expected that buildings will be equipped with a range of wireless sensors functioning as part of an overall building management system. Included in this set of sensors will be devices to monitor fire and smoke, allowing detection, localization and tracking of fires. It is expected such information could be used for a variety of purposes, including guiding building occupants to the nearest safe exit, and helping fire fighting personnel to decide on how to best tackle the disaster. Fire/smoke sensors are expected to be programmed to report periodically and also when they detect a sensor input that exceeds a threshold. In the latter case, there is a need for an emergency-adaptive, real-time and robust message delivery toward the sink. For example, a fire-fighter relies on timely temperature updates to remain aware of current fire conditions. In addition, as the fire spreads throughout the building, it becomes likely that the sensing devices may become disconnected from the network or indeed be destroyed, so the network routes have to be changed or re-discovered to adapt to these emergency conditions in order for the network to continue operating. Most existing routing protocols consider the energy efficiency and lifetime of the networks as the foremost design factor. The routing mechanisms used in general wireless sensor networks and even routing for forest fire applications are not well suited for in-building disaster situations, where timeliness and reliability are much more critical. For forest fires the focus is on tracking of fires, rather than evacuation or guidance of fire personnel. This combination of real-time requirements coupled with dynamic network topology in a critical application scenario provides the motivation for our research. In this paper, we propose an emergency-adaptive routing mechanism (EAR) designed especially for building fire emergencies using wireless sensor networks (WSN), which provides timely and robust data reporting to a sink. We do not need to know the exact localization of each sensor and also no time synchronization is needed. To the best of our knowledge, this is the first time a real-time and robust routing mechanism adaptive to building fire emergency using WSNs has been proposed. Also, this protocol could be easily used in other similar emergency applications.
Section 2 presents the related work. In Section 3 we outline the routing problem. We present an emergency-adaptive routing mechanism in Section 4. In Section 5, we present a preliminary analysis. In Section 6, we give ns2 simulation results. Finally, Section 7 concludes this paper.
2. Background and Related Work
Most routing protocols for WSNs focus on energy efficiency and link node lifetime related explicitly to its energy resources, i.e., a node is assumed to fail when the battery is depleted. Some WSN applications require real-time communication, typically for timely surveillance or tracking. Real-time routing protocols in WSNs are not new. For example, SPEED [1], MM-SPEED [2], RPAR [3] and RTLD [4] were all designed for real-time applications with explicit delay requirements. He et al. [1] proposed an outstanding real-time communication protocol binding the end-to-end communication delay by enforcing a uniform delivery velocity. Felemban et al. proposed [2] a novel packet delivery mechanism called MMSPEED for probabilistic QoS guarantee. Chipara et al. proposed [3] a real-time power aware routing protocol by dynamically adapting transmission power and routing decisions. But these routing protocols are not well suited for routing in emergency applications such as building fires, where critical and dynamic network scenarios are key factors. Amed et al. proposed [4] a novel real-time routing protocol with load distribution that provides efficient power consumption and high packet delivery ratio in WSN.
There are many robust routing protocols proposed for WSNs. Zhang et al. [5] proposed a framework of constrained flooding protocols. The framework incorporates a reinforcement learning kernel, a differential delay mechanism, and a constrained and probabilistic retransmission policy. The protocol takes the advantages of robustness from flooding. Deng et al. [6] presented a light-weight, dependable routing mechanism for communication between sensor nodes and a base station in a wireless sensor networks. The mechanism tolerates failures of random individual nodes in the network or a small part of the network. Boukerche et al. [7] presented a fault tolerant and low latency algorithm, which refer to as periodic, event-driven and query-based protocol that meets sensor networks requirements for critical conditions surveillance applications. The algorithm uses a publish/subscribe paradigm to disseminate requests across the network and an ACK-based scheme to provide fault tolerance. In building fires, network topology changes rapidly because of hazard and node failure, so general robust protocols are not suitable for such scenarios. Here, we want to design protocols that can be adaptive to the occurrence of fire, expanding, shrinking or diminishing, etc. So, “robustness” in this paper means “adaptive to fire situations”.
In this regard, the work by Wenning et al. [8] is interesting, as they propose a proactive routing method that is aware of the node’s destruction threat and adapts the routes accordingly, before node failure results in broken routes, delay and power consuming route re-discovery. They pay attention to the aspect of node failures caused by the sensed phenomena themselves.
However, in their work, they focus on disasters such as forest fire that are very different from design issues in building situations. Fire emergencies using wireless sensor networks within buildings are more challenging because of the complex physical environment and critical factors of fire hazards. In [9], we proposed a fire emergency detection and response framework for building environments using wireless sensor networks. We presented an overview of recent research activity including fire detection and evacuation, in addition to providing a testbed especially designed for building fire applications. Other researchers have worked on emergency guidance and navigation algorithms with WSNs for buildings. Tseng et al. [10] proposed a distributed 2D navigation algorithm to direct evacuees to an exit, while helping them avoid hazardous areas. Their design allows multiple exits and multiple emergency events in the sensing field. Sensors are used to establish escape paths leading to exits that are as safe as possible. When surrounded by hazards, sensors will try to guide people as far away from emergency locations as possible. Based on this, Pan et al. [11] proposed a novel 3D emergency service that aims to guide people to safe places when emergencies happen. In their work, when emergency events are detected, the network can adaptively modify its topology to ensure transportation reliability; quickly identify hazardous regions that should be avoided and find safe navigation paths that lead people to exits. Barnes et al. [12] presented a novel approach for safely evacuating persons from buildings under hazardous conditions. A distributed algorithm is designed to direct evacuees to an exit through arbitrarily complex building layouts in emergency situations. They find the safest paths for evacuees taking into account predictions of the relative movements of hazards, i.e., fires and evacuees. Tabirca et al. [13] solve a similar problem, but under conditions where hazards can change dynamically over time.
When fire expands in an inner building, there may cause a lot of segmentation in the network. In this case, a lot of routing holes occur that lead to data routing failure. The “Routing Hole Problem” is a very important and well-studied problem, where messages get trapped in a “local minimum”. Some existing “face routing” algorithms have been developed to bypass routing holes using geo-routing algorithms. GPSR [14] recovers holes by using the “right-hand rule” to route data packets along the boundary of the hole, combining greedy forwarding and perimeter routing on a planar graph. The authors of [15] proposed the first practical planarization algorithm with a reasonable message overhead, lazy cross-link removal (LCR). Fang et al. [16] presented an interesting approach, the BOUNDHOLE algorithm, which discovers the local minimum nodes and then “bounds” the contour of the routing holes. In the building fire situation, holes feature prominently and can be expected to grow in size rapidly as a fire spreads, thus demanding solutions that are robust and low complexity for quick reactions.
3. Definitions
Given a homogeneous WSN deployed in a building for fire hazard applications with N sensors and M sinks, each sensor can adjust its maximal transmission ranges to one of the k levels: r0, r1… rk−1 = rmax by using different transmission power levels from p0, p1, till pk−1 = pmax. Initially, all sensors work in p0. From the application aspect, real-time and robustness are two main challenges. Tmax is the maximum acceptable delay in reporting such a fire event to a sink node. It is required that each sensor i will report data packets to a sink node, such that:
A communication path from sensor to the sink can be found if such a path exists.
The end-to-end delay of the path is no more than Tmax.
The choice of route is adaptively changed in response to failed nodes (assumed to be caused by fire damage).
A suitable minimized power level (min {p0, p1 …pk−1}) is selected to ensure transmission to satisfy (1), (2), (3) without unnecessary power dissipation.
Each node in the network exists in one of four states (listed in the order of health degree from best to worst):
“safe”: initial state while no fire occurs.
“lowsafe”: one-hop away from an “infire” node.
“Infire”: when detects fire.
“unsafe”: detects that it cannot work correctly any longer due to a definite fire
There is a STATE message recording current change of node state to notify its neighborhood nodes in a fire.
STATE (INFIRE) message: If a sensor detects fire, it enters “infire” by broadcasting a message out to denote a new local fire source.
STATE (LOWSAFE) message: The nodes in “safe” state that receive a STATE (INFIRE) message will become “lowsafe”, and then broadcast a STATE (LOWSAFE) message to notify its neighbors. The nodes that hear the STATE (LOWSAFE) message will get to know the new state of its neighbors about fire and do nothing.
STATE (UNSAFE) message: An “infire” node works until it cannot work correctly. Before it cannot work any longer, it enters into “unsafe” state and broadcast a message. Any nodes that detect its residual energy is too low to work will enter into “unsafe”. And then broadcast a STATE (UNSAFE) message.
Thus each sensor may change its state autonomously in response to the fire and messages it receives, as shown in Figure 1.
4. Protocol Description
4.1. Initialized Routing Structure
Initialized Sink Beacon:
The purpose of routing initialization is to form an initialized neighborhood and routing construction after the sensors are deployed and connected as a WSN in the building. We assume that sinks are deployed in a relatively safe place such that they are less likely to be destroyed, for example due to walls collapsing. Once the network is deployed, each sink generates a HEIGHT message using power level p0. This serves to advertise to neighbor nodes and includes a “height” parameter that represents the hop count toward the sink, and is initialized to 0. The height value is incremented by each forwarding hop. Each node records the height information in its local neighborhood table when it receives the first HEIGHT message. The message contains a sequence number so that a node can determine if it has seen the message already, in which case it ignores it. If it is the first time that it receives a HEIGHT message, the node forwards the HEIGHT message out. As explained below this process serves to ensure that each node will know a minimal delay route path from itself toward one of the sinks.
End-to-End HEIGHT Delay Estimate:
In this HEIGHT message broadcasting process, the end-to-end delay from a node to the sink could be approximated by the cumulative delay on each hop. We use “delay estimate” in our EAR routing mechanism to make the forwarding choice. We denote delay (sink, i) as the delay experienced from the sink to each node, and then we could use delay (sink, i) as a bound to guide a real-time delivery from the node to the sink. The delay in transmitting a packet is estimated by:
(1) delay(sink,i)=∑n=1hAvg_delay=∑n=1h(Tc+Tt+Tq)*R
In formula (1), n is the hop count from the sink to node i, Tc is the time it takes for each hop to obtain the wireless channel with carrier sense delay and backoff delay. Tt is the time to transmit the packet that determined by channel bandwidth, packet length and the adopted coding scheme. Tq is the queuing delay, which depends on the traffic load, and R is the retransmission count. Among them, we omit the propagation delay, as in a WSN this is negligible due to the use of short-range radios. In the delay calculation, the delay of MAC layer with MAC protocol used is counted in.
The average end-to-end delay from each node to the sink can be computed as the cumulative hop-by-hop delay, and the delay experienced in the current hop is calculated and updated locally, and then recorded in the HEIGHT message. Then delay (sink, i) is recorded into the neighborhood table of each node. We use a periodic HEIGHT message update to calculate an average end-to-end delay (from multiple end-to-end delay estimates) as reference. Since packets in WSNs always tend to be relatively small, we consider it reasonable to ignore any impact of delay differences related to packet size. Furthermore, delay estimate utilizes Jacobson’s algorithm [17] to make adjustment by considering both the weighted average and variation of the estimated variable and as a result provides a good estimate of the delay. It can work well when link quality and network load varies. The calculation of average end-to-end delay and variation avoids a large number of deadline misses due to high variability in communication delays.
Since the traffic from the node to the sink is usually heavier than the traffic from the sink to the node under the same radio situation according to sensor applications, we can say that queuing delay Tq(sink, i) ≤ Tq(i, sink). This is bounded by the maximum queuing delay, i.e., Tq(i, sink) ≤ Tqmax. When assuming the same radio and link quality for downstream and upstream links on the counterpart route path, we can get that: delay (i, sink) ≤ delayqmax(sink, i). delayqmax(sink, i) is the delay experienced from sink to i with the maximal delay on queuing. Then our delay estimate and realistic delay on the route path T satisfy: delay (sink, i) ≤ T ≤ delayqmax(sink, i). We can use delay (sink, i) as a “bound” to guide the real-time routing forwarding selection. If the delay and slack time (defined as time left for routing) meets the estimated delay time for data delivery, the packet has a high probability to arrive before deadline and thus ensures real-time communications.
Periodic Sink Update:
With the HEIGHT message broadcast process, an initial neighborhood is formed by each sensor for which it records neighbor ID, height, state, estimated delay, residual energy of all neighbors, as well as the transmission power that the node uses to communicate with its neighbor on the path to the sink. Each sensor records its own ID, state, and residual energy. In addition, each node maintains sink ID with its minimal-delay sink. In a fire scenario, the sink may become disabled and the network’s topology will be changed by the fire. To ensure robust connectivity, each sink will periodically send out a HEIGHT message to refresh the network. The refresh rate is a protocol design parameter that trades off overhead for increased robustness to lost HEIGHT messages and path changes. In a fire situation one would expect to decrease the period, although the impact on network traffic load must also be examined.
4.2. Routing Mechanism Details
Forwarding Choice:
For a given application-specific Tmax, we use slack to remember the time left on the path from the current node to the sink. Each node in the neighborhood table is associated with a forward_flag and a timeout. The flag is used to identify the next hop as a best forwarding choice, i.e., when a node is chosen as the best forwarding choice, the forward_flag is set to 1. The timeout value is the valid time for the current forwarding node and used to prevent stale neighborhood information (introduced in Section 4.3.) If “timeout” of a forwarding choice is due, its forwarding flag is set to 0 to evict the stale relay node.
To select the best forwarding choice from local neighborhood table, we use the following criteria:
Firstly, we filter the forwarding choices by “height” to choose the nodes with lower height.
Secondly, choose the node with enough slack time according to delay estimate on the path.
Thirdly, we filter the remaining forwarding choices by node state in the priority from “safe” to “infire”.
If there is more than one node satisfied, we select the best forwarding choice with higher residual energy. If there is still a tie, we choose the lower ID.
If we cannot find a best forwarding choice with the current transmission power, we say that a “hole” has occurred (i.e., stuck in local minimum).
Hole Problem Handler by Adapting Power level:
If a sensor node cannot find a next hop that satisfies the real-time constraint with current power level, it means that the node is stuck in a local minimum. The solution is to increase the transmission power gradually by levels to find another neighbor or invoke a new neighbor discovery. Otherwise, a notify message is sent back to its upstream node (i.e., parent) to stop sending data packets to the current node; and then a routing re-discovery is invoked by the upstream node.
If we could find another node existing in the neighborhood table by adapting the transmission power, then we increase the power level and name this neighbor as a forwarding choice. Otherwise, a new neighbor discovery is invoked by increasing the transmission power gradually by levels. We increase power level gradually but not to the maximal power level directly by considering of the big interference incurred by larger power. We know that there are only two to three power levels provided on existing MICA motes and most of the motes currently used. So, it converges very quickly to the optimal power level. Figure 2 shows an example of a new neighbor discovery, where sink1 and sink2 are two sinks, and the other nodes are sensors. Node i reports and routes data to the sink. The number on each node represents the “height” of each node toward the sink. As the route path {i, a, sink1} with p0 is invalid because slack cannot satisfy the estimated end-to-end delay, node i is in the “hole”. If there are no existing eligible neighbors, then i will increase its power to p1 to reach node j and delivers the packets to another sink sink2 by route path {i, j, sink2} when “slack” on this route is no less than delay estimate.
Each sensor has k levels of power setting: {p0, p1, p2…pk−1} and could be in k levels of maximal transmission range as: {r0, r1…rk−1}. We defined a function to find appropriate transmission power by increasing the power as follows:
(2) p=pcur+ι+1,ι=1, 2, 3….k−1 where, cur is the current number of transmission range level among k levels, ı is the count of unsuccessful attempts. A sensor will increase its transmission power gradually in levels if it cannot find an eligible new neighbor.
A node increases its power according to formula (2) until one of the following conditions is satisfied:
It finds a node as a forwarding choice in “safe” state according to the height and delay estimate.
If p = pmax; in this case, it finds the new neighbor as a forwarding choice by the height and delay estimate in a priority from “safe”, “lowsafe” to “infire”; otherwise, no eligible new neighbor can be found.
In the new neighbor discovery, sensor i will broadcast out a Routing Request (RTR) message. In this process, sensor i piggybacks height, slack and the newly adapted power pi in RTR message. For a node j that hears the message, if the estimated end-to-end delay is no more than slack and its height is lower than height(i, sink), as well as its state is “safe”, then j is selected as a new neighbor. If sensor j hears the RTR with pmax, and if its height is lower than height (i, sink), then j is selected a new neighbor when j is not in “unsafe” state. The new neighbor will reply to node i with the same power that node i is using, after a random backoff to avoid collisions. The forwarding choices send reply message with pi only as necessary for reaching node i, otherwise reverting to their previous power level. Upon receiving the reply, node i inserts the new neighbors into its neighbor table. During RTR and reply message exchange, we could calculate the delay between i and its new neighbor j as follows:
(3) Ave_delay(i,j)=Round_trip_time/2
For meeting real-time requirements, the forwarding choices should satisfy that: “slack” is no less t han the average delay between i and j plus the delay estimate at node j, i.e.,
(4) slack(i)≥Ave_delay(i,j)+delay(sink, j)
If there is more than one new neighbor found, a best forwarding node is selected by the priority of state from “safe”, “lowsafe” to “infire”. If there is still a tie, the best relay is selected by the node with higher residual energy and lower ID number.
For a node that works in a larger transmission range could still be adapted to decrease the transmission power to improve energy efficiency and network capacity, while delay deadline is loose. So we define when a node detects a good connectivity with safe neighborhood that is larger than a predefined threshold, i.e., |Neighborsafe| > N_threshold, power decrease process is invoked.
We defined a function to find appropriate transmission range by decreasing transmission power as follows:
(5) p=pcur−ι′, ι= 1, 2, 3….k−1
In formula (5), cur is the current number of transmission power level among k levels. ı′ is the count of decrement.
A node is eligible for power decrease until:
The minimum power has been reached.
There are two consecutive power levels such that at the lower level the required delay is not met but at the higher power level the required delay is met.
There are two consecutive power levels such that at the lower level the required safe neighborhood connectivity N_threshold is not met but at the higher power level it is.
Neighborhood Table Management:
The neighborhood table records information including transmission power for reaching the neighbor nodes, and is updated by periodic HEIGHT messages from sinks. For power adaptation and new neighbor discovery, the neighborhood table will be updated with the new neighbors and new transmission power. The node also updates its neighborhood with the neighbors and new states as they change. If it receives a STATE (UNSAFE) message, the unsafe neighbor is removed from the table.
4.3. Routing Reconfiguration
In building fire emergencies, robust routing is crucial due to the impact of quickly moving fire on node liveness. In this section, we explain how we reconfigure to deal with failures. We assume that: (1) the minimal time interval between “infire” and “unsafe” state of a node is chosen as a parameter known beforehand and denoted as tunsafe. (2) We use necessary transmission range for connectivity between nodes (according to selected power level) to approximate the minimal fire spreading time between two nodes. In practice, there are well-known guidelines for estimating the rate of fire spread [18–19], taking into account of building materials, building geometry, etc. It’s also the case that obstacles, such as walls, that mitigate radio propagation also have the effect of slowing fire spread.
When a forwarding choice is used for relaying, we add “timeout” and avoid using stale and unsafe paths, i.e., every node on the path from source s to destination d has “timeout” to record the valid time of each link on this route. The timeout is updated when node state changes occur among the neighborhood. The forwarding choice that exceeds the timeout value is considered invalid and then evicted.
We assign an initialized large constant value to represent the estimated valid time for the node in “safe” state.
When a neighbor node j is caught in fire, a STATE (IN-FIRE) message is broadcast. If a “safe” node i receives a STATE (IN-FIRE) message from its neighbor, node i will enters into the “lowsafe” state. The timeout of node i is updated, i.e., the valid time of node i is updated, as the minimal time that this node may be caught in fire until it is out of function:
(6) timeout (i)=min(spread_time(i,j)+tunsafe
Then the timeout value of both downstream and upstream links that are adjacent to node i are also updated accordingly. If node i becomes “infire”, the timeout of adjacent links are updated as tunsafe, i.e., timeout (i) = tunsafe.
Otherwise, if node i becomes “unsafe” by local sensed data and threshold, then timeout (i) is updated as 0 and the timeout of the adjacent links are also updated to 0.
The link timeout value is updated as the state of the node adjacent to the link changes. When a node state is changed for fire, the “timeout” on upstream and downstream links that are adjacent to this node will both be updated. For path link (i, j) on each route path, the timeout value for this link is calculated as:
(7) timeout (link(i,j))=min(timeout(i), timeout(j))
In formula (7), timeout (i) and timeout (j) represents the valid time for node i, j of the route in fire, respectively.
In a building fire, node failures because of fire damage will trigger routing tree reconfiguration. In case of a path link timeout value that is lower than a threshold (i.e., the route path will be invalid very soon), a route reconfiguration is invoked to find another available route path before the current one becomes invalid. The reconfiguration is only invoked by an upstream node i of the path link (i, j) whose valid time is no less than the timeout of the link, i.e., timeout (i) ≥ timeout (link (i, j)). The routing reconfiguration of the node is invoked as a routing re-discovery by broadcasting a RTR message to set up a new route path search. The search of the forwarding choice is invoked in its neighborhood table to find if one of the existing neighbors is eligible to act as a relay or not by adapting the power to the setting recorded in local neighborhood. Otherwise, we will start a neighbor re-discovery process by increasing its power level gradually.
The re-discovery process stops when it finds another new forwarding choice with a valid route path cached toward one of the sinks (that could be a different sink from current one).
Figure 3 shows an example for timeout update in fire. For sensor f, it reports to sink by route path: {f, b, i, j, sink}. After working for a while, sensor i (colored red) senses the fire occurrence. Then sensor i broadcasts a STATE (IN-FIRE) message to notify its communication neighbors (colored yellow): a, b, d, j, and c. When these nodes receive the message, they will enter into the “lowsafe” state. For the state change of sensor i, then timeout (i) is updated as tunsafe. Accordingly, sensor i will update the timeout of its upstream and downstream link, i.e., link (b, i) and link (i, j). As our designed condition for reconfiguration, when timeout (link (b, i)) and timeout (link (i, j)) is lower than a predetermined threshold, the routing reconfiguration is invoked by the upstream node whose timeout is no less than the link timeout. Then sensor b will broadcast a RTR message to find a new relay to the sink, i.e., route path {f, b, c, e, sink}. When it comes to path link (i, j), sensor i is the upstream node of the link with the lower valid time. It will still work on this path (to forward data from sensor i to the sink) until sensor i becomes “unsafe”.
It is assumed that data packet acknowledgements are sent at the link layer (not end-to-end). When a node does not receive an acknowledgement after a given time, we say the downstream link becomes invalid and then reconfigure routing.
5. Analysis
Lemma1. The EAR routing of the sensor network graph is loop-free.
Proof: Suppose that there exists a loop “A→B→C→D→E→…→A” in the network graph by EAR routing. Each node selects its next node which has less height towards the sink. When a node is stuck in local minimum, i.e., in a routing hole, the node could increase its transmission range to find another node that has less height towards the sink if exists. According to this, we could get: height(A) <…< height(E) < height(D) < height(C) < height(B) < height(A). This is a contradiction, so we conclude that the EAR routing of the network graph is loop-free.
Theorem1. If there exists a route within delay bound from a node to one of the sinks, EAR can find this route.
Proof: From Lemma 1, we know that there is no loop in the routing graph. Since the number and height of sensor nodes is limited, so the routes will lead to the sink eventually as long as the real-time route exists.
Theorem2. For a given delay bound Tmax, the routing path found by EAR is within the delay requirement.
Proof: We denote delay (sink, i) as the delay estimate that is the minimal delay from the sink to the node, while delay (i, sink) as the delay from the node to the sink on the counterpart route path. We denote T (i, sink) as the realistic delay experienced from a node to the sink. For queuing delay in wireless sensor networks, data packets are always reported from the node to the sink, while less traffic (usually control command) is delivered from the sink to the node. So Tq(sink, i) ≤ Tq(i, sink). When assuming the same link quality of upstream and downstream links, there exists: delay (sink, i) ≤ delay (i, sink) ≤ T (i, sink). In EAR, we use delay (sink, i) as estimate of delay time form the node to the sink in routing discovery to find a route that meets the lower delay threshold, i.e., using delay (sink, i) to estimate T (i, sink). In this way, we could improve the real-time delivery ratio from the node to the sink. Since we measure the average delay with HEIGHT using power p0, we get the maximal delay estimate time delay (sink, i) on the minimum delay route path from the sink to the node within different power levels. In EAR, we find a relay node i that delay T from i to the sink with this route path should satisfy that it is no larger than the delay estimation on the route path, i.e., T (i, sink) ≤ delay (sink, i). Otherwise, we increase the power level to find another forwarding choice j, and such a node j (with increasing power) exists by satisfying: delay (sink, j) + Ave_delay (i, j) ≤ Tslack; where Tslack = Tmax − T(s, i). The end-to-end delay time T satisfies: T(s, sink) = T(s, i) + T(i, sink) ≤ T(s, i) + Ave_delay(i, j) + delay(sink, j) ≤ T(s, i) + Tslack ≤ Tmax. So, we find a route from node s to the sink that satisfies T(s, sink) ≤ Tmax.
From the above situations, if a real-time route exists, EAR can find a route path satisfying that the end-to-end delay is within the delay requirement Tmax.
6. Simulations
We verify our routing by simulations using the ns2 network simulator [20]. To create a realistic simulation environment, we simulated EAR based on the characteristics and parameters of the MICAz motes, as shown in Table 1. All nodes could be used to work with three power levels and they will work in the minimal power level as the default transmission power. Many-to-one traffic pattern is used, which is common in WSN applications. This traffic is typical between multiple source nodes and one of the sinks. There are 100 nodes distributed in a 100 m × 100 m region as shown in Figure 4. We randomly select four nodes as source nodes, and place 1–4 sinks in the simulation areas as node 99, 98, 97 and 96 respectively. Each source generates constant bit rate (CBR) traffic periodically. The real-time packet miss ratio and packet dismiss ratio by delay estimate as well as energy consumption are assigned as the main metrics for evaluating the performance of EAR. The real-time packet miss ratio (we use “miss ratio” in the following paragraph) is the ratio of all packets missed because of the delay bound to the total packets sent out.
The packet dismiss ratio by delay estimate (we use “dismiss ratio” in the following paragraph) is defined as the ratio of packets discarded by delay estimate and the total sending packets. The energy consumption is the average energy consumed for each sensor during the simulation. Within the simulated area, a fire breaks out 30 seconds after the simulation is started which means the first 30 seconds of the simulation. The node in the network is static. At 30 seconds after the simulation begins, a fire occurs randomly in the network area and then spreads to its neighbors continuously every 10 seconds. When the fire reaches a sensor node, it will lead to a terminal node failure after 10 seconds.
We compare our protocol with minimal hop count routing and RPAR protocol to make performance evaluation. The two comparing routing mechanisms are operated with the initial power as the default transmission power in EAR. RPAR is a real-time power-aware routing mechanism that achieves this by dynamically adapting transmission power and routing decisions based on packet velocity calculated by geographical distance and time left.
6.1. EAR Performance When Sink Number Increases
We simulate EAR performance when increase the sink number from 1 to 4 as the delay bound is set from 10 to 100. Figure 5 shows the end-to-end delay as delay bound increases.
We could see that end-to-end delay decreases as the sink number increases, because more sinks incur more packet delivery within the bound. For a given number sink, the end-to-end delay increases slowly as we relax the bound. For one sink, the end-to-end delay is very small as the bound is 10 ms, because very seldom packet can be delivered within the bound. Figure 6 shows the miss ratio when we decrease delay bound. The packet miss ratio according to delay bound decreases as the sink number increases from 1 to 4. Because more sinks increases the real-time packet delivery probability. Figure 7 illustrates the packet dismiss ratio according to delay estimate. From the result, the dismiss ratio decreases as the sink number varies from 1 to 4. And we can see that EAR provides a good delay estimate and guide packet delivery towards the real-time direction when compared with miss ratio results in Figure 6. Figure 8 shows the average residual energy for node in the simulation time from 0 to 300 s when the delay bound is 70 ms. The average node energy does not vary greatly as we increase the number of sinks. Since increase the sink number, more packets are delivered by more energy consumption and also less routing trials with increased power. The node energy decreases as we relax the bound because more packets are delivered within the given delay bound.
6.2. EAR Performance Compared with No Power Adaptation and No Fire Situations
We evaluate EAR routing when using 3 power levels adaptation and no power adaptation situations. Figure 9 illustrates the end-to-end delay with power adaptation and without power adaptation situations. We get the results with 1 sink and 3 sinks respectively. It is obviously the end-to-end delay decreases a lot when we use power adaptation. By the benefit of power level adaptation, we could increase the network connectivity in fire and also help to find lower delay route path to guarantee a real-time packet delivery under the given delay bound.
Figure 10 shows the miss ratio with/without power adaptation. The miss ratio increases greatly if we adapt power level in the network to increase the probability of real-time packet delivery.
We then evaluate energy efficiency of EAR routing when fire happens and no fire happens situations. Figure 11 illustrates the average node energy in the simulation time when delay bound is set to 50 ms. From the results, it is obvious that average node energy decreases in fire situation. But until 250 s of simulation, the average node energy is larger than 0. For delay bound chosen as 50 ms, the network is still effective until close to the end of the simulation in building fire situations.
6.3. Performance Compared with Other Protocols in Fire Hazard
We then compare EAR with two related routing mechanisms: RPAR and minimal hop count routing. Figure 12 shows the end-to-end delay as delay bound increases from 10 to 100 ms when there is one sink (node 99). We can see that EAR has the minimal end-to-end delay as we relax the bound, then RPAR, and minimal hop count routing has the worst result.
Because EAR adapt power level to try to increase the probability of real-time delivery and it is adaptive to fire spreading by choosing the real-time route path avoiding the dangerous area in fire. RPAR also uses power adaptation to try to increase the real-time delivery, but they are not suitable for fired, and easily chooses a minimal delay path but in the fire area. There is no real-time guarantee mechanism in minimal hop count routing and it is not suitable for fire situations.
Figure 13 shows the miss ratio of real-time packet delivery with one sink. EAR achieves the best real-time data delivery. RPAR is not suitable for fire hazard. Even it adapts power level to try to find a real-time delivery path, but the performance is bad in fire. Figure 14 shows the average node energy in simulation time when delay bound is 50 ms. From the results, three routing mechanisms have similar energy efficiency. EAR has no obvious better energy efficiency, because it increase its power level to increase real-time packet delivery and incur energy consumption.
7. Conclusions and Future Work
We present a novel real-time and robust routing mechanism that is designed to be adaptive to emergency applications such as building fire hazards. The probability of end-to-end real-time communication is achieved by maintaining a desired delay based on a message propagation estimate and power level adaptation. The design is be adaptive to realistic hazard application characteristics including fires expanding, shrinking and diminishing. Our routing mechanism is designed as a localized protocol that makes decisions based solely on one-hop neighborhood information. Our ns-2 simulation results prove that the EAR routing mechanism achieves a good real-time packet delivery adaptive to fire emergency when compared with other related works. We have implemented our protocol into a 4-node TinyOS testbed. Future work will include implementation on a 100-node testbed we have deployed at our university to monitor and help to handle building fires.
The project is funded by the 45th Chinese Postdoc General Program Foundation
Figure 1. State transition diagram for each node.
Figure 2. New neighbor discovery to solve routing “hole”.
Figure 3. Timeout update in fire and route reconfiguration.
Figure 4. Simulation grid.
Figure 5. End-to-end delay as delay bound increases.
Figure 6. Miss ratio percentage as delay bound increases.
Figure 7. Dismiss ratio percentage as delay bound increases.
Figure 8. Average node energy when delay bound = 70 ms.
Figure 9. End-to-end delay with/without power adaptation.
Figure 10. Miss ratio with/without power adaptation.
Figure 11. Average node energy when delay bound = 50 m.
Figure 12. End-to-end delay as delay bound increases.
Figure 13. Miss ratio as delay bound increase.
Figure 14. Average node energy when delay bound = 50 ms.
Table 1. Simulation parameters.
Parameter Value
Propagation model Shadowing
Shadowing deviation 4.0
Reference distance 1.0
PhyType Phy/WirelessPhy/802_15_4
MacType Mac/802_15_4
CSThresh_ (carrier sense threshold) 5.29754e-11
RXThresh_ (reception threshold) 5.29754e-11
Pt_(transmit power) 5.35395e-05/0.000214158/0.000481855
Freq_ 2.4e+9
Traffic CBR
Traffic packetSize_ 70
Traffic Interval_ 0.0969
Node Initial energy 3.6J
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PLoS Comput BiolPLoS Comput. BiolplosploscompPLoS Computational Biology1553-734X1553-7358Public Library of Science San Francisco, USA 22241970PCOMPBIOL-D-11-0127210.1371/journal.pcbi.1002308Research ArticleBiologyComputational BiologyEcologyBehavioral EcologyTheoretical EcologyTheoretical BiologyZoologyAnimal BehaviorMathematicsApplied MathematicsComplex SystemsProbability TheoryBayes TheoremMarkov ModelProbability DensityProbability DistributionStochastic ProcessesPhysicsStatistical MechanicsMulti-scale Inference of Interaction Rules in Animal Groups Using Bayesian Model Selection Multi-scale Inference of Interaction RulesMann Richard P.
1
*
Perna Andrea
1
Strömbom Daniel
1
Garnett Roman
2
Herbert-Read James E.
3
Sumpter David J. T.
1
Ward Ashley J. W.
3
1 Department of Mathematics, Uppsala University, Uppsala, Sweden2 Robotics Institute, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States of America3 School of Biological Sciences, University of Sydney, Sydney, AustraliaSporns Olaf EditorIndiana University, United States of America* E-mail: [email protected] and designed the experiments: AJWW. Performed the experiments: AJWW JEH-R. Analyzed the data: RPM AP DJTS DS RG AJWW. Contributed reagents/materials/analysis tools: RPM AP DS DJTS. Wrote the paper: RPM DJTS AP.
1 2012 5 1 2012 8 1 e100230825 8 2011 31 10 2011 Mann et al.2012This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Inference of interaction rules of animals moving in groups usually relies on an analysis of large scale system behaviour. Models are tuned through repeated simulation until they match the observed behaviour. More recent work has used the fine scale motions of animals to validate and fit the rules of interaction of animals in groups. Here, we use a Bayesian methodology to compare a variety of models to the collective motion of glass prawns (Paratya australiensis). We show that these exhibit a stereotypical ‘phase transition’, whereby an increase in density leads to the onset of collective motion in one direction. We fit models to this data, which range from: a mean-field model where all prawns interact globally; to a spatial Markovian model where prawns are self-propelled particles influenced only by the current positions and directions of their neighbours; up to non-Markovian models where prawns have ‘memory’ of previous interactions, integrating their experiences over time when deciding to change behaviour. We show that the mean-field model fits the large scale behaviour of the system, but does not capture fine scale rules of interaction, which are primarily mediated by physical contact. Conversely, the Markovian self-propelled particle model captures the fine scale rules of interaction but fails to reproduce global dynamics. The most sophisticated model, the non-Markovian model, provides a good match to the data at both the fine scale and in terms of reproducing global dynamics. We conclude that prawns' movements are influenced by not just the current direction of nearby conspecifics, but also those encountered in the recent past. Given the simplicity of prawns as a study system our research suggests that self-propelled particle models of collective motion should, if they are to be realistic at multiple biological scales, include memory of previous interactions and other non-Markovian effects.
Author Summary
The collective movement of animals in a group is an impressive phenomenon whereby large scale spatio-temporal patterns emerge from simple interactions between individuals. Theoretically, much of our understanding of animal group motion comes from models inspired by statistical physics. In these models, animals are treated as moving (self-propelled) particles that interact with each other according to simple rules. Recently, researchers have shown greater interest in using experimental data to verify which rules are actually implemented by a particular animal species. In our study, we present a rigorous selection between alternative models inspired by the literature for a system of glass prawns. We find that the classic theoretical models can accurately capture either the fine-scale behaviour or the large-scale collective patterns of movement of the prawns. However, none are able to reproduce both levels of description at the same time. To resolve this conflict we introduce a new class of models wherein prawns ‘remember’, their previous interactions, integrating their experiences over time when deciding to change behaviour. These outperform the traditional models in predicting when individual prawns will change their direction of motion and restore consistency between the fine-scale rules of interaction and the global behaviour of the group.
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Introduction
The most striking features of the collective motion of animal groups are the large-scale patterns produced by flocks, schools and other groups. These patterns can extend over scales that exceed the interaction ranges of the individuals within the group [1]–[4]. For most flocking animals, the rules dictating the interactions between individuals, which ultimately generate the behaviour of the whole group, are still not known in any detail. Many ‘self-propelled’ particle models have been proposed for collective motion, each based on a relatively simple set of interaction rules between individuals moving in one, two or three dimensions [2], [5]–[8]. Typically these models implement a simple form of behavioural convergence, such as aligning the focal individual's velocity in the average direction of its neighbours or attraction towards the position of those neighbours. Generally such rules are explicitly kept as simple as possible while remaining realistic, with the aim of explaining as much as possible of collective motion from the simplest constituent parts.
Each of the models in the literature is capable of reproducing key aspects of the large-scale behaviour of one or more biological systems of interest. Together these models help explain what aspects of inter-individual interactions are most important for creating emergent patterns of coherent group motion. With this proliferation of putative interaction rules has come the recognition that some patterns of group behaviour are common to many models, and that different models can have large areas of overlapping behaviour depending on the choice of parameters [3]. Common patterns of collective behaviour are also observed empirically across a diverse range of animal and biological systems. For example, a form of phase transition from disorder to order has been described in species as diverse as fish [9], ants [10], locusts [11], down to cells [12] and bacteria [13]. In all these systems, as density of these species is increased there is a sudden transition from random disordered motion to ordered motion with the group collectively moving in the same direction. These studies indicate that a great deal can be understood about collective behaviour without reduction to the precise rules of interaction.
In many contexts however the rules of interaction are of more interest than the group behaviour they lead to. For example, when comparing the evolution of social behavior across different species, it is important to know if the same rules evolved independently in multiple instances, or whether each species evolved a different solution to the problem of behaving coherently as a group [1]. Recently researchers in the field have become interested in using tracking data from real systems on the fine scale to infer what precise rules of motion each individual uses and how they interact with the other individuals in the group [14]–[19]. This is an important trend in the field of collective motion as we move from a theoretical basis, centred around simulation studies, to a more data-driven approach.
The most frequent approach to inferring these rules has been to find correlations between important measurable aspects of the behaviour of a focal individual and its neighbours. For example, Ballerini et al.
[14] looked at how a focal individual's neighbours were distributed in space relative to the position of the focal individual itself in a group of starlings. Significant anisotropy in the position of the -th nearest neighbour, averaged over all individuals, was regarded as evidence for an interaction between each bird and that neighbour. More recently Katz et al.
[18] and Herbert-Read et al.
[19] investigated how the change in velocity of each individual in groups of fish was correlated to the positions and velocities of the neighbouring fish surrounding the focal individual. This provides evidence not only for the existence of an interaction between neighbours but also estimates the rules that determine that interaction.
In these studies the rules of interaction are presented non-parametrically and cannot be immediately translated into a specific self-propelled particle model. Nor are these models validated in terms of the global schooling patterns produced by the fish. An alternative model-based approach that does fit self-propelled particle and similar models to data is proposed by Eriksson et al.
[16] and Mann [17]. Under this approach, the recorded fine-scale movements of individuals are used to fit the parameters of, and select between, these models in terms of relative likelihood or quality-of-fit. This approach has the advantage of providing a parametric ‘best-fit’ model and can provide a quantitative estimate the relative probability of alternative hypotheses regarding interactions.
What all previous empirical studies have lacked is a simultaneous verification of a model at both the individual and collective level. Either fine scale individual-level behaviour is observed without explicit fitting of a model [18], [19] or global properties, such as direction switches [11], [20], speed distributions [21], [22] or group decision outcome [23] have been compared between model and data. Verification at multiple scales is the necessary next step now that inference based on fine-scale data is becoming the norm. Just as simulations of large-scale phenomena can appear consistent with observations of group behaviour without closely matching the local rules of interaction, so can fine-scale inferred rules be inconsistent with large-scale phenomena if these rules of inferred from too limited a set of possible models or from correlations between the wrong behavioural measurements. The closest that any study so far has come to finding consistency between scales has been Lukeman et al.
[15]. In their study the local spatial distribution of neighbouring individuals in a group of scoter ducks was used to propose parametric rules of interaction, with some parameters measured from the fine-scale observables, but with others left free to be fitted using large-scale data. We suggest that if group behaviour emerges from individual interactions, then the form of these interactions should be inferable solely from fine-scale data without additional fitting at the large-scale. An inability to replicate the group behaviour using a selected model demonstrates that the model space has been insufficiently explored. When faced with alternative hypothesised interaction rules, model-based parametric inference provides the best means of quantitatively selecting between them.
In this paper we study the collective motion of small groups of the glass prawn, Paratya australiensis. Paratya australiensis is an atyid prawn which is widepsread throughout Australia [24]. Although typically found in large feeding aggregations, it does not appear to form social aggregations and has not been reported to exhibit collective behaviour patterns in the wild. We conduct a standard ‘phase transition’ experiment [9], [11], [12], studying how density affects collective alignment of the prawns. We complement this approach by using Bayesian inference to perform model selection based on empirical data at a detailed individual level. We select between models by calculating the probability of the fine scale motions using a Bayesian framework specifically to allow fair comparison between competing models of varying complexity. Comparison of the marginal likelihood, the probability of the data conditioned on the model, integrating over the uncertain parameter values, is a well developed and robust means of model selection that forms the core of the Bayesian methodology [25]–[28]. In adopting this approach, we reject the dichotomy of model inference based on either fine scale behaviour of the individuals or the motion of the group. Instead we use reproduction of the large scale dynamics through simulation as a necessary but not sufficient condition of the correct model.
Results
We study the positions and directions of co-moving prawns in a confined annular arena (See Methods and Materials and Figure 1). We tracked, using semi-automated software, the position of each prawn through the duration of the experiments. We pre-processed those raw tracking data by using a Hidden Markov Model to classify the movements of each prawn into a binary sequence of clockwise (CW) and anti-clockwise orientation (see Methods and Materials).
10.1371/journal.pcbi.1002308.g001Figure 1 Schematic of the experimental setup.
Prawns moving within an annulus of 200 mm external diameter and 70 mm internal diameter. Red coloured prawns indicate a clockwise orientation, blue prawns a counter-clockwise orientation. In this instance the total number of prawns , number of clockwise-moving oriented prawns , the polarisation , and the excess polarisation .
We then calculated the number of prawns travelling CW or anti-CW at each time step of each experiment involving three, six or twelve prawns. From this we calculated the average number of CW and anti-CW prawns at a given time across experiments. Figure 2A shows how the number of CW prawns, , changes over time, taken as a distribution over all trials with six prawns. There is a transition from an initially random configuration, with most trials having , to a final configuration where most experiments have either or . The final stable distribution is further shown in Figure 2B along with the final distribution for three and twelve prawn experiments. Steady state polarisation increases as a function of prawn number. The polarisation, can be defined as (1) The expected polarisation in randomly oriented groups varies with the number of individuals in the arena, being larger for smaller groups and obeying a binomial distribution. We adjust the measured polarisation by this expectation, , to obtain the excess polarisation, . Figure 2C shows this measure of polarisation over time for experiments with three, six and twelve prawns, confirming that the excess polarisation increases over time and is greater for larger groups.
10.1371/journal.pcbi.1002308.g002Figure 2 Large-scale behaviour of the experimental system.
(A) The proportion of six-prawn experiments () with a given number of CW moving prawns over time. For each point in time we calculated the distribution over all trials of the number of CW prawns. This distribution is then plotted as a heat map. (B) The final distribution of experiments with number of CW moving prawns, for three-, six- and twelve-prawn experiments ( respectively). Error bars represent the mean and standard deviation for each proportion as calculated from the final ten seconds of the experiments. (C) The average polarisation of experiments with three, six and twelve prawns over time, adjusted by the expected polarisation of randomly oriented prawns.
At a group level we see that prawns tend to align over time, producing a polarised stable state, which is higher for larger group sizes. We define the reproduction of these global patterns as the global consistency condition of our model. We insist that any realistic model for the prawns' interactions must reproduce this large-scale behaviour.
Model selection
Next we investigated a series of interaction models as to their ability to reproduce the fine scale interactions of the prawns. We predict the probability, , that a focal prawn will change its orientation, given one of a number of potential models. The direction changes are determined by the data from the six-prawn treatment. This treatment provides the best balance between the number of data points, density of direction changes, clear large scale behaviour and tracking accuracy.
Each model specifies the probability that a focal prawn will change its direction in the next time step conditioned on the relative positions and directions of the other individuals in the arena. We use a logistic mapping to ensure probabilities remain between zero and one, so each model uses the relevant variables to determine a latent ‘turning-intensity’, , such that, (2) where is a function of the relative positions and directions of the other prawns, both now and potentially in the recent past, and the model parameters.
The models are, in increasing degree of complexity, as follows. Firstly to consider models that do not include zones-of-interaction – non-spatial models. We establish a baseline with a Null model. This simply posits that direction changes occur at random, at the rate established from the single prawn data, and the prawns do not interact in any way that changes this direction-changing probability. Therefore is given simply by a baseline constant, , which is determined by the rate of direction changing in single prawns. (3) We also consider two models where the interaction is independent of absolute spatial separation. The Mean Field (MF) model includes interactions between all prawns regardless of position, such that their relative directions alter the probability of changing direction. Since the number of prawns in the experiment is fixed, the probability for a direction change is influenced by the number of individuals moving in the opposite direction (negative prawns), . Each negative prawn increases the turning intensity by an amount , (4) A Topological (T) model restricts these interactions to a limited number of nearest-neighbours, , the individuals closest to the focal prawn. The turning intensity is now influenced by the number of negative prawns, within the set of nearest-neighbours. (5)
Secondly we consider a class of Spatial models (S1–S4). These models closely resemble the classic one-dimensional self-propelled particle models from the literature [5]. The focal prawn interacts with neighbours within a spatial zone-of-interaction, . The number and directions of individuals within this interaction zone determine the probability of changing direction. A number of further variations are possible; interactions can be limited to prawns ahead of the focal prawn and/or to prawns travelling in the opposite direction to the focal prawn. We consider four variations, indicated in Table 1. The general form for this model is given by, (6) where and are the number of negative and positive (travelling in the same direction) prawns within the interaction zone, and and parameterise the influence of each individual on the turning intensity. Interactions can occur with negative prawns only, , or with both negative and positive oriented prawns, . The spatial interaction zone is either a symmetrical area centred on the focal prawn, of width radians around the ring (spatial symmetric models in Table 1), or is only directed radians ahead of the focal prawn (spatial forward models).
10.1371/journal.pcbi.1002308.t001Table 1 Model comparison.
Model Interaction zone
/rads
KL/bits
/bits Consistent?
Null None −7.5 N/A N/A N/A N/A N/A 10.2 −944 No
MF Global −7.5 N/A N/A 0.51 N/A N/A 0.6 −863 Yes
T K nearest-neighbours −7.5 N/A 3.4 0.72 N/A N/A 2.0 −864 No
S1 Spatial, symmetric −7.5
N/A 1.42 N/A N/A 1.3 −847 No
S2 Spatial, forward −7.5
N/A 1.71 N/A N/A 7.2 −834 No
S3 Spatial, symmetric −7.5
N/A 1.80 −0.01 N/A 2.0 −856 No
S4 Spatial, forward −7.5
N/A 1.74 0.20 N/A 2.3 −859 No
D1 Spatial, symmetric −7.5
N/A 1.62 N/A 0.47 0.7 −827 Yes
D2 Spatial, forward −7.5
N/A 1.50 N/A 0.46 1.5 −830 No
D3 Spatial, symmetric −7.5
N/A 1.44 0.23 0.87 0.4 −828 Yes
D4 Spatial, forward −7.5
N/A 1.44 0.37 0.82 0.8 −830 Yes
The interaction zone structure of each model, along with the (mean a posteri ) inferred values of model parameters, an indicator for the model's consistency with the observed large scale behaviour of the real system, The KL divergence between experimental and model results indicating large-scale goodness-of-fit and the log marginal likelihood () of the model calculated from the fine scale dynamics (as shown in Figure 3). N/A indicates that the model does not include the indicated parameter. The interaction zone indicates whether prawns interact with others in a spatial zone around themselves, which may be oriented either forwards or symmetrically around their centre, or with their nearest-neighbours or globally with all other individuals. Reported parameters are: , the baseline direction-change intensity; , the interaction radius for spatial models; , the number of interacting nearest-neighbours for topological models; and , the strength of interaction with negative and positive prawns respectively; and , the decay factor determining how long interaction effects persist.
Visual inspection of the movements of the prawns suggests that interactions often follow a particular pattern. Two prawns, travelling in the opposite directions, collide. After the prawns have passed each other one of the prawns may subsequently decide to change direction. Self-propelled particle and other models of collective motion do not capture this type interaction. Such interactions are non-Markovian, i.e. the change in direction is not just the result of the environment now, but of the past environment as well. We proposed a third class of models (D1–D4), simple non-Markovian extensions of the basic spatial models, where each prawn would ‘remember’ the other individuals it encountered, with those memories fading at an unknown rate after the interaction was complete. As such the prawn would integrate those interactions over time, building up experiences which would alter its chance of changing direction. Mathematically this means that the turning intensity is now auto-regressive, depending on its own value at the previous time step as well as the current positions and directions of the neighbouring individuals. We introduce a decay parameter, , which determines how quickly the turning intensity returns to normal after an interaction with a neighbour has occurred. The same variations of interaction are allowed as for the spatial models, giving a general form for the non-Markovian turning intensity as, (7) where now indicates the turning intensity at time , which depends on the value of the turning intensity at the previous time step, . The number of prawns still in the interaction zone from time is indicated by , while the number of new arrivals in the interaction zone is given by . Hence raised (or lowered) turning intensities persist over time, with a duration controlled by the value of . After the focal prawn changes direction the turning intensity is reset to the baseline, , at the next time step.
Table 1 specifies the interaction zone structure for each of eleven alternative models, grouped according to the description given above. For each model we calculate the marginal likelihood of the data, conditioned on the interaction model (see Methods and Materials). The marginal likelihood is the appropriate measure for performing model selection, especially between models of varying complexity. More complex models, by which we mean models with a larger number of free parameters, are penalised relative to simpler models when integrating over the parameter space, since less probability can be assigned to any particular parameter value a priori. The marginal likelihood indicates how likely a particular model is, rather than a model and an chosen optimal parameter value (see, for example, Mackay [29] Chapter 28 and other standard texts for discussions on this topic). The marginal likelihoods of each model are shown in Figure 3.
10.1371/journal.pcbi.1002308.g003Figure 3 The marginal-likelihood of different models calculated from the fine scale dynamics.
Each marginal-likelihood is calculated by importance sampling. The figure shows the mean and standard error from 10 instances, each of 5000 samples. Grey markers indicate models that are consistent with the observed large scale behaviour of the system, black markers indicate those that are not. Consistency is determined by alignment of the prawns towards CW or anti-CW movement in simulations.
The Null model, in which prawns do not interact, performs significantly worse than the mean-field model. Figure 4 shows that the mean-field model fulfills our global consistency condition, reproducing an increase in polarization with time and prawn number. These results show that the prawns interactions involve matching their directions to that of others, producing alignment.
10.1371/journal.pcbi.1002308.g004Figure 4 Simulation results for model MF.
(A) Proportion of six-prawn simulations () of mean-field model MF with a given number of prawns moving CW over time. (B) Final distribution of simulations by number of CW moving prawns for simulations with three, six and twelve prawns. Error bars represent the mean and standard deviation for each proportion as calculated from the final ten seconds of the simulations. (C) The average polarisation over time, adjusted by the expected polarisation of randomly oriented prawns, for simulations of three, six and twelve prawns. The KL divergence between the experimental and simulated results is 0.60 bits.
Are local spatial interactions important in reproducing observed direction changes? We note first that a topological interaction zone, where the focal prawn interacts with its nearest neighbours, has a marginal likelihood slightly lower than the mean field model. The topological model is ‘punished’ for having more parameters than the mean-field model. However, interactions between prawns are local. Figure 5 shows how the probability of changing direction depends on the position of the nearest opposite facing neighbour. An opposite facing neighbour within approximately radians ( average body lengths) of a focal prawn strongly increases the chance that the focal prawn will change direction.
10.1371/journal.pcbi.1002308.g005Figure 5 Evidence for short-range interactions.
The empirical frequency of direction changing as a function of the distance to the nearest opposite facing prawn (grey markers) and the probability of changing direction when interacting with one (solid red line) or two (dashed red line) opposite facing prawns according to the optimal model (D1). The empirical data clearly shows the spatially localised interaction, which is confined to within approximately radians, one-half body length of the average prawn. The model predicts a consistently lower probability of changing direction than the observed frequency when accounting only for instantaneous interactions. This is compensated by the accumulation and persistence of interactions over time.
This observation is further reflected in the marginal likelihood of the spatial models (S1–S4) in Figure 3. These models all significantly outperform the Mean Field model. In all four of these models the inferred interaction zone is small, approximate or half of the average prawns body length (Table 1). Model S2 has the highest marginal likelihood of these models, indicating a forward-directed interaction zone both ahead of the focal prawn, with the prawn interacting only with individuals with an opposite orientation (Figure 5).
However, simulations of the spatial models using the inferred interaction parameters (mean a posteri estimate, see Table 1) reveal that these models are not globally consistent with the data. For example, Figure 6A shows the average number of prawns travelling CW over time in 100 simulated instances of model S2. Rather than a clear movement towards full alignment either CW or anti-CW we see only a weak drift away from the original random configuration, with most simulations retaining an equal mixture of CW and anti-CW moving prawns. This is in contrast to the mean-field model, which, though far less supported by the fine-scale data, does produce a good replication of the large scale behaviour (Figure 4). As a result of this inconsistency, we cannot accept any of the spatial models as the true interaction rule for the prawns.
10.1371/journal.pcbi.1002308.g006Figure 6 Simulation results for model S2.
(A) Proportion of six-prawn simulations () of spatial model S2 with a given number of prawns moving CW over time, showing no change from the initial random configuration. (B) Final distribution of simulations by number of CW moving prawns for simulations with three, six and twelve prawns. Error bars represent the mean and standard deviation for each proportion as calculated from the final ten seconds of the simulations. (C) The average polarisation over time, adjusted by the expected polarisation of randomly oriented prawns, for simulations of three, six and twelve prawns. The KL divergence between the experimental and simulated results is 7.20 bits.
The models incorporating a non-Markovian delayed response together with a spatial interaction zone (models D1–D4) outperformed the Markovian spatial models (Figure 3) as well as the Mean Field model. Model D1 was the optimal model from those tested, indicating a symmetric short range interaction zone and interactions with only opposite oriented individuals (Table 1). Simulations of this model produce weak global consistency. Most six-prawn simulations have either five or six prawns moving in the same direction in the final state (Figure 7A). This alignment is weaker than seen in the real experiments but more consistent with the observed behaviour than any of the Markovian models. In the final distributions (Figure 7B) and mean polarisation plot (Figure 7C) we see the same increase in alignment with increasing group size as in the experimental data.
10.1371/journal.pcbi.1002308.g007Figure 7 Simulation results for model D1.
(A) Proportion of six-prawn simulations () of non-Markovian model D1 with a given number of prawns moving CW over time, showing weak bifurcation to either a CW or an anti-CW polarised state, with most experiments ending with five or six prawns travelling in the same direction. (B) Final distribution of simulations by number of CW moving prawns for simulations with three, six and twelve prawns. Error bars represent the mean and standard deviation for each proportion as calculated from the final ten seconds of the simulations. (C) The average polarisation over time, adjusted by the expected polarisation of randomly oriented prawns, for simulations of three, six and twelve prawns. The KL divergence between the experimental and simulated results is 2.32 bits.
The difference in marginal likelihood between model D3 and model D1 is within the error of the sampling method, and therefore D3 should be considered as an alternative optimal model. Moreover, model D3 is globally more consistent with experiments when simulated. Figure 8A–C give the results of simulations from this model, showing a much stronger bifurcation in the prawn directions over time (Figure 8A), and more accurate scaling with group size (Figure 8B and C).
10.1371/journal.pcbi.1002308.g008Figure 8 Simulation results for model D3.
(A) Proportion of six-prawn simulations () of non-Markovian model D3 with a given number of prawns moving CW over time, showing rapid bifurcation to either a CW or an anti-CW polarised state, with most experiments ending with six prawns travelling in the same direction. (B) Final distribution of simulations by number of CW moving prawns for simulations with three, six and twelve prawns. Error bars represent the mean and standard deviation for each proportion as calculated from the final ten seconds of the simulations. (C) The average polarisation over time, adjusted by the expected polarisation of randomly oriented prawns, for simulations of three, six and twelve prawns. The KL divergence between the experimental and simulated results is 1.46 bits.
For each model we report a measure of large-scale consistency with the experimental results, in terms of the final distribution of the proportion of CW-moving prawns. We use the Kullback-Leibler (KL) divergence [30] to measure the distance from the experimental distribution to the simulated distribution, summed over three, six and twelve prawns results (reported in Table 1 and Figures 4, 6, 7 and 8). This goodness-of-fit measure indicates that of the models discussed, the Mean Field model and non-Markovian model D3 are most consistent with the large-scale results, non-Markovian model D1 is somewhat less consistent and Markovian model S2 is very inconsistent.
Discussion
A number of physical [31]–[33], technological [34] and biological systems, including animals [9]–[11], [35], tissue cells [12], microorganisms [13], [36] are known to increase their collective order with density. Glass prawns are one additional example of such a system, which is particularly interesting since they are not known as gregarious or social species. By confining the prawns to a ring we facilitated their interactions and in doing so generated collective motion. This adds further support to the idea that collective motion is a universal phenomenon independent of the underlying interaction rules [3], [11], [37]. While we do not expect that prawns often find themselves confined in rings in a natural setting, they and other non-social animals do aggregate in response to environmental features such as food and shelter. Such environmental aggregations can, above a certain density, result in an apparently ‘social’ collective motion.
The true value of this study, however, is found not in the addition of one more species to this growing list, but in demonstrating a rigorous methodology for selecting an optimal and multi-scale consistent model for the interactions between individuals in a group. We have used a combination of techniques to identify the optimal model for our experiments: Bayesian model selection and validation against global properties. We applied Bayesian model selection to identify the model that best predicts the fine-scale interactions between prawns. This approach allows us to perform model selection in the presence of many competing hypotheses of varying complexity, while avoiding over fitting [17]. The selected models indicate that interactions between prawns are modulated primarily by the spatial separation of individuals and are localised to a very short perceptual range which is symmetric about the focal individual. This may indicate that physical contact rather than vision is the dominant mechanism, especially as the inferred size interaction zone (approximately radians) is consistent with the average body length of the prawns (approximately radians). Since in the optimal models the interaction zone is symmetric and the tracking algorithm detects a point approximately midway along the prawn's length, this suggests that the prawns may interact for as long as they remain in physical contact.
The other approach we have employed in validating our model is consistency with large-scale dynamics. Reproduction of the large-scale dynamics is frequently used to validate mathematical models of biological systems, but presents only a necessary and not a sufficient condition for model validation. Indeed, all of the models we have assessed in this work can, with the appropriate parameters, generate aligned motion consistent with experiment. The fact that our mean-field model reproduces global dynamics, but fails at a fine scale level is not particularly surprising. Mean-field models are not designed to reproduce spatially local dynamics [1]. More illuminating, however, is the failure of Markovian spatial models to the reproduce the polarisation seen in the empirical data. Models S1–S4 are variants of the standard one dimensional Vicsek self-propelled particle model [38], which has previously been validated against the global alignment patterns of marching locusts [11]. For the prawns, model parameter values which produce simulations consistent with global alignment patterns were not consistent with those inferred from fine scale observations. This inconsistency allowed us to reject standard self-propelled particle models as a good model of the data.
To identify a better model we first visually inspected the interactions between the prawns. These observations suggested a ‘memory effect’, whereby a prawn would remain influenced by individuals beyond the moment of interaction. The resulting models, D1–D4, are both consistent with the polarisation condition and superior at predicting the fine-scale interactions, providing strong evidence for non-Markovian dynamics within this system. More generally, we would expect other examples of animal motion to be non-Markovian, with individuals taking time to react to others, to complete their own actions and also potentially reacting through memory of past situations. In this context, it is important to consider the limitations of recent studies identifying rules of interaction of fish [18], [19]. These studies concentrated on quantifying local interactions, but do not try to reproduce global properties. It may be that non-Markovian and other effects are needed to produce these properties.
In what circumstances can we expect non-Markovian effects to play an important role in collective behaviour? Inference based on a Markovian model must account for behavioural changes of a focal individual in terms of their current environment. As such the crucial factor is how much the local environment changes between when the animal receives information and when it responds. Large changes in the local environment can be caused by long response times or by rapid movements of other animals relative to the focal individual. Where behavioural changes are strongly discontinuous, such as the binary one-dimensional movement in this study, non-Markovian effects may become especially important. This is because the focal individual may have to execute a number of small changes (such as stopping and turning through a several small angles) in order to register as having changed its direction of motion. Over the course of making many adjustments the environment can change dramatically from the moment that the change was initiated.
We have used qualitative replication of the large scale motion as a necessary condition for the correct model, and assigned zero probability to inconsistent models. A more subtle approach would be to give a weighting to global consistency. For example, D1 and D3 are both consistent at a global level and indistinguishable according to marginal-likelihood. As such, they should then be considered as equally viable alternative models for the real behaviour of the prawns. However, a visual inspection of global consistency favours D3 over D1 (see Figures 7 and 8). Future work could attempt to define a probability distribution over large scale outcomes, allowing fully probabilistic integration of both fine scale and large scale inference. A ‘distance’ between the summary statistics of large scale simulated behaviour and the same statistics extracted from experimental data, such as the KL divergence measure reported here, could be used to construct a Bayesian inference framework [39]. The research presented here provides a first step towards the use of multi-scale inference in the study of collective animal behaviour and in other multi-level complex systems.
Materials and Methods
Glass prawns (Paratya australiensis) were collected from Manly Dam, Sydney, Australia and transported back to aquaria facilities at the University of Sydney. They were held in 20 glass aquaria and fed green algae and fish food ad libitum. Prawns were housed for at least 2 days prior to experimentation. An annulus arena (200 mm external diameter, 70 mm internal diameter) was constructed from white plastic and filled to a depth of 25 mm with freshwater. The arena was visually isolated inside an opaque white box and filmed from above using a G10 Canon digital camera at a frame rate of 15 Hz. Data was subsequently down-sampled to 7.5 Hz by removing every second frame for computational efficiency. For each trial, we haphazardly selected one, three, six or twelve prawns and placed them in the arena. We filmed each trial for six minutes, after which we removed the prawns, emptied, and then refilled the arena with freshwater. Prawns were only used once on each day of trials. A schematic of this setup is shown in Figure 1.
Hidden Markov model
The frame-by-frame movements of the prawns are imperfect representations of the true orientation, since a prawn will often stop or even drift slightly backwards without physically turning around. A Hidden Markov Model (HMM) allows the underlying orientation of the prawns to be discovered from the noisy frame-by-frame movements by demanding a higher degree of ‘evidence’ for a direction change, in essence only identifying direction changes when the prawn makes a sustained movement in the new direction. This gives a better estimate of the true orientation than given by the instantaneous velocity alone.
We constructed a two-state HMM [40] for the observed changes in position of the prawn, as shown in Figure 9. The two states represent clockwise (CW) or anti-clockwise (anti-CW) orientation. In a CW oriented state it is assumed that the prawn will normally move in CW direction over the course of one frame, but because the prawns movements are noisy it may move in the reverse direction over short time periods while remaining oriented CW. We model the distribution of these movements as a Gaussian distribution. We further assume a symmetrical model, such that the distribution of movements in the CW state is anti-symmetric to the distribution of movements in the anti-CW state. Thus a movement of zero is equally probable in either state. We use the Baum-Welch algorithm [40], [41] to learn the transition probability and the mean and standard deviation of the Gaussian observation probability distribution, using data from single-prawn experiments. We then apply this learnt model to identify the most probable state sequence for each of the prawns in the three-, six- and twelve-prawn experiments, using the Viterbi algorithm [40], [42].
10.1371/journal.pcbi.1002308.g009Figure 9 Graphical description of a two-state Hidden Markov Model.
At any point in time the prawn is in a state of either CW or anti-CW orientation. The precise state is hidden but we make observations , the actual frame-by-frame movements of the prawn, which give information about the relative probabilities of the two states. We assume a fixed probability of transition between the states which is inferred from the data and allows for the persistence of orientation over time.
Calculation of marginal likelihoods
A given model, describes the probability of a change of direction for the focal prawn at time , conditioned on the current, and potentially past, positions of the other prawns, and and the parameters of the model . The likelihood for a given parameter set of the model is the probability of the data, , conditioned on the parameters and the model and is the product over both time steps and focal prawns of the probability for the observed outcome - either a change of direction or no change. Let equal one when prawn in experiment changes direction at time , and is zero otherwise, then, (8) where and indicate the number of experiments and the number of prawns in each experiment respectively. The marginal likelihood of the model is given by integration over the space, , of unknown parameters, (9) The prior distribution of the parameters, is chosen to represent the available knowledge about the parameters before the experiments and is split into independent parts. The prior for the same parameter over different models is the same to allow fair comparison. (10) where indicates a continuous uniform distribution, indicates a discrete uniform distribution and is the Dirac delta function. Numerical integration over the appropriate parameters was performed using importance sampling (see Mackay [29] Chapter 29), with 10,000 parameter samples generated from the prior parameter distribution. The importance sampling was repeated ten times for each model to improve estimates of the marginal likelihood and provide an estimate of the associated uncertainty.
Johannes Alneberg provided assistance with figure creation. Three anonymous reviewers gave valuable advice to improve the manuscript.
The authors have declared that no competing interests exist.
This study was partly funded by an ERC grant to DJTS (ref: IDCAB - 220/104702003) and a DVC grant from the University of Sydney to AJWW. RPM is supported by the Centre for Interdisciplinary Mathematics at Uppsala University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22253711PONE-D-11-0198210.1371/journal.pone.0029321Research ArticleBiologyBiochemistryEnzymesMetabolismDevelopmental BiologyStem CellsMedicineAnatomy and PhysiologyModulation of the Pentose Phosphate Pathway Induces Endodermal Differentiation in Embryonic Stem Cells Pentose Phosphate Pathway and EndodermManganelli Genesia
1
2
Fico Annalisa
1
Masullo Ugo
1
Pizzolongo Fabiana
3
Cimmino Amelia
1
Filosa Stefania
1
2
*
1
Stem Cell Fate Lab, Institute of Genetics and Biophysics “A. Buzzati Traverso” CNR, Naples, Italy
2
IRCCS Neuromed, Pozzilli, Italy
3
Faculty of Agriculture, University of Naples Federico II, Portici, Naples, Italy
Appanna Vasu D. EditorLaurentian University, Canada* E-mail: [email protected] and designed the experiments: GM SF. Performed the experiments: GM UM FP. Analyzed the data: GM UM FP SF. Contributed reagents/materials/analysis tools: GM AF AC SF. Wrote the paper: GM AF UM SF.
2012 12 1 2012 7 1 e2932126 1 2011 24 11 2011 Manganelli et al.2012This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Embryonic stem (ES) cells can differentiate in vitro into a variety of cell types. Efforts to produce endodermal cell derivatives, including lung, liver and pancreas, have been met with modest success. Understanding how the endoderm originates from ES cells is the first step to generate specific cell types for therapeutic purposes. Recently, it has been demonstrated that inhibition of Myc or mTOR induces endodermal differentiation. Both Myc and mTOR are known to be activators of the Pentose Phosphate Pathway (PPP). We found that, differentely from wild type (wt), ES cells unable to produce pentose sugars through PPP differentiate into endodermal precursors in cell culture conditions generally non-permissive to generate them. The same effect was observed when wt ES cells were differentiated in presence of chemical inhibitors of the PPP. These data highlight a new role for metabolism. Indeed, to our knowledge, it is the first time that modulation of a metabolic pathway is described to be crucial in determining ES cell fate.
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Introduction
Endoderm-derived organ diseases include cystic fibrosis, chronic hepatitis, and diabetes; they affect more than 150 million people worldwide. Existing transplantation-based therapies are currently limited by the availability of donor-derived tissues.
Embryonic stem (ES) cells have the potential to give rise to any of the hundreds of cell types in the human body, raising exciting new prospects for biomedical research and for regenerative medicine [1]. Indeed, ES cells are a promising, renewable source of material for transplantation, because they can be expanded indefinitely in culture and can differentiate into all cell types of the body. Researchers are now taking advantage of the understanding of endoderm organogenesis to successfully direct the differentiation of ES cells into pancreas, liver, lung and thyroid cells [2]. The potential to virtually generate any differentiated cell type from ES cells offers the possibility to establish new models of mammalian development and to create new sources of cells for regenerative medicine. To realize this potential, it is essential to be able to control ES cells differentiation and to direct the development of these cells along specific pathways [1]. The molecular events regulating the induction and tissue-specific differentiation of endoderm are central to our understanding of the development and function of many organ systems [3].
Myc transcription factor and mTOR (Mammalian Target of Rapamycin) are both key regulators of cell growth and proliferation, and both have been described to control ES cells fate. In particular, Myc and mTOR repress endoderm differentiation of ES cells [4], [5]. Furthermore, both mTOR and Myc regulate the Pentose Phosphate Pathway (PPP). Indeed, it has been described that mTOR complex 1 activation leads to induction of genes encoding the enzymes of the PPP [6] and cMyc induces the production of ribose sugars, the product of the PPP [7].
We have generated mouse ES cells with a G6pd gene deletion (G6pdΔ). G6PD is the first and key enzyme of the PPP that, oxidizing glucose-6-phosphate, produces NADPH and pentose sugars. We have previously shown that these cells are extremely sensitive to oxidative stress, in keeping with the notion that G6PD is essential for production of high levels of NADPH, required for detoxification of reactive oxygen species [8], [9], [10], [11]. In addition, it has been reported that severe G6PD deficiency is lethal for mouse embryo. Severely G6PD-deficient hemizygous male embryos stop growing between E7.5 to E8.5 and show severe abnormalities, indicating that the role of G6PD is quite basic in mammalian development [12].
In this report we show, using engineered ES cells, that modulation of the PPP is necessary to drive ES cells differentiation into endodermal precursor cells. The data were confirmed in wt ES cells using two chemical inhibitors of the PPP. Moreover, we show that the mechanism does not involve the role of the PPP in providing reducing equivalent but rather its function in the production of pentose sugars.
Results
Analysis of gene expression during wt and G6pdΔ ES cells differentiation
We differentiated wt and G6pdΔ ES cells, using the previously described protocol to differentiate ES into neuronal cells [13], and analyzed the expression profiles of undifferentiated cells and three germ layers specific markers. As shown by RT-PCR, after 6 days of differentiation the expression of Oct4 and Nanog, markers of undifferentiated ES cells, are undetectable in both cell lines (Figure 1A). Moreover, no differences in the expression profile of Nestin (neuronal precursor marker), NF-L (marker of neurons), GFAP (glial cell marker), and Nkx2.5 were observed between wt and G6pdΔ ES cells (Figure 1A); αMHC (cardiomyocyte specific marker) and TDO (hepatocyte specific marker) were not expressed in both cell lines (data not shown). Instead, whereas endoderm was never formed during wt ES differentiation, from day 8 of differentiation in G6pdΔ ES cells we observed the expression of GATA4 (mesendodermal marker) and Sox17 (endodermal precursor marker) (Figure 1A). The expression of Sox17 was confirmed by immunofluorescence analysis on wt and G6pdΔ ES cells at 10 days of differentiation using anti-Sox17 antibody (Figure 1B). Since GATA4 was previously seen expressed in neurons and astrocytes [14], we analyzed, by immunofluorescence, the co-expression of GATA4 with βIII-tubulin (neural marker) or GFAP and we never observed co-expression of these markers (Figure S1A, B). Sox17 has been described to be expressed also in oligodendrocytes [15]; by western blot, we analyzed the expression of Olig2 (oligodendrocytes specific marker), but our cell culture method does not allow the differentiation of oligodendrocytes (Figure S1C). These data strengthen our hypothesis that GATA4 and Sox17 are expressed in endodermal precursors during G6pdΔ ES cells differentiation.
10.1371/journal.pone.0029321.g001Figure 1 Endodermal induction in G6pdΔ ES cells.
(A) Analysis of different markers in wt and G6pdΔ ES cells during neural differentiation. Expression profiles of undifferentiated ES cells (Oct4 and Nanog), neural precursors (Nestin), neurons (NF-L), astrocytes (GFAP), mesendodermal precursors (GATA4), endodermal precursors (Sox17), and cardiac precursors (Nkx2.5) markers were analyzed by RT-PCR. RNA was isolated from cells at different days of differentiation. Lane C, positive control, RNA isolated from 14dpc embryos. Amplified HPRT is shown as a positive control. (B) Double immunostaining Sox17/βIII-tubulin/DAPI of cells at 10 days of differentiation showed areas of immunoreactive cells for Sox17 only in G6pdΔ ES cells. Scale bars, 50 µm. (C) RT-PCR analysis of GATA4, Sox17, NF-L (neural marker), TH (dopaminergic neuron marker) and GAD65 (gabaergic neuron marker) on wt, two different G6pdΔ ES cell lines, and G6pdΔ
pG6pd during differentiation. Lane C, positive control, on RNA isolated from 14dpc embryos. Amplified HPRT is shown as a positive control. (D) Western blot analysis with anti-Cripto and anti-Actin antibodies performed on protein extracts from wt and G6pdΔ ES cells during neural differentiation. Actin was analyzed as loading control. Below each lane the relative quantities (RQ) with respect to related undifferentiated embryonic stem cells are indicated. (E) Western blot analysis with anti-phospho-Smad2 and anti-Actin antibodies performed on protein extracts from wt and G6pdΔ ES cells during neural differentiation. Actin was analyzed as loading control. Below each lane the relative quantities (RQ) with respect to related undifferentiated embryonic stem cells are indicated.
To verify that the expression of endodermal specific markers was caused by inactivation of G6pd gene, and not by accidentally produced abnormalities, we confirmed, after differentiation, the expression of those markers in two different ES cell lines, G6pdΔ1 and G6pdΔ2, carrying the deletion of G6pd gene (Figure 1C). Moreover, in G6pdΔ
pG6pd ES cells, G6pdΔ ES cells transfected with an expression vector containing a puromycin resistance gene in which the expression of G6pd is driven by the β-actin promoter [11], we never observed the expression of GATA4 and Sox17 (Figure 1C) although they differentiate in neuronal cell lines as proved by the expression of specific neuronal markers.
Nodal signalling participates in the nervous system patterning but also in mesendoderm induction. Smad2 is an essential intracellular transducer of the Tgf-β/Nodal signal. Nodal signalling through type I (ALK4 and ALK7) and type II (ActRIIA and ActRIIB) receptors in conjunction with its co-receptor, Cripto, is crucial for generating mesendoderm precursor cells. Following the engagement of Nodal with its receptor, Smad2 becomes phosphorylated and induces mesendodermal differentiation [16], [17], [18]. During differentiation we confirmed that Nodal was expressed both in wt and G6pdΔ ES cells although at higher level in the last one (Figure S1D). We also analyzed Cripto expression in wt and G6pdΔ ES cells. Although Cripto is already switched off at day 4 of differentiation in wt ES cells, its expression is still present at day 6 of differentiation in G6pdΔ ES cells (Figure 1D, Figure S1E). Moreover, we found that Smad2 phosphorylation is induced in differentiating G6pdΔ compared to wt until day 6 of differentiation, although this pathway is active also in wt ES cells during neuronal differentiation (Figure 1E).
Induction of Definitive and Extraembryonic Endoderm
Sox17 is expressed in definitive endoderm but also in extraembryonic endoderm. Borowiak et al. (2009) found that Sox17+ cells can have two distinct morphologies: as dispersed Sox17+ cells or clustered populations [19]. They demonstrated that the dispersed Sox17+ cells also expressed extraembryonic endoderm markers. Positive identification of definitive endoderm is hindered by lack of unique markers that are expressed exclusively in these cells. Borowiak et al. (2009) concluded that clustered populations of Sox17+ cells are definitive endoderm, indeed these cells do not express extraembryonic markers [19]. Although we used a different protocol to differentiate G6pdΔ ES cells, we observed Sox17+ cells with both clustered and dispersed morphologies (Figure 2A). These data let us hypothesize that both extraembryonic and definitive endodermal cells could be differentiated from G6pdΔ ES cells.
10.1371/journal.pone.0029321.g002Figure 2 Definitive and extraembryonic endoderm differentiated from G6pdΔ ES cells.
(A) Immunofluorescent staining of the differentiated mouse G6pdΔ ES cells for Sox17 (red) and nuclei (blue) at 8 days of neural differentiation. PH, phase contrast images. Scale bars, 25 µm. (B) RT-PCR analysis of the endodermal markers GATA4 and extraembyonic endodermal markers Sox7 during differentiation. Lane C, positive control, RNA isolated from embryos and yolk sacs at 9,5 dpc. Amplified HPRT is shown as a positive control. (C) qRT-PCR for Pdx1 in wt and G6pdΔ ES cells at 13 days after treatment with Indolactam V from day 8 during differentiation. Values are means ± SD (n = 2). *P<0.05; **P<0.01; ***P<0.001.
To better define whether both cell populations are formed during G6pdΔ ES cell differentiation, we confirmed the presence of extraembryonic marker Sox7 by RT-PCR analysis (Figure 2B) [20]. Moreover, Chen et al. (2009) identified a small molecule, Indolactam V, that can induce differentiation of endodermal precursor cells (Sox17+) into pancreatic progenitor cells [21]. Wt and G6pdΔ ES cells at 8 days of differentiation were grown for 5 more days in presence of Indolactam V; the analysis by real-time RT-PCR (qRT-PCR) of mRNA extracts from both cell lines revealed the presence of Pdx1, a marker of pancreatic progenitors, exclusively in G6pdΔ ES cells (Figure 2C).
The presented data support our hypothesis that both extraembryonic and definitive endoderm Sox17+ cells are induced during G6pdΔ ES cells differentiation.
Analysis of the mechanism inducing endodermal cell differentiation
Redox status mediates ES differentiation [22]. G6PD, a NADPH-producing dehydrogenase, is an enzyme essential for the defense of the cells against oxidative stress. To analyze whether oxidant formed in absence of G6PD have a role in establishing the mechanism that drives differentiation of G6pdΔ ES cells into endodermal cells, we differentiated wt and G6pdΔ ES cells with the previously described protocol in presence of a lower oxygen concentration described to be a physiological oxygen level during development (normoxia) [23], 5% instead of the 20% used in the normal culture conditions. Although in these culture conditions a reduced amount of ROS is formed (Figure S2A), we observed expression of GATA4 and Sox17 indicating that endodermal precursor cells are still differentiated (Figure 3A). Moreover, differentiating ES cells in presence of N-acetylcysteine (NAC), a well-known antioxidant molecule, G6pdΔ ES cells are still able to differentiate into endodermal cells, in fact they expressed GATA4 and Sox17, differently from wt ES cells (Figure S2B).
10.1371/journal.pone.0029321.g003Figure 3 Mechanism inducing endodermal cell differentiation.
(A) RT-PCR of different lineage-specific marker genes in wt and G6pdΔ ES cells in presence of a lower oxygen concentration (5%) and in normal culture conditions (20%). (B) RT-PCR of different lineage-specific markers in differentiated wt E14 and Pgd+/− ES cells at 8, 10 and 13 days of neural differentiation. (C) Double immunostaining Sox17/βIII-tubulin/DAPI of cells at 10 days of differentiation showed areas of immunoreactive cells for Sox17 only in Pgd+/− ES cells. Scale bars, 75 µm. (D) qRT-PCR for Sox17 and GATA4 in wt and G6pdΔ ES cells at day 10 after treatment with D-(-)-ribose during neural differentiation. Values are means ± SD (n = 3). *P<0.05; **P<0.01; ***P<0.001.
Being the first and key enzyme of the PPP, G6PD is essential also for the production of pentose sugars. To analyze whether the pentose sugars have a role in establishing the differentiation fate of G6pdΔ ES cells into endodermal precursors, we differentiated heterozygous knockout ES cells for Phosphogluconate Dehydrogenase (Pgd+/− ES cells), the second enzyme of the PPP, using the previously described protocol. These cells, compared to wt ES, have a reduced amount of Pgd mRNA (Figure S3A), a reduced flow of glucose carbon through the oxidative arm of the PPP (Figure S3B) and moreover, differently from G6pdΔ ES cells, they are not sensitive to oxidative stress (Figure S3C). As previously observed in G6pdΔ, also Pgd+/− ES cells are able to differentiate into endodermal cells (Figure 3B, C, Figure S3D, E, F). These data suggest that modulation of the PPP is important to drive endodermal fate.
Moreover, to confirm the hypothesis that the amount of pentose sugars present in the cells can influence the differentiation fate, we differentiated wt and G6pdΔ ES cells in presence of D-(-)-ribose. Although the addition of D-(-)-ribose had no effect on wt ES cells differentiation, we observed a reduction in the amount of Sox17 mRNA in G6pdΔ differentiated ES cells (Figure 3D), confirming our hypothesis. We observed the same effect differentiating G6pdΔ ES cells in presence of L-(-)-ribose, in contrast, addition of other sugars like L-Arabinose or Sucrose had no effect on Sox17 mRNA (Figure S3G)
The addition of D-(-)-ribose during differentiation to G6pdΔ ES cells had no effect on carbon flow through Glycolysis and citric acid cycle, Maillard reaction and did not increase the amount of ROS (data not shown).
Chemical inhibitors of the PPP induce wt ES cells to differentiate into endodermal cells
DHEA and 6AN have been described to inhibit respectively G6PD and PGD activity [24], [25]. To confirm that DHEA and 6AN were inhibiting the PPP in ES cells, we measured, in wt ES cells the flow of glucose carbon through the oxidative arm of the PPP, at maximum non toxic concentration of both substances (DHEA and 6AN) and observed a statistically significant reduction of the pathway (Figure 4A); moreover, to verify that DHEA was acting on G6PD while 6AN was acting on PGD, we tested the capability to respond to oxidative stress of treated cells compared to wt and we observed that, as expected, DHEA treated wt ES cells were more sensitive to oxidizing agents as G6pdΔ ES cells [10], while 6AN treated cells show the same resistance as wt and Pgd+/− ES cells (Figure S4B).
10.1371/journal.pone.0029321.g004Figure 4 Inhibitors of the PPP induce endodermal differentiation.
(A) Double immunostaining Sox17/βIII-tubulin/DAPI of cells at 10 days of differentiation showed areas of immunoreactive cells for Sox17 in wt ES cells differentiated in presence of DHEA or 6AN. Scale bars, 50 µm. (B) qRT-PCR for Sox17 and GATA4 in wt ES cells at day 10 after treatment with DHEA or 6AN during neural differentiation. Values are means ± SD (n = 3). *P<0.05; **P<0.01; ***P<0.001.
Since both chemicals were able to inhibit the PPP in ES cells, we investigated their effect on wt ES cells differentiation. Immunofluorescence analysis revealed that both substances are able to induce differentiation of wt ES cells in Sox17+ cells (Figure 4A). Moreover, by qRT-PCR we observed an increase in the amount of GATA4 and Sox17 mRNA in DHEA and 6AN treated wt ES cells (Figure 4B).
Discussion
Understanding how the endoderm forms from ES cells is the first step towards the ultimate goal, generating specific cell types for therapeutic purposes. Studies in Xenopus laevis, zebrafish, and mice collectively, suggest a conserved mechanism for mesoderm/endoderm lineage commitment involving the transforming growth factor-β (TGF β) family member Nodal, and a common set of downstream effector molecules [16], [17], [26].
One promising differentiation strategy is to recapitulate, in vitro, the developmental signals that guide cells towards specific lineages during development [19]. In vitro it has been shown that addition of Activin A or Nodal during ES cells differentiation leads to endodermal induction.
Recently, two groups [19], [27] illustrate high throughput screening to discover novel small molecules able to induce embryonic stem cell differentiation into definitive endoderm. However, the molecules reported have still necessity of serum presence in the differentiation protocols. This means that we are still not acquainted with all the pathways involved in endodermal differentiation and, moreover, we still don't know how to control them.
The PPP has been described to be essential in the cell during the defence against oxidative stress. Our results show, for the first time, that modulation of this metabolic pathway could influence stem cell differentiation. In fact, we observed that cells unable to produce pentose sugars through the PPP, under chemically defined conditions, spontaneously differentiate into endodermal precursor cells. In the same culture conditions, as we showed before [13], wt ES cells only differentiate into neuronal cells.
The specification of mesendodermal is postulated to be dependent on Tgf-β/Nodal pathway [16], [17]. Although Smad2 seems to be active also in wt ES cells, we observed an increased activation of this pathway in G6pdΔ ES cells; furthermore, expression of Cripto, the Nodal co-receptor, was persistent in G6pdΔ ES cells until day 6 of differentiation. These data suggest that mesendodermal differentiation could be dependent on Tgf-β/Nodal pathway in G6pdΔ ES cells. Moreover, modulation of the PPP seems to be required upstream Tgf-β/Nodal pathway activation during mesendodermal induction.
We showed that the mechanism implicated in this process involves the role of the PPP in the production of pentose sugars either than the previously described function in the oxidative stress defense. Indeed, we observed that reduction of oxidative stress, during the differentiation process in G6pdΔ ES cells, did not inhibit the Sox17 activation. Moreover, Pgd+/− ES cells, also having a deficit in the PPP, are able to generate endodermal precursor cells even if they have a normal resistance to oxidative stress due to the normal G6PD activity.
In contrast, addition of D-(-)-ribose, a pentose sugar produced by PPP, to the cell culture differentiation medium decreases the amount of Sox17 transcript, suggesting that the concentration of pentose sugars in the cells could be the signal that induces endodermal differentiation.
Furthermore, we identified two substances, DHEA and 6AN, known to inhibit the PPP, that act as endodermal inductors.
G6pd is an X-linked gene. In female heterozygous for G6PD mutation causing severe deficiency, once X inactivation has produced mosaicism for the G6PD cellular phenotype, there is a strong selection against G6PD(−) cells both in mouse and in human [12], [28]. Nevertheless, analysis of G6PD(+/−) heterozygous female showed that, differently from other tissues, intestinal crypts deriving each from an endodermal stem cell were severely G6PD deficient [12]. These data confirmed ours, in fact they suggested that also in vivo in absence of G6PD, cells show the intrinsic ability to colonize endodermal derived tissues and to differentiate into endodermal cells.
Pgd has been identified in ES cells as a direct target of Myc in two different ChIP-on-chip analysis [5], [29]. In a different experiment, cMyc has been described to induce the production of the pentose sugar [7]. Recent data has reported that genes of the PPP are among the most prominently induced by mTOR [6]. Inhibition of mTOR or simultaneous inactivation of c- and N-Myc induce endodermal differentiation [4], [5]. Our results raise the possibility that Myc and mTOR can repress endodermal differentiation activating the PPP.
The data described will help direct experiments aimed at ES cell differentiation into therapeutically relevant endodermal derivatives.
Materials and Methods
ES cell culture
AK7 (wt), E14 Tg2A (MMRRC; wt E14), G6pdΔ, G6pdΔ
pG6pd and Pgd+/− (MMRRC Strain ID: 081091) ES cell lines were maintained in an undifferentiated state by culturing them on a mitomycin-C-inactivated fibroblast monolayer in presence of leukemia-inhibiting factor (LIF) [30]. Under these conditions the cell population remained undifferentiated, as determined by visual inspection under phase-contrast microscopy.
In vitro differentiation
In vitro differentiation was performed according to Fico et al. (2008) [13]. Briefly, at 48h before inducing differentiation wt AK7, wt E14, G6pdΔ, G6pdΔ
pG6pd and Pgd+/− ES cells were seeded on gelatin-coated plates. At day 0, ES cells were dissociated in a single-cell suspension and 1500 cells/cm2 were plated on gelatin-coated plates. The culture medium was replaced daily during differentiation process. Culture medium for neuronal differentiation (serum-free Knockout Serum Replacement (KSR)-supplemented medium) contained knockout Dulbecco minimal essential medium supplemented with 15% KSR (Invitrogen), 2 mM glutamine, 100 U/ml penicillin/streptomycin, and 0.1 mM β-mercaptoethanol.
Endoderm differentiation was performed adding 1.5 g/l of D-(-)-ribose in the differentiation medium. Addition of higher concentration of D-(-)-ribose during the differentiation induced cell death, lower concentration had no effect on endodermal differentiation.
To block the PPP we added DHEA and 6AN, at 100 µM and 10 µM final concentration respectively, in the differentiation medium starting from the plating and carrying on for the entire duration of the experiment.
RNA isolation and RT-PCR
Total RNA was isolated through PerfectPure RNA Cultured Cell kit (5 PRIME). Reverse transcription-PCR (RT-PCR) was performed with the Perkin-Elmer RT-PCR kit, as recommended by the manufacturer. cDNA was amplified by PCR. The number of cycles was chosen to select PCR conditions on the linear portion of the reaction curve to avoid the saturation effects of PCR. Sequence of specific primers, number of cycles, annealing temperature, and the length of the amplified products have been reported in Table S1.
Real-time RT-PCR
First-strand cDNA was synthesized from 500 ng of total RNA using oligo-dT primers, random hexamers and SuperScript II (Invitrogen). Real-time RT-PCR analysis was performed on Biorad CFX 96 Real time System using the SYBR Green PCR Master Mix (Biorad). The PCR reaction consists of 10 µl of SYBR Green PCR Master Mix, 120 ng of forward and reverse primers, and 4 µl of 1∶30 diluted template cDNA in a total volume of 25 µl and 40 cycles of amplification (95°C 10 s; 62°C 30 s; 72°C 10 s). Primer specificity was determined by melting curve analysis and standard curves were generated to check primer efficiency. The relative expression of each gene was normalized against GAPDH.
GATA4 forward 5′-CACTATGGGCACAGCAGCTCC-3′,
GATA4 reverse 5′-TTGGAGCTGGCCTGCGATGTC-3′;
Sox17 forward 5′-GGAGGGTCACCACTGCTTTA-3′,
Sox17 reverse 5′-AGATGTCTGGAGGTGCTGCT-3′;
Pdx1 forward 5′-TCACGCGTGGAAAGGCCAGT-3′,
Pdx1 reverse 5′-GTGTAGGCAGTACGGGTCCT-3′;
GAPDH forward 5′-TCTTCTGGGTGGCAGTGATG-3′,
GAPDH reverse 5′-TGCACCACCAACTGCTTAGC-3′;
Three independent PCR reactions were performed for any analyzed gene. Data were represented as mean ± SD of at least two independent experiments. Differences between control values and experimental values were compared by Student's t test.
Immunocytochemistry
Cells were fixed in 4% paraformaldehyde and 1× phosphate-buffer saline (PBS) at room temperature for 30 min. Following fixation, samples were washed three times with 1× PBS for 5 min and then incubated with 10% normal goat serum (Dako Cytomation, Glostrup, Denmark, http://dakocytomation.com) and 0.1% triton X-100 in 1× PBS for 15 min at room temperature. The cells were then washed three times in 1× PBS for 5 min and incubated with primary antibodies (monoclonal anti βIII-tubulin, 1∶400, Sigma-Aldrich; polyclonal anti-GFAP, 1∶300, Dako Cytomation) in 10% normal goat serum and 1× PBS. For antibody anti-Sox17 and anti-GATA4 after fixation the cells were permeabilized with 0.5% triton X-100 in 1× PBS for 5 min, blocked with 0.1% Triton, 10% BSA and 1× PBS for 1 hr and incubated with primary antibody (goat polyclonal anti-Sox17, 1∶20, R&D; goat polyclonal anti-GATA4, 1∶100, Santa Cruz Biotecnology Inc.) in 0.1% triton, 10% BSA in 1× PBS at 4°C overnight. Following primary antibody incubation, cells were rinsed three times in 1× PBS and further incubated with secondary antibodies: either anti-mouse IgG FITC-conjugated (1∶400; Molecular Probe) or anti-rabbit IgG FITC-conjugated (1∶200; Santa Cruz Biotechnology) in 10% normal goat serum and 1× PBS in for 30 min at room temperature; anti-goat Alexa Fluor 594 (1∶400; Invitrogen) in 0.1% triton, 0,1% BSA for 30 min at room temperature. Finally, samples were washed three times in 1× PBS and counterstained with 4′, 6′-diamido-2-phenylindole (DAPI, 250 ng/ml; Sigma-Aldrich). Labelling was detected by fluorescent illumination using an inverted microscope (DMI 6000B, Leica Microsystems); images were acquired on a DCF 360 FX B/W camera (Leica).
Western Blot Analysis
Cells were lysed in 1× RIPA lysis buffer in presence of protease inhibitor mixture (Roche)/1% phosphatase inhibitor mixture (Roche). Proteins were separated by 10% Tris-Glycine SDS/PAGE (Bio-rad) under denaturing conditions and transferred to a PVDF membrane. After blocking with 5% milk in 1× PBS/0.1% Triton X, the membrane was incubated with antibodies against phospho-Smad2 (1∶1000, Cell Signaling), Cripto (1∶1000), Olig2 (1∶1000, Dana Faber Cancer Institute), PARP (1∶1000, New England BioLabs) or β-actin (1∶1000 Cell Signaling) overnight at 4°C. The membrane was then washed, incubated with anti-mouse/rabbit peroxidase-conjugated secondary antibody (1∶1000, Cell Signaling) at room temperature for 1 hr, and developed by ECL plus (Amersham).
Measurement of 14CO2 production
14CO2 produced by PPP or Glycolysis was determined as previously described [10]. Differences between control values and experimental values were compared by Student's t test.
Measurement of ROS generation
ROS were detected using the fluorescent dye 2′,7′-dichlorofluorescein diacetate (DCFDA), (Molecular Probes, Eugene, OR) as previously described [31]. Differences between control values and experimental values were compared by Student's t test.
Furosine Analysis
D-(-)-ribose treated and untreated G6pdΔ ES cells at day 13 of differentiation were lysed in water (32 µg/µl) and hydrolyzed in 8N hydrochloric acid (HCl) for 23 hr at 110°C. The hydrolysates were filtered through a 0.45 µm Whatman filter paper. A 0.5 mL aliquot of the filtered hydrolysate was purified by a C18 cartridge from which furosine was eluted by 3 mL of 3N HCl and 20 µL of the mixture was injected into the HPLC. Furosine was determined by ion-pair RP-HPLC as previously described [32]. The separation of furosine was performed in a C8 column (250×4.6 mm i.d.) (Alltech furosine- dedicated) (Alltech Associates, Laarne, Belgium).
Supporting Information
Figure S1
Evidence that GATA4+ and Sox17+ cells, formed during
G6pdΔ
ES cells differentiation, are endodermal precursors. (A) Double immunostaining GFAP/GATA4 of cells at 13 days of differentiation show absence of co-localization between the two markers, indicating that GATA4 positive cells are not astrocytes. (B) Double immunostaining βIII-tubulin/GATA4 of cells at 13 days of differentiation show absence of co-localization between the two markers, indicating that GATA4 positive cells are not neurons. (C) Western blot analysis with anti-Olig2 and anti-Actin antibodies performed on protein extracts from wt and G6pdΔ undifferentiated ES cells (day 0) and at 10 days of neural differentiation. Actin was analyzed as loading control. (D–E) qRT-PCR for Nodal and Cripto in wt and G6pdΔ ES cells during differentiation. Values are means ± SD (n = 2). *P<0.05; **P<0.01; ***P<0.001.
(TIF)
Click here for additional data file.
Figure S2
Differentiation in antioxidant conditions. (A) Reduced amount of reactive oxygen species (ROS) is detected in G6pdΔ differentiated ES cells in presence of 5% oxygen compared with the ones cultured at 20% oxygen concentration. Values are means ± SD (n = 2). *P<0.05; **P<0.01; ***P<0.001. (B) RT-PCR of different lineage-specific markers in wt and G6pdΔ ES cells differentiated for 10 days in presence of NAC.
(TIF)
Click here for additional data file.
Figure S3
Characteritazation of heterozygous knockout
Pgd
ES cells (
Pgd
+/−). (A) RT-PCR analysis of Pgd mRNA level in undifferentiated Pgd+/− and wt E14 ES cells. Amplified HPRT is shown as a positive control. (B) Activity of the PPP determined by [1-14C] glucose and the cmp/mg of protein of 14CO2 released in wt and Pgd+/− ES cells. Values are means ± SD (n = 3). *P<0.05; **P<0.01; ***P<0.001. (C) wt, G6pdΔ, wt E14, and Pgd+/− undifferentiated ES cells were incubated with 0, 300 or 800 µM of Diamide, a thiol-oxidizing agent, for 30 min. After 8 hr, total proteins were extracted and separated on SDS-PAGE, and their respective content in cleaved PARP was analyzed by Western blotting. (D–E) Double immunostaining GFAP/GATA4 of wt E14 and Pgd+/− ES cells at 13 days of differentiation show absence of co-localization between the two markers, indicating that GATA4 positive cells are not astrocytes. (F) qRT-PCR for GATA4 in wt E14 and Pgd+/− ES cells during neural differentiation. Values are means ± SD (n = 3). *P<0.05; **P<0.01; ***P<0.001. (G) qRT-PCR for Sox17 in G6pdΔ ES cells at day 10 during neural differentiation in presence of different sugars: D-ribose (D-rib), L-ribose (L-rib), L-arabinose (L-arab) and sucrose (Sucr) at 10 mM final concentration. Values are means ± SD (n = 3). *P<0.05; **P<0.01; ***P<0.001.
(TIF)
Click here for additional data file.
Figure S4
Characterization of ES cells treated with DHEA or 6AN. (A) Activity of pentose phosphate pathway determined by 1-14C glucose and the cmp/mg of protein of 14CO2 released in wt ES cells treated with DHEA or 6AN. Values are means ± SD (n = 3). *P<0.05; **P<0.01; ***P<0.001. (B) ES cells treated with DHEA and 6AN were incubated with 300 µM of Diamide for 30 min. After 8 hr, total proteins were extracted and separated on SDS-PAGE, and their respective content in cleaved PARP was analyzed by Western blotting. Actin was analyzed as loading control.
(TIF)
Click here for additional data file.
Table S1
Primers used and PCR conditions.
(DOC)
Click here for additional data file.
We thank Rosanna Dono and Flavio Maina for helpful discussions; Daniela Ruggiero for statistical analysis assistance; Gabriella Minchiotti for gift of Cripto antibody; Alfredo Franco and Chiara Lepore for their skilful laboratory assistance; the Integrated Microscopy Facilities of IGB-CNR for technical support. Anna Maria Aliperti is acknowledged for proofreading the manuscript. Patent n. MI2011A001096.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by Regione Campania L.R. 5/2007 grant n° 2477 and by Ministero dell'Istruzione, dell'Università e della Ricerca (MIUR) Decreto Dirigenziale MIUR n° 3338/2005. GM was supported by “Assegno di Ricerca” of “Sviluppo delle esportazioni di prodotti agroalimentari del mezzogiorno” Intesa MIUR/Mezzogiorno. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22272240PONE-D-11-1991010.1371/journal.pone.0029649Research ArticleBiologyBiochemistryMolecular Cell BiologyCellular TypesMedicineCardiovascularEndocrinologyTyrosine Nitration of PA700 Links Proteasome Activation to Endothelial Dysfunction in Mouse Models with Cardiovascular Risk Factors Proteasome Links Endothelial DysfunctionXu Jian
1
*
Wang Shuangxi
2
Zhang Miao
2
Wang Qilong
2
Asfa Sima
3
Zou Ming-Hui
2
1
Division of Endocrinology and Diabetes, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America
2
Division of Molecular Medicine, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America
3
Division of Gastroenterology, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America
Xu Aimin EditorUniversity of Hong Kong, China* E-mail: [email protected] and designed the experiments: JX M-HZ . Performed the experiments: JX SW MZ QW SA . Analyzed the data: JX SW MZ M-HZ. Wrote the paper: JX M-HZ.
2012 17 1 2012 7 1 e296499 10 2011 2 12 2011 Xu et al.2012This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Oxidative stress is believed to cause endothelial dysfunction, an early event and a hallmark in cardiovascular diseases (CVD) including hypertension, diabetes, and dyslipidemia. However, the targets for oxidative stress-mediated endothelial dysfunction in CVD have not been completely elucidated. Here we report that 26S proteasome activation by peroxynitrite (ONOO−) is a common pathway for endothelial dysfunction in mouse models of diabetes, hypertension, and dyslipidemia. Endothelial function, assayed by acetylcholine-induced vasorelaxation, was impaired in parallel with significantly increased 26S proteasome activity in aortic homogenates from streptozotocin (STZ)-induced type I diabetic mice, angiotensin-infused hypertensive mice, and high fat-diets -fed LDL receptor knockout (LDLr−/−) mice. The elevated 26S proteasome activities were accompanied by ONOO−-mediated PA700/S10B nitration and increased 26S proteasome assembly and caused accelerated degradation of molecules (such as GTPCH I and thioredoxin) essential to endothelial homeostasis. Pharmacological (administration of MG132) or genetic inhibition (siRNA knockdown of PA700/S10B) of the 26S proteasome blocked the degradation of the vascular protective molecules and ablated endothelial dysfunction induced by diabetes, hypertension, and western diet feeding. Taken together, these results suggest that 26S proteasome activation by ONOO−-induced PA700/S10B tyrosine nitration is a common route for endothelial dysfunction seen in mouse models of hypertension, diabetes, and dyslipidemia.
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Introduction
Peroxynitrite (ONOO−) is formed by the reaction of superoxide anions with nitric oxide (NO) at diffusion-controlled rate. It represents a crucial pathogenic mechanism in CVD when excessively produced [1], [2]. Among many chemical reactions, ONOO− is well known by its impact on proteins through tyrosine nitration and leaving its footprint as 3-nitrotyrosine [2]. Many studies have elegantly demonstrated endogenous ONOO− generation and its diverse downstream targets, such as lipids, DNA, and proteins [2], in CVD. Although the roles of ONOO− in the pathogenesis of endothelial dysfunction and atherosclerosis have been well established, the protein targets of ONOO− in CVD have been largely unidentified.
The ubiquitin proteasome system (UPS) functions as the major non-lysosomal intracellular proteolytic system responsible for degradation of most proteins, particularly those of short-lived and regulatory nature [3]. The essential role of this system includes control of protein quality, cell cycle, transcription factor regulation, gene expression, cell differentiation, and immune response [4]. Degradation of proteins by the UPS occurs in two steps, including targeting of proteins and successive degradation by the 26S proteasome, the major proteolysis complex in the system. The 26S proteasome is a multi-catalytic protease consisting of a 20S catalytic core and two 19S regulatory particles (PA700) [4]. PA700 is first characterized as an ATP-dependent 20S proteasome activator for 26S proteasome activation [5]. Later, sub-complexes of PA700 important for substrate selection and processing have also been identified [6], [7]. Regardless modes of action, increasing evidence demonstrate that PA700 is crucial in functional regulation of the UPS [8]. Alterations in UPS have been shown to contribute to the pathogenesis of cancer, neurodegenerative, and immune diseases [9]. An emerging role has been implicated in the pathogenesis of atherosclerosis [10].
Endothelial dysfunction, defined by impaired endothelium-dependent relaxation, is an early marker for atherosclerosis. Many of the risk factors such as diabetes, hypertension, and dyslipidemia that predispose to atherosclerosis can also cause endothelial dysfunction, and the presence of multiple risk factors has been found to predict endothelial dysfunction. Available data suggest that oxidant stress-activated 26S proteasome mediated endothelial dysfunction in streptozotocin-induced diabetic mice [11] and angiotensin II (Ang II) induced hypertensive mice [12], as well as in experimental hypercholesterolemia pig [13]. It remained to be established if oxidative stress-activated 26S proteasomes is an early and a common pathogenic phenomenon for cardiovascular risk factors and cardiovascular diseases (CVD). Here we report that ONOO− tyrosine nitrates PA700/S10B resulting in activation of 26S proteasome and consequent endothelial dysfunction in mouse models of diabetes, hypertension and dyslipidemia.
Materials and Methods
Materials
Mouse GTPCH I antibody was purchased from Ascenion GmBH (Munich, Germany); ubiquitin antibody from Santa Cruz Biotechnology (Santa Cruz, CA); mouse PA700/S10B antibody from Abcam (Cambridge, MA); MG132 and purified 26S proteasome from BioMol (Plymouth Meeting, PA); fluorogenic proteasome substrates from Calbiochem (San Diego, CA); tetrahydro-L-biopterin dihydrochloride (BH4) from Cayman (Ann Arbor, MI). HUVECs and HMVEC were obtained from Cascade Biologics (Walkersville, MD) and ScienCell (Carlsbad, CA), respectively. Human GTPCH I antibody was kindly provided by Dr. Gabriele Werner-Felmayer (Innsbruck Medical University, Austria). All the other antibodies and reagents, including angiotensin II (Ang II) and streptozotocin (STZ), were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) or Fisher Scientific (Pittsburgh, PA).
Mice
Ten-week-old male C57BL/6J mice and low density lipoprotein receptor knockout (LDLr−/−) mice were obtained from the Jackson Laboratory (Bar Harbor, Me). Mice were housed in temperature controlled cages with a 12-hour light/dark cycle and given free access to water and chow. The mice were euthanized with inhaled isoflurane at the end of the animal experiments. Aortas were then removed for endothelial function assay or immediately frozen in liquid nitrogen for other assays. The animal protocols for models of diabetes, hypertension and dyslipidemia used in this paper were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Oklahoma Health Sciences Center. The approved IACUC protocol numbers are: 10-153-H, 11-072-H and 11-045.
Assays of the 26S proteasome activity
26S proteasome activity was assayed by measuring ATP dependent degradation of proteasome fluorescence substrate, as described previously [11], [14].
Streptozotocin-induced diabetes mellitus in mice
A low-dose of STZ induction regimen was used to induce pancreatic islet cell destruction and persistent hyperglycemia as described by the Animal Models of Diabetic Complications Consortium (http://www.amdcc.org). Hyperglycemia was defined as a random blood glucose level of >450 mg/dL for >2 weeks after injection. One additional group of STZ mice received Tempol (Sigma; 1 mmol/L) in their drinking water for an additional 2 weeks. Aortas were all harvested 3 weeks after STZ injection.
Ang II-induced hypertension and blood pressure measurement
Ang II was continuously administered in 10-week-old C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) at a rate of 0.8 mg/kg/d for 14 days using a mini-osmotic pump (Alzet®) as previously reported [15]. Arterial blood pressure was determined using a carotid catheter method as previously described [16]. High blood pressure (hypertension) was defined and achieved as previously reported [16]. Transfection with siRNA was performed as previously described [16].
LDLr−/− dyslipidemia model
The LDLr−/− mice were fed a Western diet or high fat diet (HFD) containing 0.21% cholesterol and 21% fat (Research Diets Inc, D12079B) for 8 weeks. Two weeks after HFD, an MG132 osmotic pump (delivered at rate of 0.72 mg/kg per day; DURECT Corporation, Model 2006) or the inhibitor-diluents (DMSO), as a negative control, was implanted subcutaneously in LDLr−/− or the control WT mice for 6 weeks. Dyslipidemia was defined as previously reported [17].
Assays of endothelium-dependent and endothelium-independent vasorelaxation
Vessel Aortic rings isolated from the treated mice were subjected to organ chamber assay of endothelium-dependent and -independent vasodilatation as described previously [11].
Statistical analysis
Comparison of vasodilatation or data from other experiments involving more than two factors (such as endothelial dependent vessel relaxation assay) was performed with a two-way ANOVA, and intergroup differences were determined using the Bonferroni inequality method. All other results were analyzed with a one-way ANOVA. Values are expressed as mean ± SEM. P<0.05 was accepted as significant.
Results
Exogenous ONOO− increase PA700 tyrosine nitration and 26S proteasome activity in vitro
To test the impacts of ONOO− on the 26S proteasome, purified 26S proteasomes were exposed to low concentration (1 µM) of chemically synthesized ONOO−
in vitro. As depicted in Fig. 1, ONOO− markedly increased 3-nitrotyrosine staining of PA700/S10B, the regulatory unit of 26S proteasome, without changing the apparent integrity of the proteasome (Fig. 1A). The increment of tyrosine nitration in PA700/S10B was correlated with an over 2-fold increase in 26S proteasome activity (Fig. 1B). Increased 26S proteasome activity was further confirmed by an in situ substrate-in-gel assay, in which the vehicle- or ONOO− - treated 26S proteasomes were separated on a native-PAGE (3–14% gradient gel) followed by fluorogenic substrate incubation and fluorescence capturing under UV (Fig. 1C: in-gel substrate).
10.1371/journal.pone.0029649.g001Figure 1 ONOO− nitrates PA700/S10B and increases 26S proteasome activity both in vitro and in intact cell.
In vitro (A–C): ONOO− (1 µM) was incubated with the purified 26S proteasome for 5 min; in intact cell (D–E): HUVEC was incubated with ONOO− for 0.5 h, in the presence or absence of uric acid (50 µM pre-incubation for 1 h). Cell free system (in vitro) was subjected to (A) Western blot to detect levels of PA700/S10B and the tyrosine nitration of 26S proteasome/PA700/S10B, (B) 26S proteasome activity (chymotrypsin-like activity), (C) an alternative 26S proteasome activity assay: a substrate-in-gel assay with a fluorogenic substrate followed by fluorescence capturing under the UV light. HUVEC cell lysate was subjected to (D) Western blotting of PA700/S10B tyrosine nitration and (E) assay of 26S proteasome activity (chymotrypsin-like activity). All blots shown are representative of three independent experiments. All results (n = 3) were analyzed with a one-way ANOVA.
ONOO− nitrates PA700/S10B and increases 26S proteasome activity in intact endothelial cells
We next investigated if the effects of ONOO− on proteasome activity could be recapitulated in HUVEC. As depicted in Figure 1D, exogenous addition of ONOO− in HUVEC significantly enhanced tyrosine nitration of PA700/S10B compared to those treated with vehicle. Pre-incubation of uric acid (UA), a known ONOO− scavenger, abolished the ONOO- enhanced PA700/S10B tyrosine nitration (Fig. 1D). Further, UA abrogated ONOO− enhanced proteasome activation in HUVEC (Fig. 1E). In sum, these data suggest that exogenous ONOO− causes tyrosine nitration of PA700/S10B resulting in consequent activation of 26S proteasome in HUVEC.
ONOO− enhances 26S proteasome assembly in vitro and in intact endothelial cells
The assembly of 19S and 20S proteasomes into 26S proteasome is considered as a key step in controlling 26S proteasome activity [5]. Thus, it was interesting to evaluate if ONOO− increased the assembly of 19S and 26S proteasome sub-complexes. We first tested this on the purified proteasomes. We used native PAGE gel to separate the treated 26S proteasomes, like Fig. 1C, and then either performed a Western blotting (Fig. 2A: IB) or a direct staining with Coomassie Brilliant Blue (CBB) (Fig. 2A: CBB staining). Compared to the vehicle-treated (Fig. 2A), ONOO−-treated 26S proteasomes presented an increase in PA700/S10B IB staining as well as the 26S proteasome CBB staining, suggesting an increased 26S proteasome assembly (Fig. 2A: IB and CBB staining), which may contribute to the enhanced 26S proteasome activity (Fig. 1B and 1C).
10.1371/journal.pone.0029649.g002Figure 2 ONOO− promotes 26S proteasome assembly both in vitro and in intact cell.
In vitro (A): ONOO− (1 µM) was incubated with the purified 26S proteasome for 5 min; in intact cell (B): HUVEC was incubated with ONOO− for 0.5 h, in the presence or absence of uric acid (50 µM pre-incubation for 1 h). Cell free system (in vitro) was subjected to (A) separation on a native gradient (3–14%) PAGE gel followed either by Western-blotting (IB) or a direct staining with coomassie brilliant blue (CBB staining) for 26S proteasome assembly. HUVEC cell lysate was subjected to (B) Western blotting of the PA700/S10B co-immunoprecipitates with a β7 antibody. All blots shown are representative of three independent experiments. All results (n = 3) were analyzed with a one-way ANOVA.
We further tested if the ONOO−-mediated 26S proteasome assembly could be reproduced in intact cells. To avoid the confound effects exerted by the proteasome purification and enrichment which is required in the native gel approach, we adapted a previously described approach [12] by measuring the association of proteasome representative subunits to estimate 26S proteasome assembly. These subunits represent the19S (PA700/S10B) and the 20S (β7) sub-complex, respectively. As shown in Fig. 2B, ONOO− increased 26S proteasome assembly, as evidenced by increased association of PA700/S10B and β7 subunits, compared to those treated with the vehicle. Pre-incubation of uric acid abrogated ONOO−-enhanced proteasome association in HUVEC (Fig. 2B). Collectively, these data suggest that exogenous ONOO− promotes 26S proteasome assembly in intact cell.
The 26S proteasome is activated in aortic homogenates from mouse models of diabetes, hypertension, and dyslipidemia
We sought to exploit if ONOO−-mediated 26S proteasome activation could be recapitulated in whole animals when cardiovascular risk factors were present. We first assayed 26S proteasome activity in mouse model of STZ-induced diabetes, Ang II-induced hypertension, and HFD induced dyslipidemia, which are all recognized cardiovascular risk factors and associated with oxidative stress. As shown in Fig. 3, homogenates from STZ-diabetic aorta presented a ∼2 fold increase in 26S proteasome activity compared to those of the vehicle-treated control mice (Fig. 3A). Injection of a potent proteasome inhibitor MG132 (5 mg/kg body weight, i.p. 2d., as reported [11], [18]) significantly decreased 26S proteasome activity in STZ-induced diabetic mice (Fig. 3A). In aortic preparation from Ang II-infused hypertensive mice, about 2-fold of increase in 26S proteasome activity were detected (Fig. 3B); however, knockdown of PA700/S10B, which were confirmed in Fig. 4B, suppressed the 26S proteasome activation (Fig. 3B). Similarly, compared to normal chow fed mice, aortic tissues prepared from HFD fed mice (dyslipidemia) presented approximately 2 fold increase in 26S proteasome activity (Fig. 3C); the enhanced 26S proteasome activation was blocked when MG132 was administrated (through the implanted osmotic pump with infusion rate of 0.72 mg/kg/d, 6 wks, as reported [19]) (Fig. 3C).
10.1371/journal.pone.0029649.g003Figure 3 The 26S proteasome is activated and results in degradation of the target proteins, which can be prevented either by ONOO− inhibition or by MG132 administration, in aortic homogenates from mouse models of diabetes, hypertension, and dyslipidemia.
Mouse models of (A) diabetes (STZ: 50 mg/kg/d, sham: sodium citrate, i.p., 5d; MG132, 5 mg/kg/d, i.p., 2d; n = 5/group); (B) hypertension (angiotensin II: 0.8 mg/kg/d, sham: saline; osmotic pump infusion, 14d.; PA700/S10B/control siRNA, i.v. 7d; n = 5/group) and (C) high fat-diets-induced atherosclerosis (LDLr−/− mice, normal chow or HFD, 8 wks; MG132: 0.8 mg/kg/d; sham: saline; osmotic pump infusion, 2 wks after HFD, 6 wks; n = 5/group). AT the end of the animal experiment, aortas were removed and their homogenates were either subjected to 26S proteasome activity assay (chymotrypsin-like activity) (A–C), or Western blotting with the corresponding antibodies as indicated. All results (n = 5) were analyzed with a one-way ANOVA. * indicates significant vs. control; NS: not significant vs. control.
10.1371/journal.pone.0029649.g004Figure 4 PA700/S10B tyrosine nitration and 26S proteasome sub-complex association (assembly), but not the PA700/S10B protein levels, are increased in aortic homogenates from mouse models of diabetes, hypertension, and dyslipidemia.
Mouse models of (A) diabetes (STZ: 50 mg/kg/d, sham: sodium citrate, i.p., 5d; Tempol, 1 mmol/kg/drinking water, 2 wks.; n = 5/group); (B) hypertension (angiotensin II: 0.8 mg/kg/d, sham: saline; osmotic pump infusion, 14d.; PA700/S10B/control siRNA, i.v. 7d; n = 5/group) and (C) high fat-diets-induced dyslipidemia (LDLr−/− mice, normal chow or HFD, 8 wks; MG132: 0.8 mg/kg/d; sham: saline; osmotic pump infusion, 2 wks after HFD and for 6 wks; n = 5/group). AT the end of the animal experiment, aortas were removed and their homogenates were subjected to immunoprecipitation and Western blot. The immunoprecipitation assay was performed using either an anti-PA700/S10B or anti-3-NT antibody. All blots shown are representative for mice n = 5. All results were analyzed with a one-way ANOVA. * indicates significant vs. control; NS: not significant vs. control.
Pharmacologic or genetic inhibition of 26S proteasome prevents GTPCH I and thioredoxin from degradation in mouse models of diabetes, hypertension, and dyslipidemia
We next exploited the functional outcomes of the altered 26S proteasome activity by examining the turnover of proteins that are essential to endothelial cell homeostasis and are recognized substrates of or related to the 26S proteasome. GTP cyclohydrolase I (GTPCH I) is one of the very few known proteins in this category. As a potential substrate of the 26S proteasome [11], [20], [21], [22]_ENREF_23, GTPCH I is a rate limiting enzyme for de novo synthesis of tetrahydrobioptin (BH4), a key cofactor of eNOS [23]. Indeed, aortas from diabetic (Fig. 3D), hypertensive (Fig. 3E) mice, but not the control mice, presented decreased GTPCH I levels, as we previously reported [11], [12]. Interestingly, protein levels of GTPCH I were also found decreased in aortic tissues from the HFD-induced dyslipidemia mice, compared to normal chow fed mice (Fig. 3F). Furthermore, protein levels of thioredoxin (Trx), a protein important to maintain a cellular reducing environment [24], [25], was also decreased in aortas from the mice of diabetes (Fig. 3D), hypertension (Fig. 3E), and dyslipidemia (Fig. 3F). Importantly, inhibition of ONOO− generation by Tempol administration (Fig. 3D), or inhibition of the 26S proteasome by siRNA-mediated PA700/S10B knockdown (Fig. 3E), or by MG132 treatment, restored protein levels of both GTPCH I and Trx (Fig. 3D, 3E, and 3F).
Enhanced PA700/S10B tyrosine nitration and 26S proteasome assembly in mouse models of diabetes, hypertension, and dyslipidemia
We then investigate the potential mechanisms underlying 26S proteasome activation which would be shared by all studied models. To this end, we used an anti-PA700/S10B antibody to pull down PA700/S10B so that the levels of nitrated PA700/S10B could be assessed with an anti-nitrotyrosine antibody in Western blot. As shown in Fig. 4A, compared to those of vehicle-treated mice, aorta from the STZ-treated mice presented higher levels of PA700/S10B tyrosine nitration, accompanied by enhanced 26S assembly, as evidenced by increased association of 26S proteasome sub-complexes (PA700/S10B, the 19S proteasome sub-complex and the 20S proteasome core), but not PA700/S10B protein levels (Fig. 4A). Importantly, these augments were abolished in mice treated with Tempol, a potent superoxide scavenger therefore an inhibitor of ONOO− generation (Fig. 4A).
Like the effect of STZ-induced diabetes, Ang II also increased PA700/S10B tyrosine nitration and 26S proteasome assembly, but not PA700/S10B protein levels (Fig. 4B), consistent with our previous studies [12]. Furthermore, siRNA-mediated PA700/S10B knockdown prevented association of 26S proteasome sub-complexes as expected (Fig. 4B). Such a loss of 26S proteasome assembly was in line with the blockage of Ang II-induced 26S proteasome activation (Fig. 2B) and of the reduction of GTPCH I and Trx protein levels (Fig. 3B).
Importantly, HFD, but not the normal chow feeding, elevated both PA700/S10B tyrosine nitration and 26S proteasome assembly (Fig. 4C). Interestingly, MG132 infusion for 6 weeks reversed these increase, without affecting PA700/S10B protein levels (Fig. 4C). Of note, the levels of PA700/S10B tyrosine nitration and 26S proteasome assembly were closely related to those of the 26S proteasome activity (Fig. 3C).
Nitration of PA700/S10B-mediated 26S proteasome induces endothelial dysfunction
Finally, we monitored the endothelial function of isolated vessel, a valuable surrogate endpoint to assess the impact of therapeutic interventions [26]. As presented in Fig. 5, aortas from diabetic but not control mice, exhibited impaired acetylcholine-induced vessel relaxation (Fig. 5A) in parallel with a down-regulation of both GTPCH I and Trx proteins (Fig. 3D), which were associated with enhanced PA700/S10B tyrosine nitration, 26S proteasome assembly (Fig. 4A) and activation (Fig. 3A). However, these effects were abolished when Tempol, a SOD mimetic, was co-administrated (Fig. 3D, 4A, and 5A), a reminiscence of the protective effect of MG132 administration previously observed [11]. In contrast, aortas from all group of mice presented no significant differences in vessel relaxation evoked by sodium nitroprusside (SNP) (Fig. 5B), an NO donor that can induce endothelium-independent vessel relaxation [27]. This indicates that the impairment of acetylcholine-induced vessel relaxation is mainly due to endothelial dysfunction.
10.1371/journal.pone.0029649.g005Figure 5 Inhibition of the 26S proteasome either by ONOO− inhibition or by MG132 administration rescues endothelial dysfunction in mouse models of diabetes, hypertension, and dyslipidemia.
Mouse models of (A/B) diabetes (STZ: 50 mg/kg/d, sham: sodium citrate, i.p., 5d; Tempol, 1 mmol/kg/drinking water, 2 wks.; n = 5/group); (C/E) hypertension (angiotensin II: 0.8 mg/kg/d, sham: saline; osmotic pump infusion, 14d.; PA700/S10B/control siRNA, i.v. 7d; n = 5/group) and (D/F) high fat-diets-induced dyslipidemia (LDLr−/− mice, normal chow or HFD, 8 wks; MG132: 0.8 mg/kg/d; sham: saline; osmotic pump infusion, 2 wks after HFD and for 6 wks; n = 5/group). AT the end of the animal experiment, aortas were removed for endothelial function assay. The removed aortas were cut into 3-mm rings, and precontracted with 30 nmol/L of U46619 in organ chambers (PowerLab, ADInstruments, Colorado Springs, Co). (A/C/D) Endothelium-dependent vasodilator responses were determined in the presence of acetylcholine (0.01 to 100 µmol/L). (B/E/F) Endothelium-independent vasodilator responses were determined in the presence of sodium nitroprusside (SNP) (0.0001 to 1 µmol/L). All results were analyzed with a one-way ANOVA. * indicates significant v.s. control; NS: not significant v.s. control.
Similarly, aortas from the mice of Ang II-induced hypertension (Fig. 5C) and of HFD-induced dyslipidemia (Fig. 5D) shared the same pathway, manifesting as an impaired acetylcholine-induced vessel relaxation (Fig. 5C and 5D) which were associated the PA700/S10B-mediated 26S proteasome activation (Fig. 3B, 3C, 4B, and 4C). Most importantly, inhibition of the 26S proteasome either by siRNA-mediated PA700/S10B knockdown (Fig. 5C) or by MG132 administration (Fig. 5D) significantly ameliorated the acetylcholine-induced vessel relaxation. Likewise, there were no significant differences in SNP-evoked vessel relaxation among groups (Fig. 5E and 5F), further indicating that endothelial dysfunction contributes to the impaired vessel relaxation.
Discussion
In this study, we have defined a mechanism shared by different models of cardiovascular diseases, in which tyrosine nitration of PA700/S10B- mediated 26S proteasome deregulation is linked to endothelial dysfunction, a key surrogate marker for CVD. To the best of our knowledge, this is the first report on the impacts of oxidative stress on a major subcellular system in multiple animal models with risk factors of cardiovascular disease. Proteasome deregulation could alter essential cellular targets, resulting in diseased conditions (Fig. 6). Therefore, the current study will open new avenue to proteasome-related mechanisms in CVD.
10.1371/journal.pone.0029649.g006Figure 6 Tyrosine nitration of PA700/S10B-mediated proteasome activation is a common pathway leading to endothelial dysfunction in mouse models with cardiovascular risk factors.
Oxidative stress plays an essential role in the parthenogenesis of cardiovascular diseases including diabetes, hypertension and dyslipidemia. ONOO−, formed by the reaction of superoxide with nitric oxide at diffusion-controlled rate, when overproduced, has been demonstrated to affect various pathophysiological events. The presented evidence support a shared mechanism in mouse models with cardiovascular risk factors, in which deregulation of 26S proteasome caused by ONOO−, likely via tyrosine nitration of PA700/S10B, the regulatory complex of 26S proteasomes. Proteasome deregulation could alter essential cellular targets, resulting in early diseased conditions, such as endothelial dysfunction (the initial damaging stage), which may either adaptively improve or worsen the conditions (arrows). Identification of mechanisms underlying these alterations may help to define proper intervention to bring clinic benefit to the patients.
In supporting the notion that oxidative stress links 26S proteasome activation to endothelial dysfunction, we have provided evidence at various setting ranging from cell free and cell culture to whole animal models with common CVD risk factors. In highlights, we found that (1) ONOO− reacts directly with the isolated 26S proteasome, which results in increased 26S proteasome assembly and activation, likely through the enhanced tyrosine nitration of PA700/S10B, the key regulatory complex of 26S proteasomes; (2) such a ONOO−-mediated biochemical process occurs in intact culture cells, because pretreatment of the culture cells with UA, a ONOO− scavenger, prevents the 26S proteasome activation pathway; (3) independent of the type of CVD risk factors in the studied models, aortic 26S proteasome activation are all present; (4) the 26S proteasome activation in animal study manifests as the increased 26S proteasome assembly and activity, which are all accompanied by augmented PA700/S10B tyrosine nitration; (5) activation of aortic 26S proteasome decreases proteins which are important to endothelial homeostasis; such as GTPCH I, which is directly related to NO bioavailability and key to the vascular endothelial function, and Trx, which is related to endothelial function maintenance [24] through reactive oxygen species scavenging [28], apoptosis suppression [29], or survival promotion [30]; (6) intervention through ONOO− inhibition (Tempol administration) or through 26S proteasome inhibition, via pharmaceutical (MG132 administration) or genetic approaches (siRNA knockdown of PA700/S10B), restore GTPCH I and Trx, the proteins that have been shown important for endothelial homeostasis; (7) these data are further validated in vivo in that either inhibition of endogenous ONOO− or 26S proteasome activation could ameliorate the otherwise impaired endothelial function (restoration of acetylcholine-induced vessel relaxation). Emerging data support that proteasome assembly, including 26S complex assembly [6], [31], [32] and individual sub-complex assembly [33], [34], is important for its function. Post-translational modifications of PA700 have been linked to the regulation of proteasome assembly [35] and function [36], although it remains to be established how tyrosine nitration observed in present study would affect the assembly. Therefore, the increased 26S assembly, induced by oxidative stress (e.g. ONOO−) mediated proteasome modifications (e.g. tyrosine nitration), is likely the mechanism for deregulated 26S proteasome, which is linked to endothelial dysfunction shared by the studied mouse models.
A cardinal feature of endothelial dysfunction is impaired endothelium-dependent vasodilatation caused by loss of NO bioavailability [37]. Not a single mechanism alone can explain endothelial dysfunction. Rather, an interplay among multiple regulatory pathways results in pathogenesis of this vascular disorder [38], [39], [40]. Oxidant such as ONOO− attacks various molecules in vascular endothelium, vascular smooth muscle and myocardium, eventually leading to endothelial dysfunction in CVD [2]. In the present study, GTPCH I degradation is attributed to ONOO− activated 26S proteasome in several models. However, we did not exclude the possibility that ONOO− also makes it a good substrate for proteasome, since mild modification by ONOO− results in selective recognition and degradation by proteasome [41]. In either case, activated 26S proteasome would accelerate GTPCH I degradation. It is crucial to maintain appropriate levels of GTPCH I protein for vascular health, since GTPCH I deficiency has been demonstrated in animal models to cause endothelial dysfunction [11] and high blood pressure [16]. In contrast, restoration of GTPCH I has been found beneficial [42], which is due, at least in part, to the improved endothelial function [42], [43] or the suppression of oxidative stress [44]. It is intriguing that the proteins responsible for redox regulation, such as Trx, are in the list of the proteasome substrates in the present study. These proteins have been shown to be endothelial function protective. For instance, the predominant role of Trx to limit oxidative stress directly has been demonstrated in various disease models [45]. Although we presented limited target proteins as evidence of functional proteasome activation, we expect other key molecules may undergo the same pathway which warrants further investigation. In fact, increasing evidence revels that UPS involves in the turn-over of eNOS [46], [47], [48], [49], one of the most endothelial protective molecule, and several other factors essential to endothelium homeostasis [50]. It is widely believed that imbalance between the generation of endothelial-derived relaxing factors (EDFR) and contracting factors (EDCF) may contribute to endothelial dysfunction [51]. Therefore, it would be important to examine if UPS also affects the turnover of these factors globally, an under-examined dimension of proteomics in protein stability in humans [52]. It is also important to note that global activation of 26S proteasome does not guarantee target degradation, a complex process that may require additional (co)factors. However, given the fact that PA700/S10B siRNA knockdown abolishes the effects mediated by proteasomes, 26S proteasome activation does play a decisive role in the degradation of its substrates. In any case, the observed activation of 26S proteasome may be one of the crucial partners for the whole degradation process. It merits further investigation to identify new partners in this unified mechanism.
In summary, the preclinical data presented here indicate that oxidative stress (ONOO−) might be the common linker that connects 26S proteasome activation to endothelial dysfunction, which is prevalent in most types of CVD. Although targets of ONOO− vary [2]_ENREF_2, manipulating the shared one as demonstrated in this study may bring overall beneficial clinic outcome. The findings in this study also may provide insight to drug design for CVD in that targeting specific components of the 26S proteasome might improve the outcomes of medical intervention.
A part of this paper has been presented at the ATVB 2010 Scientific Sessions, San Francisco, CA: April 8–10. 2010.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by National Institutes of Health (NIH) grants (HL079584, HL074399, HL080499, HL105157), a research award from the American Diabetes Association, and funds from the Warren Chair in Diabetes Research of the University of Oklahoma Health Sciences Center (All to MH-Z). MH-Z is a recipient of the National Established Investigator Award of American Heart Association (AHA). JX is supported by a Scientist Development Grant (AHA, 10SDG2600164), a COBRE grant (NIH/National Center for Research Resources: P20 RR 024215), and a research award from the Oklahoma Center for Advancement of Science and Technology (HR11-200). The funders had no role in study design, data collection and analysis.
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BMC CancerBMC Cancer1471-2407BioMed Central 1471-2407-11-5272220427510.1186/1471-2407-11-527Research ArticleBaicalein mediates inhibition of migration and invasiveness of skin carcinoma through Ezrin in A431 cells Wu Bin [email protected] Ji [email protected] Damao [email protected] Weiwei [email protected] Yu [email protected] Youxiang [email protected] Xiaowei [email protected] Hongfu [email protected] Faqing [email protected] Department of Dermatology, Xiangnan College, Chenzhou 423000, P.R. China2 Department of Dermatology, Xiangya Hospital, Central South University, Changsha 410008, P.R. China3 Department of Clinical Laboratory, Xiangya Hospital, Central South University, Changsha 410008, P.R. China4 Metallurgical Science and Engineering, Central South University, Changsha 410008, P.R. China5 Department of Clinical Laboratory, Zhuhai Hospital, Jinan University, Zhuhai 519000, P.R. China2011 28 12 2011 11 527 527 23 7 2011 28 12 2011 Copyright ©2011 Wu et al; licensee BioMed Central Ltd.2011Wu et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
Ezrin is highly expressed in skin cancer and promotes tumor metastasis. Ezrin serves as a promising target for anti-metastasis therapy. The aim of this study is to determine if the flavonoid bacailein inhibits the metastasis of skin cancer cells through Ezrin.
Methods
Cells from a cutaneous squamous carcinoma cell line, A431, were treated with baicalein at 0-60 μM to establish the non-cytotoxic concentration (NCC) range for baicalein. Following treatment with baicalein within this range, total Ezrin protein (both phosphorylated and unphosphorylated forms) and phosphorylated-Ezrin (phos-Ezrin) were detected by western blotting, and Ezrin RNA was detected in A431 cells using reverse transcription-polymerase chain reaction (RT-PCR). Thereafter, the motility and invasiveness of A431 cells following baicalein treatment were determined using wound-healing and Boyden chamber invasion assays. Short-interfering RNA (si-RNA) specifically targeting Ezrin was transfected into A431 cells, and a si-RNA Ezrin-A431 cell line was established by G418 selection. This stable cell line was transiently transfected with Ezrin and mutant Ezrin plasmids, and its motilityand invasiveness was subsequently determined to clarify whether bacailein inhibits these processes through Ezrin.
Results
We determined the range of NCCs for baicalein to be 2.5-40 μM in A431 cells. Baicalein displayed a dose- and time-dependent inhibition of expressions of total Ezrin and phos-Ezrin within this range NCCs. In addition, it exerted this inhibitory effect through the reduction of Ezrin RNA transcript. Baicalein also inhibited the motility and invasiveness of A431 skin carcinoma cells within the range of NCCs, in a dose- and time-dependent manner. A431 cell motility and invasiveness were inhibited by 73% and 80% respectively when cells were treated with 20 μM baicalein. However, the motility and invasiveness of A431 cells containing the Ezrin mutant were not effectively inhibited by baicalein.
Conclusions
Baicalein reduces the migration and invasiveness of A431 cells through the inhibition of Ezrin expression, which leads to the suppression of tumor metastasis.
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Background
Ezrin is a member of the ezrin-radixin-moesin (ERM) protein family that crosslinks the epithelial cell membrane with cytoskeleton. Ezrin helps maintain cell shape and motility, binds to adhesion molecules and participates in the regulation of intracellular signal transduction [1-4]. It is reported that Ezrin has an abnormal expression and a modified subcellular localization in tumor cells. Ezrin serves as a crosslinking molecule between the membranes of keratinocytes and cytoskeleton. Interacting with other adhesion molecules, Ezrin plays an essential part in the development of tumors, by promoting the proliferation and infiltration of tumor cells, metastasis, neovascularisation, and other biological mechanisms involved in malignancy [5-10]. In addition, Ezrin is considered an important potential anti-tumor drug target molecule [8-10].
One important mechanism for regulating the function of Ezrin is through phosphorylation of a conserved threonine residue in the C terminus of Ezrin protein (Thr-567) [11-14]. Non-phosphorylated Ezrin exists in a folded conformation, which results in the masking of its binding sites for other molecules. Phosphorylation at the conserved threonine residue causes conformational changes in Ezrin, unmasking its binding sites [11,14]. Phosphorylation of Ezrin at Thr 567 keeps it open and active, and prolongs its half-life [11]. Phosphorylated-Ezrin (phos-Ezrin) may be involved in various functions, including cell adhesion and motility, as well as the organization of cell surface structure.
Baicalein (5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one) is one of four major flavanoids found in Scutellaria baicalensis Georgi, an herb widely used to treat various inflammatory diseases and ischemia [15]. In addition to its effectiveness against free radicals, baicalein has been reported to have a variety of other functions [16,17]. Recently, baicalein was discovered to have anti-cancer activity through inhibition of the Phosphoinositide 3-kinase (PI3K) pathway [18]. It also exerts proapoptotic activity through reactive oxygen species (ROS)-mediated and Ca2+-dependent mitochondrial dysfunction pathways in various cell types [19]. Bacailein has an inhibitory effect on lung cancer [20], colorectal cancer [21], gastric cancer [22], ovarian cancer [23], breast cancer [24], prostate cancer, and skin cancer [25,26]. Baicalein was also shown to inhibit the Epstein-Barr virus (EBV) early antigen activation induced by 12-O-tetradecanoylphorbol-13-acetate, and inhibit mouse skin tumors in an in vivo two-stage carcinogenesis model [27]. In particular, it was found that its anti-tumor effects in skin cancer were associated with inhibition of the p12-LOX pathway [28]. However, little is known about the molecular mechanisms of its anti-metastatic effects. Here, we show a novel anti-metastatic mechanism for baicalein in skin cancer cells, through inhibition of Ezrin and phos-Ezrin in A431 cells.
Methods
Reagents and antibodies
Chemical reagents, including dimethyl sulfoxide (DMSO), Tris, HCl, sodium dodecyl sulfate, and MTT [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxyme-thoxyphenyl)-2-(4-ulfophenyl)-2H-tetrazolium] were purchased from Sigma-Aldrich (St.Louis, MO). Baicalein was purchased from Sigma-Aldrich (St.Louis, MO), and stored at 4°C under dark conditions. The stock solution of baicalein for incubation with cells was prepared in DMSO and further diluted in the culture medium. The final DMSO concentration in the medium was 0.1% (in control or treated samples), which did not affect cell viability. TRIozl reagent was purchased from Invitrogen. Antibody against Ezrin was purchased from Covance (Berkeley, CA), antibody against phosphorylated Ezrin at Thr-567 (phos-Ezrin Thr-567) was purchased from Cell Signaling Technology (Danvers, MA) and antibodies against β-actin and normal mouse immunoglobulin G (IgG) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The secondary antibodies horseradish peroxidase-linked anti-mouse IgG and anti-rabbit IgG were purchased from Santa Cruz Biotechnology, Inc. The protein assay kit was purchased from Bio-Rad (Herndon, VA).
Cell culture and baicalein treatment
A431 cells (human squamous carcinoma cell line) were purchased from the Shanghai Cell Biological Institute of the Chinese Academy of Science (Shanghai 200007, China). The cell line was cultured as a monolayer in RPMI-1640 medium containing 10% fetal bovine serum, 2 mM L-glutamine, 100 μg/ml penicillin, 100 mg/ml streptomycin (Invitrogen, Carlsbas, CA), and maintained in an incubator with a humidified atmosphere of 95% air and 5% CO2 at 37°C. For baicalein treatment, appropriate amounts of stock solution of baicalein were added to the cultured cells to achieve the indicated concentrations and then incubated for the indicated time points. Following baicalein treatment, cell viability was determined using MTT assays. To determine if baicalein inhibited Ezrin and phos-Ezrin in a dose-dependent manner, A431 cells were treated with 10, 20, and 40 μM baicalein for 24 h. To determine if baicalein inhibited Ezrin and phos-Ezrin in a time-dependent manner, A431 cells were treated with 20 μM baicalein for 24, 48, and 72 h. After treatment with baicalein, the cells were harvested, and proteins were extracted from the cell samples. Expressions of Ezrin and phos-Ezrin were detected by western blotting.
Determination of cell viability (MTT assay)
To evaluate the cytotoxicity of baicalein, MTT assays were performed to determine cell viability. A431 cells were seeded in 96-well plates at a density of 3.5 × 103 cells/well and treated with baicalein at 0-60 μM concentrations at 37°C for 48 h. After the exposure period, cell media was removed, and cells were washed with phosphate-buffered saline (PBS). Thereafter, the media was changed and cells were incubated with 100 μl MTT (5 mg/ml) for 4 h. The total number of viable cells per dish is directly proportional to the production of formazan, which was solubilized in isopropanol, and measured spectrophotometrically at 563 nm [29].
Western blotting analysis
After treatment with baicalein, cell samples were disrupted with 0.6 ml lysis buffer (1 × PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 100 μg/ml phenylmethanesulfonyl fluoride, 10 μg/ml aprotinin, and 1 mM sodium orthovanadate). The cell lysate was then subjected to a centrifugation of 10,000× g for 10 min at 4°C. The supernatant protein concentration of each sample was determined using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Inc., Hercules, CA). Protein (40 μg) from each sample was separated using a 10% polyacrylamide gel and transferred onto a nitrocellulose membrane. The blot was subsequently incubated with 5% non-fat milk in PBS for 1 h to block non-specific binding, and incubated with specific antibodies against Ezrin (Covance) or phos-Ezrin (Cell Signaling Technology) for 2 h and incubated with an appropriate peroxidase-conjugated secondary antibody (Sigma, St. Louis, MO) for 1 h. All incubations were carried out at 37°C. The blot was washed 3 times in PBS, and the signal was developed using 4-chloro-1-napthol/3,3-o-diaminobenzidine. The relative photographic density was quantified by scanning the photographic negative using a gel documentation and analysis system. β-actin was used as an internal control to verify basal protein expression levels of Ezrin and phos-Ezrin, as well as equal protein loading.
RT-PCR for detecting Ezrin RNA
To detect Ezrin RNA in A431 cells following baicalein treatment, we performed reverse transcription-polymerase chain reaction (RT-PCR). A431 cells were initially treated with baicalein at various concentrations (5, 10, 20, and 40 μM) for 48 h. The cell samples were then harvested, and RNAs in these samples were extracted using TRIozl and following the manufacturer's suggested protocol (Invitrogen). For detecting Ezrin RNA, primers for PCR were designed based on GenBank sequences for full-length human Ezrin and β-actin cDNA. The following primer sequences were used: for ezrin, primer 1 (sense) 5'-CTCATCCAGGACATCACCCA-3', primer2 (antisense) 5'-TCACTCCAAGGAAAG CCAAT 3'. The corresponding PCR product was 450 bp. For β-actin, primer 3 (sense), 5'-GACAGGATGCAGAAGGAGAT-3', primer2 (antisense) 5'-TGTGTGGACTTGGGAG AGGACT-3'. The corresponding PCR product was 550 bp. The PCR products were visualized using agarose gel electrophoresis. Following electrophoresis, the relative PCR product band densities were quantified by densitometry using ImageQuant image analysis system (Storm Optical Scanner, Molecular Dynamics). β-actin was used as an internal control to verify the basal expression level of Ezrin and equal RNA loading.
Wound-healing assays
A431 and si-RNA Ezrin-A341 cells (2 × 106) were seeded in 10-mm plates at 37°C for 24 h. Confluent monolayer of A431 was then wounded using a plastic tip. Cells were treated with baicalein at 20 μM, and then photographed after 48 h. Cells moving cross the boundaries lines were counted.
Cell invasion and motility assay
For cell invasion assay, A431 cells were treated with different concentrations of baicalein. After 24 h of treatment, cells were removed by trypsinization, and their invasiveness was tested in vitro using a Boyden chamber invasion assay [30]. Matrigel (Collaborative Biomedical Products, Bedford, MA) was diluted to 0.5 mg/ml with cold filtered distilled water and applied to 8-mm pore size polycarbonate membrane filters. Treated cells were seeded in a Boyden chamber (Neuro Probe, Cabin John, MD) at a density of 1.5 × 104 cells/well in 50 μl of serum-free-medium in the top well of the chamber and then incubated for 12 h at 37°C. The bottom well contained standard medium with 20% fetal bovine serum. The cells that invaded the lower surface of the membrane were fixed with methanol and stained with hematoxylin and eosin. Random fields were counted for cells that had invaded the membrane, using an optics microscope. To determine the effect of baicalein on cell motility, cells were seeded in a Boyden Chamber on membrane filters, which were not coated with Matrigel. The motility of cells treated or untreated with baicalein was measured as previously described [30]. The statistical analysis was corrected for cell viability to clarify the effect of baicalein.
Construction of expression vectors
A full-length Ezrin DNA fragment was generated by PCR and subcloned into a pcDNA3.1 vector (Amersham Biosciences Corp., Piscataway) to generate a pcDNA3.1-Ezrin plasmid [29]. A plasmid containing a mutant form of Ezrin (pcDNA3.1-Ezrin M) was generated with the QuickChange II site-directed mutagenesis kit and Ezrin mutant primers: Primer 1 (sense), 5'-CAGGGCAACGCCAAGCAGCGCAT-3'; Primer 2 (antisense), 5'-ATGCGCTGCTTGGCGTTGCCCTG-3' (Thr567 was muted into Ala 567). The pU6pro vector was used to construct a non-specific control vector containing a scrambled sequence (si-mock), as well as two si-RNA vectors specifically targeting Ezrin (si-Ezrin). The pU6pro-si-mock and pU6pro-si-Ezrin vectors were generated following the manufacturer's recommended protocol. Primers were synthesized for si-mock (general scramble: sense, 5'-TTTGACTACCGTTGTTATAGGTGTTCAAGAGACACC TATAACAACGGTAGTTTTTT-3'; antisense, 5'-CTAGAAAAAACTACCGTTGTTAT AGGTGTCTCTTGAACACCTATAACAACGGTAGT-3') and si-Ezrin (Set 1, 5'-CCCCAAAGATTGG CTTTCC-3' (position in the open reading frame, 704-722); Set 2, 5'-TCCACTATGTGGATAATAA-3' (open reading frame, 140-158) (Ambion, Texas) [29]. All constructs were confirmed by restriction enzyme mapping and DNA sequencing.
Generation of stable cell lines
A431 cells (5.0 × 105) were transfected with pU6pro-si-Ezrin constructs using Lipofectamine2000 reagent (Life Technologies, Inc.) following the manufacturer's suggested protocol. si-RNA Ezrin-A341 stably-transfected cell lines were obtained by selection for G418 resistance (400 μg/ml). Ezrin knockdown was confirmed by assessing Ezrin expression. si-RNA Ezrin-A341 cell lines were transiently transfected with 4 μg of pcDNA3.1, pcDNA3.1-Ezrin, or pcDNA3.1-Ezrin M. After baicalein treatment, the invasion and motility of these stably-transfected cell lines were determined using a Boyden chamber invasion assay.
Statistical analysis
Each assay was performed in triplicate. Data are expressed as the mean ± standard deviation (SD). The statistical significance of the data obtained were evaluated using the Student's t-test (* p < 0.05).
Results
Cytotoxicity assay for baicalein in skin cancer cells
Baicalein is a flavonoid found in Scutellaria baicalensis Georgi, which is the aglycone compound of baicaline. Its chemical structure is showed in Figure 1a. In this study, we determined the cytotoxicity of baicalein by treating A431 cells with various concentrations of baicalein for 48 h. Compared with the control treatment (0.1% DMSO), the cell viability of the samples treated with baicalein at concentrations between 2.5-40 μM was not significantly altered (Figure 1b), indicating that baicalein was not cytotoxic to A431 cells at these dosages. Hence, 2.5-40 μM was determined to be a range of the non-cytotoxic concentration (NCC) of bacailein on A431 cells. This range of concentrations was therefore applied in all subsequent experiments.
Figure 1 Effect of baicalein on the viability of A431 cells. a. Structure of baicalein, which is the aglycone compound of baicaline. b. A431 cells were treated with 0, 2.5, 5, 10, 20, 40, 50, or 60 μM of baicalein for 48 h before being subjected to an MTT assay for cell viability. Data were represented as the mean ± SD from three independent experiments. Baicalein had no cytotoxic effect on A431 cells at a range of 2.5-40 μM. Results were statistically analyzed with a Student's t-test (*p < 0.05). The error bars represent SDs.
Suppression of Ezrin expression by baicalein
To determine if baicalein had an inhibitory effect on Ezrin, a NCC of baicalein (10-40 μM) was used to treat A431 cells, and then total Ezrin and phos-Ezrin expression levels were detected with western blotting. After baicalein treatment, total Ezrin and phos-Ezrin expression levels dramatically decreased compared with the control (Figure 2a, lane 1 vs 2, 3, 4 in the upper and middle panels), and this decrease was dose-dependent. To determine if this inhibition was dependent on the length of time, 20 μM baicalein was used to treat A431 cells for 12, 24, and 48 h, and total Ezrin and phos-Ezrin expression levels were examined. The results suggest that the inhibitory effect of baicalein on total Ezrin and phos-Ezrin was time-dependent (Figure 2b, lane 1 vs 2, 3, 4 in the upper and middle panels). To determine if the inhibitory effect of baicalein on Ezrin protein expression occurs through downregulation of Ezrin RNA, Ezrin RNA expression was detected in A431 cells treated with baicalein using RT-PCR. Following baicalein treatment, Ezrin RNA expression dramatically decreased in a dose-dependent manner (Figure 3a, lane 2 vs 3, 4, 5, 6, and Figure 3b, lane 1 vs. 2, 3, 4, 5).
Figure 2 The inhibitory effects of baicalein on Ezrin and phos-Ezrin in A431 cells. a. A431 cells were treated with baicalein at 0, 10, 20, and 40 μM for 24 h. b. A431 cells were treated with 20 μM baicalein for 12, 24, and 48 h. After baicalein treatment, total protein was extracted from these cell samples. The protein samples were then subjected to western blotting, and the levels of Ezrin and phos-Ezrin expression in these samples were determined. Baicalein inhibited the expression of Ezrin and phos-Ezrin at Thr-567 in a dose- and time-dependent manner. β-actin or Ezrin respectively served as the control for quantifying Ezrin or phos-Ezrin. Three independent experiments were carried out and representative figures are shown.
Figure 3 Inhibitory effect of baicalein on Ezrin RNA in A431 cells. A431 cells were treated with baicalein at 0, 5, 10, 20, and 40 μM for 48 h. After baicalein treatment, the cell samples were harvested, and RNAs in these samples were extracted. Ezrin RNA expression was detected by RT-PCR. 0.1% DMSO served as the blank control. β-actin served as the loading control. The expression of Ezrin RNA in A431 cells gradually decreased with increasing baicalein concentrations. Three independent experiments were carried out and representative figures are shown.
Inhibitory effect of baicalein on the motility and invasiveness of A431 cells
Ezrin is associated with cell motility in tumor invasion. As a first step towards examining the effect of baicalein on Ezrin expression and function, we investigated whether baicalein could inhibit the invasion and motility of A431 cells in vitro. We used wound-healing assays to test the migration of A431 cells with baicalein treatment. Following baicalein treatment, the motility of A431 cells was significantly inhibited, and consequently the cells were unable to migrate into the wound (Figure 4, panel a vs. b and lane 1 vs. 2 in panel c, *p < 0.05). Same as Figure 4-(b), the crawling movement of cells was also inhibited when transfected with Ezrin si-RNA (Figure 4-c). Ezrin expression dramatically decreased following baicalein treatment and Ezrin si-RNA transfection (Figure 4-e). There were virtually no scratches across the boundaries and the migration of A431 cells was not observed. Hence, we concluded that there was no significant difference in the migration of Ezrin si-RNA transfected cells and cells treated with baicalein.
Figure 4 Inhibitory effects of baicalein on A431 cell migration. A431 cells (2 × 106) were seeded in 10-mm plates and incubated at 37°C for 48 h. Confluent monolayers of A431 cells were then wounded using a plastic tip. The cells were treated with 20 μM baicalein, then photographed after 48 h. The cells migrating cross the boundaries lines in the center of the wells were counted, and Ezrin expression was examined in the cell samples. (a) A431 cells treated with 0.1% DMSO. (b) A431 cells treated with 20 μM baicalein. (c) A431 cells transfected with Ezrin si-RNA. (d) Numbers of cells that moved cross the lines (10 fields). (e) Ezrin expression in A431 cells with 0.1% DMSO, 20 μM baicalein or Ezrin si-RNA, respectively. The data are represented as the mean ± SD from three independent experiments. Results were statistically analyzed using one-way analysis of variance (ANOVA) with a post hoc Dunnett's test (* p < 0.05). The error bars represent SDs.
To examine the invasiveness of A431 cells following baicalein treatment, a Boyden chamber coated with Matrigel was used. The results suggested that the number of cells that invaded the lower chamber was significantly reduced by baicalein treatment (Figure 5A-b; Figure 5A-c, lane 2 *p < 0.05). The observed reduction was concentration-dependent, with 80% inhibition occurring when 20 μM baicalein was used (Figure 5A-c). A similar inhibitory effect was also observed for the mobility of baicalein-treated cells. To further test the time and dose-dependence of the inhibitory effects observed, A431 cells were treated with baicalein at various concentrations or time points, and then subjected to analyses for motility and invasiveness. After baicalein treatment, the motility and invasive abilities of cells were decreased in a dose-dependent manner, there was only 27% mobility and 21% invasion when cells were treated with 20 μM baicalein, and 16% motility and in 11% invasion when treated with 40 μM baicalein (Figure 5B, C, *p < 0.05). The cells also displayed a time-dependent reduction in these properties (Figure 5D, E, *p < 0.05, **p < 0.01). Hence, these results indicated that baicalein could inhibit A431 cell migration and invasiveness in a dose and time-dependent manner.
Figure 5 Inhibitory effects of baicalein on the motility and invasiveness of A431 cells. A431 cells were treated with 20 μM bacailein, and cell invasiveness was detected using Boyden Chamber with Matrigel coating. A-(a), Invasiveness of control cells (treated with 0.1% DMSO). A-(b), Invasiveness of cells treated with 20 μM baicalein. Scale bar, 10 μm. A-(c), Numbers of cells invading through the filters of chamber (10 fields). To test for dose-dependence, A431 cells were treated with baicalein at 20 or 40 μM for 48 h, and for the time-course experiment, A431 cells were treated with 20 μM baicalein for 24, 48, and 72 h. The cells were then subjected to analyses for motility and invasion abilities as described in Methods. B. Motility of A431 cells at various concentrations of baicalein. C. Invasiveness of A431 cells at various concentrations of baicalein. D. Motility of A431 cells at various time points. E. Invasion of A431 cells at various time points. The data are represented as the mean ± SD from three independent experiments. Results were statistically analyzed using one-way ANOVA with post hoc Dunnett's test (* p < 0.05). The error bars represent SDs. IR, invasion rate. MR, motility rate.
Suppression of the migration and invasiveness of A431 cells by baicalein through Ezrin
To test if the reduction of motility and invasiveness of A431 cells by baicalein occurs through Ezrin, si-RNA targeting Ezrin was transfected into A431 cells. si-RNA Ezrin-A431 cells were then transiently transfected with pcDNA3.1, pcDNA3.1-Ezrin M or pcDNA3.1-Ezrin and then treated with 20 μM baicalein for 48 h. The motility and invasiveness of the cells were then evaluated. Following baicalein treatment, the invasiveness and motility of cells transfected with pcDNA3.1-Ezrin dramatically decreased compared with the control-transfected cells (Figure 6b, lane 5 vs. 6, and Figure 6c, lane 5 vs. 6, *p < 0.01). Ezrin expression was also decreased compared with control cells (Figure 6a, lane 5 vs. 6 in the upper panel). si-RNA Ezrin-A431 cells transfected with pcDNA3.1-Ezrin M did not show decreased motility and invasiveness (Figure 6b, lane 3 vs. 4, and Figure 6c, lane 3 vs. 4). In the transfects with pcDNA3.1-Ezrin M, Ezrin expression decreased when baicalein treatment (Figure 6a, lane 3 vs. 4), and baicalein degraded Ezrin expression, but the motility and invasion of cells did not alter (Figure 6b, lane 3 vs. 4, and Figure 6c, lane 3 vs. 4). These data suggest that baicalein inhibits Ezrin expression, and consequently decreases the invasiveness and migration of skin cancer cells.
Figure 6 Suppression of motility and invasiveness of A431 cells by baicalein through Ezrin. si-RNA Ezrin-A431 cells were transiently transfected with pcDNA3.1, pcDNA3.1-Ezrin M, or pcDNA-Ezrin and then treated with bacailein. a. Ezrin expression in the transfected cells was detected by western blotting. β-actin served as a loading control. The transfected cells were then subjected to analyses for motility (b) and invasion (c) as described in Methods. Data are represented as the mean ± SD from three independent experiments. Results were statistically analyzed using one-way ANOVA with post hoc Dunnett's test (* p < 0.05). The error bars represent SDs.
Discussion
Ezrin is overexpressed in a variety of neoplastic cells including skin cancers [31], and is involved in the later stages of tumor progression and metastasis. It is expressed in most primary melanomas of the skin and in all metastatic tumors. Ezrin expression correlates with tumor thickness and level of invasion, which suggests an association between Ezrin expression and tumor progression [32]. The intensity of Ezrin immunoreactivity was found to increase with tumor size, as measured by tumor thickness (Breslow classification) and invasion to dermal layers (Clark classification) [33]. The assessment of Ezrin expression may be exploited as a new tool to evaluate the malignancy of human melanoma. In addition, gene therapy or drug treatments aimed at inhibiting actin assembly to the phagosomal membranes may be proposed as a new strategy for the control of tumor aggressiveness [34].
Baicalein, a major flavonoid from a traditional Chinese herb Scutellaria baicalensis Georgi (Huangqin), which is the aglycone compound of baicaline, possesses potent anti-cancer properties. It has been reported that baicalein inhibited mouse skin tumors in an in vivo two-stage carcinogenesis model [27]. Pretreatment of mouse skin with various amounts of baicalein caused inhibition of H2O2 and myeloperoxidase formation by 12-O-tetradecanoylphorbol-13-acetate. These results indicate that baicalein serves as a potential cancer-chemopreventive agent against tumors [25]. In the present work, A431, a human epithelial carcinoma cell line with high malignancy and high expression of Ezrin, was used to investigate the inhibitory mechanism of baicalein. To distinguish anti-cancer effects from cytotoxicity to cells, we first measured the NCCs of baicalein that could be used on A431 cells. We determined 0-40 μM to be the range of NCCs that could be used. We next set out to test if baicalein had an inhibitory effect on Ezrin. We found that Ezrin expression was effectively inhibited by baicalein. In addition, Ezrin function was previously reported to be regulated by phosphorylation of Thr-567 at its C-terminus [11-14]. We found that baicalein could effectively inhibit the phosphorylation of Ezrin at Thr-567 of C terminus. Furthermore, baicalein exerted its inhibitory effect by suppressing the Ezrin RNA transcript. Based on the above results, we believe that baicalein specifically inhibits Ezrin expression and phosphorylation.
Ezrin is involved in a variety of cellular functions, including cell adhesion, motility, and the organization of cell surface structure [3,4]. We speculate that baicalein represses Ezrin expression at RNA transcript, and reduces Ezrin and phos-Ezrin protein expression, inhibits cell migration and tumor invasion. Interestingly, baicalein had no effect on the motility and invasiveness of A431 cells transfected with a mutant form of Ezrin Ala 567. Although Baicalein also inhibited Ezrin expression in the transfect with pcDNA3.1-Ezrin M, baicalein may degrade Ezrin expression, the motility and invasion of cells did not alter after bacailein treatment. Baicalein may inhibit 6-10B cell migration and invasion mainly through reducing phos-Ezrin at Thr 567. These results indicate that baicalein mediates the reduction of migration and invasiveness of A431 cells through phos-Ezrin at Thr567. Baicalein may serves as a novel drug for skin cancer therapy in the future.
Conclusion
Here, we provide evidence that baicalein inhibits the invasive abilities of skin cancers through Ezrin. Baicalein inhibits the migration and invasiveness of A431 cells, following the reduction of Ezrin, phos-Ezrin and Ezrin RNA. However, baicalein had no effect on A431 cells transfected with an Ezrin mutant at 567, suggesting that its inhibitory effect on cell migration and tumor invasiveness occurs mainly through phos-Ezrin at Thr 567.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
BW performed the NCC assay, cell culture and statistical analyses, and wrote the paper. JL performed RT-PCR, designed the PCR primers and controlled the quality of the PCR reactions. DMH constructed the plasmids and tested cell motility and invasion. WWW performed the western blots. YC performed the cell transfections and established the stable cell lines. XWT performed statistical analyses and revised the paper. HFX performed cell culture. FQT coordinated the study and revised the paper. All authors read and approved the final manuscript.
Pre-publication history
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2407/11/527/prepub
Acknowledgements
This work was in part, supported by the National Natural Science Foundation of China (81071718, 81000881, 30973400, and 30670990), Fundamental Research Funds for the Central Universities (21611612), Program for New Century Excellent Talents in University, NCET (NCET-06-0685), the Traditional Chinese Medicine Foundation of Hunan (2008039), the Science and Technology Foundation of Hunan (08FJ3176), and the Science Foundation of Central South University (08SDF07).
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22292084PONE-D-11-2572310.1371/journal.pone.0030980Research ArticleBiologyAnatomy and PhysiologyNeurological SystemDevelopmental BiologyEvolutionary BiologyEvolutionary ProcessesGeneticsModel OrganismsAnimal ModelsNeuroscienceNeurophysiologyFunctional Conservation of the Drosophila gooseberry Gene and Its Evolutionary Alleles Conservation of Drosophila gooseberry FunctionsLiu Wei
1
3
Xue Lei
2
3
*
1
College of Veterinary Medicine, Northwest Agriculture & Forest University, Yangling, Shaanxi, China
2
School of Life Science and Technology, Tongji University, Shanghai, China
3
Institute for Molecular Biology, University of Zürich, Zurich, Switzerland
Singh Shree Ram EditorNational Cancer Institute, United States of America* E-mail: [email protected] and designed the experiments: LX. Performed the experiments: WL LX. Analyzed the data: WL LX. Wrote the paper: WL LX.
2012 23 1 2012 7 1 e3098021 12 2011 30 12 2011 Liu, Xue.2012This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.The Drosophila Pax gene gooseberry (gsb) is required for development of the larval cuticle and CNS, survival to adulthood, and male fertility. These functions can be rescued in gsb mutants by two gsb evolutionary alleles, gsb-Prd and gsb-Pax3, which express the Drosophila Paired and mouse Pax3 proteins under the control of gooseberry cis-regulatory region. Therefore, both Paired and Pax3 proteins have conserved all the Gsb functions that are required for survival of embryos to fertile adults, despite the divergent primary sequences in their C-terminal halves. As gsb-Prd and gsb-Pax3 uncover a gsb function involved in male fertility, construction of evolutionary alleles may provide a powerful strategy to dissect hitherto unknown gene functions. Our results provide further evidence for the essential role of cis-regulatory regions in the functional diversification of duplicated genes during evolution.
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Introduction
During early Drosophila embryogenesis, the antero-posterior axis is progressively defined by the activities of four classes of segmentation genes: maternal coordinate genes, zygotic gap genes, pair-rule genes, and segment-polarity genes [1]–[4]. In addition to their roles in patterning the embryonic epidermis, many segmentation genes participate in other developmental programs like neurogenesis [5], myogenesis [6], and development of imaginal discs [7].
The Drosophila gooseberry (gsb) gene, initially identified as a member of the segment-polarity gene class [1], is required after germ band extension to maintain the ventral epidermal expression of wingless (wg), which suppresses ubiquitous denticle formation, through a wg-gsb autoregulatory loop [8]. In the central nervous system (CNS), gsb is essential for the activation of gooseberry neuro (gsbn) in a segmentally repeated pattern [9], for the differentiation of certain neuroblasts, and for the formation of the posterior commissure in each segment [10]–[14]. Since all known gsb mutant alleles are embryonic lethal [11], possible postembryonic functions of gsb remain largely unknown. Recently, gsb has been found to sustained expression of synaptic homeostasis, indicating the existence of postembryonic functions [15].
gsb encodes a transcription factor including two DNA binding domains in its N-terminal moiety, a paired-domain and a prd-type homeodomain [16]–[18]. Both domains are highly conserved in the N-terminal halves of the Drosophila Paired (Prd) and mouse Pax3 proteins, whose C-terminal halves, however, seem unrelated in their primary sequences to the C-terminal portion of Gsb [17], [19]. prd is a member of the pair-rule gene class, specifying position along the antero-posterior axis with a double-segment periodicity and regulating the expression of segment-polarity genes [20]. The Pax3 gene, a mutation in which is responsible for the Splotch phenotype in mice [21] and Waardenburg's syndrome I in humans [22], [23], plays a pivotal role in myogenesis [24].
Despite their divergent developmental functions, Gsb and Pax3 proteins are able to substitute for most functions of Prd when expressed under the control of the complete prd cis-regulatory region in prd-Gsb and prd-Pax3 transgenes [25]. While prd-Pax3 is able to rescue the cuticular phenotype of prd mutants, prd-Gsb can further rescue prd mutants to adulthood [25], though the rescued males show reduced accessory glands and are sterile [26]. Taken together, these results indicate that Gsb, and Pax3 proteins have retained most functions of Prd despite their highly diverged C-terminal halves and further point to the cis-regulatory region as an important determinant for the functional diversification of these three genes. However, these experiments left unanswered the question of whether Prd and Pax3 proteins could substitute for the normal functions of Gsb.
To address this question, we produced two “evolutionary alleles” [25] of gsb, namely gsb-Prd and gsb-Pax3, which express Prd or Pax3 proteins under the control of the complete gsb cis-regulatory region. We show that both transgenes are able to rescue gsb mutants to fertile adults, albeit at reduced efficiencies, which suggests that both Prd and Pax3 proteins have conserved all normal functions of Gsb. We conclude that the divergent functions of gsb, prd, and Pax3 genes are predominantly determined by their different cis-regulatory regions and are further modified by their protein coding regions. These results provide additional evidence to our previous model that the acquisition of different cis-regulatory elements is the primary mechanism in the evolution of new functions [25]. Since some of the rescued males are sterile, gsb is important for male fertility. This discovery of a male fertility function of gsb suggests that the construction of “evolutionary alleles” may serve as a powerful tool to reveal the hitherto unknown functions of a gene.
Results
Characterization of two hypomorphic gsb alleles
The gsb gene was initially uncovered by two large deficiencies, Df(2R)IIX62 and Df(2R)KrSB1, obtained in a screen for embryonic segmentation mutants [1]. Transheterozygotes of the two deficiencies have lost at least two genes in addition to gsb (
Figure 1A
). Their cuticle shows a strong segment-polarity phenotype (Fig. 2C), which is indistinguishable from that of homozygous Df(2R)IIX62 embryos (
Figure 2B
) [1].
10.1371/journal.pone.0030980.g001Figure 1 Locus of the gsb gene.
(A) gsb mutant alleles. The two deficiencies, Df(2R)IIX62 and Df(2R)SB1, as well as the two hypomorphic alleles, gsb525 and gsbP1155, are depicted. Neighboring genes uncovered by Df(2R)IIX62, zip, uzip, CG3441, and gsbn upstream of gsb, gol and dTKR downstream of gsb, and their direction of transcription are indicated (the rigth telomere of the second chromosome is to the right). Exons are marked by black boxes in the enlarged portion of (A) and also in (B). (B) Map of gsb0-525 abd gsb0-ΔHC transgenes. Both transgenes contain the upstream epidermis enhancers of gsb, GEE and GLE (
Fig. 1A
; Li et al., 1993), the gsb promoter, and the entire 3′ UTR of gsb. In gsb0-ΔHC, 519 bp of coding region between the gsb525 mutation and a SacII site are deleted, resulting in a shift of the open reading frame after the gsb525 nonsense mutation. (C) Sequence surrounding the gsbP1155 insertion site. The negative numbers refer to nucleotides upstream of the transcription start site. The eight nucleotides, duplicated during insertion of the P-element, are underlined.
10.1371/journal.pone.0030980.g002Figure 2 Cuticular phenotypes of gsb mutants.
(A) Df(2R)IIX62/CyO, (B) Df(2R)IIX62, (C) Df(2R)IIX62/Df(2R)KrSB1, (D) Df(2R)IIX62/gsb525, (E) gsb525, (F) gsbP1155, (G) Df(2R)IIX62; gsb0-525 (H) Df(2R)IIX62; gsb0-ΔHC. Note in strong gsb mutants (B, C), the ventral naked cuticle region of each segment is transformed into denticle belt, generating an overall denticle pattern, which is in contrast to wild-type (A). Scale bar: 50 um.
Two alleles affecting only the gsb gene were identified late, including a point mutation, gsb525, and a P-element insertion, gsbP1155
[11]. In gsb525, the codon of the first amino acid of the homeodomain is mutated to a TAA stop codon. In gsb525 embryos, the gsb mRNA level is much reduced by stage 11, presumably because gsb activity depends on the wg-gsb autoregulatory loop [8], and no Gsb protein is detected by immunostaining, while the protein product of the Gsb target, Gsbn, is barely detectable. The fact that gsb525/Df(2R)IIX62 embryos exhibit only a weak cuticular phenotype (
Figure 2D
), while that of gsb525 embryos (
Figure 2E
) is nearly wild-type (
Figure 2A
) implies that gsb525 is not a null allele [11]. Its hypomorphic nature might be explained in two not mutually exclusive ways: the cuticular function of gsb is provided either by a Gsb525 protein truncated before the homeodomain but including the entire paired domain, or by undetectable levels of wild-type Gsb protein generated by a low probability of read-through at the ochre nonsense mutation. To elucidate this question, we prepared two rescue constructs. gsb0-525 contains the same mutation as gsb525, whereas gsb0-ΔHC encodes only the truncated Gsb525 protein (
Figure 1B
). Both of these two constructs are under the control of the gsb upstream region including the gsb cuticle enhancers GEE and GLE [26]. Evidently, only gsb0-525 can rescue the cuticle phenotype (
Figure 2G
), whereas gsb0-ΔHC cannot (
Figure 2H
). This demonstrates that in gsb525 embryos an undetectable level of wild-type Gsb protein is produced that is nearly enough to rescue the cuticular function of gsb.
gsbP1155 is also an interesting allele. It is an insertion of a P element located only 54 bp upstream of the gsb transcription start site (
Figure 1C
). This P-element insertion leads to largely reduced gsb mRNA and protein levels in homozygous embryos. While these mutants show a wild-type cuticular phenotype (
Figure 2F
) and only mild CNS defects [11], we observed a strongly reduced expression of gsbn (data not shown). It follows that gsbP1155 is a weaker allele than gsb525.
Generation of gsb-Prd and gsb-Pax3 transgenic flies
Previous work demonstrated that a gsb rescue construct, gsb-res, was able to perform all the known gsb functions and rescue gsb mutants to adulthood [9], [11], which suggests that all essential gsb enhancer elements are included in this gsb transgene (
Figure 3
). To examine whether and to what extent the Prd and Pax3 proteins are able to substitute for the normal functions of Gsb, two rescue constructs, namely gsb-Prd and gsb-Pax3, were obtained by replacing the gsb coding region in gsb-res by that of prd and Pax3, respectively (
Figure 3
). Transgenic files were generated by P-element-mediated transformation in the Drosophila germlines [31]. Several independent lines were obtained for each construct. Only transgenic lines that were homozygous viable were selected for further investigation.
10.1371/journal.pone.0030980.g003Figure 3 Map of gsb-res, gsb-Prd and gsb-Pax3 transgenes.
The gsb-res transgene corresponds to the enlarged 20-kb genomic fragment in
Fig. 1A
, which includes the gsb transcribed region as well as adjacent 14-kb upstream and 3-kb downstream sequences [9]. The upstream sequence also contains the 5′ portion of the gsbn up to part of the third exon. In gsb-Prd and gsb-Pax3 transgenes, the gsb coding region (except of a small region encoding the C-terminus) is replaced by prd and Pax3 cDNAs, while upstream and downstream regions are retained. The gsb intron is also retained by inserting it between sequences of the gsb and prd or Pax3 leaders. Coding regions are indicated as black boxes except for the paired-domain (PD) and the prd-type homeodomain (HD) which are hatched. The gsb and gsbn introns are indicated as open boxes. The transcription start of gsb is marked by 0, and poly(A) addition signals AATAAA are indicated.
It has been previously shown that in wild-type embryos, Gsb protein is initially expressed during blastoderm at the end of cellularization in eight stripes in every other segment, which correspond to the odd-numbered Gsb stripes [9]. At gastrulation, the even-numbered Gsb stripes emerge between the odd-numbered stripes to generate a segmentally repeated expression pattern. Toward the end of germ band extension, Gsb protein reaches its highest levels in the ectoderm and becomes laterally restricted to the neuroectodermal region (
Figure 4A
). As expected, in gsb-Prd and gsb-Pax3 embryos, the Prd protein (
Figure 4B
) and Pax3 mRNA (
Figure 4C
) are expressed in patterns that are indistinguishable from that of endogenous Gsb protein (
Figure 4A
). At this time of development the endogenous Prd protein is barely detectable in the epidermis [27].
10.1371/journal.pone.0030980.g004Figure 4 Expression of Gsb and Prd proteins and Pax3 mRNA under control of the gsb cis-regulatory region.
Expression of Gsb protein in wild-type embryos (ry506; A), of Prd protein in transgenic gsb-Prd embryos (B), and of Pax3 mRNA in transgenic gsb-Pax3 embryos (C) at the extended germ band stage. Wild-type embryos were stained with anti-Gsb antiserum and transgenic embryos, collected from homozygous gsb-Prd or gsb-Pax3 stocks, were stained with anti-Prd antiserum or hybridized in situ with digoxigenin-labeled Pax3 cDNA. Unfolded embryos are shown and oriented with their anterior to the left. Scale bar: 100 um.
Rescue of gsb target gene expression by gsb-Prd and gsb-Pax3
Previous work has shown that Gsb is required to maintain late wg expression in the ventral epidermis through a wg-gsb autoregulatory loop [8]. In homozygous Df(2R)IIX62 embryos, Wg starts to decay in the ventral epidermis after 6 hours [8] and is no longer detectable at stage 13 (
Figure 5B
), while it remains expressed in wild-type embryos (
Figure 5A
). By introducing gsb-Prd or gsb-Pax3 transgenes into such gsb mutant embryos, the Wg expression pattern is fully rescued by one copy of either transgene (
Figure 5C, D
).
10.1371/journal.pone.0030980.g005Figure 5 Rescue of Wg and Gsbn expression in gsb mutant embryos by gsb-Prd and gsb-Pax3 transgenes.
Expression of Wg (A–D) and Gsbn (E–H) proteins in wild-type (A, E), homozygous Df(2R)IIX62 (B–D) or transheterozygous Df(2R)IIX62/gsb525 (F–H) gsb mutant embryos carrying no (B, F), one copy of the gsb-Prd (C, G) or gsb-Pax3 (D, H) transgene. Embryos at stage 13 (A–D) or stage 10 (E–H) are oriented with their anterior to the left and dorsal side up. Embryos were collected from crosses between Df(2R)IIX62/CyO, hb-LacZ; gsb-Prd/+ or Df(2R)IIX62/CyO, hb-LacZ; gsb-Pax3/+ males and Df(2R)IIX62/CyO, hb-LacZ (A–D) or gsb525/CyO, hb-LacZ females (E–H), and double stained for ß-galactosidase and or Gsbn protein with rabbit antiserum against ß-galactosidase and anti-Wg monoclonal antibodies or rabbit anti-Gsbn antiserum. Embryos stained with ß-galactosidase have at least one copy of wild-type gsb allele and were used as control (A, E). One quarter of the embryos did not stain for ß-galactosidase. Half of these embryos did not express Wg in the ventral epidermis and Gsbn in the CNS as expected for gsb mutants. The other half displayed rescued expression patterns, which suggested the presence of the transgenes. Scale bar: 100 um.
Beginning with stage 9, Gsb is expressed in delaminating neuroblasts, where it is required for the activation of gsbn
[9]. This is apparent from a complete loss of Gsbn expression in Df(2R)IIX62/gsb525 embryos at the extended germ band stage (
Figure 5F
), while Gsbn expression is strongly expressed in the CNS of wild-type embryos at this stage (
Figure 5E
). Gsbn expression in such mutants are rescued by gsb-Prd or gsb-Pax3 transgene, respectively (
Figure 5G, H
). Taken together, these results demonstrate that Prd and Pax3 proteins can substitute for Gsb function in the transcriptional activation of two essential target genes.
Rescue of gsb- cuticular phenotype by gsb-Prd and gsb-Pax3
One conspicuous feature of the Drosophila larva is the metameric ventral cuticular pattern, which crucially depends in each segment on the products of the segment-polarity genes. Embryos lacking gsb function exhibit a segment-polarity cuticle defect [1], which consists of mirror image duplications of denticle belts into the posterior portions of each segment where naked cuticle would develop in wild-type embryos (compare
Figure 6A, B
). This phenotype is caused by the loss of late Wg expression, which is required to repress the ubiquitous denticle formation in the ventral epidermis [8]. Consistent with the result that both gsb-Prd and gsb-Pax3 can rescue the late Wg expression in gsb mutants, both transgenes are able to fully rescue the cuticular phenotype of homozygous Df(2R)IIX62 embryos when present as a single copy (
Figure 6C, D
). It follows that Prd and Pax3 proteins are able to perform the cuticular function of Gsb.
10.1371/journal.pone.0030980.g006Figure 6 Rescue of the cuticular phenotype of gsb mutant embryos by gsb-Prd and gsb-Pax3 transgenes.
Ventral view of cuticle preparations of wild-type (ry506; A) and homozygous Df(2R)IIX62 embryos without (B) and with one copy of the gsb-Prd (C) or gsb-Pax3 transgene (D) are shown under dark-field illumination (anterior is up). Wild-type and gsb mutant embryos were collected from the Df(2R)IIX62/SM1 stock, while gsb mutant embryos carrying one copy of the transgenes were collected from crosses between Df(2R)IIX62/SM1; gsb-Prd or Df(2R)IIX62/SM1; gsb-Pax3 males and Df(2R)IIX62/SM1 females. gsb mutants were distinguished from wild type by the presence of the zip phenotype, a deformed head structure resulting from the deletion of the zip gene, which is uncovered by Df(2R)IIX62
[30]. Scale bar: 50 um.
Rescue of gsb- CNS phenotype by gsb-Prd and gsb-Pax3
In addition to its function in patterning the epidermis, gsb plays an important role in the development of the embryonic CNS [9]–[12]. Most prominently, posterior commissures (
Figure 7A
) are missing or reduced in each segment of Df(2R)IIX62/gsb525 embryos (
Figure 7B
). This CNS phenotype can be fully rescued by one copy of the gsb-Prd (
Figure 7C
) or gsb-Pax3 transgene (
Figure 7D
), which indicates that Prd and Pax3 proteins are able to replace the Gsb function in the CNS.
10.1371/journal.pone.0030980.g007Figure 7 Rescue of the CNS phenotype of gsb mutant embryos by gsb-Prd and gsb-Pax3 transgenes.
Patterns of longitudinal and commissural axons in the CNS of wild-type (ry506; A) and Df(2R)IIX62/gsb525 embryos without (B) and with one copy of the gsb-Prd (C) or gsb-Pax3 transgene (D). Embryos at stage 15 were collected from crosses between Df(2R)IIX62/CyO, hb-LacZ; gsb-Prd/+ or Df(2R)IIX62/CyO, hb-LacZ; gsb-Pax3/+ males and gsb525/CyO, hb-LacZ females, and double stained with rabbit antiserum against ß-galactosidase and monoclonal antibody BP102. One quarter of the embryos did not stain for ß-galactosidase as expected. Half of these embryos have missing or reduced posterior commissures as expected for gsb mutants, the other half displays fully rescued commissural patterns as in wild-type embryos. Scale bar: 10 um.
Rescue of gsb mutants to adulthood by gsb-Prd and gsb-Pax3
To test if Prd and Pax3 proteins are able to substitute for all Gsb functions, we tested the ability of gsb-Prd and gsb-Pax3 transgenes to rescue gsb mutants to adulthood. For this purpose, a deficiency, Df(2R)IIX62, and two strong alleles of gsb, gsb525 and gsbP1155, were used. Homozygous or heterozygous combinations of these three alleles are lethal during embryogenesis, which shows that gsb is required for postembryonic viability. Although rescue efficiencies are less than half of that of gsb-res, one copy of gsb-Prd or gsb-Pax3 is able to rescue about a quarter of Df(2R)IIX62/gsb525 embryos to adulthood (
Table 1
). For all three transgenes, two copies result in 50% higher rescue efficiencies than one copy (
Table 1
), which suggests that at least one gsb function required for the viability is dosage dependent. Consistent with this interpretation, one copy of the transgenes is able to rescue a much higher proportion of Df(2R)IIX62/gsbP1155 or gsb525/gsbP1155 embryos to adulthood (
Table 1
). Therefore, both Prd and Pax3 proteins are able to substitute for all Gsb functions required for survival to adulthood, albeit at lower efficiencies.
10.1371/journal.pone.0030980.t001Table 1 Rescue of gsb mutant embryos to viable adults by gsb-Prd and gsb-Pax3 transgenes.
gsb-res (%)
gsb-Prd (%)
gsb-Pax3 (%)
1 copy 2 copies 1 copy 2 copies 1 copy 2 copies
Df(2R)IIX62/gsb525
62 (238/385) 96 (194/203) 21 (90/429) 31 (104/339) 27 (88/326)
41 (96/234)
Df(2R)IIX62/gsbP1155
86 (607/707) nd 51 (144/284) nd 74 (192/260)
nd
gsb525/gsbP1155
99 (344/346)
nd
61 (230/376)
nd
77 (226/293)
nd
Percentage of rescued gsb- flies harboring one or two copies of gsb-res, gsb-Prd or gsb-Pax3 transgenes (actual numbers of rescued flies per total number of expected gsb mutants are given in parentheses). Df(2R)IIX62/gsb525 flies carrying one or two copies of the transgenes were obtained as offspring from the crosses between Df(2R)IIX62/SM1; P/P (P stands for the transgenes) males and gsb525/SM1 or gsb525/SM1; P/P females. Df(2R)IIX62/gsbP1155 and gsb525/gsbP1155 flies carrying one copy of the transgenes were obtained from the crosses between Df(2R)IIX62/SM1; P/P or gsb525/SM1; P/P males and gsbP1155/SM1 females. nd, not determined.
gsb is required for male fertility
Since all known gsb mutant alleles are lethal during embryogenesis [1], [9], [11], the adult functions of gsb remain unknown. Interestingly, most of the Df(2R)IIX62/gsb525 males rescued by one copy of gsb-Prd or gsb-Pax3 are sterile (
Table 2
), while females are fully fertile (data not shown). Therefore, gsb is endowed with a function that is essential for male fertility. Two copies of gsb-Prd or gsb-Pax3 result in significantly enhanced fertilities of Df(2R)IIX62/gsb525 males (
Table 2
), which suggests that this male fertility function is also dosage dependent. Consistent with this explanation, one copy of gsb-res rescues fertility in 39% of the Df(2R)IIX62/gsb525 males, while two copies rescue male fertility almost completely (
Table 2
). In addition, one copy of gsb-Prd or gsb-Pax3 is able to rescue fertility in about half of the Df(2R)IIX62/gsbP1155 males and in three quarters of the gsb525/gsbP1155 males (
Table 2
), whereas one copy of gsb-res suffices to fully rescue male fertility in these two mutant combinations (
Table 2
). We conclude that gsb is required for male fertility, a function for which both Prd and Pax3 proteins are able to substitute.
10.1371/journal.pone.0030980.t002Table 2 Rescue of fertility of gsb mutant males by gsb-Prd and gsb-Pax3 transgenes.
gsb-res (%)
gsb-Prd (%)
gsb-Pax3 (%)
1 copy 2 copies 1 copy 2 copies 1 copy 2 copies
Df(2R)IIX62/gsb525
39 (36/92) 91 (20/22) 9 (2/23) 30 (6/20) 15 (3/20)
35 (6/17)
Df(2R)IIX62/gsbP1155
92 (90/98) nd 43 (16/37) nd 48 (16/33)
nd
gsb525/gsbP1155
95 (74/78)
nd
77 (23/30)
nd
75 (21/28)
nd
Percentage of fertile males among gsb mutant males that were rescued by one or two copies of gsb-res, gsb-Prd or gsb-Pax3 transgenes (actual numbers of fertile males per total number of rescued males are given in parentheses). Rescued males were obtained from crosses described in legend of table 1 and were placed individually with at least three wild-type virgin females in fresh vials to score fertility. nd, not determined.
Discussion
Evolutionary alleles of gsb
The Drosophila gsb and prd and mouse Pax3 genes encode transcription factors that share in their N-terminal moieties two DNA binding domains, a paired-domain and a prd-type homeodomain [16]–[19]. The homology between the N-terminal parts of the three proteins suggests that they were derived from a common ancestor, and thus might have retained some same abilities, despite their divergent C-terminal sequences and apparently distinct developmental functions [32]. Indeed, gsb-Prd and gsb-Pax3, which express Prd or Pax3 protein under the control of the gsb cis-regulatory region, are able to execute all in vivo functions of gsb, though less efficiently. Hence, both Prd and Pax3 may be considered as leaky mutant proteins of Gsb, whereas gsb-Prd and gsb-Pax3 are hypomorphic or ‘evolutionary’ alleles of gsb, as the coding regions of the three genes have been derived from a common ancestral gene during the course of evolution. These two ‘evolutionary’ alleles are weaker than the weakest previously known gsb allele, gsbP1155, which generates a normal cuticular pattern but displays a weak CNS phenotype and is homozygous lethal during embryogenesis [11]. As these two new alleles have uncovered the previously unknown function of gsb required for male fertility, construction of evolutionary alleles may serve as an additional approach to discover unknown functions of a gene [25].
Although the N-terminal portions of the three proteins are rather conserved, their C-terminal parts have diverged to an extent that no obvious similarity in the primary sequences could be perceived [17], [19]. Thus, it is particularly interesting that both Prd and Pax3 proteins have retained the potential to perform all the normal functions of Gsb, which suggests that all the important functional motives in the C-terminal part of Gsb have been conserved in the C-termini of Prd and Pax3, presumably in the 3-D structures. It follows that the functional diversification of gsb, prd, and Pax3 reside in their cis-regulatory rather than their divergent C-terminal coding regions. Therefore, our results are consistent with, and add further weight to, the hypothesis that the acquisition of new enhancer elements by a gene plays a dominant role in evolution [25], [26].
Evolutionary relationship between Gsb, Prd and Pax3 proteins
Our previous work has shown that Pax3 can perform only the cuticle function, but not the viability and male fertility functions of Prd [25], [33]. Here we report that Pax3 is able to substitute for all Gsb functions in promoting embryonic CNS and cuticle development, postembryonic viability, and male fertility. Thus, in terms of functional conservation, Pax3 seems to be more closely related to Gsb than to Prd. It follows that Gsb and Pax3 are functionally also closer to the common ancestor than Prd. As an independent test of this conclusion, it would be interesting to see if Gsb is a better substitute for Pax3 functions than Prd.
In support of this hypothesis, Pax3 resembles Gsb better than Prd in primary sequences. For Gsb and Pax3, but not Prd, share an octapeptide that is located between the paired-domain and the prd-type homeodomain [16], [19], [32]. In addition, Prd possesses near its C-terminal end a PRD repeat [34], which is also found in the products of several other genes that are important for early development [34], [35], but not in Gsb and Pax3. Therefore, the common ancestor of Gsb, Prd, and Pax3 probably included, in addition to the paired-domain and the prd-type homeodomain, the octapeptide in between. After duplication and separation during the course of evolution, Gsb and Pax3 retained these three motives while Prd lost the octapeptide, but instead, obtained the PRD repeat.
In addition to its embryonic functions, gsb is also required for male fertility. This function appears to be dosage dependent, as better rescue efficiencies were achieved by either increasing the copy number of the transgenes or using weaker gsb mutant alleles (
Table 2
). Interestingly, prd is also required for male fertility, in particular for the development of accessory glands [33], [34]. Since Gsb is able to substitute for all Prd functions that are required for survival to adulthood [25], but not its male fertility function [28], the male fertility function of Prd might have evolved after its separation from Gsb or have been subjected to strong selection during the course of evolution.
Dosage effect of Pax genes
Pax genes encode transcription regulators characterized by the presence of the paired-domain [32]. In vertebrates, Pax genes exhibit strong dosage effects, as most Pax genes are haploinsufficient [36], and overexpression of Pax6 in mice leads to severe eye abnormalities [37]. In Drosophila, prd shows haploinsufficiency in an adult segmentation phenotype, and the prd evolutionary allele prd-Gsb displays strong dosage effects for all prd functions required for survival to adulthood [25]. In addition, overexpression of eyeless, the Drosophila homolog of Pax6, results in a small eye phenotype [38]. Here we show that one copy of the gsb rescue construct, gsb-res, is able to rescue only 62% of the Df(2R)IIX62/gsb525 mutants to adulthood (
Table 1
), of which only 39% of the males are fertile (
Table 2
). However, higher rescue efficiencies were scored in both cases by two copies of the transgene (
Table 1
,
2
), which indicates a dosage dependence of gsb functions in promoting viability and male fertility. This interpretation was confirmed by the use of two different combinations of gsb mutants, and by two gsb evolutionary alleles, gsb-Prd and gsb-Pax3 (
Table 1
,
2
). A dosage effect was also reported for gsb functions in embryonic cuticle and CNS development, as reflected by differences in penetrance of the cuticle and CNS phenotypes in various combinations of different gsb mutant alleles [11]. Since the hypomorphic gsb mutants, gsb525 and gsbP1155, display a normal cuticle but defects in the CNS [11], and one copy of gsb-res is able to fully rescue the CNS phenotype but to rescue the viability and male fertility functions only partially (
Table 1
,
2
) in Df(2R)IIX62/gsb525 mutants, the cuticle function is least sensitive while the viability and male fertility functions are most sensitive to a decrease in the level of Gsb activity. The incomplete rescue of the viability and male fertility functions in Df(2R)IIX62/gsb525 mutants by one copy of gsb-res may result from two effects. First, the deficiency Df(2R)IIX62, which deletes, in addition to gsb, several other genes including gsbn
[17], [30], which is downstream of gsb, might affect the viability and male fertility. Second, gsb-res expresses Gsb protein at a subnormal level [9], [11], which may result from a position effect of the P-element insertion or from the absence of additional gsb enhancer element(s) from the transgene.
The male fertility function of gsb
In addition to its embryonic functions, gsb is also required for the male fertility. This function appears dosage dependent, for better rescue efficiencies were achieved by either increasing the copy number of the transgenes or using weaker gsb mutant alleles (
Table 2
). gsb may get involved in male fertility via several means. First, Gsb plays pivotal role in the development of ejaculatory duct that is required for the transfer of accessory gland secretions and sperm to females during copulation. Ejaculatory duct also secretes components of seminal fluid that might be essential for sperm fertility [39]. Second, Gsb is expressed in the secondary cells of adult accessory glands, suggesting a role of Gsb in the regulation of accessory gland secretions that are crucial for the male fertility (33). Third, males heterozygous for Df(2R)IIX62, which deletes gsb and its downstream gene gsbn, behave less aggressive in copulation (data not shown). This phenotype can be rescued by adding one copy of gsb-res (data not shown), implying the impaired Gsb-Gsbn pathway is responsible for this behavioral defect. In support of this interpretation, both Gsb and Gsbn are expressed in the leg and antenna imaginal discs (W.L., L.X. and M.N., unpublished observation), suggesting a role of gsb and gsbn in the development of leg and antenna, both of which have been shown to be important for eliciting proper male sexual behavior [40].
Interestingly, prd is also required for male fertility, for prd mutant males rescued by two differently modified prd transgenes, prd-Gsb [25] and prdRes [41], are sterile, despite their capabilities to copulate and transfer sperm to females [33]. These males have severely reduced or no accessory glands [33], [41], suggesting prd is essential for accessory gland development. Hence, prd and gsb, though both are required for male fertiltiy, are involved in distinct developmental programs during metamorphosis. Since Gsb is able to substitute for all Prd functions that are required for survival to adulthood [25], but not its male fertility function [33], the male fertility function of Prd might have evolved after its separation from Gsb or have been subjected to strong selection during the course of evolution.
Materials and Methods
Plasmid constructions and generation of transgenic flies
Mutations were introduced into gsb0-525 and gsb0-ΔHC by PCR mutagenesis. Taking pKSpL5-Gsb [27] as template, the following primers were used: gsb-8 (5′-GTC GTC CGG GCT AGC CTT TAT TTC CT-3′), gsb-11 (5′-GGA AAT AAA GGC GAT CGC GGA CG -3′, gsb-12 (5′-CGT CCG CGA TCG CCT TTA TTT CC-3′), T3 primer, and T7 primer. Fragments containing the mutations were cloned into gsb-0 [27], the gsb complete leader region and intron were also recovered.
The gsb-Prd and gsb-Pax3 constructs were derived from gsb-res
[9] in three steps. First, the 1-kb gsb intron was obtained as a PCR product with the primer gint1 (5′-GTC TAG AGT AAG CAC CGA CAG ATA GA-3′) and gint2 (5′-GTC TAG ACT GGA AGA ATT AGA GAA ACA-3′), digested with XbaI and inserted into the SpeI site of pKSpL5-Prd and pKSpL5-Pax3 [27] to generate pKSgint-Prd and pKSgint-Pax3, respectively. Subsequently, the 3.4-kb XbaI fragments from pKSgint-Prd and pKSgint-Pax3 were cloned into the AvrII site of gsb-0 to produce gsb-int-Prd and gsb-int-Pax3. Finally, gsb-Prd and gsb-Pax3 were constructed by replacing the 5.6-kb NheI-XbaI fragment in gsb-res with the corresponding fragments from gsb-int-Prd and gsb-int-Pax3, respectively.
The gsb-Prd and gsb-Pax3 constructs were injected together with pUChspΔ2-3 helper plasmid into ry506 embryos and ry
+ transformants were selected.
Immunostaining and in situ hybridization of embryos
Embryo collection, fixation, and immunostaining were carried out as described [28]. Polyclonal antibodies against Prd (1∶500) [28] Gsb, and Gsbn (1∶1000) [9], monoclonal antibody against Wg (1∶100) [29], and monoclonal antibody BP102 (1∶50), which reveals the patterns of the longitudinal and commissural axons in the CNS [11], have been described. Polyclonal anti-ß-galactosidase antibody (1∶1000) was obtained from Cappell.
In situ hybridization with digoxigenin-labeled Pax3 cDNA was performed essentially as described [25].
Cuticle preparation
Embryos were collected and allowed to develop for 24 h at 25°C before cuticles were prepared as described [1].
Fly strains and rescue experiments
Three gsb alleles were used in this work: Df(2R)IIX62, a gsb null allele that deletes gsb, gsbn, and five additional genes [17], [30]; gsb525, a strong hypomorphic allele in which the first amino acid of the homeodomain is converted to a stop codon [11]; and gsbP1155, a hypomorphic allele with a P-element inserted into the gsb promoter region [11]. To rescue the cuticle, CNS, viability, and male fertility functions of gsb by the transgenes, we used the following fly stocks: (1) Df(2R)IIX62/SM1, (2) gsb525/SM1, (3) gsbP1155/SM1, (4) Df(2R)IIX62/SM1; gsb-res, (5) gsb525/SM1; gsb-res, (6) Df(2R)IIX62/SM1; gsb-Prd, (7) gsb525/SM1; gsb-Prd, (8) Df(2R)IIX62/SM1; gsb-Pax3, and (9) gsb525/SM1; gsb-Pax3.
We are deeply indebted to Markus Noll for his invaluable advice, support, and encouragement throughout this project. We are grateful to Thomas Gutjahr for technical assistance and Fritz Ochsenbein for expert artwork. We thank P. Gruss for a Pax3 cDNA, S. Cohen for anti-Wg monoclonal antibody, and C. S. Goodman for BP102 monoclonal antibody. We are obliged to Hans Noll for comments on the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work has been supported by the following three funds: 1. Swiss National Science Foundation, Grant No. 31-40874.94; 2. National Natural Science Foundation of China, Grant No. 30971681; 3. Fund from NWAF (No. Z11021005). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22291934PONE-D-11-2176910.1371/journal.pone.0030322Research ArticleMedicineCritical Care and Emergency MedicineGastroenterology and HepatologyLiver DiseasesInfectious HepatitisInfectious DiseasesViral DiseasesHepatitisSurgeryDowngrading MELD Improves the Outcomes after Liver Transplantation in Patients with Acute-on-Chronic Hepatitis B Liver Failure Downgrading MELD Improves the Outcomes after LTLing Qi
1
Xu Xiao
1
Wei Qiang
1
Liu Xiaoli
2
Guo Haijun
1
Zhuang Li
1
Chen Jiajia
2
Xia Qi
2
Xie Haiyang
1
Wu Jian
1
Zheng Shusen
1
*
Li Lanjuan
*
1
Key Lab of Combined Multi-Organ Transplantation, Ministry of Public Health, Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
2
State Key Lab for Diagnosis and Treatment of Infectious Diseases, Department of Infectious Diseases, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
Gluud Lise Lotte EditorCopenhagen University Hospital Gentofte, Denmark* E-mail: [email protected] (LL) (LL); [email protected] (SZ) (SZ)Conceived and designed the experiments: QL XX SZ LL. Performed the experiments: QW XL HG LZ JC QX HX JW. Analyzed the data: QL XX. Contributed reagents/materials/analysis tools: SZ LL. Wrote the paper: QL.
2012 24 1 2012 7 1 e3032230 10 2011 19 12 2011 Ling et al.2012This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
High score of model for end-stage liver diseases (MELD) before liver transplantation (LT) indicates poor prognosis. Artificial liver support system (ALSS) has been proved to effectively improve liver and kidney functions, and thus reduce the MELD score. We aim to evaluate whether downgrading MELD score could improve patient survival after LT.
Methodology/Principal Findings
One hundred and twenty-six LT candidates with acute-on-chronic hepatitis B liver failure and MELD score ≥30 were included in this prospective study. Of the 126 patients, 42 received emergency LT within 72 h (ELT group) and the other 84 were given ALSS as salvage treatment. Of the 84 patients, 33 were found to have reduced MELD score (<30) on the day of LT (DGM group), 51 underwent LT with persistent high MELD score (N-DGM group). The median waiting time for a donor was 10 for DGM group and 9.5 days for N-DGM group. In N-DGM group there is a significantly higher overall mortality (43.1%) than that in ELT group (16.7%) and DGM group (15.2%). N-DGM (vs. ECT and DGM) was the only independent risk factor of overall mortality (P = 0.003). Age >40 years and the interval from last ALSS to LT >48 h were independent negative influence factors of downgrading MELD.
Conclusions/Significance
Downgrading MELD for liver transplant candidates with MELD score ≥30 was effective in improving patient prognosis. An appropriate ALSS treatment within 48 h prior to LT is potentially beneficial.
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Introduction
One-third of global individuals infected with hepatitis B virus (HBV) reside in China, with 130 million carriers, 30 million chronically infected, and 300 thousand per year HBV-related deaths [1]–[3]. Because of the high prevalence of hepatitis B, acute exacerbation of chronic hepatitis B and acute deterioration of cirrhosis are the most common causes of liver failure, contributing to especially high mortality in China. Most recently, these types of liver failure has been considered as acute-on-chronic liver failure (ACLF), which was clearly defined by Asian Pacific Association for the Study of the Liver as ‘acute hepatic insult manifesting as jaundice and coagulopathy, complicated within 4 weeks by ascites and/or encephalopathy in a patient with previously diagnosed or undiagnosed chronic liver disease’ [4]. When ACLF progresses to multi-organ dysfunction such as hepatorenal syndrome and hepatic encephalopathy, the prognosis is dismal unless liver transplantation (LT), the only definitive therapy to salvage these patients, is performed [4]. Since model for end-stage liver diseases (MELD) score was used for organ allocation, candidates with ACLF have the priority to gain the donor liver and receive emergency LT because patients with ACLF usually have high MELD score [5], [6]. However, ‘high-grade’ (≥30) MELD scores in ACLF indicate poorer prognosis after liver support treatment even LT [6]–[8]. For ACLF patients with MELD score≥30, the 30-day survival was less than 10% after ALSS salvage [7], and the 1-year survival after LT was much lower than that of patients with MELD score <30 (33.3% vs. 77.8%) [9]. Thus when the MELD score is <30 it may be the optimal time to perform LT for patients with ACLF [9].
Artificial liver support system (ALSS) has been proved to be an effective way to improve liver function and thus serve as a bridge to LT [10]. After such treatment, total bilirubin, international normalized ratio, encephalopathy, and serum creatinine can be remarkably improved and thus MELD score was reduced [11]. According to the treatment guidelines for ALSS formulated by Artificial Liver Group, Chinese Association of Infectious and Parasitic Diseases and Chinese Medical Association [12], the management of ACLF patients were principally supportive with ALSS treatment prior to LT. In this study, we performed ALSS to salvage ACLF patients with ‘high-grade’ MELD score. We aim to evaluate whether downgrading pre-transplant MELD score could improve patient survival after LT, and to determine the possible influence factors of downgrading MELD.
Methods
Patient characteristics
A total of 189 adult patients with acute-on-chronic hepatitis B liver failure (ACLF-HBV) underwent primary LT between January 2001 and June 2010 at the First Affiliated Hospital, Zhejiang University School of Medicine, China. Informed consent was obtained from all donors and recipients before LT. Each organ donation or transplant in our centre was strictly selected according to the guidelines of the Ethical Committee of our hospital, the regulation of Organ Transplant Committee of Zhejiang province and the Declaration of Helsinki. Of the 189 liver transplant candidates, 126 (113 male and 13 female) representing MELD score ≥30 in the waiting list were enrolled in this prospective study. The study protocol was approved by the Ethics Committee, and written informed consent was obtained from all study patients. Recipients with liver cancer were excluded from the study population. Patient characteristics were summarized in Table S1.
All patients received lamivudine combined with low-dose intramuscular hepatitis B immunoglobulin therapy according to our anti-virus protocol [13]. All patients were given standard medical treatments including energy supplements, intravenous infusion of albumin and plasma, and preventive treatment of complications. Of the 126 patients, 42 gained prompt donor livers and received emergency LT without any prior treatment (ELT group), the other 84 were given ALSS treatment before LT. The 84 patients were further divided into two sub-groups according to the MELD score on the day of transplant: decreased MELD group (DGM group, n = 33) (MELD score decreased to a level of <30) and non-decreased MELD group (N-DGM group, n = 51) (persistent high MELD score ≥30).
There are indications for ACLF patients receiving ALSS treatment according to Artificial Liver Group, Chinese Association of Infectious and Parasitic Diseases and Chinese Medical Association [12]. The methods of ALSS included plasma exchange with or without continuous hemodiafiltration or plasma perfusion, and molecular adsorbents recirculating system, as described previously [14]. The specialists from Infectious Diseases Department chose therapies and carried out ALSS treatment 1–3 times per week based on the condition of patients and the facility (availability of plasma or machine). The decision to initiate hemodiafiltration were made by consultant nephrologists to prevent uremia or immediate death from the adverse complications of renal failure [15]. In principle, patients with coagulopathy were indicated for plasma exchange (PE); patients with hepatic encephalopathy were given PE plus plasma perfusion or continuous hemodiafiltration. For patients complicated with hepatorenal syndrome or water – electrolytes imbalance, we applied PE plus continuous hemodiafiltration or molecular adsorbents recirculating system. In DGM group, 106 sessions of ALSS were applied to 33 patients with PE 61 times, PE plus plasma perfusion 21 times, PE plus continuous hemodiafiltration 14 times and molecular adsorbents recirculating system 10 times. In N-DGM group, 149 sessions of ALSS were applied to 51 patients, with PE 69 times, PE plus plasma perfusion 39 times, PE plus continuous hemodiafiltration 20 times and molecular adsorbents recirculating system 21 times. Emergency LT was performed within 72 h after patients became LT candidates for the purpose of our protocol [16].
United Network for Organ Sharing status was used to stratify the patients on the waiting list and allocate donor organs before December 2002, and then substituted by MELD score after January 2003. Post-reperfusion liver biopsies were obtained after liver implantation for histological evaluation of donor liver steatosis. According to the grade of macrovesicular steatosis, liver grafts were categorized into four groups: no steatosis, mild steatosis (<30%), moderate steatosis (30–60%), and severe steatosis (>60%) [17]. Deceased donor liver transplantation (DDLT) and living donor liver transplantation (LDLT) were performed in 93 and 33 cases, respectively. Of 93 donations after cardiac death, 3 were controlled and 90 were uncontrolled. Operation techniques of both DDLT and LDLT were described previously [18], [19]. The primary immunosuppressive regimen was triple therapy incorporating tacrolimus or cyclosporine, mycophenolate and steroid [20]. An IL-2 receptor blocker was used in selected patients.
Data collection
All patients were followed up closely in the outpatient clinic and data were collected for analysis. Pre-transplant data included age, gender, underlying liver disease, complications, MELD score, serum potassium and sodium, need for ALSS, and need for intensive care. Post-transplant complications, organ function and patient survival were also collected.
MELD was calculated according to the UNOS formula: MELD = 3.78×ln (bilirubin [mg/dl])+9.57×ln (creatinine [mg/dl])+11.20×ln (international normalized ratio)+6.43 and the range of the MELD score is 6–40 [21]. Delta-MELD = MELD score calculated on the day of transplant−MELD score calculated when patients were listed as LT candidates. The post-transplant model for predicting mortality (PMPM) score was calculated at 24 h following transplantation: PMPM score = −5.359+1.988×ln (serum creatinine [mg/dl])+1.089×ln (total bilirubin [mg/dl]) [22].
According to Asian Pacific Association for the Study of the Liver, ACLF was defined as ‘acute hepatic insult manifesting as jaundice (serum bilirubin >5 mg/dl) and coagulopathy (international normalized ratio >1.5 or prothrombin time activity <40%), complicated within 4 weeks by ascites and/or encephalopathy in a patient with previously diagnosed or undiagnosed chronic liver disease’ [4]. The diagnosis of acute exacerbation of chronic hepatitis B was based on findings of fibrous bands and ductular proliferation using by biopsy [4]. The diagnosis of cirrhosis with acute deterioration was based on the presence of hepatocyte necrosis and features of acute hepatitis under the background of cirrhosis [4]. The diagnostic criteria for hepatorenal syndrome was based on the International Ascites Club consensus [23]. The diagnosis of hepatic encephalopathy was based on the clinical manifestations and the signs of brain edema. The severity of hepatic encephalopathy was evaluated by the criteria for grading mental status [14]. Early allograft dysfunction was defined by the presence of at least one of the following characteristics: total bilirubin >10 mg/dl, prothrombin time ≥17 s and hepatic encephalopathy from days 2 to 7 post-transplantation [24]. Acute kidney injury was defined as an elevated level of serum creatinine (>1.5 mg/dL) or/and need for hemodiafiltration during the first post-transplant week [25]. Acute rejection was diagnosed routinely in liver biopsies according to the Rejection Activity Index criteria. Bacterial and fungus infection was diagnosed on the basis of primary culture, while viral infection was diagnosed on the basis of PCR result in the blood sample. Biliary complications were classified as bile leakage and stenosis. Bile leakage was primarily diagnosed on the basis of bilirubin in abdominal drainage, newly inserted pigtail, or cholangiography. Biliary stenosis was diagnosed on the basis of an overt dilatation of the intra-hepatic duct according to the imaging findings.
Statistical methodology
The Kolmogorov-Smirnov test was used to evaluate normality. Quantitative variables were presented as mean ± SD. Categorical variables were expressed as values and percentages. Student's t test or Mann-Whitney test was used to compare quantitative variables, while Chi-square test was used to compare categorical variables. Kaplan-Meier method with log-rank test and COX regression analysis were used for survival analysis. Logistic regression analysis was used for influence factors analysis. Variables with statistically significance in univariate analysis were taken for a forward stepwise multivariate analysis. SPSS for Windows version 11.0 (SPSS Inc., Chicago, IL) was used to complete all the analyses, and a P value of <0.05 was considered statistically significant.
Results
Pre-transplant ALSS treatment
The liver and kidney functions were improved temporarily after each ALSS session in all treated patients, presenting a significantly decreases in total bilirubin (33.4±12.7 mg/dL vs. 16.3±9.7 mg/dL, P<0.001), alanine aminotransferase (169±135 U/L vs. 78±67 U/L, P = 0.012), aspartate aminotransferase (202±107 U/L vs. 101±80 U/L, P<0.001), prothrombin time (32.6±10.3 s vs. 21.5±6.4 s, P = 0.024), international normalized ratio (3.36±0.95 vs. 2.17±0.76, P = 0.018) and serum creatinine (2.04±1.04 mg/dL vs. 1.21±0.83 mg/dL, P = 0.041). However, after all sessions of ALSS treatment, DGM group showed better liver and kidney functions than N-DGM group (
Table 1
).
10.1371/journal.pone.0030322.t001Table 1 Changes of biochemistry parameters after all sessions of ALSS treatment.
DGM group N-DGM group
(n = 33) (n = 51)
Total bilirubin
On the day of listing 28.4±11.8 26.6±10.2
On the day of transplant*
19.6±8.6 29.2±12.4
Alanine aminotransferase
On the day of listing 132±123 148±102
On the day of transplant*
67±46 169±97
Aspartate aminotransferase
On the day of listing 195±112 202±96
On the day of transplant*
98±76 213±105
Prothrombin time
On the day of listing 28.4±11.2 30.1±10.6
On the day of transplant*
20.2±7.3 31.2±9.8
International normalized ratio
On the day of listing 2.8±0.9 2.9±1.1
On the day of transplant*
2.1±0.7 3.0±1.2
Serum creatinine
On the day of listing 1.2±0.6 1.5±1.0
On the day of transplant*
1.1±0.7 1.7±1.1
Abbreviations: ALSS, artificial liver support system; DGM, downgraded MELD; MELD, model for end-stage liver disease; N-DGM, non-downgraded MELD;
*: N-DGM group vs. DGM group, P<0.05;
Complications which occurred in 31 patients (24.6%) during ALSS therapy included skin rash (11.1%), hypotension (6.3%), blood coagulation in perfusion cartridges (3.2%), local bleeding (3.2%) and infection (2.4%).
The median interval time from the last ALSS treatment to LT was 48 h (range: 24 h–8 d) in DGM group and 72 h (24 h–25 d) in N-DGM group (P = 0.147). The median waiting time for a donor liver was 10 days (range: 4–43 d) in DGM group and 9.5 days (range: 4–70 d) in N-DGM group, (P = 0.792).All recipients and donors experienced uneventful operative procedure.
Post-transplant complications
The incidence of post-transplant hemorrhage, early allograft dysfunction, acute kidney injury, acute rejection, infection and biliary complication was 10.3%, 28.6%, 15.9%, 12.7%, 29.4% and 13.5%, respectively. Most of complications (82.4%) developed in the first post-transplant month. In N-DGM group there was a significantly higher prevalence of acute kidney injury than that in ELT group (39.2% vs. 14.3%, P = 0.008) and DGM group (39.2% vs. 9.1%, P = 0.002), and higher incidence of infection than that in ELT group (43.1% vs. 19.0%, P = 0.013) and DGM group (43.1% vs. 21.2%, P = 0.039).
Patient survival
Of all 126 patients, 34 passed away during a median of 1.53 (0.03–9.86) years follow-up (
Figure 1
). The 1-month, 1-year and 5-year mortality was 16.7%, 24.6% and 27.0%, respectively, in the whole study population. There was a significantly higher overall mortality in N-DGM group than that in ELT group (43.1% vs. 16.7%, P = 0.007) and DGM group (43.1% vs. 15.2%, P = 0.006).
10.1371/journal.pone.0030322.g001Figure 1 Outcomes of all 126 ACLF patients with pre-transplant MELD ≥30.
ALSS, artificial liver support system; DGM, downgraded MELD; ELT, emergency liver transplantation; GVHD, graft versus host disease; MELD, model for end-stage liver disease; MODS, multi-organ dysfunction syndrome; N-DGM, non-downgraded MELD.
Most of deaths (21/34) occurred during the first post-transplant month. The causes of early death (<30 d) were multi-organ dysfunction syndrome (MODS, n = 15), hemorrhage (n = 4) and infection (n = 2). The early mortality was 14.3%, 9.1%, and 23.5% in the ELT group, DGM group and N-DGM group, respectively.
Patient cumulative survivals were presented in
Figure 2
and did not differ significantly between patients undergoing LDLT and those receiving DDLT (P = 0.338), patients with acute exacerbation of chronic hepatitis B and those with acute deterioration of cirrhosis (P = 0.655), or DGM group and ELT group (P = 0.901). There was a significantly lower patient cumulative survival in N-DGM group than that in NDM group (P = 0.008) and ELT group (P = 0.006). Patients with PMPM score <−1.4 had a remarkably higher cumulative survival than those with PMPM score >−1.4 (P<0.001).
10.1371/journal.pone.0030322.g002Figure 2 Comparison of patient cumulative survivals.
Kaplan-Meier analysis was used to compare survivals between patients underwent LDLT and those received DDLT (A), patients with acute exacerbation of chronic hepatitis B and those with acute deterioration of cirrhosis (B), ELT group, DGM group and N-DGM group (C), patients with PMPM score <−1.4 and those with PMPM score >−1.4 (D). LDLT, living donor liver transplantation; DDLT, deceased donor liver transplantation; ELT, emergency liver transplantation; DGM, downgraded MELD; N-DGM, non-downgraded MELD; PMPM, post-transplant model for predicting mortality.
Risk factors of death
Univariable analysis showed the following pre-transplant factors that were significantly related to the early death (<30 d): N-DGM (vs. ECT and DGM), delta-MELD, hepatorenal syndrome, infection, and serum sodium. These factors were then entered into the multivariable COX analysis and the independent risk factors of early death were N-DGM (vs. ECT and DGM) (RR = 2.426, P = 0.049) and hepatorenal syndrome (RR = 2.422, P = 0.039) (
Table 2
).
10.1371/journal.pone.0030322.t002Table 2 COX regression for pre-transplant influence factors of patient death.
Univariable analysis Multivariable analysis
Risk ratio (95% CI)
P
Risk ratio (95% CI)
P
Early mortality (<30 d)
N-DGM (vs. ECT and DGM) 3.112 (1.331–7.273) 0.009 2.426 (1.002–5.881) 0.049
Delta-MELD 1.086 (1.006–1.173) 0.035
Hepatorenal syndrome 3.136 (1.404–7.005) 0.005 2.422 (1.047–5.600) 0.039
Infection 2.372 (1.065–5.281) 0.034
Serum sodium 0.927 (0.875–0.981) 0.008
Overall mortality
N-DGM (vs. ECT and DGM) 3.196 (1.623–6.294) 0.001 3.209 (1.499–6.869) 0.003
Delta-MELD 1.083 (1.018–1.152) 0.011
Hepatorenal syndrome 2.015 (1.050–3.867) 0.035
Serum sodium 0.938 (0.895–0.984) 0.009
Abbreviations: CI, confidence interval; DGM, downgraded MELD; ELT, emergency liver transplantation; MELD, model for end-stage liver disease; N-DGM, non-downgraded MELD.
N-DGM (vs. ECT and DGM), delta-MELD, hepatorenal syndrome and serum sodium were found significantly associated with overall death, however, only N-DGM (vs. ECT and DGM) (RR = 3.209, P = 0.003) was the independent risk factor of overall death (
Table 2
).
Influence factors of downgrading MELD
Compared with DGM group, N-DGM group showed older age, more hepatic encephalopathy, more hepatorenal syndrome, and more infection (
Table 3
). Logistic regression univariable analysis demonstrated age >40 y, hepatic encephalopathy, hepatorenal syndrome, infection and the interval from last ALSS to LT >48 h were negative influence factors of downgrading MELD. Then these factors were entered into multivariable analysis and the independent negative factors influencing the reduction of MELD were age >40 y (OR = 0.240, P = 0.015) and the interval from last ALSS to LT >48 h (OR = 0.261, P = 0.022).
10.1371/journal.pone.0030322.t003Table 3 Logistic regression for influence factors of downgrading MELD.
Univariable analysis Multivariable analysis
Odds ratio (95% CI)
P
Odds ratio (95% CI)
P
Age >40 y (vs. ≤40 y) 0.292 (0.112–0.759) 0.012 0.240 (0.076–0.760) 0.015
Hepatic encephalopathy (yes vs. no) 0.400 (0.162–0.986) 0.047
Hepatorenal syndrome (yes vs. no) 0.250 (0.088–0.708) 0.009
Infection (yes vs. no) 0.250 (0.088–0.708) 0.009
Interval from last ALSS to LT >48 h (vs. ≤48 h) 0.307 (0.106–0.888) 0.029 0.261 (0.083–0.824) 0.022
Abbreviations: ALSS, artificial liver support system; CI, confidence interval; MELD, model for end-stage liver disease; LT, liver transplantation.
Discussion
There is a lack of a clear definition of ACLF until the Asian Pacific Association for the Study of the Liver consensus meeting in 2009. In China, where there is particularly high prevalence of hepatitis B and huge population of hepatitis B patients, ACLF has long been considered as a kind of severe viral hepatitis according to the Viral Hepatitis Protection and Cure Guideline established by the Chinese Infection and Hepatology Association. Although ACLF is believed to be reversible, the reversibility depends on the severity and nature of the acute insulting and the degree of underlying chronic liver disease [4]. For ACLF-HBV patients with ‘high-grade’ MELD score (≥30), resolving liver failure and sustaining life can be hardly achieved [7]. Recent Studies demonstrated extremely high short-term mortality of >90% in ACLF patients with MELD scores ≥30 under conventional medications [7], [8]. ALSS treatment could slightly decrease the mortality (68–91%) and therefore serve as a bridge to LT [7], [8]. Thus LT was considered as the only curative therapy for these patients. Since MELD score has been widely used for donor organs allocation, ACLF patients usually have the priority to gain a donor liver and receive emergency LT, which can effectively resolve endotoxemia and liver failure before ACLF greatly progresses [26]. As a result, the short-term and long-term survivals were satisfactory for ACLF patients receiving emergency LT. This indicated that LT should be considered as the first-line treatment option in these patients. But timely LT is not always available because of the donor shortage. The acute loss of liver function on the basis of chronic hepatitis or cirrhosis can dramatically accumulate massive metabolic toxins, leading to organ impairment and then causing severe complications such as infection and organ failure [4], [27]. Therefore, we performed ALSS as a salvage treatment in all study patients who had no chance to receive emergency LT as recommended by Artificial Liver Group, Chinese Association of Infectious and Parasitic Diseases and Chinese Medical Association.
For patients receiving ALSS treatment prior to LT, only those with decreased MELD score showed encouraging long-term survival after LT. Persistently high MELD score before LT was identified as the major independent risk factor of both early death and overall death. ALSS could create good environment for the self-regeneration of remained hepatocytes and thus led to great amelioration in encephalopathy, total bilirubin, international normalized ratio and creatinine, as well as a decrease in MELD score [11], [14]. In this sense, pre-transplant salvage treatment could be considered as ‘valid’ and ‘invalid’ in patients with decreased MELD and non-decreased MELD, respectively. For patients with non-reduced MELD, high levels of circulating endotoxins could be even elevated throughout the transplantation procedure and during the early post-transplant period, and then contribute to high morbidity and mortality [28]. For patients with decreased MELD, ALSS treatment improved patient conditions and enhanced the surgical tolerance. Although their waiting time was much longer than patients receiving emergency LT, patients experienced uneventful procedure during peri-operative period and showed comparable incidence of post-transplant complications and low early mortality. Since a reduced MELD score played a crucial role in the patient outcome, further identification of the potential influence factors of downgrading MELD was essential.
Age, hepatic encephalopathy, hepatorenal syndrome, infection and treatment interval (from last ALSS to LT) were found to be associated with reduction of MELD score. The cut-off values of age and treatment interval were chosen according to the clinical experience. Old age has been considered as a risk factor of patient prognosis after LT especially in patient with liver failure [29]. However, the impact of old age on the efficacy of ALSS has been rarely studied and need clarification in the further research. As well known, ALSS could only substitute a few elementary liver functions but not replace the entire spectrum of hepatic function. It is beneficial to ameliorate the microenvironment of the liver, but the function recovery is basically dependent on the self-regeneration of remained hepatocytes. In this study, hepatic encephalopathy, hepatorenal syndrome and infection, which reflected clinical severity of end-stage liver disease, were not independent influence factors. Consequently, the ability of ALSS to improve the MELD score might not be determined by the severity of underlying diseases. For ACLF patients with ‘high-grade’ MELD scores, whose livers have virtually little chance of self-regeneration [7], our results revealed the efficacy of ALSS was more likely determined by the therapeutic timing. The multivariate analysis showed long treatment interval (last ALSS to LT >48 h) played central role in the reduction of MELD score. A recent study investigated the dynamic change of total bilirubin, international normalized ratio and creatinine levels after ALSS, showing a significant improvement at 24 h, however, deterioration at 72–120 h post-ALSS [11] in these parameters. Thus many patients received several sessions of ALSS treatment before LT because their conditions deteriorated soon after one session of salvage treatment. These results suggested that consecutive sessions of ALSS were indeed effective in bridging critically ill patients to LT. An appropriate or even additional ALSS treatment within 48 h prior to LT was beneficial for improving patient's condition and downgrading MELD, thus further reducing the post-transplant mortality.
Hepatorenal syndrome was found to be another independent risk factor of early deaths. Hepatorenal syndrome occurs predominantly in advanced cirrhosis and also develops in severe liver failure, and may accompany the worst prognosis among all the complications of cirrhosis. There has been a consensus that pre-transplant renal dysfunction was a strong predictor of poor prognosis after LT, especially in patients with high MELD score [15], [30], [31]. In some LT candidates with severe kidney impairment, renal function maybe deteriorated after LT and combined liver kidney transplantation should be considered [15], [32]. Our results were consistent with the previous studies that high prevalence of hepatorenal syndrome contributed to the high incidence of post-transplant acute kidney injury and high mortality. The findings indicated that management of pre-transplant renal dysfunction was of vital importance for reducing the morbidity and mortality in patients with ACLF.
In the present study, we found several potential influencing factors including the quality of donors, HBV DNA load and cirrhosis background did not affect the patient survival. We have previously reported that moderately steatotic liver grafts provide adequate function in the first phase after transplantation and can be used for transplantation [17]. The shortage of donor organs has required us to use moderately but not severe steatotic liver grafts in order to expand the donor pool. HBV DNA load has been reported to be a predictor of poor prognosis among ACLF patients with high MELD score [8]. However, no good prognostic ability was seen among those critically ill patients after liver transplantation. Since lamivudine combined with low-dose intramuscular hepatitis B immunoglobulin therapy were routinely used in our center, HBV has been well controlled during the peri-operative period and the HBV recurrent rate has largely been reduced [13]. Other prognostic factors rather than HBV DNA load may play key roles in patient survival. Post-transplant model for predicting mortality (PMPM) was proven once again to be a good survival predictor even in this special study population [22]. In our centre, we use PMPM as an alarm bell for early recognition and prediction of poor outcome.
There were several limitations in this study. First, the endotoxin levels were not measured in all study population and thus not included in the data analysis. The comparison of circulating endotoxin levels during the peri-operative period among ELT, DGM and N-DGM groups, and the possible negative effect of high circulating endotoxin level on patient outcomes should be further evaluated in ACLF patients with high MELD score. Second, this study was limited in a Chinese population with severe hepatitis B. These study results should be further verified in a heterogeneous Western population with a relatively low incidence of hepatitis B. Third, this was not a randomized study because the selection of patients for emergency LT had to follow the organ allocation system. Only the critically ill patients received timely ALSS treatment which was limited by the availability of plasma and machine. Therefore, there were several confounding variables which may affect the results. A randomized study between groups that have and do not have prompt ALSS (within 48 h prior to LT) to reduce MELD scores should be conducted to verify our results.
In summary, for ACLF patients with ‘high’ MELD score, emergency LT was the choice and reduction of the MELD score before LT was effective in improving the patient prognosis. An appropriate ALSS treatment within 48 h prior to LT can be beneficial.
Supporting Information
Table S1 Demographic comparisons among the ELT, DGM and N-DGM groups.
(DOC)
Click here for additional data file.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by the Chinese High Tech Research & Development (863) Program (2011AA020104), Science Fund for Creative Research Groups of the National Natural Science Foundation of China (81121002) and the Technology Group Project for Infectious Disease Control of Zhejiang Province (2009R50041). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22299051PONE-D-11-2340210.1371/journal.pone.0031093Research ArticleBiologyGeneticsMolecular GeneticsMolecular Cell BiologyMedicineOncologyCancer Risk FactorsCancer TreatmentCancers and NeoplasmsHead and Neck TumorsOral MedicineOtorhinolaryngologyGrape Seed Proanthocyanidins Inhibit the Invasiveness of Human HNSCC Cells by Targeting EGFR and Reversing the Epithelial-To-Mesenchymal Transition Proanthocyanidins Inhibit Invasion of HNSCC CellsSun Qian
2
Prasad Ram
2
Rosenthal Eben
3
4
Katiyar Santosh K.
1
2
4
*
1
Birmingham Veterans Affairs Medical Center, Birmingham, Alabama, United States of America
2
Department of Dermatology, University of Alabama at Birmingham, Alabama, United States of America
3
Department of Surgery-Otolaryngology, University of Alabama at Birmingham, Alabama, United States of America
4
Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama, United States of America
Roemer Klaus EditorUniversity of Saarland Medical School, Germany* E-mail: [email protected] and designed the experiments: QS RP SKK. Performed the experiments: QS RP. Analyzed the data: SKK ER RP. Contributed reagents/materials/analysis tools: SKK ER. Wrote the paper: SKK.
2012 27 1 2012 7 1 e3109322 11 2011 2 1 2012 Sun et al.2012This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Head and neck squamous cell carcinoma (HNSCC) is responsible for approximately 20,000 deaths per year in the United States. Most of the deaths are due to the metastases. To develop more effective strategies for the prevention of metastasis of HNSCC cells, we have determined the effect of grape seed proanthocyanidins (GSPs) on the invasive potential of HNSCC cell and the mechanisms underlying these effects using OSC19 cells as an in vitro model. Using cell invasion assays, we established that treatment of the OSC19 cells with GSPs resulted in a dose-dependent inhibition of cell invasion. EGFR is over-expressed in 90% of HNSCCs and the EGFR inhibitors, erlotinib and gefitinib, are being explored as therapies for this disease. We found that GSPs treatment reduced the levels of expression of EGFR in the OSC19 cells as well as reducing the activation of NF-κB/p65, a downstream target of EGFR, and the expression of NF-κB-responsive proteins. GSPs treatment also reduced the activity of ERK1/2, an upstream regulator of NF-κB and treatment of the cells with caffeic acid phenethyl ester, an inhibitor of NF-κB, inhibited cell invasion. Overexpression of EGFR and high NF-κB activity play a key role in the epithelial-to-mesenchymal transition, which is of critical importance in the processes underlying metastasis, and we found treatment with GSPs enhanced the levels of epithelial (E-cadherin, cytokeratins and desmoglein-2) and reduced the levels of mesenchymal (vimentin, fibronectin, N-cadherin and Slug) biomarkers in the OSC19 cells. These results indicate that GSPs have the ability to inhibit HNSCC cell invasion, and do so by targeting the expression of EGFR and activation of NF-κB as well as inhibiting the epithelial-to-mesenchymal transition.
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Introduction
Squamous cell carcinoma of the head and neck (HNSCC) is the sixth most commonly occurring malignancy world-wide. It is responsible for approximately 20,000 deaths and affects more than 40,000 people in the United States annually [1], [2]. HNSCCs exhibit aggressive behavior with a high incidence of secondary primaries in the head and neck (5–7% per year) together with a high incidence of distant metastases. This potent metastatic capacity is associated closely with the high mortality rate. Advances in surgical and medical therapies for HNSCC have resulted in only a modest improvement in the mortality rate, which has remained at approximately 50% for the last three decades [3]–[6]. Currently available therapies including conventional chemotherapy and surgical resection are often associated with severe morbidity due to the involvement of vital structures of the head and neck, side effects and therapeutic resistance. Therefore, elucidation of the molecular pathways essential for metastasis and identification of approaches that minimize the invasive and metastatic behaviors of cancer cells is of high priority in the treatment of patients with HNSCC and in chemoprevention.
Naturally occurring bioactive dietary phytochemicals that are non-toxic and devoid of major side effects are candidates for the prevention of recurrence or metastasis of cancer cells. Such agents can be utilized as complementary and alternative medicine and/or as adjuvant therapy for conventional cytotoxic therapies. The seeds of grapes (Vitis vinifera L.) are a rich source of proanthocyanidins and these grape seed proanthocyanidins (GSPs) are promising bioactive phytochemicals that have shown anti-carcinogenic effects in some tumor models with no apparent signs of toxicity in these animal models [7]–[9]. In spite of the anti-carcinogenic effects of GSPs [7], their chemotherapeutic effects on the invasive potential of HNSCC cells have not been assessed.
Epidermal growth factor receptor (EGFR) appears to play a critical role in HNSCC. It is overexpressed in almost all HNSCC tumors (>90%) and its overexpression is associated with poor prognosis [10]–[12]. It has been reported that small molecule inhibitors of EGFR, such as erlotinib and gefitinib, can prevent the growth and progression of HNSCCs [13]. A downstream target of EGFR, the transcription factor nuclear factor-kappaB (NF-κB), also has been shown to be essential for metastasis in models of cancer progression [14]. The overexpression of EGFR, enhanced EGFR-associated signaling, and enhanced NF-κB activity have all been linked to the epithelial-to-mesenchymal transition (EMT). During EMT, cancer cells lose expression of proteins that promote cell-cell contact, such as E-cadherin, and acquire mesenchymal markers, such as vimentin, fibronectin and N-cadherin, that promote tumor progression, cell invasion and metastasis [15], [16]. In the present communication, we explored the chemotherapeutic effects of GSPs on the invasive potential of human HNSCC cells and ascertained whether EGFR and its associated pathways are involved in this process. Comparison of the invasive potential of several different HNSCC cell lines derived from different sub-sites indicated that the OSC19 cell line exhibited the greatest invasive potential and this cell line was used in all subsequent studies. We report that GSPs inhibit the invasive potential of the OSC19 HNSCC cells through inhibition or reversal of EMT and that this GSPs-induced inhibition is accomplished through a process that involves a reduction in the levels of EGFR expression, inactivation of NF-κB and inactivation of Erk1/2.
Materials and Methods
Source of the grape seed proanthocyanidins
The GSPs preparation was received from the Kikkoman Company, Noda, Japan (no financial conflict of interest) and used throughout the study. Quality control of the GSPs preparation is maintained by the company on lot-to-lot basis. The GSPs preparation contains approximately 89% proanthocyanidins, with dimers (6.6%), trimers (5.0%), tetramers (2.9%) and oligomers (74.8%), as described earlier [7]–[9]. The product is stable for at least two years when refrigerated at 4°C.
Chemicals, reagents and antibodies
Boyden Chambers and polycarbonate membranes (8 µm pore size) for cell migration/invasion assays were obtained from Neuroprobe, Inc. (Gaithersburg, MD). The antibodies specific for N-cadherin, keratin 8, keratin 18, fibronectin, EGFR, ERK1/2, cyclooxygenase-2 (COX-2), matrix metalloproteinase (MMP)-2, MMP-9, inducible nitric oxide synthase (iNOS) and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), while antibodies for vimentin, E-cadherin, Slug, NF-κB, IκB kinase α (IKKα), IκBα and vascular endothelial growth factor (VEGF) were purchased from Cell Signaling Technology (Beverly, MA), while desmoglein 2 was obtained from Abcam (Cambridge, MA). The appropriate secondary antibodies conjugated with horseradish peroxidase were procured from Invitrogen (Carlsband, CA). UO126, an inhibitor of the mitogen-activated/extracellular protein-regulated kinase (MEK), was purchased from Sigma Chemical Co. (St. Louis, MO). Erlotinib was procured from Santa Cruz Biotechnology and gefitinib from Toronto Research Chemicals, Inc. (North York, ON, Canada).
Cell lines and cell culture conditions
HNSCC cell lines derived from the oral cavity (UM-SCC1), larynx (UM-SCC5), pharynx (FaDu) and tongue (OSC19) were obtained from Dr. Rosenthal (University of Alabama at Birmingham, Birmingham, AL). The OSC19 cell line was developed from tumor cells that had metastasized to the lymph node from an HNSCC of the tongue. The cells were cultured as monolayers in DMEM supplemented with 10% heat-inactivated fetal bovine serum and 100 µg/mL penicillin-streptomycin (Invitrogen), and kept in a humidified atmosphere of 5% CO2 at 37°C. Cells were seeded at a density of 1×106 cells per culture dish and allowed to attach for 24 h, at which time they were sub-confluent, before treatment with GSPs or other agents. The GSPs, erlotinib or gefitinib were dissolved in a small amount of dimethylsulfoxide (DMSO), which was added to the complete cell culture medium. The maximum concentration of DMSO in media was 0.1% (v/v). Cells treated with DMSO only served as a vehicle control.
Cell proliferation assay
The effect of GSPs on the viability or cell proliferation of normal human bronchial epithelial cells or human HNSCC cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Sigma) or MTT assay, as previously described [17]. A total of 1×104 cells per well in 200 µL complete medium were seeded in a 96-well plate and treated with varying concentrations of GSPs for 48 h. The cells were treated with 50 µL of 5 mg/mL MTT and the resulting formazan crystals were dissolved in dimethylsulfoxide (200 µL). Absorbance was recorded at 540 nm with a reference at 650 nm serving as the blank. The effect of GSPs on cell viability was assessed as percent cell viability compared to vehicle-treated control cells, which were arbitrarily assigned 100% viability. All treatment concentrations were repeated in six wells.
Cell death assay
The trypan blue dye exclusion assay was used to determine the cytotoxic effect of GSPs on the cells. Briefly, 5×104 cells were cultured into each well of a six-well culture plates. After overnight incubation, the cells were treated with varying concentrations of GSPs (0, 10, 20, 40 µg/mL) for 48 h. Thereafter cells were harvested, treated with 0.25% trypan blue dye and the cells that had taken up the dye were counted under a microscope using a hemocytometer, as detailed previously [17]. The cytotoxic effects of GSPs are expressed as the mean±SD percentage of dead cells in each treatment group from three repeated experiments.
Cell invasion assay
The invasion capacity of HNSCC cells was determined in vitro using Boyden Chambers (Gaithersburg, MD). In this assay, the two chambers were separated with Matrigel-coated Millipore membranes (6.5 mm diameter filters, 8 µM pore size), as detailed previously [18], [19]. Briefly, cells (1.5×104 cells/100 µL serum-reduced [0.5% FBS] medium) were placed in the upper chamber of the Boyden chambers and the test agents were added alone, or in combination, to the upper chamber (200 µL). The lower chamber contained the medium alone (150 µL). The chambers were assembled and kept in a cell culture incubator for the specified periods of time. After incubation, cells from the upper surface of the Millipore membranes were removed with gentle swabbing and the cells on the lower surface of membranes were fixed and stained with crystal violet. The membranes were examined microscopically and cellular invasion was determined by counting the migrating/invasive cells on each membrane in at least 4–5 randomly selected fields using an Olympus BX41 microscope. Representative photomicrographs were obtained using a Qcolor5 digital camera system fitted to an Olympus BX41 microscope. Each experiment was repeated three times and the resultant cell invasion data are presented in terms of the mean number of invasive or migrating cells±SD/microscopic field (magnification, ×10) from three independent experiments.
Assay for NF-κB/p65 activity
The NF-κB TransAM Activity Assay Kit (Active Motif, Carlsbad, CA) was used for quantitative analysis of NF-κB/p65 activity following the manufacturer's protocol. For this purpose, the nuclear extracts of cells from various treatment groups were prepared using the Nuclear Extraction Kit (Active Motif, Carlsbad, CA) following the manufacturer's instructions and as described previously [20]. Briefly, this assay kit is an ELISA-based kit to detect and quantify NF-κB activation. By using an antibody that is directed against the NF-κB/p65 subunit, the activated NF-κB/p65 subunit bound to the oligonucleotide is detected. Addition of a secondary antibody conjugated to horseradish peroxidase provides sensitive colorimetric readout that is easily quantified by spectrophotometer. The manufacturer suggests that this NF-κB/p65 TransAM activity assay kit is more sensitive than EMSA. Absorbance was recorded at 450 nm using absorbance at 650 nm as the reference. The results are expressed as the percentage of the optical density of the non-GSPs-treated control group.
Western blot analysis
After incubation of cells for the indicated time periods with or without the treatment of GSPs or other agents, the cells were harvested, washed with cold PBS and lysed with ice-cold lysis buffer supplemented with protease inhibitors, as detailed previously [18], [19]. Cytoplasmic and nuclear protein fractions were prepared separately for the analysis of respective proteins. The purity of cytoplasmic and nuclear fractions was tested. The absence of β-actin in nuclear fraction confirms its purity while absence of Lamin B or Histone H3 proteins in cytoplasmic fraction suggests that this fraction is free from nuclear fraction. The purity was confirmed using western blot analysis. Proteins (30–50 µg) were resolved on 10% Tris-Glycine gels and transferred onto a nitrocellulose membrane. After blocking the non-specific binding sites, the membrane was incubated with the primary antibody at 4°C overnight. The membrane was then incubated with the appropriate peroxidase-conjugated secondary antibody and the specific-protein bands were visualized using the enhanced chemiluminescence reagents. The equal loading of protein samples on the gel was verified after stripping and re-probing of the membrane with anti-β-actin antibody. Representative blots are shown from three independent experiments.
Statistical analysis
For statistical analysis of cell invasion assays, the control, gefitinib, erlotinib or GSPs treatment groups or combined-treatment groups separately were compared using one-way analysis of variance (ANOVA) followed by the post hoc Dunn's test using GraphPad Prism version 4.00 for Windows software (GraphPad Software, San Diego, California. www.graphpad.com.) All quantitative data for cell migration are shown as the mean number of migrating cells±SD/microscopic field from at least three independent experiments. In each case P<0.05 was considered statistically significant.
Results
Analysis of the invasive potential of human HNSCC cells
First, we assessed the invasion capacity of various HNSCC cell lines that originated from different sub-sites of the head and neck, including the oral cavity (SCC1), larynx (SCC5), pharynx (FaDu) and tongue (OSC19) using a modified Boyden chamber assay. Incubation of the HNSCC cells for 48 h resulted in a greater number of invasive cells than incubation for 24 h. Representative photographs of crystal violet-stained membranes are shown in Figure 1A. As shown in Figure 1B, calculation of the cell invasion data in terms of the mean number of invasive cells±SD/microscopic field (magnification, ×10), the invasion capacity of OSC19 cells was found to be greater (380±16 cells/microscopic field) than SCC1 (75±7), SCC5 (38±4) and FaDu (58±5) cells. The invasion capacity of OSC19 cells was significantly higher (P<0.001) than that of SCC1, SCC5 and FaDu cells. Under identical conditions, the migration of normal human bronchial epithelial cells was hardly detectable (data not shown). As the invasive potential of OSC19 cells was significantly greater than other HNSCC cell lines tested under the conditions of this assay, the OSC19 cell line was selected for further studies.
10.1371/journal.pone.0031093.g001Figure 1 Analysis of invasive potential of HNSCC cell lines.
(A) The invasion potential of several human HNSCC cell lines including SCC1 (derived from the oral cavity), SCC5 (derived from the larynx), FaDu (derived from the pharynx) and OSC19 (derived from a lymph-node metastases from the tongue) were assessed using a Boyden chamber cell invasion assay. Equal numbers of cells were loaded and after 48 h incubation, invasive cells were detected on the membrane by staining with crystal violet. (B) The invasive cells were counted and the results expressed as the mean number of invasive cells±SD/microscopic field, magnification: ×10. Significantly higher versus other cells, *
P<0.001.
GSPs inhibit the invasive potential of OSC19 cells
To determine whether bioactive phytochemicals have the ability to inhibit the invasive potential of HNSCC cells, we used GSPs as a test agent in the in vitro cell invasion assay described above. As shown in Figure 2, as compared to non-GSPs-treated control cells, the treatment of cells with GSPs at the concentrations of 10, 20 and 40 µg/mL for 48 h reduced the invasive potential of OSC19 cells in a concentration-dependent manner. The density of the migrating cells on the membrane after staining with crystal violet is shown in Figure 2A, and a summary of the mean numbers of invasive cells±SD/microscopic field is provided in Figure 2B. The cell invasion potential of the OSC19 cells was inhibited by 41–81% (P<0.05−0.001) in a concentration-dependent manner after treatment with GSPs. To verify that the inhibition of invasion of OSC19 cells by the GSPs was due to a direct effect on migration ability, and was not due to a reduction in cell viability, a trypan blue assay was performed using cells that were treated identically to those used in the cell invasion assays. Treatment of OSC19 cells with various concentrations of GSPs (0, 10, 20 and 40 µg/mL) for 48 h had no significant effect on cell viability or induction of cell death (data not shown). Similarly, we also checked the toxic effects of GSPs on normal bronchial epithelial cells in vitro. These cells were treated with various concentrations of GSPs for 24 and 48 h under identical conditions. As shown in Figure 2C, treatment of cells with GSPs did not reduce significantly the proliferation ability or viability of cells as well as could not induce significant cell death under the experimental conditions used in this study. These data suggest that GSPs are not toxic to normal bronchial epithelial cells.
10.1371/journal.pone.0031093.g002Figure 2 Effect of GSPs on HNSCC cell invasion and EGFR expression.
(A) OSC19 cells were treated with the indicated concentrations of GSPs for 48 h and the effects on their invasiveness assessed as described in Figure 1. Crystal violet staining of the membranes showed that, as compared to non-GSPs-treated control cells, the GSPs inhibited invasion of cells in a concentration-dependent manner. (B) The migratory cells were counted and the results expressed as the mean number of invasive cells±SD/microscopic field, magnification: ×10. The values are reported from three separate experiments. Significant inhibition versus non-GSPs-treated control, *
P<0.001, ¶
P<0.01. (C) The non-toxic effect of GSPs on normal human bronchial epithelial cells was determined in terms of cell viability and cell death. The data on cell viability are expressed in terms of percent of control cells (non-GSPs-treated) as the mean±SD of 6 replicates. Similarly, the cytotoxic effect of GSPs on normal bronchial epithelial cells was determined using trypan blue dye exclusion assay as described in Materials and Methods and is expressed in terms of percent of dead cells as mean±SD from three experiments. (D) Dose-dependent effect of GSPs on EGFR expression in OSC19 cells. The levels of EGFR in whole cell lysates of OSC19 cells treated with different concentrations of GSPs for 48 h were determined using western blot analysis. Representative blots are shown from three independent experiments.
Treatment of cells with GSPs reduces the level of EGFR expression
As it has been shown that EGFR is overexpressed in over 90% of HNSCC tumors [10]–[12], we determined whether inhibition of cell invasion of OSC19 cells by GSPs is associated with a reduction in the expression of EGFR. For this purpose, whole cell lysates from different treatment groups were analyzed by western blotting. As shown in Figure 2D, treatment of OSC19 cells with GSPs for 48 h resulted in a reduction in the levels of EGFR expression in a concentration-dependent manner as compared to the expression of EGFR in non-GSPs-treated controls. These results suggest that the GSPs-induced reduction in EGFR expression may be associated with an inhibitory effect of the GSPs on the invasive potential of these cells.
The EGFR inhibitors, erlotinib and gefitinib, inhibit the invasive potential of OSC19 cells
We next examined the effects of the erlotinib, a selective inhibitor of EGFR, and gefitinb, a kinase inhibitor that inhibits EGFR, on the cell invasion potential of the OSC19 cells. For this purpose, cells were incubated with various concentrations of erlotinib (0, 0.1, 1.0 and 10.0 µM) for 48 h in Boyden chambers. As shown in Figure 3A, treatment of the cells with erlotinib resulted in a dose-dependent reduction in the cell invasion capacity of OSC19 cells as reflected by the presence of invasive cells on the membrane compared with non-erlotinib-treated controls. The resultant data on cell invasion was determined in terms of the number of invasive cells/microscopic field±SD at different concentrations of erlotinib and are summarized in Figure 3B. Similar results were obtained when OSC19 cells were treated with gefitinib. Resultant data on cell invasion which are shown in Figure 3C and are summarized in Figure 3D, demonstrated that the treatment of OSC19 cells with gefitinib for 48 h under identical conditions resulted in a dose-dependent inhibition of cell invasion. These results support the concept that the GSPs may act to inhibit the invasion of OSC19 HNSCC cells by targeting the EGFR.
10.1371/journal.pone.0031093.g003Figure 3 Effect of the EGFR inhibitors, gefitinib and erlotinib, on HNSCC cell invasion.
(A) OSC19 cells were treated with the indicated concentrations of erlotinib, a small molecule inhibitor of EGFR, for 48 h and the effects on invasion assessed as described in Figure 1. Crystal violet staining of the membrane shows that erlotinib inhibited invasion of OSC19 cells in a concentration-dependent manner. (B) The invasive cells were counted and the results expressed as the mean number of invasive cells±SD/microscopic field, magnification: ×10. (C) Similarly, treatment of OSC19 cells with the indicated concentrations of gefitinib, a small molecule inhibitor of tyrosine kinase activity and, thus, of EGFR, inhibited cell invasion in a dose-dependent manner as indicated by crystal violet staining of the membrane. (D) The invasive cells from the experiment described in Figure 3C were counted and the results expressed as the mean number of invasive cells±SD/microscopic field, magnification: ×10. Significant reduction of cell invasion versus untreated control cells, ¶
P<0.05, *
P<0.001.
GSPs reduce the level and activity of NF-κB/p65 in HNSCC cells: NF-κB is an important mediator of cell invasion
As NF-κB is a down-stream target of EGFR, we assessed whether GSPs affect the levels and activation of NF-κB in OSC19 cells. OSC19 cells were incubated with various concentrations of GSPs (10, 20 and 40 µg/mL) for 48 h, and thereafter the cells were harvested and cytoplasmic and nuclear fractions were prepared for western blot analysis. As shown in Figure 4A, western blot analysis of the nuclear fraction revealed that treatment of cells with GSPs reduced the translocation of NF-κB/p65 into the nucleus in a concentration-dependent manner. The results also indicated that treatment with GSPs resulted in the downregulation of IKKα whereas increases the levels of IκBα, which leads to the inactivation of NF-κB and its translocation to the nucleus. The activity of NF-κB/p65 was significantly reduced in a concentration-dependent manner (30–75%, P<0.05, P<0.001) after treatment of the cells with GSPs (Figure 4B). Similar results were observed when the OSC19 cells were treated with erlotinib under identical experimental conditions (Figures 4C, 4D). To further assess whether NF-κB has a role in HNSCC cell invasion, OSC19 cells were treated with caffeic acid phenethyl ester (0, 5.0 and 10.0 µg/mL), a potent inhibitor of NF-κB, and cell invasion was determined. As shown in Figure 4E, treatment of OSC19 cells with caffeic acid phenethyl ester resulted in a significant reduction of cell invasion (38% and 64%; P<0.05, and P<0.001) compared to non-caffeic acid phenethyl ester-treated control cells, and these results are similar to that observed on treatment of the cells with GSPs (Figure 2A, 2B).
10.1371/journal.pone.0031093.g004Figure 4 Effect of GSPs and erlotinib on NF-κB activation in OSC19 cells.
(A) Treatment of OSC19 cells with GSPs decreases the basal levels of NF-κB/p65 and IKKα while inhibiting the degradation of IκBα. After treatment of cells for 48 h with various concentrations of GSPs the cells were harvested, cytosolic and nuclear fractions were prepared and these were subjected to western blot analysis. Representative blots are shown from three independent experiments with identical observations. (B) The activity of NF-κB/p65 in the nuclear fraction of cells after treatment with and without GSPs was measured using an NF-κB/p65-specific activity assay kit, n = 3. Activity of NF-κB/p65 is expressed in terms of percent of the control (non-GSPs-treated) group. Significant decrease versus control: ¶
P<0.05, *
P<0.001. (C) Similarly, treatment of OSC19 cells with erlotinib reduced the levels of NF-κB and IKKα. Representative blots are shown from two independent experiments. (D) The activity of NF-κB/p65 in the nuclear fraction of cells after treatment with and without erlotinib for 48 h was measured using the NF-κB/p65-specific activity assay kit, n = 2. Activity of NF-κB/p65 is expressed in terms of percent of control (non-erlotinib-treated) group. Significant decrease versus control: *
P<0.001. (E) Treatment of OSC19 cells with caffeic acid phenethyl ester (CAPE), an inhibitor of NF-κB, for 48 h inhibited cell invasion in a concentration-dependent manner. Data on cell invasion are summarized as the mean number of invasive cells±SD/microscopic field from three independent experiments, magnification: ×10. Significant inhibition versus non-CAPE-treated cells, *
P<0.001, ¶
P<0.05. (F) GSPs reduce the levels of NF-κB-targeted proteins, such as MMP-2, MMP-9, COX-2, iNOS and VEGF, in OSC19 cells. After treatment of cells with and without GSPs for 48 h, cells were harvested, and cell lysates were subjected to the analysis of the levels of proteins using western blot analysis. Representative blots are shown from three independent experiments.
GSPs inhibit the expression of NF-κB-targeted proteins in OSC19 cells
As the functional activity of NF-κB is mediated through its targeted proteins, we further determined whether GSPs affect the levels of NF-κB-responsive proteins associated with cancer cell invasion, including MMPs, COX-2, iNOS and VEGF, in HNSCC cells. As shown in Figure 4F, western blot analysis revealed that treatment of OSC19 cells with GSPs resulted in a markedly reduced levels of MMP-2, MMP-9, COX-2, iNOS and VEGF proteins as compared to non-GSPs-treated controls, which is indicated by the visual intensity of the respective bands.
GSPs as well as MEK inhibitor (UO126) inhibit the phosphorylation of ERK1/2 in OSC19 cells: UO126 reduces the invasive potential of OSC19 cells
Mitogen-activated protein kinases (MAPKs) are down-stream targets of EGFR signaling as well as upstream regulators of NF-κB, and have been implicated in cancer cell metastasis [21]. Therefore, we examined the effect of GSPs on activation of the extracellular-signal regulated kinase (ERK1/2) in HNSCC cells. Western blot analysis revealed that treatment of OSC19 cells with GSPs for 48 h inhibited the phosphorylation of ERK1/2 in a dose-dependent manner, as shown in Figure 5A. Similarly, the treatment of cells with UO126 also inhibited the phosphorylation of ERK1/2 (Figure 5B). We then determined the role of activated ERK1/2 on OSC19 cell invasion. The cell invasion assay revealed that treatment of OSC19 cells with UO126 for 48 h significantly inhibited (68%, P<0.001) the invasiveness of cells (Figure 5C, 5D). A summary of data obtained from three independent experiments is shown in Figure 5D.
10.1371/journal.pone.0031093.g005Figure 5 Both GSPs and MEK inhibitor (UO126) inhibit OSC19 cell invasion by reducing activation of ERK1/2.
(A) GSPs inhibit the activation of ERK1/2 in OSC19 cells in a dose-dependent manner. OSC19 cells were treated with indicated concentrations of GSPs for 48 h, the cells harvested and whole cell lysates subject to western blotting using antibodies specific for total ERK1/2 and phosphorylated ERK1/2. (B) Similarly, the treatment of OSC19 cells with UO126, a MEK inhibitor, (80 µM) for 48 h inhibited the activation of ERK1/2 in cells as determined by western blot analysis. Representative blots are shown from three separate experiments. (C) OSC19 cells were treated with UO126 (80 µM) for 48 h and cell invasion assessed as described in Figure 1. Crystal violet staining of the membrane indicated that UO126 inhibited the invasive potential of cells as compared to non-UO126-treated control cells. (D) The data on cell invasion are summarized and expressed as the mean number of invasive cells±SD/microscopic field, magnification: ×10. Experiments were repeated two times. Significant difference versus control *
P<0.001.
GSPs promote transition of the mesenchymal state to the epithelial state in HNSCC cells
Upregulation of EGFR and activation of downstream targets like ERK1/2 and NF-κB play critical roles in the EMT [15], [16], [21], which has been implicated in cancer cell invasion and metastasis. Therefore, we checked the effect of GSPs on the EMT in OSC19 cells. For this purpose, OSC19 cells were incubated with varying concentrations of GSPs and erlotinib separately for 48 h. Thereafter, cells were harvested and cell lysates were prepared for the analysis of epithelial and mesenchymal biomarkers using western blot analysis. Western blot analyses revealed that GSPs increased the levels of epithelial biomarkers, such as E-cadherin, keratin 8, keratin 18 and desmoglein 2 in OSC19 cells in a dose-dependent manner compared to untreated controls (Figure 6A). In contrast, the levels of mesenchymal biomarkers, such as N-cadherin, vimentin, fibronectin and Slug, were reduced in OSC19 cells after treatment with GSPs in a dose-dependent manner (Figure 6B). Similar to GSPs, the treatment of OSC19 cells with erlotinib resulted in an increase in the levels of epithelial biomarkers (Figure 6C) and a decrease in the levels of mesenchymal biomarkers in OSC19 cells (Figure 6D).
10.1371/journal.pone.0031093.g006Figure 6 Treatment of OSC19 cells with GSPs or erlotinib results in reversal of epithelial-to-mesenchymal transition.
(A) Treatment of OSC19 cells with GSPs for 48 h enhanced the levels of epithelial biomarkers in the cells as assessed by western blotting, including the levels of E-cadherin, keratin-8, keratin-18 and desmoglein-2. (B) Simultaneously, the levels of mesenchymal biomarkers in cells, such as, N-cadherin, vimentin, fibronectin and Slug, were decreased in a dose-dependent manner. (C and D) Under identical conditions, treatment of OSC19 cells with erlotinib also resulted in reversal of epithelial-to-mesenchymal transition in a dose-dependent manner. Presented blots are representative of three independent experiments with similar results.
Discussion
The therapeutic potential, including antitumor activity, of GSPs has been reported in various preclinical models [7]–[9], [22], [23]; however, the effect of GSPs on the invasive potential of cancer cells is less explored. In the current study, we investigated the potential utility of GSPs in the prevention of invasiveness of HNSCC cells. HNSCCs can arise from several different sub-sites. In our preliminary studies, we tested the invasiveness of HNSCC cell lines generated from the oral cavity, larynx, and pharynx. The results suggested that, under the experimental conditions used in these studies, these cell lines exhibited greater invasive potential than normal bronchial epithelial cells. The invasive potential of the metastatic OSC19 cell line that originated from the tongue was greater than the other cell lines (SCC1, SCC5, FaDu).
GSPs were found to inhibit the invasiveness of OSC19 cells in a dose-dependent manner and this inhibitory effect of GSPs was associated with the downregulation of EGFR expression in the OSC19 cells. The OSC19 cells overexpress EGFR and the inhibition of EGFR by GSPs may contribute to the inhibition of cell invasion of these cells. This concept is supported by the evidence that treatment of the OSC19 cells with gefitinib or erlotinib, small molecule inhibitors of EGFR, resulted in a reduction in the cell invasion ability. It has been reported that inhibitors of EGFR can prevent the growth and progression of HNSCCs; however, their long term use may also induce some form of toxicity [13]. Notably, significant toxicity has not been associated with the use of GSPs in animal models [7]–[9], [22].
NF-κB is a downstream target of EGFR, and activation of NF-κB has been identified as an important regulator of cancer cell invasion, metastasis and angiogenesis [14], [24], [25]. Therefore, we checked the effect of GSPs on the basal levels of NF-κB in OSC19 cells and found that treatment of these cells with GSPs results in downregulation as well as inactivation of the NF-κB pathway in a dose-dependent manner. GSPs decrease the levels of IKKα which is responsible for inactivation of NF-κB. Treatment of cells with caffeic acid phenethyl ester, an inhibitor of NF-κB, resulted in an inhibitory effect on the invasion of HNSCC cells. NF-κB-targeted proteins, such as MMPs, COX-2, iNOS and VEGF, have been implicated in tumor angiogenesis and tumor cell migration. Treatment of OSC19 cells with GSPs down-regulates the expression of these NF-κB-targeted proteins, which supports the evidence that NF-κB has a role in invasion of HNSCC cells, and that the inhibitory effect on cell invasion by GSPs is mediated, at least in part, through the inactivation of NF-κB. It is important to mention that all these effects of GSPs may not be solely caused by the inhibition of EGFR; other factors or targets may also play a role and that need to be identified. Proteins of the MAPK family are also downstream targets of EGFR and have been shown to play a crucial role in cancer cell migration/invasion. Activation of the proteins of MAPK family leads to the activation of NF-κB. Our results show that inhibition of invasiveness of OSC19 cells by GSPs is associated with the inhibition of ERK1/2 phosphorylation. The use of MEK inhibitor (UO126) blocked the cell invasion capacity of OSC19 cells, and this function of UO126 is similar to the action of GSPs. These observations suggest a possible involvement of the ERK1/2-NF-κB pathway in inhibition of the invasive potential of HNSCC cells by GSPs.
In addition to the role of NF-κB in cancer biology, such as in tissue invasion, cell migration and metastasis, NF-κB has been identified as an important regulator of EMT in several cancer cell types [14], [24], [25]. EMT plays a major role in invasion and metastasis of epithelial tumors. EMT can render tumor cells migratory and invasive through the involvement of all stages, invasion, intravasation and extravasation [16], [26]. During the EMT stage, cells change from an epithelial to a mesenchymal phenotype. They lose their characteristic epithelial traits and instead gain properties of mesenchymal cells. This process is coordinated primarily by the loss of epithelial biomarkers such as E-cadherin and certain cytokeratins, concomitant with the acquisition of mesenchymal markers, such as vimentin, fibronection, N-cadherin, etc.. In the present study, we found that GSPs treatment of OSC19 cells resulted in the suppression or loss of mesenchymal biomarkers while restoring the levels of epithelial biomarkers, which suggests that GSPs have the ability to reverse the EMT process in HNSCC cells. This may be one of the possible mechanisms through which GSPs reduce the invasiveness of HNSCC cells thereby inhibiting their invasion in our in vitro model.
Biological activity of phytochemicals largely depends on their bioavailability. The study of bioavailability and metabolism of any agent or phytochemical is an important part of all investigations. Some studies have been performed to examine the bioavailability and metabolism of proanthocyanidins. Studies have shown that polymeric proanthocyanidins are not absorbed as such in the gut [27]. The detection of proanthocyanidin dimers B1 and B2 in human plasma was reported in some studies [28], [29]. The absorption of these dimers was significantly lower than that of the monomeric flavanols [29]. However, these compounds were found to protect the intestinal mucosa against oxidative stress or the actions of carcinogens. Additionally, proanthocyanidins have been found to be quite stable, and GSPs are stable at least 2 years if stored in refrigerator at 4°C.
Together, the results from this study have identified for the first time that bioactive proanthocyanidins from grape seeds inhibit the invasiveness of human HNSCC cells and that this effect involves: (i) the inhibitory effect of GSPs on EGFR expression, (ii) the inhibitory effect of GSPs on the activation of the ERK1/2 proteins and inactivation of NF-κB, and (iii) reversal of the EMT. These events and observations are summarized in Figure 7. Further in vitro and in vivo studies may help to develop GSPs as a pharmacologically safe bioactive chemotherapeutic or chemopreventive agent when used either alone or in combination with other anti-metastatic drugs for the treatment of HNSCC in humans.
10.1371/journal.pone.0031093.g007Figure 7 Summary of the action of GSPs on the prevention of HNSCC cell invasion.
GSPs inhibit the expression levels of EGFR in human HNSCC cells, which plays a critical role in inactivation of ERK1/2, NF-κB and reversal of epithelial-to-mesenchymal transition. Collectively, these effects lead to GSPs-induced inhibition of cancer cell invasion. However, all these effects of GSPs may not be solely caused by the inhibition of EGFR expression; other targets may also play a role and that need to be further identified.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by funds from the Veterans Administration Merit Review Award (SKK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22319601PONE-D-11-2177410.1371/journal.pone.0031019Research ArticleBiologyDevelopmental BiologyMolecular DevelopmentMolecular Cell BiologySignal TransductionSignaling CascadesMedicineCardiovascularDrugs and DevicesUrologyEndogenous Urotensin II Selectively Modulates Erectile Function through eNOS eNOS Involvement in U-II Induced Penile Erectiond'Emmanuele di Villa Bianca Roberta
1
2
Mitidieri Emma
2
Fusco Ferdinando
1
D'Aiuto Elena
3
Grieco Paolo
4
Novellino Ettore
4
Imbimbo Ciro
1
Mirone Vincenzo
1
Cirino Giuseppe
1
2
Sorrentino Raffaella
1
2
*
1
Interdepartmental Research Centre for Sexual Medicine (CIRMS), University of Naples, Federico II, Naples, Italy
2
Department of Experimental Pharmacology, University of Naples, Federico II, Naples, Italy
3
Section of Clinical Immunology, Department of Clinical and Experimental Medicine, Second University of Naples, Naples, Italy
4
Department of Pharmaceutical and Toxicological Chemistry, University of Naples, Federico II, Naples, Italy
Bonini Marcelo G. EditorUniversity of Illinois at Chicago, United States of America* E-mail: [email protected] and designed the experiments: GC RddVB RS. Performed the experiments: ED RddVB FF PG CI EM. Analyzed the data: GC RddVB FF EM VM EN RS. Wrote the paper: GC RddVB RS.
2012 2 2 2012 7 2 e310192 11 2011 30 12 2011 d'Emmanuele di Villa Bianca et al.2012This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
Urotensin II (U-II) is a cyclic peptide originally isolated from the neurosecretory system of the teleost fish and subsequently found in other species, including man. U-II was identified as the natural ligand of a G-protein coupled receptor, namely UT receptor. U-II and UT receptor are expressed in a variety of peripheral organs and especially in cardiovascular tissue. Recent evidence indicates the involvement of U-II/UT pathway in penile function in human, but the molecular mechanism is still unclear. On these bases the aim of this study is to investigate the mechanism(s) of U-II-induced relaxation in human corpus cavernosum and its relationship with L-arginine/Nitric oxide (NO) pathway.
Methodology/Principal Findings
Human corpus cavernosum tissue was obtained following in male-to-female transsexuals undergoing surgical procedure for sex reassignment. Quantitative RT-PCR clearly demonstrated the U-II expression in human corpus cavernosum. U-II (0.1 nM–10 µM) challenge in human corpus cavernosum induced a significant increase in NO production as revealed by fluorometric analysis. NO generation was coupled to a marked increase in the ratio eNOS phosphorilated/eNOS as determined by western blot analysis. A functional study in human corpus cavernosum strips was performed to asses eNOS involvement in U-II-induced relaxation by using a pharmacological modulation. Pre-treatment with both wortmannin or geldanamycinin (inhibitors of eNOS phosphorylation and heath shock protein 90 recruitment, respectively) significantly reduced U-II-induced relaxation (0.1 nM–10 µM) in human corpus cavernosum strips. Finally, a co-immunoprecipitation study demonstrated that UT receptor and eNOS co-immunoprecipitate following U-II challenge of human corpus cavernosum tissue.
Conclusion/Significance
U-II is endogenously synthesized and locally released in human corpus cavernosum. U-II elicited penile erection through eNOS activation. Thus, U-II/UT pathway may represent a novel therapeutical target in erectile dysfunction.
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Introduction
Urotensin II (U-II) is a cyclic peptide hormone derived from pre-pro-U-II by urotensin-converting enzyme. It was first isolated from teleost fish and homologues subsequently were identified across the evolutionary spectrum, including mammals and man. U-II causes both vasoconstriction and vasodilation depending by the vascular district and the species considered [1]–[6]. Its vasoactive effect is mediated by binding to a GPR14 (UT receptor), a G protein-coupled receptor [7]. U-II is secreted from heart and several other tissues into the circulation [8]. However, the source of U-II production in the human body remains to be elucidated. Both U-II and UT receptor are expressed widely within the cardiovascular system, and their expression is up-regulated in human cardiovascular disease, including congestive heart failure, hypertension, type II diabetes and diabetic nephropathy [9]–[11]. Collectively, these data indicate U-II as potential modulator of cardiovascular homeostasis in human.
Recently, we have demonstrated the involvement of U-II/UT pathway in erectile function [12]. Indeed, an intra-cavernous injection of U-II in rats causes an increase in intra-cavernous pressure without affecting systemic blood pressure. It has also been demonstrated that UT receptor is present in human corpus cavernosum (HCC). It is located on the endothelium and it mediates an endothelium-dependent relaxation involving the L-arginine/nitric oxide (NO) pathway [12]. It is well established that the L-arginine/NO pathway plays a major role in erectile function in man [13], [14]. NO is produced by a group of enzymes called nitric oxide synthase (NOS) that by converting L-arginine into L-citrulline produce NO [15], [16]. The endothelial NOS (eNOS) is constitutively expressed within the vascular system, it is tightly regulated and produces physiologically relevant levels of NO.
The regulation of eNOS involves multiple molecular mechanisms that act in concert to both positively or negatively affect the function of this enzyme. eNOS is classified as a constitutive and strictly calcium/calmodulin-dependent enzyme [17]. The calcium levels as well as the heath shock protein 90 (Hsp90) recruitment increase the catalytic activity of eNOS [18], [19]. The eNOS-associated Hsp90 may also serve as a scaffolding protein, facilitating the organization of additional associated regulatory proteins. In addition, fluid shear stress or other stimuli by phosphorylation can shift eNOS to an higher state of activation [20]. For example, bradykinin enhances eNOS phosphorylation; this effect is maximal after 5 minutes and it is maintained for at least 20 minutes in cultured endothelial cells [21]. In recent years, it has been reported that eNOS phosphorylation at serine1177 by phosphatydilinositol 3 kinase (PI3K)/protein kinase B (Akt) is critical for the maintenance of full penile erection [22], [23]. Thus eNOS activity is finely regulated and can shift to an higher degree of activation following molecular modulation [17]–[23].
The present study investigates the relationship between U-II/UT and L-arginine/NO pathway in human corpus cavernosum. Our data demonstrate that U-II pro-erectile response relies on eNOS-derived NO, contributing to the maintenance of full penile erection.
Results
U-II is present as mRNA in HCC
The RT-PCR analysis (Figure 1) clearly demonstrated the U-II presence in HCC samples. Since it has been reported that U-II is over-expressed in human tumoural cell lines SW-13 [24], a positive control by using SW-13 cells was performed, too. No amplifications were observed when PCR was performed in same conditions but without cDNA.
10.1371/journal.pone.0031019.g001Figure 1 Quantitative RT-PCR for U-II in HCC.
U-II is expressed as mRNA in HCC samples. Human tumoural cells SW-13 were used as positive control. Data were normalized on the basis of GAPDH and expressed as mean ± standard error of the mean (SEM) of three different human specimens.
Taken together, these data demonstrate that U-II can be locally synthesized and endogenously released in HCC.
U-II vasorelaxant effect involves eNOS activation
As previously described U-II causes an endothelium- and NO-dependent relaxation in pre-contracted HCC strips [12]. A pharmacological eNOS modulation was operated by using inhibitors that either interfere with Hsp90 recruitment (geldanimycin) or inhibit phosphorylation (wortmannin). Both wortmannin (0.1 µM) or geldanamycin (1 µM) significantly reduced U-II induced relaxation of human tissues (Figure 2 A, ***p<0.001 and **p<0.01).
10.1371/journal.pone.0031019.g002Figure 2 Panel A: U-II induced concentration-response curve (0.1 nM–10 µM) in HCC strips was significantly inhibited by pre-treatment with wortmannin (0.1 µM), PI3K inhibitor, or geldanamycin (1 µM) Hsp90 inhibitor (***p<0.001 and ** p<0.01 versus vehicle).
Data were expressed as the mean ± standard error of the mean (SEM) of six different specimens. Data were analyzed by ANOVA followed by Bonferroni post test. Panel B: NOx (total nitrite) production in HCC tissue incubated with U-II (1 nM-10 µM) or vehicle for 30 min. U-II caused a significant increase in NO production compared with vehicle (*p<0.05, **p<0.01, ***p<0.001). Data were expressed as mean ± standard error of the mean (SEM) from four different specimens and analyzed by ANOVA followed by Bonferroni post test.
U-II promotes NO production in HCC
In order to confirm the NO involvement in U-II/UT signaling we evaluated the NO production in HCC homogenate tissues stimulated with U-II (1 nM–10 µM). Data, expressed as total nitrite, clearly showed that U-II caused a significant increase in NOx production compared with vehicle (Figure 2 B, *p<0.05, **p<0.01, ***p<0.001).
U-II induces eNOS phosphorylation in HCC
To further asses the U-II involvement in eNOS activation we performed a western blot study. The protein expression of eNOS as well as phosphorylated eNOSSer 1177 (p-eNOS) in tissues incubated with U-II (10 µM), at scheduled time, was evaluated. U-II induced eNOS phosphorylation in a time-dependent manner, reaching its maximum at 30 minutes (Figure 3 A, *p<0.05). Thus 30 minutes of incubation has been chosen as the time point to perform the experiments by using inhibitors. Wortmaninn (0.1 µM), an irreversible inhibitor of PI3K, but not geldamycin, reverted significantly eNOS phosphorylation induced by U-II challenge (***p<0.001, Figure 3 B).
10.1371/journal.pone.0031019.g003Figure 3 Western blot analysis for eNOS and p-eNOSSer-1177 in HCC tissue.
Panel A: U-II (10 µM) caused an increase in eNOS phosphorylation expressed as p-eNOS/eNOS ratio in a time-dependent manner (*p<0.05 vs vehicle). Panel B: U-II-induced eNOS phosphorylation (**p<0.01 vs vehicle), was significantly reverted by wortmannin (0.1 µM), PI3K inhibitor, (°p<0.05 vs U-II) but not by geldanamicin (1 µM), Hsp90 inhibitor. Data were expressed as the mean ± standard error of the mean (SEM) of four different specimens and were analyzed by ANOVA followed by Bonferroni post test.
U-II induces the recruitment of eNOS to UT receptor in HCC
The co-immunoprecipation assay allows to identify protein-protein/enzyme interaction. Thus, to confirm the relation between U-II/UT pathway and eNOS, we performed the eNOS precipitation and monitored the co-precipitation of UT receptor in HCC tissues incubated for 30 minutes with U-II (10 µM) or vehicle. U-II induced the formation of the immunocomplex between UT and eNOS (Figure 4).
10.1371/journal.pone.0031019.g004Figure 4 Co-immunoprecipitation analysis of UT receptor and eNOS.
Tissues were stimulated with either vehicle (A) or U-II (C), lysates were incubated with mouse anti-eNOS. Lanes B and D correspond to the negative control of A and C, respectively. The western blot was probed with rabbit anti-GPR14 (UT receptor). U-II but not vehicle caused the co-immunoprecipation between eNOS and UT receptor.
Discussion
Human penile erection is a resultant of several complex mechanisms. A key issue is the balance between contracting and relaxant factors which are instrumental in order to achieve penile erection. It is now well accepted that NO plays a major role in induction and maintenance of erection [25]–[28]. Recently we have reported that U-II-induced relaxation in HCC is endothelium- and NO-dependent [12]. On this basis we have proposed that U-II, physiologically circulating in our body [8], contributes to penile homeostasis. Our present finding further extends the importance of this signaling pathway and confirms that endothelium is necessary.
We have demonstrated that mRNA for U-II is present in the human tissue. This finding indicates that U-II can be locally endogenously synthesized within the HCC and that U-II/UT pathway is involved within erection physiology. The exclusive presence of UT receptor on the endothelial cells in human penile tissue [12] further corroborates this concept. Based on this and our previous finding, we have hypothesized an interaction between UT receptor and eNOS at molecular level within the endothelium. Following this hypothesis we performed a co-immunoprecipitation study. The formation of the immunocomplex demonstrated that a link exists between eNOS and U-II/UT pathway within the human penile tissue. This molecular evidence was functionally confirmed. Indeed, the incubation of HCC homogenates with U-II caused a concentration-dependent increase in NOx production. Interestingly, the highest U-II concentration elicited a six fold increase in NOx generation. Thus, U-II released within the corpus cavernosum binds its receptor on endothelial cells leading to eNOS activation and in turn to NO production. Basically, U-II-induced relaxation contributes to eNOS-derived NO generation in HCC. But how activation of this pathway leads to an increase production of eNOS-derived NO? To date it is well consolidate the concept that neuronal NOS (nNOS) and eNOS mediate the initiation and maintenance of penile erection, respectively [22]. Neuronal signal initiates penile erection by activating nNOS that elicits a rapid and transient NO release causing an increase in blood flow [29]. The resulting shear stress force on the endothelium activates the PI3K/Akt/eNOS phosphorylation cascade, causing more sustained NO release and relaxation [22], [30]. Indeed, Akt-phoshorylated eNOS results 15 to 20 fold more active that un-phospohorylated eNOS [31]. In other words, the phosphorylation of eNOS shifts the enzyme at higher state of activation, boosting NO production [32].
In the human body, the penis is one of the organ physiologically subjected to shear stress and the resultant eNOS phosphorylationSer1177 plays a key role in the maintenance of full penile erection [22]. Therefore, we performed a pharmacological modulation of the eNOS phosphorylation in order to investigate if U-II activity relies on this mechanism. U-II caused a significant increase in eNOS phosphorylation Ser1177 in a time-dependent manner. The irreversible inhibition of PI3K/Akt operated by wortmannin, reverted the phosphorylation induced by U-II. Also in this case the molecular finding has been confirmed through a functional study. Indeed, U-II relaxant effect on HCC was virtually abolished by wortmannin.
Another recognized key player in the mechanism of eNOS activation is Hsp90. In fact, shear stress as well as some other endogenous substances enhances the interaction between Hsp90 and eNOS [18], [19]. The increased association of eNOS to Hsp90 shifts the enzyme to an higher active state, too. Basically, Hsp90 acts as an allosteric modulator of eNOS by inducing conformational changes in the enzyme that result in an increased activity [33]. Geldanamycin, an Hsp 90 inhibitor, blocked U-II induced relaxation of HCC strips at the same extent of wortmannin. Experimental evidence suggests that Hsp90-eNOS hetero-complex occurs simultaneously with other signaling events such as the mobilization of intracellular calcium and/or protein phosphorylation [34]. Therefore, it appears feasible the U-II/UT pathway contributes to the maintenance rather than triggering erection. Indeed, the pro-erectile effect of U-II is strictly dependent upon eNOS derived-NO generation. The obligatory role for eNOS-derived NO in U-II effect is supported by the finding that i) blockade of eNOS phosphorylation and Hsp90 coupling abrogates U-II effect ii) incubation of HCC tissue with U-II causes a notable increase in NO generation.
In conclusion , U-II/UT pathway contributes to the physiology of erection through eNOS-derived NO. UII/UT pathway once activated, most likely by the shear stress due to the erection, causes eNOS phosphorylation and Hsp90 recruitment shifting eNOS activity to a more activate state. This in turn leads to a sustained NO release which contribute to the maintenance of the ongoing erection.
Thus, U-II/UT pathway could represent an important novel target in order to find new pharmacological approaches in erectile dysfunction. Indeed, it is known that a certain percent of patients (about 35%) do not respond to PDE5 inhibitors [35]. As PDE5 inhibitors and U-II share the same target NO, it is plausible that in the next future U-II agonists might be used in combination with PDE5 inhibitors in order to enhance cGMP signaling. Indeed, it could be that in some cases there is only a limited amount of endothelium functionally working and the PDE5 inhibition by itself will not be sufficient to sustain the erection triggered. In this case the combination with the UII agonist could exert a synergistic effect.
Methods
Peptide
The human U-II was synthesized and purified at the Department of Pharmaceutical and Toxicological Chemistry of the University of Naples, Federico II. The peptide was obtained by solid-phase peptide synthesis as previously reported [36]. Purification was achieved using a semi-preparative reversed phase high-performance liquid chromatography (HPLC) C18 bonded silica column (Vydac 218TP1010; The Separations Group Inc., Hesperia, CA, USA). The purified peptide was 99% pure as determined by analytical reversed-phase HPLC. The correct molecular weight were confirmed by mass spectrometry and amino acid analysis.
Human Tissue
In male-to-female transsexuals undergoing surgical procedure for sex reassignment, the penis and testicles were amputated and a neo-vagina was created to simulate female external genitalia. All the surgical procedures were performed at the Department of Urology, University of Naples, Federico II, Naples, Italy [37]. The corpora cavernosa were carefully excised from the penis immediately after amputation and placed in ice-cold oxygenated Krebs' solution [37]. All patients were informed of all procedures and gave their written consent. Local Ethical Committee (Faculty of Medicine and Surgery; University of Naples Federico II, via Pansini, 5; 80131, Naples, Italy) approved the use of human corpus cavernosum tissue for in vitro studies.
Real-Time Quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
The presence of U-II was determined by quantitative PCR. Briefly, total RNA from omogenated HCC tissue was extracted by using TRIzol reagent (Invitrogen, Milan, Italy), to eliminate genomic DNA contamination 1 µg of above RNA was treated with RQ1 RNase-free DNase I (Promega Corporation, Madison, USA), according to the manufacturer's recommendations. Reverse transcription was performed using M-MLV Reverse Transcriptase (Invitrogen, Milan, Italy) according to the manufacturer's recommendations, and 20 ng of cDNA samples were used for quantitative PCR. Samples were run in triplicate in 25-µL reactions using an 7500 Real Time PCR System (Applied Biosystems, Foster City, CA). Amplification was done using Sybr Green PCR Master Mix (Applied Biosystems, Monza, Italy). U-II-specific forward primers 5′-GCACTGTTTGCTTTGGACTCC-3′and reverse primer : 5′-TGGTCGTCCATGCACAGATT-3′, and human GAPDH forward primer 5′-AACGGATTTGGTCGTATTGGGC- 3′ and reverse primer 5′-TCGCTCCTGGAAGATGGTGATG-3′ were specifically designed using Primer Express Software 2.0 and validated for their specificity. Samples were incubated at 50°C for 2 min and at 95°C for 10 min followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. Differences in cDNA input were corrected by normalizing signals obtained with primers specific for glyceraldehydes-3-phosphate dehydrogenase (GAPDH). To exclude nonspecific amplification and/or the formation of primer dimers, control reactions were performed in the absence of target cDNA. In order to validate the results we used human tumoural cell lines SW-13 as positive control [24]. Gene expression levels were calculated using the 2−ΔCT method and are presented as ratio between mean fold change of target gene and GAPDH ± standard error. Data were expressed as mean ± standard error of the mean (SEM) from three different specimens.
NOx determination
HCC tissues were incubated with U-II at different concentration (1 nM–10 µM) or vehicle for 30 minutes at 37°C. The reaction was stopped in liquid nitrogen. Homogenate tissues were incubated in a microplate with cadmium (50 mg/well) for 1 h to convert the inorganic anions nitrate (NO3) to nitrite (NO2). After centrifugation at 14,000 rpm for 15 min, total nitrite (NOx) content was determined fluorometrically in microtiter plates (PerkinElmer Instruments, LS55; UK) using a standard curve of sodium nitrite [38]. NOx content was calculated by using the internal standard curve. Data were expressed as mean ± standard error of the mean (SEM) from four different specimens and analyzed by using analysis of variance (ANOVA) followed by Bonferroni post hoc test. The level of statistical significance was taken as p<0.05.
Functional reactivity of HCC Strips
Longitudinal strips (2 cm) of HCC were dissected and isolated from the trabecular structure of the penis [25]. Krebs' solution had the following composition (mM): 115.3 NaCl; 4.9 KCl; 1.46 CaCl2; 1.2 MgSO4; 1.2 KH2PO4; 25.0 NaHCO3; 11.1 glucose (Carlo Erba, Milan, Italy). HCC strips were mounted in organ bath containing oxygenated (95% O2 and 5% CO2) Krebs' solution at 37°C. HCC strips were connected to isometric force-displacement transducers (model 7002, Ugo Basile, Comerio, Italy) and changes in tension were recorded continuously by using a software (Datacapsule, Basile, Comerio, Italy). Tissues were preloaded with 2 g of tension and allowed to equilibrate for 90 minutes in Krebs' solution. After equilibration, tissues were standardized by performing repeated phenylephrine (PE; 3 µM; Sigma, Milan, Italy) contractions until three equal responses were obtained. Endothelial integrity was assessed by using acetylcholine (Ach; 0.01–10 µM; Sigma, Milan, Italy) and tissues that showed a relaxation effect less that 80% were discarded. A concentration response curve to U-II (0.1 nM–10 µM) was obtained in the presence of endothelium, using HCC strips pre-contracted with PE (3 µM). In order, to investigate the involvement of L-arginine/NO pathway in U-II-induced relaxation we operated a pharmacological modulation. HCC strips were incubated for 30 minutes with either wortmannin (0.1 µM, Tocris, UK) an irreversible inhibitor of PI3K or geldanamycin (1 µM, Sigma, Milan, Italy) an Hsp90 inhibitor before U-II challenge. The doses of wortmannin (0.1 µM) and geldamycin (1 µM) were selected by a preliminary dose-finding study (data not shown).
Data were calculated as % of relaxation to PE tone and expressed as the mean ± standard error of the mean (SEM) of six different specimens. The results were analyzed by using analysis of variance (ANOVA) followed by Bonferroni post hoc test. The level of statistical significance was taken as p<0.05.
Western Blot Analysis
In order to evaluate the effect of U-II on the eNOS phopsphorylation -Ser 1177 (p-eNOS Ser 1177), HCC tissues were incubated with the peptide (10 µM) at scheduled time (5, 15, 30 and 60 minutes). In another set of experiments, tissues were pre-treated with wortmannin (0.1 µM, Tocris, UK) or geldamycin (1 µM, Sigma, Milan, Italy) and thereafter incubated with U-II (10 µM) or vehicle for 30 minutes. Next, HCC tissues were homogenized in modified RIPA buffer (Tris-HCl 50 mM, pH 7.4, Triton 1%, sodium deoxycholate 0.25%, NaCl 150 mM, ethylenediaminetetraacetic acid 1 mM, phenylmethylsulphonyl fluoride 1 mM, aprotinin 10 µg/mL, leupeptin 20 µM, NaF 1 µM, sodium orthovanadate 1 µM) by liquid nitrogen. Protein concentration was estimated by the Bio-Rad protein assay using bovine serum albumin (BSA) as standard. Equal amounts of protein (50 µg) of the tissue lysates were separated on 8% sodium dodecyl sulfate polyacrylamide gels and transferred to a polyvinylidene fluoride membrane. Membranes were blocked by incubation in phosphate-buffered saline (PBS) containing 0.1% v/v Tween 20, non fat dry milk (5%) and NaF (50 mM) for 1 hours, followed by overnight incubation at 4°C with rabbit anti-p-eNOSSer-1177 polyclonal antibody (1∶1000, Cell Signaling, DBA, Italy) and with mouse eNOS monoclonal antibody (1∶1000; BD Transduction Laboratories). The filters were washed extensively in PBS containing 0.1% v/v Tween 20, before incubation for 2 hours with horseradish peroxidase-conjugate anti rabbit secondary antibody (1∶5000). Membranes were then washed and developed using enhanced chemiluminescence substrate (Amersham Pharmacia Biotech, San Diego, CA, USA). The data were evaluated by densitometric analysis and expressed as p-eNOS/eNOS ratio.
Data were expressed as mean ± standard error of the mean (SEM) of four different specimens. The results were analyzed by using analysis of variance (ANOVA) followed by Bonferroni post hoc test. The level of statistical significance was taken as p<0.05.
UT receptor and eNOS co-immunoprecipitation
HCC samples were homogenized (500 µg) and incubated with 20 µl of Protein A–coupled sepharose beads and 1 µg/ml of IgG mouse at 4°C for 3 h. The samples were centrifuged (600 rpm for 15 sec) and the supernatants were incubated overnight at 4°C with 20 µl of mouse eNOS monoclonal antibody (Cell Signaling, DBA, Milan, Italy) or normal mouse serum (to evaluate non-specific binding) on a rotating wheel.Next, the samples were incubated with 40 µl of Protein A–coupled sepharose beads at 4°C for 2 h and centrifuged (600 rpm for 15 sec). The pellets were extensively washed and then suspended in 3× Laemmly buffer. After heating at 95°C for 5 minutes, samples were subjected to Western blot analysis developed for UT (1∶1000; GPR14 (H-90), Santa Cruz Biotechnology Inc., Heidelberg, Germany).
Competing Interests: The authors have declared that no competing interests exist.
Funding: The present study was supported by European Society for Sexual Medicine (ESSM) grant for Medical Research 2009 and by Regional grant for research n.5/2002, 2007. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22319627PONE-D-11-0808210.1371/journal.pone.0031388Research ArticleBiologyBiochemistryGeneticsMolecular GeneticsMolecular Cell BiologyMedicineCardiovascularNephrologyHomocysteinylated Albumin Promotes Increased Monocyte-Endothelial Cell Adhesion and Up-Regulation of MCP1, Hsp60 and ADAM17 Protein Homocysteinylation and Endothelial DamageCapasso Rosanna
1
Sambri Irene
1
Cimmino Amelia
4
Salemme Sofia
1
Lombardi Cinzia
3
Acanfora Filomena
3
Satta Ersilia
3
Puppione Donald L.
5
Perna Alessandra F.
2
3
*
Ingrosso Diego
1
2
1
Department of Biochemistry and Biophysics “F. Cedrangolo”, School of Medicine, Second University of Naples, Naples, Italy
2
Cardiovascular Research Centre, School of Medicine, Second University of Naples, Naples, Italy
3
First Division of Nephrology, School of Medicine, Second University of Naples, Naples, Italy
4
Institute of Genetics and Biophysics, National Research Council, Naples, Italy
5
The Molecular Biology Institute, University of California Los Angeles, Los Angeles, California, United States of America
Bearden Shawn E. EditorIdaho State University, United States of America* E-mail: [email protected] and designed the experiments: AFP DI. Performed the experiments: RC IS AC DLP SS CL FA ES. Analyzed the data: RC DLP AFP DI. Contributed reagents/materials/analysis tools: AFP DI. Wrote the paper: AFP DI.
2012 3 2 2012 7 2 e313889 5 2011 6 1 2012 Capasso et al.2012This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Rationale
The cardiovascular risk factor homocysteine is mainly bound to proteins in human plasma, and it has been hypothesized that homocysteinylated proteins are important mediators of the toxic effects of hyperhomocysteinemia. It has been recently demonstrated that homocysteinylated proteins are elevated in hemodialysis patients, a high cardiovascular risk population, and that homocysteinylated albumin shows altered properties.
Objective
Aim of this work was to investigate the effects of homocysteinylated albumin - the circulating form of this amino acid, utilized at the concentration present in uremia - on monocyte adhesion to a human endothelial cell culture monolayer and the relevant molecular changes induced at both cell levels.
Methods and Results
Treated endothelial cells showed a significant increase in monocyte adhesion. Endothelial cells showed after treatment a significant, specific and time-dependent increase in ICAM1 and VCAM1. Expression profiling and real time PCR, as well as protein analysis, showed an increase in the expression of genes encoding for chemokines/cytokines regulating the adhesion process and mediators of vascular remodeling (ADAM17, MCP1, and Hsp60). The mature form of ADAM17 was also increased as well as Tnf-α released in the cell medium. At monocyte level, treatment induced up-regulation of ICAM1, MCP1 and its receptor CCR2.
Conclusions
Treatment with homocysteinylated albumin specifically increases monocyte adhesion to endothelial cells through up-regulation of effectors involved in vascular remodeling.
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Introduction
Hyperhomocysteinemia is a cardiovascular risk factor both in the general population and in selected patient groups [1]. Previous evidence showed that high homocysteine increases cell adhesion and induces a proinflammatory state in the vessel wall by promoting adhesion molecule expression and monocyte recruitment. In particular, nuclear factor (NF)-kB activation and intercellular adhesion molecule-1 (ICAM-1) stimulation have been shown [2]. High homocysteine also up-regulates monocyte chemoattractant protein-1 (MCP1), interleukin-8 (IL-8) expression and secretion in cultured human endothelial cells, smooth muscles cells, and monocytes [3]–[6]. Also VCAM-1 expression is up-regulated [7].
Plasma homocysteine is mainly protein-bound, accounting for >90% of total homocysteine (the remainder is found as free low-molecular weight disulfide forms, including homocystine, that is the homocysteine homodimer, and the homocysteine-cysteine mixed disulfide). Only 1.5–4% of homocysteine in circulation is present in its reduced form [8]. In many of previous studies, homocysteine was simply added to cell culture medium in its rather unphysiological free reduced form, thus rising concerns about the possibility of artifactual effects mediated through intervening formation of adducts with unpredictable protein targets.
Under physiological conditions protein homocysteinylation occurs through acylation of free amino groups (protein-N-homocysteinylation), [9]–[11] and thiol group oxidation (protein-S-homocysteinylation) [12].
It has been shown that hyperhomocysteinemia elicits its effects on the vasculature mainly through homocysteinylated proteins [13]. In this respect, the functional properties of human serum albumin are altered by homocysteine binding [14].
Homocysteine is commonly elevated in end-stage renal disease (ESRD) patients on hemodialysis. This fact has attracted much scientific interest, because of the high cardiovascular risk in these patients, which is not exhaustively explained by the presence of conventional risk factors and/or specific uremic toxins [15]–[17]. We recently showed that plasma protein homocysteinylation is increased in uremic patients on hemodialysis and significantly reduced, although not normalized, after folate supplementation [14]. Concerning this issue, it can be mentioned that some emphasis has been given in the literature on the role played by protein-bound uremic toxins [18].
Protein homocysteinylation could be one of the principal mediators of homocysteine toxicity, contributing to detrimental structural and functional alterations at the molecular and cellular level [19]–[21]. We therefore investigated the role of homocysteinylated albumin in eliciting cell adhesion, and monitored, starting with a genome-wide approach, the expression of relevant mediators in the adhesion process.
Methods
Synthesis and characterization of homocysteinylated albumin
Homocysteinylated albumin was produced according to a modification of the protocol published by Jakubowski [9] by incubation with homocysteine thiolactone, determining the formation of mainly N-homocysteinylated albumin, and a smaller amount of S-homocysteinylated albumin. Homocysteinylated albumin was HPLC-analyzed [14]. The N-homocysteinylated albumin adduct was largely prevalent, while a very small amount of S-homocysteinylated albumin. N-homocysteinylated albumin concentrations used in the experiments were in the range of 0.80 nmol Hcy/mg albumin and, in order to facilitate comparison with equivalent concentrations of circulating N-protein bound homocysteine, are referred to as equivalent to concentration of homocysteine in mol/L [14].
Homocysteinylated and unmodified albumin, purified by reversed phase HPLC [14], were then characterized by electrospray mass spectrometry (ESI-MS) at the Pasarow Mass Spectrometry Laboratory, UCLA, Los Angeles California, U.S.A., as described by Puppione et al. [22]. Briefly, samples were separated by size-exclusion chromatography and mass spectrometry (ESI-MS) was performed using a triple quadrupole instrument (API III, Applied Biosystems). Data were processed using MacSpec 3.3, Hypermass and BioMultiview 1.3.1 software (Applied Biosistems).
Cell cultures and treatments
Human endothelial cell line EAhy926 (ATCC) were grown in high glucose concentration DMEM (Gibco), containing 10% fetal bovine serum (Gibco), 1% glutamine, 1% penicillin/streptomycin (Gibco), 1% fungizone (Gibco). Human monocytoid cell line U937 (ATCC) were grown in RPMI-1640 (Gibco) containing 10% bovine fetal serum (Gibco), 1% penicillin/streptomycin (Gibco).
EAhy926 were incubated for 18 h, in the presence of 0.3 or 1.0 µmol/L Homocysteinylated albumin equivalent. Negative controls were: a) cells incubated with unmodified human serum albumin at a comparable protein concentration; b) cells incubated without human serum albumin (i.e. untreated negative control); c) cells incubated with carboxylmethylated human serum albumin, as a control of differently-modified albumin at cysteine levels [23]. This control was included in order to rule out that any covalent modification of human serum albumin could trigger adhesion, irrespective of the kind of amino acid modification involved. Cells incubated with Tnf-α 10 ng/ml for 4 h represented positive controls.
U937 (ATCC) were incubated for 18 h, in the presence of 1.0 µmol/L Homocysteinylated albumin equivalent. Negative controls were: a) cells incubated with unmodified human serum albumin at a comparable protein concentration; b) cells incubated without human serum albumin (i.e. untreated negative control).
Adhesion assay
EAhy926 were plated to 90% confluence in 24-well multiwell plates and treated with homocysteinylated albumin or the appropriate control for 18 h at 37°C. Treatment medium was removed and saved; cells were washed once with incomplete DMEM medium and 8×105 monocytoid cells per well were co-incubated, for 30 min at 37°C in the endothelial treatment medium. Non-adherent U937 were removed by gently washing thrice with PBS. Finally PBS containing 1% glutaraldehyde was added to fix monocytes to the endothelial monolayer. Adherent monocytes were counted directly using a Zeiss Axiovert 10 inverted photomicroscope (Carl Zeiss S.p.A., Milan, Italy) on three randomly selected high magnification microscopic fields per well, for each independent experiment. Ten independent experiments were performed. Results were expressed as both mean of the absolute number of adherent monocytes per field, and percentage of adherent monocytes relevant to samples treated with Tnf-α (100% adhesion).
RNA extraction
RNA extraction was performed, on endothelial cells, treated with homocysteinylated albumin, utilizing Trizol reagent (Invitrogen), according to the supplier's protocols. RNA concentration was measured by NanoDrop UV/VIS micro- spectrophotometry (ND-1000; NanoDrop Technologies, Wilmington, DE, USA).
Expression profile
Microrray hybridization and data analysis were carried out using Human Genome U133A Plus 2.0 GeneChip arrays (Affymetrix, Santa Clara, CA, U.S.A.), containing 54000 hybridized genes, essentially as described by Calin et al [24]. Transcriptome data were compliant with the MIAME (Minimum Information About a Microarray Experiment) standard and registered in suitable format on the ArrayExpress Archive database (http://www.ebi.ac.uk/microarray-as/ae/).
Real time PCR
cDNA synthesis from 1 µg of total RNA was made using the QuantiTect reverse transcriptase kit (Qiagen, Life Sciences, Milan, Italy). Amplifications were performed with the iCycler thermalcycler (Bio-Rad Laboratories S.r.l., Segrate Milan, Italy) with the fluorescence detection system iCycler iQ real time PCR. The amplification mix contained 1 µl cDNA, 0.3 µM of each primer, 12.5 µl of master mix QuantiTECT SYBR green (Quiagen), and H2O DEPC, for a final volume of 25 µl. Primers pair and amplification condition are described in Table 1. Relative expression was calculated using the delta Cq method. The value of 2−delta delta Cq>1 reflects increased expression of the relevant gene, and a value of 2−delta delta Cq<1 points to a decrease in gene expression.
10.1371/journal.pone.0031388.t001Table 1 Amplification conditions and primer pairs for PCR experiments.
MCP1 (NM_002982.3) 95°C for 30 sec, 58°C for 30 sec, 72°C for 30 sec
5′-CAGCAGCAAGTGTCCCAAAG-3′
5′-GAGTGAGTGTTCAAGTCTTCGG-3′
ICAM1 (NM_000201) 95°C for 30 sec, 62°C for 30 sec, 72°C for 30 sec
5′-GGTGTATGAACTGAGCAATGTG-3′
5′-TGGCAGCGTAGGGTAAGG-3′
ADAM17 (NM_003183) 95°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec
5′-GTATCTGAACAACGACACCTG-3′
5′-CTCCTGGCACTTCTTCTGG-3′
NRP1 (NM_003873) 95°C for 30 sec, 60°C for 30 sec, 72°C for 30 sec
5′-GGAACTTGGTGGATGAATGTGATG-3′
5′-CTCTGATTGTATGGTGCTGTCTATG-3′
TFP1 (NM_006287) 95°C for 30 sec, 57°C for 30 sec, 72°C for 30 sec
5′-ATAACTCCCTGACTCCGCAATC-3′
5′-TGGCACGACACAATCCTCTG-3′
VCAM1 (NM_001078) 95°C for 30 sec, 63°C for 30 sec, 72°C for 30 sec
5′-TCTACGCTGACAATGAATCCTG-3′
5′-CTTGACTGTGATCGGCTTCC-3′
CCR2 (NM_001123396) 95°C for 30 sec, 54°C for 30 sec, 72°C for 30 sec
5′-TTCTTCATCATACTCCTGACAATCG-3′
5′-AGCAAACACAGCCACCAACC-3′
Hsp60
95°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec
Quatitect primer assay (Quiagen)
All conditions are relevant to real time PCR except for VCAM1, where tradition PCR has been employed. 35 amplification cycles have been performed.
For VCAM1 transcript amplification, 2 µl cDNA were employed and mix was replaced by a mixture containing: 0.5unit of Taq Polymerase (Fermentas Inc., MD, USA), 0.2 mM each dNTP, 2 µl buffer, in a 20 µl final volume and reactions carried out in a Mastercycler gradient (Eppendorf s.r.l., Milan, Italy).
Protein analyses
Western Blot analyses were performed as previously described [25], using anti-human HSP60 (BD Pharmingen), anti-human ADAM17 (Abcam), anti-human MCP1 (Santa Cruz), anti-human ICAM1 (BD Pharmingen) and anti-human CCR2 (Abcam) as appropriate.
ICAM1, VCAM1, MCP1 and Tnf-alpha concentrations in cell culture media were determined utilizing the relevant ELISA kits (R&D Systems) according to the supplier's protocols.
Immunoprecipitation of Hsp60 was performed by magnetic bead separation using DYNAL Beads (Invitrogen) crosslinked to anti-human Hsp60 antibody (ABCAM) according to the manufacturer. The protein eluted from the crosslinked beads was revealed by Western blotting.
Cytofluorometric analysis
Cell pellets, harvested and washed with cold PBS containing 0.1% BSA were treated with Phycoeritrine-labeled anti-VCAM1 antibodies or Allophycocyanin-labeled anti-ICAM1 (BD Pharmingen, Milan, Italy) and incubated in ice for 1 h in the dark. At the end of incubation, 1 ml of cold PBS/0.1% BSA was added and cells were pelleted. Finally, 500 µl of a PBS/0.1% BSA containing 1 µl of 0.2 µg/ml propidium iodide (Sigma-Aldrich, Milan, Italy) were added to the cell pellets prior to analysis. cytofluorimetric analysis was performed in a FACSCalibur (BD Biosciences, Milan, Italy).
Statistical analysis
An unpaired Student's t test was performed, to compare means in the homocysteinylated albumin experiments, or a two-way ANOVA to assess the timed effects of treatments as appropriate [26]. All results are presented as the mean (SD). All experiments were done in triplicate except otherwise stated.
Results
Characterization of homocysteinylated albumin by mass spectrometry
ESI-MS spectra of unmodified albumin and its homocysteinylated derivative are reported in Fig. 1. The calculated molecular mass value of native albumin was 66446 Da (Fig. 1; panel A). The homocysteinylated derivative had a calculated molecular mass of 67805 Da (Fig. 1 panel B, inset) and a calculated mass of 33903 Da for the doubly charged protein (Fig. 1; panel B). The difference between the homocysteinylated and native species is equal to 1359 Da. This difference corresponds to the acquisition of 7 N-homocysteinyl moieties (N-Lys-Hcy-SH; 117 Da), which are N-linked (amide linkage) to as many Lys residues of albumin [27], plus 4 S-homocysteinyl moieties (N-Lys-Hcy-S-S-Hcy; 133 Da), linked to N-linked homocysteine, through an S-S linkage [11], [28]–[30]. This result is highly consistent with what previously reported [27] except that the S-linked species could not be detected in this report, since the thiol groups in the N-homocysteinylated protein derivative were blocked by iodoacetamide (we omitted this step in order to prevent possible non-physiological toxic effects to cell cultures). On the other hand the reactivity of the free thiol group of the N-linked homocysteinyl moiety has been amply established by previous work [11], [28]–[30].
10.1371/journal.pone.0031388.g001Figure 1 Characterization of homocysteinylated albumin by mass spectrometry.
Panel A: ESI-MS of human serum albumin. Panel B: ESI-MS of homocysteinylated albumin. Inset: magnification on expanded scale of the signal at Da = 67805. The family of molecular ions is compatible with the structures shown in the panel.
Effects of treatment of EAhy926 monolayer with homocysteinylated albumin on monocyte adhesion
Results are presented both as the mean of adherent monocyte number per field (Fig. 2A), and as percent of adhesion with respect to the Tnf-α treated positive control (100% adhesion) (Fig. 2B). Examples of adhesion assay presentation are also given (Fig. 2C).
10.1371/journal.pone.0031388.g002Figure 2 Effects of homocysteinylated albumin on monocyte adhesion.
U937 monocytoid cells adhesion onto an endothelial monolayer (EAhy926) expressed as adherent cells (number/field; panel A) and percentage adherent cells compared to positive control (panel B). Counts are the mean of ten independent experiments, each carried out by counting five different fields/sample of triplicate samples. Examples of microscopic fields are shown on the right. C: negative control (untreated cells); A: unmodified albumin; AH: homocysteinylated albumin; AC: carboxymethylated albumin; T: positive control (Tnf-α). 0.3 or 1: homocysteine micromolar concentration present in the assay in the form of N-homocysteinyl groups bound to albumin, as comparable to the in vivo situation [14]; p<0.0001.
Results showed that treatment of endothelial cells with Homocysteinylated albumin at concentrations comparable to those detected in vivo in uremic hyperhomocysteinemic patients [14] is followed by a significant increase of monocyte adhesion onto the endothelial monolayers. Conversely, treatment with Homocysteinylated albumin, at concentrations comparable to those detected in vivo
[14] in normal subjects, does not result in any significant increase of adhesion with respect to control treatments.
Homocysteinylated albumin treatment increases adhesion molecule expression
Cell adhesion and endothelial damage are increased in uremia and paralleled by time-dependent VCAM-1 and ICAM-1 up-regulation [31], [32].
Fig. 3 and 4 show the results of ICAM1 and VCAM1 kinetic monitoring in Eahy926 treated with 1.0 µmol/L homocysteinylated albumin compared to control at both gene expression and protein levels. ICAM1 transcripts significantly increased after 1.0 µmol/L homocysteinylated albumin treatment, compared to control (Fig. 3A). Consistently, a significant increase in the surface expression of ICAM1 protein became evident within 18 h treatment (Fig. 3B), paralleled by ICAM1 release in the medium (Fig. 3C), thus mirroring the situation detected at cell surface level.
10.1371/journal.pone.0031388.g003Figure 3 ICAM1 expression in endothelial cells treated with N-homocysteinylated albumin.
Panel A: expression levels of ICAM1 transcripts quantitated by real time PCR (treated: 1 µmol/L homocysteinylated albumin; control: unmodified albumin); (p<0.001). Panel B: cytofluorimetric analysis of ICAM1 time course surface expression by EAhy926 endothelial cells treated with homocysteinylated albumin (C: unmodified albumin negative control; Tnf-α: positive control). Panel C: Time course of ICAM1 release in the culture medium, quantitated by ELISA assay. C: negative control (untreated cells); A: unmodified albumin; AH: 1 µmol/L homocysteinylated albumin; (p<0.001).
10.1371/journal.pone.0031388.g004Figure 4 VCAM1 expression in endothelial cells treated with N-homocysteinylated albumin.
Panel A: Time course of induction of VCAM1 transcripts, in EAhy926 endothelial cells, by treatment with 1 µmol/L homocysteinylated albumin. Panel B: cytofluorimetric analysis of ICAM1 time course surface expression by EAhy926 endothelial cells treated with homocysteinylated albumin. (C: unmodified albumin negative control; Tnf-α: positive control). Panel C: Time course of ICAM1 release in the culture medium, quantitated by ELISA assay. C: negative control (untreated cells); A: unmodified albumin; AH: 1 µmol/L homocysteinylated albumin. (p<0.001).
After only 2 h of treatment, as shown in Fig. 4A, the VCAM1 transcripts could be detected, and decline thereafter. This indicates that homocysteinylated albumin elicits an immediate response in the regulation of this particular gene. Also VCAM1 antigen exposure could be detected at high levels on cell surface at 18 h of treatment with homocysteinylated albumin (Fig. 4B). Consistently, a parallel increase in VCAM1 protein released in the medium could be also detected in the culture medium of cells treated with homocysteinylated albumin, compared to control (Fig. 4C).
Homocysteinylated albumin treatment determines increased expression in specific mediators of endothelial cell activation and damage
We were then prompted to investigate the alterations induced by homocysteinylated albumin on endothelial monolayers, which may explain the increased tendency of monocytoid cells to adhere. To this purpose we employed a genome-wide transcriptional analysis using microarray hybridization. RNA samples, extracted from endothelial cells treated with 1 µmol/L homocysteinylated albumin, untreated albumin, and untreated cells, were utilized. Treatment with homocysteinylated albumin significantly modifies gene expression profile of endothelial cells compared to control. In particular, among the twenty-three up-regulated genes, five are possibly implicated in endothelial activation (CCL2, HSPD1, ADAM17, TFP1, NRP1) (Table 2). Validation by real time PCR was carried out for MCP1, HSPD1, ADAM17, TFP1, NRP1, in consideration of their possible involvement in vascular remodeling processes. The increase of transcript levels, upon treatment with 1 µmol/L homocysteinylated albumin, was confirmed for all these five genes of interest (Fig. 5).
10.1371/journal.pone.0031388.g005Figure 5 Validation by Q-PCR of transcriptome results relevant to upregulated gene involved in endothelial dysfunction.
A: unmodified albumin; AH: homocysteinylated albumin. Gene expression in the AH sample group was significantly increased with respect to the corresponding genes in the A sample group (p<0.001).
10.1371/journal.pone.0031388.t002Table 2 Upregulated genes from transcriptional analysis of EAhy926 cells treated with homocysteinylated albumin compared to control.
GENE NAME AND FUNCTION GENE SYMBOL Accession number Fold change
Tissue Factor Pathway Inhibitor isoform a TFP1 215447_at 4.972
NADH Dehydrogenase subunit 5 MTND5 1553575_at 4.762
Dehydrogenase/Reductase member 2 isoform 2 DHRS2 214079_at 4.711
Neuropilin1 NRP1 234072_at 4.178
Spectrin Repeat Containing Nuclear Envelop 1 SYNE1 244144_at 3.758
Insulin-like growth factor 1 receptor IGF1R 203628_at 3.598
Secretogranin V, Variant 1 SCG5 203889_at 3.441
Xenotropic/Polytropic Retrovirus Receptor, var 1 XPR1 244755_at 3.331
Transmembr. coiled-coin domain family 1, var 3 TMCC1 237943_at 2.788
ADAM17 metalloprotease ADAM17 205745_x_at 2.664
Tau Tubulin Kinase 2 TTBK2 231610_at 2.664
Mitochondrial Ribosomal Protein S28 MRPS28 236955_at 2.609
Dehydrogenase/Reductase member 2, var 3 DHRS2 206463_s_at 2.581
Solute Carrier Family 25, member 45, var 1 SLC25A45 1563498_s_at 2.497
RAS p21 protein activator 2 RASA2 206636_at 2.474
Polypyrimidine tract binding protein 2 PTBP2 236962_at 2.446
par-3 partitioning defective 3 homolog B, var B PARD3B 244586_x_at 2.387
mucin20, cell surface associated, var S MUC20 226622_at 2.374
UDP-glucose ceramide glucosyltransferase UCGC 204881_s_at 2.265
PAP associated domain, var 1 MTPAP 238706_at 2.241
Chemokine (C-C motif) ligand 2 CCL2 216598_s_at 2.236
ubinuclein 2 UBN2 238350_at 2.140
Heat shock protein 60 kD HSPD1 200806_s_at 2.005
Homocysteinylated albumin concentration was comparable to that detected in vivo in hyperhomocysteinemic uremic patients compared to control.
Among the up-regulated genes identified in the transcriptional profile of endothelial cells treated with 1.0 µmol/L homocysteinylated albumin, we identified three genes deserving special notice for their involvement in vascular activation and damage: CCL2, ADAM17, and Hsp60. Transcriptional increase of all these genes (real time PCR), as well as the levels of the relevant protein products (ELISA and/or western blot), were also kinetically monitored.
A time-dependent increase in MCP1 transcription levels could be observed, in Eahy926 treated with 1.0 µmol/L homocysteinylated albumin with a maximum at 18 h (Fig. 6A). Consistently, ELISA assays showed a significant increase in MCP1 secreted by treated cells (Fig. 6B).
10.1371/journal.pone.0031388.g006Figure 6 Time-dependent, increased expression of ADAM17, MCP1 and Hsp60 in endothelial cells upon homocysteinylated albumin treatment.
Panel A: Real time PCR evaluation during time course of ADAM17, MCP1 and Hsp60 mRNA. Panel B: ELISA assay of MCP1 released in the culture medium of treated cells. Panel C: Western blotting analysis of intracellular levels of ADAM17, and Hsp60, and analysis of Hsp60 released in the medium by immunoprecipitation and western blotting (Hsp60 IP). A: unmodified albumin control; AH: homocysteinylated albumin treatment. Levels of transcripts or proteins in the homocysteinylated albumin sample group were significantly increased compared to control (p<0.001).
The transcript of ADAM17 is subject to time-dependent increase upon treatment of endothelial cells with 1.0 µmol/L homocysteinylated albumin (Fig. 6A). In addition, protein levels were analyzed using an antibody capable of recognizing the two forms, the precursor (110 kDa) and the mature form (80 kDa), of ADAM17 and, as shows in Fig. 6C, an increase of both ADAM17 forms could be observed, which was particularly evident in the case of the mature form. Consistently, we also found a release of Tnf-alpha in the culture medium of cells treated with homocysteinylated albumin, (168 pg/ml), while it was undetectable in the controls.
Hsp60 transcription levels also increased, in a time-dependent fashion, with a similar peak at 18 h (Fig. 6A). In consideration of the prevalent intracellular localization of Hsp60 antigen, we evaluated Hsp60 with Western blotting on endothelial cell extracts. Hsp60 is normally segregated within the intracellular compartment. We showed, by immunoprecipitation, that, upon stimulation with homocysteinylated albumin, Hsp60 increased within the intracellular compartment, paralleled by a time-dependent release of Hsp60 in the medium (Fig. 6C).
Homocysteinylated albumin treatment determines increased expression in specific mediators on U937
Cell adhesion involves changes which occur both at the endothelial and monocyte levels. We were then prompted to investigate the alterations induced by homocysteinylated albumin on monocyte U937 cell line, under conditions in which we observed an increased cell adhesion to endothelial monolayers treated with homocysteinylated albumin. We then analyzed the expression levels and the relevant protein levels of three important markers of monocyte activation, ICAM1, MCP1 and CCR2. U937 treated with homocysteinylated albumin 1.0 µmol/L showed a significant increase both of mRNA levels (Fig. 7A) and protein levels (Fig. 7B) of ICAM1, MCP1 and CCR2, thus confirming that the observed increase in monocyte adhesion, upon treatment with homocysteinylated albumin, occurs through up-regulation of some typical mediator molecules of monocyte activation.
10.1371/journal.pone.0031388.g007Figure 7 Increased expression of ICAM1, MCP1 and CCR2 in U937 upon homocysteinylated albumin treatment.
Panel A: Real time PCR evaluation of ICAM1, CCR2 and MCP1 mRNA. Panel B: Western blotting analysis of intracellular levels of ICAM1, CCR2 and MCP1. Levels of both transcripts and proteins in the homocysteinylated albumin sample were significantly increased compared to control (p<0.001).
Discussion
We investigated the effects of homocysteinylated albumin treatment on monocyte adhesion in a human endothelial cell co-culture system and relevant biomolecular alterations. We observed increased monocyte adhesion onto the endothelial monolayers, concomitantly with up-regulation of ICAM-1 and VCAM-1 after treatment with homocysteinylated proteins. It has been previously shown that high homocysteine modifies gene expression in cultured cells [33]–[35] and in vivo in animal models [36] and in humans [37]. In our present model, both endothelial and monocytoid cells showed, after treatment, a significant, specific and time-dependent increase, at both transcriptional and protein levels, of genes potentially involved in vascular remodeling processes: i.e. ADAM17, MCP1, Hsp60 as schematically summarized in Fig. 8.
10.1371/journal.pone.0031388.g008Figure 8 Summary of the effects of homocysteinylated albumin at cellular levels.
Concentrations of N-homocysteinylated albumin, comparable to those detected in vivo
[14], were used. Right panel: untreated cells. Left panel: treated cells. Numbers in circle refer to molecular markers and mediators of increased adhesion detected in the present work. At endothelial cell level. Transcriptional activation of MCP1, Hsp60, ADAM17 (no 1). Increase of the cleaved form of ADAM17 (no 2). Increased shedding of Tnfα (no 3), ICAM1 and VCAM1 (AM: adhesion molecules; no 4). Increased intracellular levels and release of Hsp60 (no 5). Increased MCP1 levels and release (no 6). At monocyte level. Transcriptional activation of MCP1, CCR2 and ICAM1 (no 7). Increased levels of MCP1 (no 8), CCR2 (no 9), ICAM1 (no 10). The prevalent molecular circulating homocysteine-protein adducts are schematically illustrated in the lower left corner of right panel (see also Fig. 1).
ADAM17 is a metalloproteinase involved in the shedding of adhesion molecules, e.g. ICAM1 [38] and Tnf-α release [39]. Transcriptional up-regulation of ADAM 17 was accompanied by an increase of its mature form, and, consistently, of Tnf-α released in the cell medium. ADAM17 activation is also consistent with the increase of ICAM1 released in the medium of treated cells. Hyperhomocysteinemia has been hypothesized to be an indicator of oxidant stress [40]. Moreover homocysteinylated, oxidized LDL-dependent increase of reactive oxygen species in the endothelium has been shown [41]. Consistently, we may hypothesize that, in our model of hyperhomocysteinemia, high homocysteinylated albumin may contribute to activation of ADAM17 through the chemical displacement of the pro-domain in the cysteine switch of this protein [42].
We also detected an up-regulation of MCP1, a protein belonging to type CC chemokine family, that mediates monocyte recruitment in proximity of endothelial lesions, by creating of a chemotactic gradient towards the inflammatory site. Consistently, we found a significant increase of MCP1 in the medium of treated cell, compared to controls.
Homocysteinylated albumin treatment also determined a transcriptional up-regulation of Hsp60, together with its protein product. Hsp60 was increased both at the cellular level and in the extracellular medium. Heat shock proteins regulate maintenance of protein conformation and stability, through their reciprocal interaction. They can be expressed constitutively or produced in response to various types of cell stress. Hsp60 was shown to be an important autoantigen in atherosclerosis [43]. Hsp60 overexpression entails its expression on cell surface and its secretion, favoring macrophage adhesion and trans-endothelial migration. Such studies also showed that Hsp60 membrane exposure participates in the pathogenesis of the endothelial lesions by binding to specific antibodies, thus eliciting a cytotoxic effect towards the endothelium. Macrophages express Hsp60 ligands and their interaction induces their activation [44]. Plasma levels of Hsp60 are significantly higher in subjects with cardiovascular disease with respect to those without [45].
The alterations we detected in the endothelial cells, in response to homocysteinylated albumin treatment, were mirrored by consistent alterations induced in the monocytoid cells. In these cell components we detected, upon homocysteinylated albumin treatment, an up-regulation of ICAM1 and MCP1 (which are known to be produced by activated monocyte to amplify inflammatory signal reinforcing monocyte recruitment) and CCR2 (the MCP1 receptor). We previously showed that, in mononuclear cells of uremic patients on hemodialysis, who are typically hyperhomocysteinemic, DNA hypomethylation is present, with alterations of the expression pattern of methylation-dependent genes [37].
It has been previously shown that homocysteine is capable of inducing vascular alterations at endothelial and vascular smooth muscle cell levels. Hyperhomocysteinemia and hypomethylation are associated with the activation of growth factors, lipid deposition, and vascular smooth muscle cell proliferation activation. High homocysteine triggers a pro-inflammatory state involving adhesion molecule expression and monocyte recruitment, through NF-kB activation and stimulation of ICAM-1 and VCAM-1 induction [46], [47]. Homocysteine induces in vitro, in endothelial and vascular smooth muscle cells, increased expression of MCP1, of interleukin-8 (IL-8) and their secretion, thus promoting monocyte adhesion [2]–[7], [48]–[51]. It has been reported that hyperhomocysteinemia due to CBS deficiency promotes monocyte activation and proinflammatory alterations in transgenic mice [52]. However, previous work was often performed utilizing concentrations of free homocysteine in the high micromolar or even millimolar range, i.e. up to one order of magnitude higher than what observed in homocystinuria, the pathological condition in which the highest levels are reached [2]–[7], [48]–[53]. In our present work, for the first time, we treated cells with high homocysteine mimicking conditions which actually take place in vivo. In fact we carried out cell treatment with homocysteinylated albumin (not free homocysteine) and its concentrations were comparable to the in vivo uremic milieu.
Homocysteine is mainly protein-bound and homocysteinylation is a widespread post-biosynthetic protein modification regarded as a major mechanism through which homocysteine induces vascular alterations. In this respect it has been shown that N-homocysteinylated derivatives of both LDL [21] and HDL [54] are detectable in human plasma, suggesting that homocysteinylation of plasma lipoproteins occurring in vivo is facilitated by lipoproteins oxidation, since these oxidized lipoprotein are more susceptible to homocysteinylation with respect to unmodified lipoproteins. It has been proposed that protein homocysteinylation could be one of the principal mediators of homocysteine toxicity [19]–[21]. We showed increased plasma protein homocysteinylation in hyperhomocysteinemic uremic patients on hemodialysis, which resulted only partially responsive to homocysteine-lowering therapy [14]. Albumin also mediates protein endocytosis, and is itself internalized, thus determining, under pathological conditions, an alteration of the expression of cytokines and relevant receptors [55], [56]. Human serum albumin displays altered functional properties (e.g. towards ligand binding) in consequence of homocysteinylation [14].
All in all our present data support the hypothesis that homocysteinylated proteins are neither secondary byproducts nor a mere biohumoral circulating marker of chronic hyperhomocysteinemia. Our data speak in favor of a mechanistic model of action according to which protein homocysteinylation, rather than free homocysteine, could exerts cell responses related to up-regulation of inflammatory chemokines which have been directly related to the pathogenesis of the early steps of atherosclerotic lesions.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This study was entirely financed by public sources, no private profit institutions provided any funding. This research was supported in part by grants PRIN2005-prot.2005062199_003 “Hyperhomocysteinemia, cardiovascular risk factor in uremia, and structure-function alterations of biological macromolecules” and PRIN2007-prot.2007EBCYYW_004 “Homocysteinylated proteins as an effector of vascular endothelial damage in uremia” to Professor Diego Ingrosso. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22363546PONE-D-11-2083810.1371/journal.pone.0031056Research ArticleBiologyAnatomy and PhysiologyPhysiological ProcessesMolecular Cell BiologyCellular TypesSignal TransductionSignaling CascadesMedicineAnatomy and PhysiologyPhysiological ProcessesCardiovascularActivation of the AMP-Activated Protein Kinase (AMPK) by Nitrated Lipids in Endothelial Cells AMPK-Dependent Angiogenesis by Nitrated LipidsWu Yong
1
2
Dong Yunzhou
1
Song Ping
1
Zou Ming-Hui
1
*
1
Section of Molecular Medicine, Department of Medicine, Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America
2
Department of Biochemistry, University of California, Riverside, Riverside, California, United States of America
Xu Aimin EditorUniversity of Hong Kong, China* E-mail: [email protected] and designed the experiments: YW MZ. Performed the experiments: YW YD PS. Analyzed the data: YW YD PS MZ. Wrote the paper: YW MZ.
2012 17 2 2012 7 2 e3105619 10 2011 31 12 2011 Wu et al.2012This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.The AMP-activated protein kinase (AMPK) is an important regulator of endothelial metabolic and functional homeostasis. Here, we examined the regulation of AMPK by nitrated oleic acid (OA-NO2) and investigated the implications in endothelial function. Treatment of bovine aortic endothelial cells (BAECs) with OA-NO2 induced a significant increase in both AMPK-Thr172 phosphorylation and AMPK activity as well as upregulation of heme oxygenase (HO)-1 and hypoxia-inducible factor (HIF)-1α. Pharmacologic inhibition or genetic ablation of HO-1 or HIF-1α abolished OA-NO2-induced AMPK phosphorylation. OA-NO2 induced a dramatic increase in extracellular signal-regulated kinase (ERK)1/2 phosphorylation that was abrogated by the HO-1 inhibitor, zinc deuteroporphyrin IX 2,4-bis-ethylene glycol (ZnBG). Inhibition of ERK1/2 using UO126 or PD98059 reduced but did not abolish OA-NO2-induced HIF-1α upregulation, suggesting that OA-NO2/HO-1-initiated HIF-1α induction is partially dependent on ERK1/2 activity. In addition, OA-NO2 enhanced endothelial intracellular Ca2+, an effect that was inhibited by the HIF-1α inhibitor, YC-1, and by HIF-1α siRNA. These results implicate the involvement of HIF-1α. Experiments using the Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor STO-609, the selective CaMKII inhibitor KN-93, and an isoform-specific siRNA demonstrated that OA-NO2-induced AMPK phosphorylation was dependent on CaMKKβ. Together, these results demonstrate that OA-NO2 activates AMPK in endothelial cells via an HO-1–dependent mechanism that increases HIF-1α protein expression and Ca2+/CaMKKβ activation.
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Introduction
Accumulating evidence indicates that reactive nitrogen species (RNS), which are inflammatory oxidants, mediate diverse physiologic and pathologic processes in cardiovascular, pulmonary, and neurodegenerative diseases [1]. RNS, such as peroxynitrite (ONOO−), nitrogen-dioxide radical, and nitronium ion, which are formed from nitric oxide (NO), are important factors in complications of obesity and diabetes [2]. RNS react with unsaturated fatty acids to form relatively stable nitrated products (nitroalkene derivatives), including the abundant and clinically important nitrated oleic and linoleic acids [3]. Although this process occurs via various mechanisms, the common denominator is a proclivity for homolytic addition of nitrogen dioxide (•NO2) to the double bond, yielding an array of regio- and stereoisomers [4]. Nitrated unsaturated fatty acids (NO2-UFAs) represent a convergence of lipid and NO signaling and have emerged as a novel class of endogenously produced vascular signaling molecules [5]. The beneficial effects of NO2-UFAs include cGMP-dependent vessel relaxation, inhibition of inflammatory cell function, adaptive and anti-inflammatory cell responses, induction of heme oxygenase-1 (HO-1) expression, inhibition of nuclear factor (NF)-κB, and activation of peroxisome proliferator activated receptor (PPAR) and Keap1/Nrf2 [6].
Plasma free and esterified nitrated oleic acid (OA-NO2) concentrations have been reported to be 619±52 and 302±369 nM, respectively, and these levels are approximately 50% greater than the level of nitrated linoleic acid (LNO2). The combined blood levels of the free and esterified fatty acid derivatives exceeds 1 µM [4]. The plasma levels of NO2-UFA derivatives in hyperlipidemic patients are elevated relative to levels in normolipidemic subjects [7]. In addition, an increase in oxidative stress in hypercholesterolemia [8] may contribute to the formation of nitrated species in the vascular wall [9]. Thus, nitrated lipids in the plasma can be used as an indicator of the chain-breaking antioxidant role of NO in lipid oxidation [10] and/or provide a footprint for the presence of oxidants/nitrating agents in the vascular system. We postulate that the presence of these nitrated products in vivo promote cardiovascular homeostasis and compensate for impaired vascular endothelial function in the context of dyslipidemia, obesity, and diabetes. The molecular target(s) and mechanisms underlying the vascular-protective and anti-inflammatory effects of NO2-UFAs remain poorly defined.
The serine/threonine kinase, AMP-activated protein kinase (AMPK), is a member of the Snf1/AMPK protein kinase family that is found in all eukaryotes [11]. This kinase is thought to act as a cellular energy sensor by stimulating ATP-producing catabolic pathways and inhibiting ATP-consuming anabolic pathways [12]. AMPK is comprised of three subunits: a catalytic α-subunit and regulatory β- and γ-subunits. Activation of AMPK requires the phosphorylation of Thr172 in the activation loop of the α-subunit by an upstream AMPK kinase (AMPKK) [13]. Interestingly, the first AMPKK to be identified was LKB1, a tumor suppressor that is mutated in humans with Peutz-Jegher syndrome [14]. Patients with this syndrome have an increased risk of developing carcinomas of the colon, stomach, and pancreas. Recently, calcium (Ca2+) calmodulin-dependent kinase kinase (CaMKK) [15] was identified as an upstream AMPKK. CaMKK is activated by a rise in intracellular Ca2+ concentrations ([Ca2+]i), and this kinase phosphorylates and activates AMPK in an AMP-independent manner [15]. Therefore, in addition to responding to an increase in the AMP-to-ATP ratio, AMPK may also be activated by a rise in [Ca2+]i in response to nutrients, drugs, or physiological stimuli. While the AMPK pathway is traditionally thought to be a regulator of metabolism, recent studies have demonstrated that AMPK may also act to maintain endothelial function [16]. AMPK exerts pleiotropic effects that are believed to be beneficial for endothelial function. These effects, which are ultimately anti-atherogenic, include induction of the endothelial nitric oxide synthase (eNOS)/NO pathway and result in an increase in NO bioavailability, suppression of endothelial ROS production following exposure to deleterious stimuli, such as hyperglycemia or high free fatty acids (FFAs), and modulation of vascular tone (see review [16]). AMPK also possesses anti-apoptotic and anti-inflammatory activities [17].
Since many of the metabolic changes and endothelial-protective effects attributable to NO2-UFAs are similar to those observed in response to AMPK activation, we hypothesized that AMPK activation is an important mediator of NO2-UFA activity. As such, AMPK activation may explain the pleiotropic beneficial effects of NO2-UFAs on the cardiovascular system in obesity and diabetes. Whether NO2-UFAs activate AMPK and, if so, by what mechanism(s) has yet to be determined. In the present study, we examined the effects of NO2-UFAs on the AMPK upstream kinases, specifically LKB1 and CaMKKβ. We also investigated whether NO2-UFAs modulate the eNOS/NO pathway, which is known to be an indicator of endothelial function and an important property of AMPK in cardiovasculature. We report that treatment with NO2-UFAs induces CaMKKβ-dependent AMPK activation through an HO-1/HIF-1α/Ca2+ pathway in vitro and show that NO2-UFAs promote p-eNOS and NO production via activation of the AMPK pathway in endothelial cells.
Results
OA-NO2 induces HO-1 protein and AMPK phosphorylation/activation in bovine aortic endothelial cells (BAECs)
To investigate activation of AMPK by OA-NO2 in endothelial cells, we treated confluent BAECs with different concentrations of OA-NO2 for 2–16 h. AMPK activation was indirectly assessed by western blot analysis of AMPK Thr172 phosphorylation, which is essential for AMPK activity [18]. As shown in Figure 1A and 1B, incubation of BAECs with OA-NO2 (0.5–2.5 µM for 16 h, or 2.5 µM for 2–16 h) resulted in a dose- and time-dependent increase in AMPKα phosphorylation at Thr172. AMPK Thr172 phosphorylation in BAECs gradually increased beginning 6 h after incubation with 2.5 µM OA-NO2 and reached peak levels after 16 h. This increased AMPK phosphorylation was associated with elevated AMPK activity, as measured by the SAMS peptide assay (Figure 1C). OA-NO2 treatment did not alter total AMPK levels, suggesting that OA-NO2-induced phosphorylation of AMPK was not due to altered expression of these proteins. Since OA-NO2 activated AMPK in both BAECs and human umbilical vein endothelial cells (HUVECs) at similar potencies, we performed the majority of our experiments with BAECs.
10.1371/journal.pone.0031056.g001Figure 1 Induction of HO-1 protein and AMPK activation by OA-NO2.
A) BAECs were incubated with OA-NO2 at the indicated concentrations or with BSA (vehicle) for 16 h, and western blot analysis was performed as described in Materials and Methods to detect HO-1 protein expression and AMPK phosphorylation at Thr172. The blot is representative of those obtained from three separate experiments. Corresponding densitometric analyses of phosphorylated AMPK and ACC are shown. *p<0.05 vs. control. B) BAECs were incubated with 2.5 µM OA-NO2 for the indicated times, and western blotting was performed as above. The blot is representative of three blots obtained from three separate experiments. *p<0.05 vs. corresponding control. C) Confluent BAECs were exposed to vehicle or OA-NO2 (2.5 µM) for 16 h. AMPKα was immunoprecipitated from cell lysates (1 mg) with a specific antibody. AMPK activity was assayed by 32P-ATP incorporation into the SAMS peptide. *p<0.05 vs. control. D) BAECs were incubated with the indicated concentrations of OA for 16 h. Western blotting was performed as described in Materials and Methods. E) BAECs were infected with Ad-DN-AMPK (MOI = 50) or Ad-GFP (control). Infected and non-infected cells were treated with 2.5 µM OA-NO2 for 16 h. AICAR and metformin were used as positive controls. The blot is representative of three blots obtained from three separate experiments.
The changes in AMPK phosphorylation were mirrored by changes in HO-1 protein expression, as evaluated by western blot analysis. Exposure of BAECs to a range of OA-NO2 concentrations resulted in an upregulation of HO-1 that was initially observed at 4 h with peak induction at 16 h. HO-1 induction and AMPK activation were specifically due to the nitroalkene moiety of OA-NO2, because oleic acid (OA) did not induce HO-1 protein expression or AMPK phosphorylation (Figure 1D). At a concentration of 2.5 µM, OA-NO2 potently activated AMPK and induced HO-1 expression without causing cellular toxicity. On the basis of these results, we chose to stimulate BAECs with 2.5 µM OA-NO2 for 16 h in subsequent experiments.
To determine whether OA-NO2-induced HO-1 expression was mediated by AMPK, we used an adenovirus encoding a dominant-negative mutant form of AMPKα (Ad-DN-AMPK) to suppress AMPK activity. AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) and metformin, two well characterized AMPK activators, were used as positive controls for AMPK phosphorylation. OA-NO2 significantly elevated AMPK phosphorylation (Figure 1E). As expected, Ad-DN-AMPK effectively suppressed AMPK phosphorylation but failed to abolish HO-1 induction by OA-NO2, suggesting that AMPK does not act upstream of HO-1 production in this system.
Activation of AMPK by OA-NO2 does not require LKB1
Two recent studies [14], [19] showed that LKB1 acts as an upstream AMPK kinase. To evaluate the role of LKB1 in OA-NO2-induced AMPK activation, we investigated whether OA-NO2 altered LKB1 Ser 428 phosphorylation, which is essential for LKB1 activity and LKB1-dependent AMPK activation. As shown in Figure 2A, OA-NO2 did not alter the levels of LKB1 Ser428 phosphorylation.
10.1371/journal.pone.0031056.g002Figure 2 Activation of AMPK by OA-NO2 does not require LKB1.
A) Phosphorylation of LKB1 Ser428 was not affected by OA-NO2 in BAECs. Confluent BAECs were exposed to 2.5 µM OA-NO2 for 16 h, and phosphorylated LKB1-Ser428 was detected by a phospho-specific antibody in western blots. The blot is a representative of three blots obtained from three independent experiments. Lower panels: summary data (n = 3). B) LKB1 is not required for AMPK activation by OA-NO2. Confluent LKB1-deficient Hela-S3 cells were exposed to 2.5 µM OA-NO2 for 16 h, and then AMPK and ACC phosphorylation were assayed as described in Materials and Methods. The blot is representative of three blots obtained from three independent experiments. Lower panels: summary data (*p<0.05 vs. control; n = 3). C) LKB1 siRNA did not abolish OA-NO2-stimulated AMPK activation in HUVECs. HUVECs were incubated with LKB1-specific siRNA or control siRNA for 48 h and then treated with OA-NO2 or vehicle for 16 h. After treatment, cell lysates were analyzed for LKB1 protein levels and AMPK phosphorylation at Thr172. Lower panels: summary data (*p<0.05 vs. control; n = 3).
We next determined whether OA-NO2 activated AMPK in Hela-S3 cells, an LKB1-deficient cell line. Similar to the results obtained in BAECs, OA-NO2 (2.5 µM) induced AMPK phosphorylation on Thr172 in Hela-S3 cells (∼3-fold, p<0.05; Figure 2B). In parallel with AMPK phosphorylation, OA-NO2 dramatically increased phosphorylation of the known AMPK substrate, acetyl-CoA carboxylase (ACC), at Ser79 (∼2.4-fold, p<0.05), providing an additional indication that AMPK was activated in the absence of LKB1. To further explore the role of LKB1, we investigated the effect of siRNA-mediated LKB1 downregulation on AMPK activation induced by OA-NO2. As shown in Figure 2C, OA-NO2-induced AMPK phosphorylation was not significantly altered in cells pre-treated with LKB1 siRNA. The suppression of LKB1 protein expression by LKB1 siRNA (∼60%) was confirmed by western analysis. Taken together, these results strongly suggest that OA-NO2-induced phosphorylation of AMPK Thr172 is independent of its upstream kinase, LKB1.
Induction of HIF-1α by OA-NO2 is dependent on HO-1
Hypoxia-inducible factor (HIF)-1 is a dimeric protein complex that plays an integral role in the body's response to hypoxia [20]. Like AMPK, HIF-1 is one of the primary genes involved in the homeostatic process that leads to increased vascularization in hypoxic areas, such as those within localized ischemia and in tumors [20]. HIF-1 and AMPK represent two main cellular pathways involved in coping with hypoxic stress and protecting cells against energy depletion and tissue reperfusion injury in times of metabolic crisis [21]. In addition, both HIF-1 and HO-1 are associated with ferrous iron metabolism [21]. Given that OA-NO2 induced both HO-1 expression and AMPK activation, we determined whether OA-NO2 also influenced HIF-1 expression. As predicted, OA-NO2 induced an increase in HIF-1α in HUVECs with a time course similar to OA-NO2-induced HO-1 expression and AMPK phosphorylation (Figure 3A). To explore the potential involvement of HO-1 in OA-NO2-induced HIF-1α expression, we pre-treated cells with the selective HO inhibitor zinc deuteroporphyrin IX 2,4-bis-ethylene glycol (ZnBG) and HO-1-specific siRNA. As shown in Figure 3A and B, ZnBG and HO-1 siRNA treatment abrogated the induction of HIF-1α protein expression and AMPK phosphorylation by OA-NO2, suggesting that OA-NO2 acts through the induction of HO-1 to increase HIF-1α expression and subsequently activate AMPK. The efficacy of the HO-1 siRNA in the downregulation of HO-1 expression was confirmed by western analysis (Figure 3B).
10.1371/journal.pone.0031056.g003Figure 3 OA-NO2-induced HIF-1α is dependent on HO-1.
A) HUVECs were treated with 1 µM ZnBG for 30 min followed by incubation with OA-NO2 for the indicated times. AMPK protein and phosphorylation levels and HIF-1α protein expression were assayed as described in Materials and Methods. The blot is representative of three blots obtained from three independent experiments. B) HUVECs were incubated with HO-1-specific siRNA or control siRNA for 48 h and then treated with OA-NO2 or vehicle for 16 h. After treatment, cell lysates were analyzed for HIF-1α and HO-1 protein levels and AMPK phosphorylation at Thr172. Lower panels: summary data (*p<0.05 vs. control; #
p<0.05 vs. OA-NO2 group; n = 3).
ERK1/2 is partially responsible for OA-NO2/HO-1-mediated HIF-1α induction
Clearly, the HIF pathway plays an important role in the regulation of metabolism under hypoxic conditions; however, a variety of HIF-1 stimuli function independently of oxygen concentration. These stimuli are primarily mediated by proteins that regulate HIF-1 translation. This pathway contrasts with hypoxic stimuli, which act upon pre-existing α-subunits [20]. HIF-1α activation by non-hypoxic stimuli has been linked primarily to the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway and the mitogen-activated protein kinase (MAPK) pathway [22]. Both kinase pathways are known to be intimately associated with the regulation of HIF-1α protein translation, stabilization, and transcriptional activity [23]. Our data indicate that OA-NO2 did not activate PI3-K/Akt (data not shown). To determine whether OA-NO2-mediated HIF-1α induction is dependent on the MAPK pathway, we examined p38 MAPK and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation following stimulation with OA-NO2. Treatment with OA-NO2 induced a dose-dependent increase in the phosphorylation of ERK1/2 and p38 MAPK (Figure 4A). ZnBG inhibited OA-NO2-induced activation of ERK1/2, suggesting a role for HO-1 in this activation (Figure 4B).
10.1371/journal.pone.0031056.g004Figure 4 OA-NO2-induced HIF-1α is partially dependent on ERK1/2.
A) BAECs were incubated with the indicated concentrations of OA-NO2 for 16 h. Protein expression and phosphorylation of p38 MAPK and ERK1/2 were assayed as described in Materials and Methods. The blot is representative of three blots obtained from three independent experiments. B) BAECs were treated with ZnBG (1 µM) for 30 min followed by incubation with OA-NO2 for 16 h. ERK1/2 phosphorylation levels were monitored by immunoblot analysis, and band density was normalized to total ERK1/2 levels (lower panel). The blot is representative of three blots obtained from three independent experiments. *p<0.05 vs. control; #
p<0.05 vs. OA-NO2 group. C) BAECs were treated with OA-NO2 alone or with the ERK1/2 signaling inhibitors, UO126 (10 µM) or PD98059 (50 µM), 1 h before the addition of OA-NO2. HIF-1α protein levels were monitored by immunoblot analysis, and band density was normalized to β-actin levels (lower panel). The blot is representative of three blots obtained from three independent experiments. *p<0.05 vs. control; #
p<0.05 vs. OA-NO2 group.
We next determined whether ERK1/2 was a potential upstream mediator of OA-NO2-induced HIF-1α upregulation using the ERK1/2 inhibitor, UO126 (10 µM), and the MAPK/ERK kinase (MEK)-specific inhibitor PD98059 (50 µM). Pre-treatment of BAECs with UO126 or PD98059 decreased HIF-1α levels by approximately 50% relative to cells treated with OA-NO2 alone (Figure 4C), suggesting that OA-NO2-mediated HIF-1α induction is due, at least in part, to MEK/ERK1/2 activity. UO126 and PD98059 did not affect basal HIF-1α expression. In addition to increasing HIF-1α expression, ERK1/2 has previously been reported to phosphorylate HIF-1α in vitro, and this post-translational modification can increase HIF-1α activity, presumably by impeding proteasome/von-Hippel-Lindau (VHL) recognition [20]. Although very little is known about the phosphorylation of HIF-1α, this phosphorylation event may be important for optimal activity of the HIF-1 pathway.
HIF-1α induction by OA-NO2 contributes to intracellular Ca2+ accumulation and AMPK activation
To examine the involvement of the HIF-1α pathway in OA-NO2-mediated AMPK activation, we employed the widely used HIF-1α inhibitor, 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1) and HIF-1α siRNA. Pre-treatment of BAECs with YC-1 or HIF-1α siRNA significantly inhibited the OA-NO2-induced increase in AMPK Thr172 phosphorylation levels relative to OA-NO2 treatment alone (Figure 5A and B). Since OA-NO2-induced AMPK activation is LKB1-independent, we postulated that the Ca2+/CaMKK pathway plays a role in the activation of AMPK by OA-NO2/HIF-1α. In addition, because HIF-1α has been reported to regulate Ca2+ homeostasis in pulmonary arterial smooth muscle cells via upregulation of store-operated Ca2+ channels and enhanced Ca2+ influx [24], we examined a possible role for HIF-1α in this process. To determine whether OA-NO2 affected endothelial cell [Ca2+]i, we measured intracellular fluorescence in Fluo-4–loaded BAECs. We found that OA-NO2 treatment produced an approximately 1.4- to 2.2-fold increase in [Ca2+]i at 6 and 16 h after treatment, respectively (Figure 5C). To confirm a role for HIF-1α in this OA-NO2-mediated [Ca2+]i increase, we used pharmaceutical- and genetic-inhibition strategies to downregulate HIF-1α. Pre-treatment of BAECs with YC-1 or transfection of these cells with HIF-1α siRNA partially suppressed the OA-NO2-induced increases in [Ca2+]i (Figure 5D), indicating that this process is at least partially dependent on HIF-1α. We further investigated whether a Ca2+ signal is involved in OA-NO2-induced AMPK activation using BAPTA-AM, a cell-permeable Ca2+ chelator that is widely used as an intracellular Ca2+ “sponge”. Our results indicate that chelation of intracellular Ca2+ by BAPTA-AM (25 µM, 30 min) significantly inhibited OA-NO2-stimulated AMPK phosphorylation (∼60%, p<0.05; Figure 5E). Together, these data show that intracellular Ca2+ is necessary for OA-NO2-induced AMPK activation.
10.1371/journal.pone.0031056.g005Figure 5 HIF-1α mediates OA-NO2-induced intracellular Ca2+ accumulation and AMPK activation.
A) BAECs were treated with YC-1 (30 µM) for 30 min followed by incubation with OA-NO2 for the indicated times. AMPK phosphorylation and protein levels were assayed as described in Materials and Methods. The blot is representative of three blots obtained from three independent experiments. B) BAECs were incubated with HIF-1α-specific siRNA or control siRNA for 48 h and then treated with OA-NO2 or vehicle for 16 h. After treatment, cell lysates were analyzed for HIF-1α and AMPK protein levels and AMPK phosphorylation at Thr172. Lower panels: summary data (*p<0.05 vs. control; #
p<0.05 vs. OA-NO2 group; n = 3). C) BAECs were incubated with OA-NO2 (2.5 µM) for the indicated times. Intracellular Ca2+ was measured with Fluo-4 fluorescent dye as described in Materials and Methods. *p<0.01 vs. control (n = 4). D) BAECs were pre-treated with YC-1 (30 µM) or BAPT-AM (25 µM) for 30 min or HIF-1α siRNA or control siRNA for 48 h followed by incubation with OA-NO2 for 16 h. After treatment, intracellular Ca2+ was measured in intact cells using a Fluo-4 NW kit. *p<0.05 vs. control; #
p<0.05 vs. OA-NO2 group (n = 3). E) BAECs were pre-loaded with 25 µM BAPT-AM for 30 min prior to incubation with 2.5 µM OA-NO2 for 16 h. AMPK protein levels and phosphorylation at Thr172 were detected as described above. Representative blots (top) and densitometric analyses (bottom) are shown. Values are means ± SD from three independent measurements. *p<0.05 vs. control; #
p<0.05 vs. OA-NO2 group.
CaMKKβ mediates OA-NO2-induced AMPK activation
Both the tumor suppressor LKB1 [14] and CaMKK [15] are important AMPK kinases as each activates AMPK by directly phosphorylating the AMPK α subunit on Thr172. As shown above (Figure 2A–C), LKB1 is unlikely involved in the activation of AMPK by OA-NO2. Because treatment with OA-NO2 elevated [Ca2+]i and OA-NO2-stimulated AMPK activation was Ca2+ sensitive, we speculate that the AMPK kinase, CaMKKβ, which is activated by Ca2+/calmodulin binding, may be involved. To test this hypothesis, we used the relatively selective CaMKKα and CaMKKβ inhibitor, STO-609 (1 µM) [25] and the competitive CaM inhibitor, KN-93 (3 µM). Either STO-609 or KN-93 was sufficient to prevent activation of AMPK by OA-NO2 (Figure 6A), suggesting the involvement of CaMKK in this activation. A specific role for CaMKKβ was indicated by the results of siRNA experiments, which showed that downregulation of CaMKKβ caused a significant reduction in OA-NO2-stimulated AMPK phosphorylation (Figure 6B). Downregulation of CaMKKβ protein was verified by western blot analysis. These results suggest that CaMKKβ is the major AMPK kinase under these conditions. To further validate this notion, we determined whether treatment with OA-NO2 enhanced the association between CaMKK and AMPK as this interaction is a prerequisite for AMPK phosphorylation/activation. Indeed, following OA-NO2 treatment, an enhanced CaMKK and AMPK association was detected by immunoprecipitation with either AMPK or CaMKK antibodies (Figure 6C). Moreover, pre-treatment with YC-1 dramatically inhibited the OA-NO2-induced increased CaMKK-AMPK association, further substantiating the indispensable role of HIF-1α in OA-NO2-induced AMPK activation.
10.1371/journal.pone.0031056.g006Figure 6 CaMKK is the upstream AMPKK that mediates OA-NO2-induced AMPK activation.
A) HUVECs were treated with STO-609 (1 µM) or KN-93 (3 µM) for 1 h followed by incubation with OA-NO2 for 16 h. AMPK and eNOS phosphorylation and protein expression were assayed as described in Materials and Methods. The blot is representative of three blots obtained from three independent experiments. Lower panels: summary data (*p<0.05 vs. control; #
p<0.05 vs. OA-NO2 group; n = 3). B) HUVECs were incubated with CaMKKβ-specific siRNA or control siRNA for 48 h and then treated with OA-NO2 for 16 h. After treatment, cell lysates were analyzed for AMPK and eNOS phosphorylation and protein levels. Lower panels: summary data (*p<0.05 vs. control; #
p<0.05 vs. OA-NO2 group; n = 3). C) HUVECs were pre-treated with YC-1 (30 µM) for 30 min followed by incubation with OA-NO2 for 16 h. An immunoblot of AMPK precipitated with an anti-CaMKK antibody is shown. The blot is representative of three blots obtained from three independent experiments. Lower panels: summary data (*p<0.05 vs. control; #
p<0.05 vs. OA-NO2 group; n = 3).
OA-NO2-induced eNOS phosphorylation is dependent on AMPK
We previously demonstrated that AMPK phosphorylates and activates eNOS in cultured endothelial cells [26]. Similarly, Zhang et al. demonstrated that infection of endothelial cells with a recombinant adenovirus expressing constitutively active AMPK resulted in eNOS activation and increased NO production [27]. Here, we used phosphorylation of Ser1177 in eNOS, which is a reported substrate of AMPK, as an indicator of AMPK activity in this system. As shown in Figure 7A, incubation of BAECs with OA-NO2 increased eNOS Ser1177 phosphorylation in a dose-dependent manner that was very similar to that for AMPK phosphorylation. The time course of OA-NO2–induced eNOS phosphorylation was also similar to that for AMPK phosphorylation (Figure 7B). To confirm that OA-NO2-stimulated eNOS phosphorylation involved AMPK, we infected BAECs with adenovirus encoding a dominant-negative form of AMPK (Ad-DN-AMPK). As expected, phosphorylation of both AMPK and eNOS was increased after treatment of control BAECs (Ad-GFP-infected or non-infected BAECs) with 2.5 µM OA-NO2 for 16 h (Figure 7C). In contrast, overexpression of Ad-DN-AMPK completely abolished OA-NO2-induced eNOS phosphorylation. Consistent with a role for AMPK in phosphorylating eNOS, treatment with STO-609 or KN-93 to inhibit the AMPK kinase CaMKK or downregulation of CaMKKβ using siRNA prevented OA-NO2-induced eNOS phosphorylation (Figure 6A and B).
10.1371/journal.pone.0031056.g007Figure 7 AMPK mediates OA-NO2-induced eNOS phosphorylation and NO production in BAECs.
BAECs were treated with (A) different concentrations of OA-NO2 for 16 h or (B) 2.5 µmol/L OA-NO2 for the indicated times. Lysates were analyzed by western blot for the indicated proteins. The blot is representative of three blots obtained from three separate experiments. C) Western blot of phosphorylated AMPK and eNOS in OA-NO2-stimulated BAECs infected with Ad-DN-AMPK (MOI = 50). Non-infected cells or cells infected with Ad-GFP served as controls. For A–C, the corresponding densitometric analyses are shown. *p<0.05 vs. control; #
p<0.05 vs. GFP with OA-NO2-treated group. D) NO release by OA-NO2-stimulated BAECs infected with Ad-DN-AMPK (MOI = 50) or Ad-GFP (control). *p<0.05 vs. non-transfected, no OA-NO2 group; #
p<0.05 vs. OA-NO2-treated, Ad-GFP group. E) AMPK activity corresponding to C and D above. *p<0.05 vs. no OA-NO2 treatment, Ad-GFP group; #
p<0.05 vs. OA-NO2-treated, Ad-GFP group. F) The proposed signaling pathway involved in AMPK/eNOS activation in response to OA-NO2 treatment in endothelial cells.
Both Akt and AMPK are capable of phosphorylating eNOS at Ser1179 [28]. Thus, we determined whether Akt also contributes to OA-NO2-induced eNOS phosphorylation. OA-NO2 did not increase basal Akt phosphorylation of Ser473 (data not shown), suggesting that OA-NO2-stimulated eNOS phosphorylation does not require Akt but depends on activation of AMPK.
Next, we determined whether OA-NO2-induced eNOS phosphorylation was associated with increased NO release. Treatment with OA-NO2 significantly increased NO release, an effect that was inhibited by Ad-DN-AMPK transfection (Figure 7D). Importantly, Ad-DN-AMPK transfection reduced AMPK activity in OA-NO2-treated cells to below control levels (Figure 7E). These results indicate that OA-NO2 increases NO release via AMPK activation.
Discussion
OA-UFAs are unique signaling mediators that are present in a variety of cell types, including endothelial cells [29]. Considerable evidence points to a role for circulating OA-UFAs in vascular-protective effects [9], although the mechanisms by which OA-NO2 are incompletely understood. In this study, we report that OA-NO2 activates AMPK in endothelial cells via a Ca2+-dependent pathway, and we implicate CaMKKβ as the responsible upstream kinase. Our results show that HIF-1α is an inducer of the intracellular Ca2+ mobilization that leads to AMPK activation. On the basis of the results of experiments with an HO-inhibitor and HO-1 siRNA, we also implicate HO-1 in OA-NO2-induced upregulation of endothelial HIF-1α.
HO is a rate-limiting enzyme in heme degradation and functions to convert heme to biliverdin, carbon monoxide (CO) and iron. Human HO occurs in two main isoforms, the inducible HO-1 form and the constitutive HO-2 form. Previous studies demonstrated that HO-1 exerts anti-inflammatory effects [30], including prolongation of cardiac xenograft graft survival [31] and inhibition of leukocyte transendothelial migration during complement-dependent inflammation [32] and in response to low-density lipoprotein (LDL) oxidation [33]. More importantly, once induced, HO-1 also confers vascular cytoprotection [30]. The importance of this result is demonstrated by the severe and persistent endothelial damage observed in the case of human HO-1 deficiency [34] and in gene-targeted mice deficient in HO-1 [35]. Furthermore, induction of HO-1 may directly regulate endothelial cell activation, preventing adhesion molecule expression and chronic graft rejection [36]. In vitro, HO-1 protects endothelial cells from hydrogen peroxide–mediated cell death [37] and from tumor necrosis factor α (TNFα) cytotoxicity [38]. In addition, HO-1 has been suggested to play a role in angiogenesis. This supposition is supported by the observation that overexpression of HO-1 induces proliferation and formation of capillary-like structures [39]. Thus, therapeutic induction of HO-1 may be beneficial in the treatment of chronic inflammatory diseases as well as cardiovascular diseases. Our results indicate that OA-NO2 potently induced HO-1 protein expression in endothelial cells. In agreement with our studies, other studies have suggested that HO-1 expression is induced in human aortic endothelial cells (HAECs) by LNO2, which shows a higher potency in this context than other established stimuli, including oxidized fatty acids and hemin. This induction of HO-1 expression by NO2-FA is not mediated by NO, NF-κB, or PPARγ [40]. A recent study conducted by Liu et al.
[41] suggested that AMPK activation regulates HO-1 gene expression in endothelial cells via the Nrf2/antioxidant responsive element signaling pathway and that HO-1 contributes to the biological actions of this kinase. Our data do not exclude the possibility that AMPK activation upregulates HO-1 expression at the gene transcription level. As shown in Figure 1E, Ad-DN-AMPK partially blocks OA-NO2-induced HO-1 protein levels, suggesting that AMPK activation may play a small role in HO-1 protein expression; however, OA-NO2 induces HO-1 expression 2.5-fold as early as 4 h after incubation when AMPK is not yet activated (Figure 1B), implying that OA-NO2 induces HO-1 directly or via another unknown pathway. Thus, AMPK does not act upstream of HO-1 production in this system. Current data supports the idea that upregulation of human HO-1 expression by NO2-FA requires synergy between the cAMP-dependent response element and the AP-1 sequences in the −4.5 kb HO-1 promoter region [6], [40]; however, the mechanisms involved in these events remain poorly characterized, and the identities of the signaling molecules downstream of HO-1 that mediate the vascular protective effects of nitrated lipids are not entirely known.
The novel finding of this study is that OA-NO2 induces HIF-1α expression via HO-1 under non-hypoxic conditions. HIF-1, which exists as a heterodimer composed of the HIF-1α and HIF-1β subunits, has been shown to mediate numerous physiological and pathophysiological responses to hypoxia. Under normoxic conditions, however, HIF-1α is ubiquitinated and rapidly degraded [42] and is thus present at very low levels under these conditions. Under hypoxic conditions, the HIF-1α subunit is induced. Because HIF-1β is constitutively expressed, HIF-1α is responsible for conferring hypoxia sensitivity to heterodimeric HIF-1. Using specific chemical inhibitors, we demonstrated that ERK1/2 partially mediated HO-1–induced HIF-1α expression (Figure 4). In addition to inducing HIF-1α expression, ERK1/2 has been reported to phosphorylate HIF-1α and to thereby increase its activity [20]. The possibility that CO, a metabolite of HO-1 [43], also plays a role in HIF-1α induction by HO-1 cannot be excluded by our data. A recent study by Chin et al.
[44] suggested that exposure of macrophages to CO resulted in rapid HIF-1α activation and stabilization, which regulates the expression of genes involved in inflammation, metabolism, and cell survival. This previous study also provided evidence that CO may serve as a signaling intermediary between HO-1 and HIF-1α.
Another important find of the present study is that HIF-1α contributes to an increase in [Ca2+]i, which is responsible for CaMKKβ-dependent AMPK activation (Figure 5 and 6). Pharmaceutical and genetic inhibitors of HIF-1α suppressed OA-NO2-induced increases in [Ca2+]i, suggesting that HIF-1α is involved in the OA-NO2-induced mobilization of Ca2+ in endothelial cells. Previous studies demonstrated that hypoxia evokes an increase in [Ca2+]i in endothelial cells [45], and presumably, the induction of HIF-1α contributes to this increase in [Ca2+]i under hypoxic conditions or under normoxic conditions in the presence of induction factors that activate HIF-1α. The mechanisms that are involved in endothelial Ca2+ homeostasis within the vasculature following HIF-1α activation are also largely unknown. Although HIF-1α has been reported to regulate Ca2+ homeostasis in pulmonary arterial smooth muscle cells via upregulation of store-operated Ca2+ channels and enhanced Ca2+ influx [24], the detailed mechanisms remain to be elucidated.
Our results also showed that OA-NO2-stimulated AMPK activation was inhibited by chelation of intracellular free Ca2+, selective inhibition of CaMKK by STO-609, selective inhibition of Ca2+/calmodulin-dependent protein kinase by KN-93, and siRNA-mediated silencing of CaMKK-β expression (Figure 6). In addition, the association between AMPK and CaMKK was enhanced by OA-NO2 treatment. Taken together, these data indicate that the HIF-1α/Ca2+/CaMKK- pathway is crucial for OA-NO2-induced AMPK activation. Furthermore, CaMKK-mediated AMPK activation in endothelium has also recently been reported in response to thrombin [46], adenosine diphosphate (ADP) [47], and bradykinin [48].
Results from previous studies indicated that endothelial AMPK may play an important physiological role in the function of both endothelial cells and the cardiovascular system as a whole; thus, activation of AMPK may provide an explanation for the beneficial effects of OA-NO2 on these systems (see review [49]). Our findings show that induction of the CaMKKβ/AMPK pathway by OA-NO2 in endothelial cells may have both physiological and therapeutic relevance. First, endothelial AMPK activation by OA-NO2 activates nitric oxide synthase (via phosphorylation on Ser 1177) and elevates NO bioavailability, and these effects may not only protect against early events in atherogenesis, such as white cell adherence [50], but may also prevent later steps in atherogenesis, including fibrous plaque formation. Endothelial NO likely represents the most important anti-atherogenic defense molecule in the vasculature [50]. Nitrated lipids act as NO donors in vitro and are widely considered to be a possible endogenous source of NO [51]. Enhanced NO production under hyperlipidemic and hypercholesterolemic conditions, such as those that occur with obesity or insulin-resistant status, however, has been associated with low NO bioactivity [52]. Therefore, it is reasonable to postulate that the presence of these nitrated products and the related AMPK/eNOS/NO pathway in vivo may actually play a compensatory role, providing an adjustable supply of NO to compensate for the impaired NO bioactivity and endothelial-dependent vasorelaxation that is characteristic of the early steps of vascular disease. Second, AMPK signaling acts as a novel regulator of angiogenesis and is specifically required for endothelial cell migration and differentiation under conditions of hypoxia [53] or in response to adiponectin [54]. Additionally, AMPK-dependent eNOS activity is required for adequate endothelial tube formation [54]. As described above, increased levels of nitrated lipids are formed in the context of the hyperlipidemia associated with obesity and insulin resistance. Thus, the induction of AMPK and angiogenesis by nitrated lipids may also represent an adaptive defense mechanism against impaired angiogenesis and/or vascular injury caused by obesity-related dislipidemia. The role that activated AMPK plays in increasing fatty acid oxidation via phosphorylation and inhibition of ACC and leading to a decrease in the concentration of malonyl-CoA is most important [55]. In addition, AMPK decreases fatty acid incorporation into glycerolipids in some tissues, either secondary to its effect on fatty acid oxidation or via phosphorylation and inhibition of sn-glycerophosphate acyltransferase, the first committed enzyme in diacylglycerol and triglyceride synthesis [56]. Furthermore, endothelial AMPK activity may inhibit glycerol-3-phosphate acyltransferase, which is required for de novo synthesis of diacylglycerol [56]. Thus, AMPK may lessen endothelial diacylglycerol production (and thus protein kinase C activation) both by reducing the availability of the FFA substrates required for this synthesis and by directly inhibiting the enzyme that catalyzes it.
In conclusion, we have demonstrated for the first time that NO2-UFAs activate AMPK in endothelial cells by a mechanism that depends on an increase in HO-1 followed by HIF-1α protein expression and Ca2+/CaMKKβ activation. Our results also indicate that AMPK activity is required for eNOS/NO production in endothelial cells (Figure 7F). The present study further suggests that AMPK activation by nitrated lipids may play an essential role in compensating or protecting vascular endothelial function against vascular injury in obesity-related dyslipidemia. AMPK might be a valid therapeutic target for treating vascular disorders in obesity and type II diabetes.
Materials and Methods
Materials
BAECs and cell culture media were obtained from Clonetics Inc. (Walkersville, MD). HUVECs and cell culture media were purchased from Cascade Biologics (Portland, OR). FFA-free bovine serum albumin (BSA) and oleic acid were obtained from Sigma (St. Louis, MO). ZnBG was purchased from Porphyrin Products, Inc. (Logan, UT). OA-NO2 was obtained from Cayman Chemical (Ann Arbor, MI), and AICAR was purchased from Toronto Research Chemicals (Toronto, Canada). The MEK inhibitors PD 98059 and UO 126 were obtained from Calbiochem (La Jolla, CA). 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1) was purchased from AG Scientific Inc. (San Diego, CA). Antibodies against phospho-ACC (Ser79), phospho-AMPK (Thr172), AMPK, phospho-LKB1 (Ser428), LKB1, and phospho-eNOS (Ser1177) were purchased from Cell Signaling (Beverly, MA). The antibodies against ACC were obtained from Alpha Diagnostic International (San Antonio, TX). All other chemicals and organic solvents were of the highest grade and were obtained from Sigma.
Cell culture and adenoviral infection
BAECs were grown in EBM supplemented with 2% fetal bovine serum and growth factors. HUVECs were maintained in Medium 200 supplemented with a low-serum growth supplement before use. All culture media were supplemented with both penicillin (100 U/ml) and streptomycin (100 µg/ml). Cells between passages 5 and 10 were used for all experiments. All cells were incubated in a humidified atmosphere of 5% CO2/95% air at 37°C. BAECs were infected with adenovirus encoding a dominant-negative mutant form of AMPKα (Ad-DN-AMPK) or green fluorescence protein (Ad-GFP) as a control. Infections were performed in 80% confluent cultures of BAECs in media containing 0.1% fetal bovine serum and recombinant adenovirus at the indicated multiplicity of infection (MOI). Cultures were incubated with adenoviruses for 48 h before experimentation. Using these conditions, infection efficiency was typically at least 80%, as determined by GFP expression.
SiRNA-mediated gene silencing in endothelial cells
HUVECs or BAECs were transfected with LKB1 siRNA, HO-1 siRNA, HIF–1α siRNA, CaMKKβ, or the corresponding scrambled siRNA (negative control) for 48 h using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's instructions. Infected cells were starved in serum-free medium for 6 h, then exposed to the indicated concentrations of OA-NO2 or vehicle for 24 h.
Measurement of NO production
For NO detection, BAECs grown in 24-well plates were incubated for 30 min in the presence of 15 µM 4,5-diaminofluorescein diacetate (DAF-2 DA) in PBS or in PBS alone (control) in the dark at 37°C. Cells were then washed with PBS to remove excess DAF-2 DA, and the change in fluorescence over 15 min was measured with excitation and emission wavelengths of 485 and 530 nM, respectively, at room temperature using a microplate reader (FL 600, Bio-Tek). Changes in fluorescence were also visualized with a fluorescence microscope (Olympus IX71), and images were captured for analysis [57].
Measurement of intracellular Ca2+
[Ca2+]i was measured using a Fluo-4 NW kit from Invitrogen according to the manufacturer's instructions. In brief, BAECs were treated with OA-NO2, control or HIF-1α siRNA, YC-1, or BAPTA-AM. The culture medium was then aspirated, cells were washed once with Hepes buffer (pH 7.4), and 1 ml of Hepes buffer with fluorescent dye was added to the cells. After the cells were incubated for 30 min, the fluorescence intensity was measured with excitation and emission wavelengths of 485 and 520 nM, respectively.
Western blot analysis
BAECs, HUVECs, or Hela-S3 cells were lysed in cold RIPA buffer. Protein concentrations were determined using a bicinchoninic acid protein assay system (Pierce, Rockford, IL). Proteins were analyzed by western blotting with ECL-Plus detection as described previously [58]. Relative PPARγ protein expression was measured in HUVEC nuclear extracts as previously detailed [59].
AMPK activity assay
AMPK activity was assayed in the presence and absence of AMP (200 µM) using the SAMS peptide, as previously described [60]. AMPK activity was calculated by determining the difference in activity between both conditions.
Statistical analysis
Statistical comparisons of vasodilation were performed using a two-way analysis of variance (ANOVA), and intergroup differences were analyzed using Bonferroni's post-hoc test. Time-course studies were analyzed using a repeated-measure ANOVA. All other results were analyzed using a one-way ANOVA. Values are expressed as mean ± SD. P-values less than 0.05 were considered significant.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by National Institutes of Health Grants, a grant from the Juvenile Diabetes Research Foundation, a grant from the Oklahoma Center for the Advancement of Science and Technology (OCAST), a grant-in-aid from the American Diabetes Association, and funds from the Travis Endowed Chair in Endocrinology at the University of Oklahoma Health Science Center (all to Dr. Zou). Dr. M.H. Zou is a receipient of National Established Investigator Award of American Heart Association. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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OncotargetOncotargetImpactJOncotarget1949-2553Impact Journals LLC 22190353Research PapersDNA-PKCS binding to p53 on the p21WAF1/CIP1 promoter blocks transcription resulting in cell death Hill Richard 1Madureira Patricia A. 2Waisman David. M. 2Lee Patrick W.K. 131 Department of Microbiology & Immunology, Dalhousie University, Halifax, Nova Scotia, Canada2 Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada3 Department of Pathology, Dalhousie University, Halifax, Nova Scotia, CanadaCorrespondence to:Patrick W.K. Lee, [email protected] 2011 20 12 2011 2 12 1094 1108 8 12 2011 9 12 2011 Copyright: © 2011 Hill et al.2011This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedA key determinant of p53-mediated cell fate following various DNA damage modalities is p21WAF1/CIP1 expression, with elevated p21 expression triggering cell cycle arrest and repressed p21 expression promoting apoptosis. We show that under pro-death DNA damage conditions, the DNA-dependent protein kinase (DNA-PKCS) is recruited to the p21 promoter where it forms a protein complex with p53. The DNA-PKCS-associated p53 displays post-translational modifications that are distinct from those under pro-arrest conditions, ablating p21 transcription and inducing cell death. Inhibition of DNA-PK activity prevents DNA-PKCS binding to p53 on the p21 promoter, restores p21 transcription and significantly reduces cell death. These data demonstrate that DNA-PKCS negatively regulates p21 expression by directly interacting with the p21 transcription machinery via p53, driving the cell towards apoptosis.
DNA-PKCsp53p21 transcription suppression
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INTRODUCTION
The most prevalent reported genetic defect observed in human cancer is the loss or inactivation of the tumor suppressor protein p53 [1, 2]. In unstressed cells, p53 is maintained at a low level by its negative regulator MDM2 [3, 4] and is at the center of a complex signalling network. In response to a broad range of oncogenic stresses including DNA damage, chemical exposure or hypoxia, p53 can facilitate either DNA repair (promoting cell survival) or trigger apoptosis (programmed cell death), thereby ensuring the removal of irreparably damaged cells. p53 is able to dictate these cell fates in large measure because it is a transcription factor with sequence-specific DNA-binding activity that regulates the expression of a plethora of genes [5]. Chromatin-immunoprecipitation (ChIP) studies revealed that p53 directly binds to approximately 1600 genes that fall primarily into three categories: cell cycle inhibition, apoptosis, and genome stability [6]. Despite this wealth of information, the mechanism regarding how p53 mediates the choice between life and death remains unclear.
The generally accepted dogma for p53-controlled cell fate holds that cell cycle arrest is predominantly mediated by the expression and activation of the cyclin-dependent kinase inhibitor CDKN1A (p21) [7-9], in contrast to apoptosis that is primarily controlled by the expression and activation of pro-apoptotic genes including Bax [10] and PUMA (p53 upregulated modulator of apoptosis) [11, 12]. While p21's ability to inhibit both the G1-S and the G2-M cell cycle transitions is well established, emerging evidence suggests that p21 also possesses potent anti-apoptotic activity to complement its pro-arrest functions. For example, it has been shown that p21 binds to and inactivates procaspase 3, thereby inhibiting apoptosis [13]. In addition, caspase 2, which acts upstream of caspase 3, is transcriptionally repressed by p21 [14]. Furthermore, p21 can also suppress the induction of pro-apoptotic genes by MYC or E2F1 by direct inhibition of their transcription functions [15]. There is also evidence that p21 protects cells from irradiation-induced apoptosis by blocking CDKs involved in the activation of the caspase cascade downstream [16] while nutrient starvation induced cell death is also suppressed by p21 [17]. Collectively, these data suggest that p21 is capable of launching a multi-level anti-apoptosis strategy, effectively counteracting the pro-apoptotic functions of Bax and PUMA. Thus, while the induction and presence of pro-apoptotic genes are required for the cell to trigger a potent apoptotic response, there is also the absolute requirement for the cell to abolish p21 expression and mediate p21 protein degradation to enable apoptosis to proceed.
Since both pro-arrest (p21) and pro-death (e.g. Bax and PUMA) elements are downstream transcriptional targets of p53, the delicate balance between their expression levels necessarily hinges on the selective activation or suppression of specific p53 transcriptional activity [18-22]. In this regard, post-translational modifications of p53 have been shown to play a central role. Depending on the nature of DNA damage or cell stress, p53 undergoes different modifications that dictate its ultimate function. A most common and critical modification of p53 is ser15 phosphorylation that prevents MDM2-mediated monoubiquitination and nuclear export, allowing p53 to accumulate in the nucleus [4]. The ser46 and ser315 residues have also attracted significant attention as following ser46 phosphorylation, p53 specifically induces pro-apoptotic gene expression [23, 24] in contrast to ser315 phosphorylation that stimulates the expression of p21 [25]. In addition to phosphorylation, acetylation of p53 has also been shown to regulate p53-dependent transcription (for a review see [26]. The acetylation of lys120 was shown as an absolute requirement for PUMA and Bax transcription after p53 promoter recruitment. In contrast, lysine 382 acetylation specifically and significantly increased p21 expression [27, 28]. Based on these studies, it is clear that there is enormous complexity regarding p53 posttranslational modifications, that many appear to be stress-specific, and that these diverse modifications can activate or repress select target genes dictating cell fate.
The phosphatidylinositol 3-kinase-like protein kinases (PI3KKs) are large proteins that include Ataxia telangiectasia mutated (ATM), ATM and Rad3-related (ATR), and the DNA-dependent protein kinase (DNA-PK) that are each activated following a range of cellular stresses and can direct p53 posttranslational modifications. The most defined cellular response is the activation of the G1 and G2 cell cycle checkpoints mediated by ATM via p53 phosphorylation that leads to p21 transcription and cell cycle arrest [29, 30]. ATR reinforces this response by the phosphorylation of signaling intermediates including checkpoint kinase 1 (Chk 1) [31]. The most classically defined DNA-PK function is V(D)J recombination that is responsible for antibody diversity and normal immune development (reviewed in [32, 33]). In addition to this well characterized role, however, there is now a significant body of data implicating DNA-PKCS as an upstream element of p53, being involved in the latter's posttranslational modification and apoptotic response to severe DNA damage [20, 34-37]. It thus appears that ATM/ATR and DNA-PK play antagonistic roles in dictating stress-induced cell fate (cell cycle arrest vs apoptosis) through their control of a common element, p53 [38, 39]. More recent data suggest that it is the control over p53-mediated p21 expression that ultimately determines cell fate, with ATM/ATR and DNA-PK promoting and suppressing p21 expression, resulting in cell cycle arrest and apoptosis, respectively [40-43].
With DNA-PK's emerging role as an important mediator of apoptosis, it is important to understand how DNA-PK activation could lead to down-regulation of p21. Here we report that under conditions that favor cell death, DNA-PKCS forms a protein complex with p53 and is recruited to the p21 promoter. This complex formation abrogates p21 transcription, preventing cell cycle arrest and as a result sets into motion a potent apoptotic response to DNA damage. DNA-PKCS does not inhibit the ability of p53 to bind to target gene promoters (pro-arrest or pro-death), however, only the transcription of the pro-arrest p21 gene is blocked. DNA-PKCS therefore directly interferes with the p53-mediated p21 transcription machinery, priming the cell for apoptosis.
RESULTS
p21 expression correlates with cell fate following DNA damage
To investigate how p53 can direct cell fate we exposed cells to various DNA damaging conditions that result in cell cycle arrest, apoptosis, or both. Gemcitabine is a chemotherapeutic and an analog of deoxycytidine that inhibits DNA synthesis [44]. Doxorubicin is a chemotherapeutic anthracycline that creates DNA breaks via the inhibition of topoisomerase II [45]. Chromium (VI) is a carcinogen that binds DNA, generating adducts, single and double strand DNA breaks [46], while γ−irradiation (IR) introduces double strand DNA breaks. All four of these DNA damaging agents activated and induced significant accumulation of the p53 protein (Fig. 1A and S1A), however, their effects on cell fate were very different. Whereas chromium treatment triggered significant apoptosis but no cell cycle arrest (measured by caspase-3 cleavage and the accumulation of a sub-G1 cell population), IR induced almost exclusively cell cycle arrest (with little to no apoptosis) (Fig. 1A, 1B and S1B). The effects of gemcitabine and doxorubicin fell between the two ends of the spectrum, with gemcitabine inducing significantly less cell cycle arrest than doxorubicin. These four agents therefore provided a full cell fate spectrum that allowed us to more precisely characterize the role of DNA-PKCS in directing p53-dependent cell fate.
Figure 1 All DNA damage modalities induce p53 but manifest different p21 expression levels that correlate with cell cycle arrest or apoptosis
(A) [p53+/+] HCT116 cells were exposed to chromium (Cr(VI)), gemcitabine (Gem), doxorubicin (Doxo) or γ-irradiation (γ-IR) for the specified times (hr). Cell extracts were subjected to immunoblot analysis for p53, caspase-3 or β-actin expression. (B) HCT116 p53+/+ cells were exposed to Cr(VI), Gem, Doxo or γ-IR. At the time points indicated DNA content was analyzed by flow cytometry of cells stained with BrdU and propidium iodide. Each plot represents the cell cycle profile at 24 h post damage. Quantification of the percentage of the sub-G1, G1 and G2 DNA content was determined for each sample and is represented in each graph (20,000 total events counted). (C) HCT116 p53+/+ cell extracts were subjected to immunoblot analysis for p21, MDM2, PUMA, Bax, and β-actin expression. (D) Trizol RNA extraction was carried out from [p53+/+] HCT116 cells 12 h post damage; cDNA was generated and the expression level for p21 was measured. The mRNA expression levels were normalized to GAPDH mRNA, and are represented as fold increase or decrease over untreated cells. Error bars represent the standard deviation obtained from three independent experiments.
We then investigated the protein expression of key p53-regulated genes over time following exposure to each agent in a range of classically studied model cancer cell lines. As exemplified by the [p53+/+] HCT116 cell line, the most dramatic difference between the four treatments was noted for p21 expression (Fig. 1C). Whereas IR and doxorubicin treatments resulted in the gradual accumulation of p21, chromium and gemcitabine caused a significant reduction of the protein at 6 and 12 h post damage. In contrast to p21, the total protein levels of MDM2, PUMA and Bax were equivalently induced by each modality investigated. The reduced p21 protein levels post gemcitabine or chromium treatment corresponded with the lowered transcription of this gene (Fig. 1D and S1C) as these agents had no effect on protein stability or the half-life of the p21 transcript [20]. Collectively, our results are compatible with the view that the reduction in p21 expression allows continued cell cycle progression which, in the presence of both DNA damage and the continuous expression of apoptotic proteins, ultimately leads to cell death.
Post-translational modifications of p53 bound to the p21 promoter
We next questioned whether p53 was recruited to the promoters of p21 under each DNA damage modality even where expression of this gene was suppressed. Accordingly, chromatin immunoprecipitation (ChIP) assays were carried out and revealed surprisingly that p53 was recruited to both the distal and proximal p21 promoters irrespective of whether there was active p21 gene transcription (Fig. 2 (top panel) and S1D). To determine if the p53 bound to the p21 promoters was modified differently following each DNA damage regime, ChIP analyses were performed using a panel of antibodies against specific modified p53 residues. The results (Fig. 2 and S2) show that while all p21 promoter-bound p53 was phosphorylated on ser15 following exposure to any of the four DNA damage agents, enhanced ser37 phosphorylation was found primarily on p53 bound to the p21 promoters under conditions (chromium or gemcitabine) that instigated cell death. Conversely, phospho-ser315 and acetyl-lys382 were found mainly on p21 promoter-bound p53 under pro-arrest (doxorubicin or IR) conditions. These results clearly indicate that depending on the DNA damage modality, the p53 bound to the p21 promoters was modified differently, leading to either active p21 transcription (mediating cell cycle arrest), or p21 transcription repression (resulting in cell death).
Figure 2 p21 promoter-bound p53 is differently modified under arrest versus apoptotic conditions
Chromatin immunoprecipitation (ChIP) was carried out with [p53+/+] HCT116 cells following mock or 6 h post DNA damage with the indicated modalities using the total or isoform-specific anti-p53 antibodies indicated. PCR was performed for either the distal (-2.2 kb) p21 or proximal (-1.3 kb) p21 promoter regions. Quantification of the bands following each DNA damage modality was determined in triplicate and represented for each promoter region. Error bars in all panels represent the standard deviation obtained from three independent experiments.
While we primarily focused on the p21 promoters, we also examined p53 modifications on the MDM2, PUMA and Bax promoters under each DNA damage condition (Fig. S3). Our results show that p53 phosphorylated at ser15 was found on the MDM2 promoter at 6 h post damage under all DNA damage conditions, but was bound to the PUMA promoter only under conditions that induced cell death (chromium or gemcitabine) and only bound to the Bax promoter following Cr(VI) exposure. Interestingly, p53-ser37 could be detected on both the MDM2 and PUMA promoters 6 h post chromium, gemcitabine or doxorubicin exposure. No p53-ser37 could be detected on the Bax promoter following exposure to any of our DNA damage agents. In contrast to other p53 modifications, we noted the enhanced recruitment of p53-phopho-ser33 and phosphor-ser46 isoforms to all the promoters examined 6 h post damage with any of our tested agents. Collectively, these results indicate that for each p53-dependent cell phenotype (pro-arrest or cell death) there are significantly different p53 post translational profiles.
Autophosphorylated DNA-PKCS and p53 form a protein complex under pro-apoptotic conditions
Having noted distinct p53 post-translational modifications (phosphorylation/acetylation) following DNA damage that triggered cell cycle arrest or cell death (due to continued cycling of DNA damaged cells) correlated with disparate p21 promoter recruitment and expression, we questioned which upstream activators of p53 mediated this response. We examined the three major PI3KK's ATM, ATR and DNA-PKCS since they dictate specific p53 responses linked primarily to either the induction of cell cycle arrest (ATM/ATR) or apoptosis (DNA-PKCS). We found that all three PI3KKs are activated (phosphoryation at ser1981 for ATM, ser428 for ATR, and thr2609 for DNA-PKCS) under any of the four DNA damage conditions (Fig. 3A and S4A). As expected, all ATM and ATR phosphorylations were inhibited by the ATM/ATR-specific inhibitor, CGK-733 [47], but not by the DNA-PK-specific inhibitor, NU-7026 [48]. However, the use of these inhibitors on DNA-PKCS thr2609 phosphorylation was revealing. It clearly showed that depending on the DNA damage treatment used, this phosphorylation event was dictated by different PI3KKs. The DNA-PK inhibitor NU-7026 effectively blocked thr2609 phosphorylation under pro-death (chromium and gemcitabine) conditions, but not under pro-cell cycle arrest (doxorubicin and IR) conditions. Conversely, the ATM/ATR inhibitor CGK-733 did not block thr2609 phosphorylation under damage conditions that lead to cell death (chromium or gemcitabine) but its inhibitory effect, albeit partial, under pro-arrest conditions (doxorubicin or IR) was evident. Our results show that the DNA-PKCS phosphorylated (likely via autophosphorylation) under DNA damage conditions that lead to cell death is intrinsically different from the DNA-PKCS activated (via ATM/ATR [49]) under cell cycle arrest conditions. This raises the hypothesis that DNA-PKCS activated under certain damage conditions is destined to induce cell death by the suppression of cell cycle arrest, whereas DNA-PKCS activated under pro-arrest conditions is poised to perform its DNA repair duty following cell cycle arrest.
Figure 3 DNA-PKCS activation under pro-apoptotic conditions is independent of ATM/ATR and forms a complex with p53
(A) [p53+/+] HCT116 cells were pre-incubated with the DNA-PK inhibitor NU-7026 (10 μM) or the ATM/ATR inhibitor CGK-733 (20 μM) and exposed to Cr(VI), Gem, Doxo or γ-IR for 12 h. Cell extracts were prepared and subjected to immunoblot analysis for total DNA-PKCS or P-thr2609 DNA-PKCS. (B) The cell extracts from (A) were subjected to immunoblot analysis for total p53. (C) [p53+/+] HCT116 cells were exposed to Cr(VI), Gem, Doxo or γ-IR for the specified times. Cell extracts were prepared, immunoprecipitated (IP) with an anti-DNA-PKCS or anti-p53 antibody, and subjected to western blot (WB) analysis for p53 or DNA-PKCS. Immunoblots for total p53 and total DNA-PKCS in the extracts are also shown. (D) (left) Extracts prepared from 12 h post damage cells were immunoprecipitated with an anti-DNA-PKCS antibody and analyzed for co-precipitated specific p53 isoforms. (Right) Extracts from [p53−/−] HCT116 cells were used to confirm antibody specificity.
The specific activation of ATM/ATR and DNA-PK under pro-death and pro-arrest conditions following each DNA damage regime was also reflected by their specific action on p53. Fig. 3B and S4B show that the ATM/ATR inhibitor effectively blocked p53 accumulation under pro-arrest conditions, but not under conditions that actuated cell death. Conversely, the DNA-PK inhibitor blocked p53 accumulation under pro-death DNA damage conditions (in the absence of cell cycle arrest) but not under pro-arrest conditions. Thus, p53 accumulation under pro-death or pro-arrest conditions is dependent on DNA-PKCS or ATM/ATR, respectively. This is again consistent with the observation that p53 is modified differently under the two cell phenotypes.
To further support this observation, we have previously shown that murine cell lines expressing adenovirus E1A (thus are unable to induce cell cycle arrest) undergo cell death post irradiation and that DNA-PKCS and p53 form a protein-protein complex [34]. To determine if this complex formation occurs in human lines and correlates strictly with cell death, co-immunoprecipitation was carried out on lysates from cells damaged under the four DNA damage conditions. We found that both gemcitabine and chromium induced the formation of a DNA-PKCS/p53 protein complex that was not detected following doxorubicin treatment or IR (Fig. 3C and S4C). These observations, together with the results shown in Fig. 3B, raise the hypothesis that p53/DNA-PKCS only form a protein/protein complex following DNA-PKCS autophosphorylation and that when DNA-PKCS is phosphorylated by ATM/ATR this protein complex does not form. In addition, the p53 bound to DNA-PKCS was found to be phosphorylated on ser15, ser37, and ser46, and acetylated at lys120, in agreement with our ChIP studies (Fig. 3D). It is noteworthy that all of these modifications have been strongly linked with enhancing p53-dependent apoptosis [24]. Furthermore and consistent with our ChIP data, we were unable to detect phospho-ser315 or acetyl-lys382 p53 in the DNA-PK/p53 complex.
DNA-PKCS/p53 interaction occurs on the p21 promoter
The observation that p21 promoter-bound p53 and p53 bound in a complex with DNA-PKCS are similarly modified led us to hypothesize that DNA-PKCS could be recruited to the p21 promoters under pro-death DNA damage conditions. This was indeed found to be the case. We noted the rapid binding of DNA-PKCS to both distal and proximal p21 promoters under pro-death (DNA damage conditions chromium or gemcitabine) but not pro-arrest conditions (doxorubicin or IR) in a range of model cancer cell lines (Fig. 4A, S5A and S5B). This recruitment was p53-dependent as we could not detect significant DNA-PKCS binding to the p21 promoters in similarly treated p53−/− HCT116 cells, and no DNA-PKCS binding was observed on the GAPDH promoter that was used throughout as our negative control. It is noteworthy that DNA-PKCS recruitment to the PUMA and Bax promoters was negligible relative to the p21 promoters (Fig. 4A), and could only be detected at late time points (24 h after chromium exposure or 48 h post gemcitabine treatment) once the cellular phenotype was markedly apoptotic. As expected, the p21 promoters-bound DNA-PKCS was found to be phosphorylated at thr2609 (Fig. 4B, S5A and S5B).
Figure 4 DNA-PKCS forms a complex with p53 on the p21 promoters under pro-apoptotic conditions
(A) ChIP was carried out with [p53+/+] HCT116 cells following mock or 6 h post DNA damage with the indicated modalities using the anti-DNA-PKCS antibody. PCR was performed for both the distal (-2.2 kb) p21 and proximal (-1.3 kb) p21 promoter regions. Quantification of the bands following each DNA damage modality was determined for triplicate studies and represented for each promoter region. (B) ChIP assays were conducted as in (A) using an anti-P-thr2609-DNA-PKCS antibody. (C) Top left: ChIP assay for p53 from [p53+/+] HCT116 cells 6 h post Cr(VI), Gem, or γ-IR exposure. Top right: ChIP assay for p53 from [p53+/+] HCT116 cells following DNA-PKCS immunodepletion. Bottom left: ChIP assay for DNA-PKCS 6 h post DNA damage. Bottom right: ChIP assay for DNA-PKCS following p53 immunodepletion. PCR was performed for the distal (-2.2 kb) and the proximal (-1.3 kb) p21 promoter regions. The error bars in all panels represent standard deviation obtained from three independent experiments.
To demonstrate that DNA-PKCS recruited to the p21 promoters represented the same population that was associated with p53, we carried out immune-depletion experiments followed by ChIP analysis. Under conditions where p21 transcription was repressed following chromium or gemcitabine treatment, immune-depletion of nuclear lysates with an anti-DNA-PKCS antibody led to the drastic reduction of p53 bound to the p21 promoters (Fig. 4C). This was not observed with nuclear lysates from IR-treated cells. Conversely, reciprocal immune-depletion with an anti-p53 antibody effectively removed DNA-PKCS bound to the p21 promoters following chromium or gemcitabine treatment while no DNA-PKCS could be detected on these promoters following IR treatment. Taken together, these studies clearly demonstrate that p53 and DNA-PKCS form a protein complex and that this complex is localized to the p21 promoter.
We next compared the temporal kinetics of p53 and DNA-PKCS recruitment to the p21 promoters. As revealed by ChIP analysis, the recruitment of p53 on the proximal and distal p21 promoter was detected as early as 30 min post DNA damage (gemcitabine or chromium) on the distal p21 promoter and 3 h on the proximal promoter (Fig. 5A, 5B, 5D and 5E). A similar recruitment profile was also observed in the Panc-1 cell line (Fig. S6A, S6B, S6C and S6D). In contrast, the temporal recruitment of DNA-PKCS was significantly slower than p53, with DNA-PKCS binding first detected on the distal p21 promoter around 3 h and on the proximal promoter approximately 6 h post damage. This shows that p53 recruitment to both p21 promoters precedes that of DNA-PKCS, suggesting that the early recruitment of p53 to the p21 promoters is likely independent of DNA-PK.
Figure 5 p53 recruitment to the p21 promoter precedes DNA-PKCS binding
(A) ChIP assay for p53 or DNA-PKCS from [p53+/+] HCT116 cells following Gem exposure for the time points indicated. + indicates 6 h pre-incubation with the ATM/ATR inhibitor CGK-733 (20 μM) or the DNA-PK inhibitor NU-7026 (10 μM). PCR was performed for the distal (-2.2 kb) p21 promoter region. (B) ChIP assays for p53 or DNA-PKCS were conducted as described in (A) and PCR was performed for the proximal (-1.3 kb) p21 promoter region. (C) The quantification of the PCR bands shown in (A) and (B) and two repeated ChIP studies was carried out and normalized to each input lane. (D) ChIP assay for p53 or DNA-PKCS from [p53+/+] HCT116 cells following Cr(VI) exposure for the time points indicated. + indicates 6 h pre-incubation with the ATM/ATR inhibitor CGK-733 (20 μM) or the DNA-PK inhibitor NU-7026 (10 μM). PCR was performed for the distal (-2.2 kb) p21 promoter region. (E) ChIP assay for p53 or DNA-PKCS was conducted as described in (D) and PCR was performed for the proximal (-1.3 kb) p21 promoter region. (F) The quantification of PCR bands shown in (D) and (E) and two repeated ChIP studies was conducted and the values normalized to input samples as described in (C). The boxed areas highlight the effect of the ATM/ATR inhibitor on recruitment of early, but not late p53 to the p21 promoter. Error bars for the graphs shown in (C) and (F) represent the standard deviation obtained from three independent experiments.
To test the possibility that the early p53 recruitment to the p21 promoters could be mediated by ATM/ATR, ChIP analyses were carried out using the ATM/ATR and DNA-PK inhibitors. We found that the inhibition of ATM/ATR by CGK-733 delayed the early, but not the late recruitment of p53 to both the distal and proximal p21 promoters under pro-death conditions (Fig. 5C and 5F, indicated on each graph in the boxed area). The temporal recruitment of p53 is now more in line with that of DNA-PKCS which was unaffected by the inhibition of ATM/ATR. This result indicates that the early recruitment of p53 to the p21 promoters is independent of DNA-PKCS and mediated by ATM/ATR. Importantly, at later time points in the presence of sustained DNA damage there was the recruitment of DNA-PKCS to the p21 promoter-bound p53. The binding of both p53 and DNA-PKCS to the p21 promoters was effectively blocked by the DNA-PK inhibitor NU-7026, consistent with significantly attenuated p53 protein accumulation (Fig. 3B). A likely explanation is that the p53 that initially binds to the p21 promoter at earlier time points becomes further modified at later times by DNA-PKCS following its recruitment to the promoter.
DNA-PKCS inhibition restores p21 expression and increases cell survival
We then questioned what the effect of DNA-PK inhibition would be on downstream p53-regulated genes under the various DNA damage conditions. Strikingly, repression of p21 transcription following chromium or gemcitabine treatment was effectively reversed upon inhibition of DNA-PK, with the transcription of p21 restored to levels comparable to those prior to damage (Fig. 6A) whereas inhibition of ATM/ATR had no effect suggesting that under these conditions DNA-PKCS represses p21 transcription. In contrast, under pro-arrest conditions (doxorubicin or IR), p21 transcription was ablated when ATM/ATR was inhibited, whereas the inhibition of DNA-PK had no effect. This is consistent with the loss of p53 protein accumulation following the inhibition of ATM/ATR under pro-arrest conditions (Fig. 3B). In contrast to p21, chromium and gemcitabine exposure elicited an increase in both PUMA and Bax transcription (Fig. S7A) that was blocked by the DNA-PK inhibitor but not the ATM/ATR inhibitor. Enhanced expression of PUMA and Bax was also evident under pro-arrest conditions however this enhancement was ATM/ATR-dependent as it was blocked by CGK-733. As expected, in the absence of p53, there was little to no p53-regulated gene expression and that following treatment with each DNA damage modality there was limited transcription of these genes which was not significantly affected by ATM, ATR or DNA-PKCS inhibition (Fig. S8B). It is noteworthy that these responses were conserved in a range of cancer cell lines (Fig. S7C and S7D).
Figure 6 Inhibition of DNA-PKCS restores p21 expression under pro-apoptotic conditions
(A) Trizol RNA extraction was carried out from [p53+/+] HCT116, A549 and Panc-1 cells 12 h post damage; cDNA was generated and the expression level for p21 was measured. The mRNA expression levels were normalized to GAPDH mRNA, and are represented as fold increase or decrease over untreated cells. The DNA-PK inhibitor (NU-7026 [10 μM]) or the ATM/ATR inhibitor (CGK-733 [20 μM]) was pre-incubated for 6 h prior to DNA damage. (B) Immunoblot for p21, PUMA or Bax in [p53+/+] HCT116 cells 12 h post DNA damage. + indicates 6 h pre-incubation with the DNA-PK inhibitor NU-7026 (10 μM) or the ATM/ATR inhibitor CGK-733 (20 μM). For each gel β-actin indicates loading control. (C) Flow cytometric analysis of [p53+/+] HCT116 cells treated as described in (A). The percentage subG1 populations indicated in each FACS profile represent the average percentage from three independent experiments (10,000 total events counted per sample). (D) EC50 values were calculated using MTS assay for the [p53+/+] HCT116, A549 and Panc-1 cell lines 48 h post Cr(VI), Gem or Doxo treatment. The error bars for (D) represent the standard deviation obtained from three independent experiments.
The protein expression levels under each DNA damage condition was also examined and was found to be consistent with our gene expression results (Fig. 6B). Following chromium or gemcitabine exposure there was the loss of the p21 protein. In contrast, there was significant accumulation of both PUMA and Bax. Inhibition of DNA-PKCS restored p21 protein levels while reducing the levels of PUMA and Bax, consistent with our transcription studies. Furthermore, as predicted from our gene expression experiments, inhibition of ATM/ATR was unable to rescue the loss of the p21 protein and had no effect on the elevated PUMA and Bax protein levels. In contrast to chromium and gemcitabine treatment, both doxorubicin and IR exposure significantly increased the levels of all three proteins (p21, PUMA and Bax) in an ATM/ATR-dependent manner. Similar observations were made using the Panc-1 cell line (Fig. S8). Taken together, these observations are congruent with the notion that cell fate is dependent more on the regulation of expression of pro-survival genes (such as p21) than on the regulation of expression of pro-death genes (such as PUMA and Bax) and that DNA-PK and ATM/ATR play antagonistic roles in dictating these events.
To test this hypothesis we exposed cells to each damage agent in the presence of either the ATM/ATR inhibitor or the DNA-PK inhibitor. At 48 hours following Cr(VI) or gemcitabine exposure we note the significant accumulation of a sub-G1 population. In contrast and as predicted, doxorubicin or IR treatment induced cell cycle arrest (Fig. 6C). The inhibition of ATM/ATR did not significantly alter the sub-G1 population following Cr(VI) or gemcitabine treatment however as expected it did increase the sub-G1 population after doxorubicin or IR treatment [50, 51]. DNA-PK inhibition significantly reduced the sub-G1 population after Cr(VI) or gemcitabine exposure but had no discernable effect 48 hours post-doxorubicin or IR treatment. To complement our FACS approach we determined cell viability (EC50) for each chemical when ATM/ATR or DNA-PK was inhibited (Fig. 6D). In support of our data shown in Figure 6C, the inhibition of DNA-PK significantly increased cell viability after Cr(VI) or gemcitabine treatment but had no effect on cell viability following doxorubicin treatment. (We were unable to determine an EC50 value following IR treatment at 48 hours post damage). The inhibition of ATM/ATR did not significantly alter (although did lower) the EC50 for Cr(VI) or gemcitabine, however, it did cause a noticeable decrease (P=0.0259) in cell viability following doxorubicin treatment. In this regard it is interesting to note that a recent study showed that upon camptothecin treatment, ATM activation leads to cell cycle arrest, however when absent or inhibited, there is hyper DNA-PKCS activation causing cell death [38]. We also addressed a previous report that demonstrated over expression of Myc can suppress p21 transcription by forming a protein complex with Miz1 on the proximal p21 promoter [52]. Our results indicate that the endogenous suppression of p21 transcription following either gemcitabine or chromium exposure is DNA-PKCS dependent and independent of Myc/Miz-1 (Fig. S9).
DISCUSSION
The present study reveals a mechanism whereby a key DNA damage sensor upstream of p53 is linked to a major effector protein downstream of p53, leading to cell death (Fig. 7). We demonstrate that under DNA damage conditions which induce p53-dependent cell death there is recruitment of DNA-PKCS to the p21 promoter. This promoter recruitment occurs by DNA-PKCS binding to p53 on the p53 responsive elements within the p21 promoter after DNA-PKCS autophosphorylation. Upon DNA-PKCS promoter binding, there is the rapid loss of p21 transcription and the p21 protein. As a result of this ablated mRNA expression and subsequent protein loss, the cell is unable to induce cell cycle arrest and continues to cycle in the presence of sustained DNA damage. In striking contrast, under the same cellular conditions there is no DNA-PKCS recruitment on pro-apoptotic promoters (PUMA or Bax) and consequently there is the elevated, continuous transcription of PUMA and Bax, producing a cellular environment that rapidly drives the cell towards apoptosis.
Figure 7 Model for DNA-PKCS-mediated repression of p21 transcription and induced cell death
Under pro-arrest conditions (e.g. IR or Doxo), ATM/ATR is activated and modifies p53 promoting p21 transcription and inducing cell cycle arrest. Concurrently, ATM/ATR phosphorylates DNA-PKCS, priming the latter for its DNA repair function. Under pro-death damage conditions (e.g. Cr(VI) or Gem), DNA-PKCS undergoes autophosphorylation independently of ATM/ATR. DNA-PKCS binds to p53 on the p21 promoter, resulting in the abrogation of p21 transcription. By preventing the accumulation of p21, DNA-PKCS suppresses cell cycle arrest and p21-mediated survival, directing the cell towards cell death.
A most important aspect of the present model is that it reconciles a number of key observations made through the years pertaining to the complex interplay between ATM, p53, p21, and importantly, DNA-PK, and amalgamates them into a single unifying mechanistic concept pertaining to the transcription regulation of p21. Under conditions that favor cell cycle arrest, the ATM/ATR signaling pathway is activated, triggering a p53 phosphorylation/acetylation cascade involving key p53 “arrest-specific” residues (e.g. phosphorylation at ser15 and ser315, and acetylation at lys382). Following p21 promoter recruitment this “modified” p53 activates p21 transcription, and cell cycle arrest ensues. Under pro-death conditions, the recruitment of DNA-PKCS to the p21 promoter apparently initiates a separate and different p53 phosphorylation/acetylation cascade (e.g. phosphorylation at ser15, ser37, ser46 and acetylation at lys120), which results in the abrogation of p21 transcription. While precisely how the cell senses pro-arrest versus pro-death stress signals is unclear at present, a glimpse of the mechanisms involved in the transition from the pro-arrest to pro-death state could be gleaned from the time course experiment presented in Figure 5. During earlier times upon exposure to Cr(VI) or Gemcitabine, the recruitment of p53 to the p21 promoter precedes that of DNA-PKCS, and that this recruitment is ATM/ATR-dependent. This “early bound” p53 likely functions as an activator of p21 transcription, priming the cell for cell cycle arrest. Concomitantly, at these earlier times, DNA-PKCS is phosphorylated, and this phosphorylation event is also ATM/ATR-dependent. It is tempting to speculate that the damaged cell is utilizing ATM/ATR to activate and couple p53's pro-arrest function with DNA-PK's DNA repair function. Indeed, recent evidence supports such a cross-talk between ATM/ATR and DNA-PK in situations where the repair of the damaged DNA is still a viable option [49, 53]. Under pro-death conditions, DNA-PK undergoes autophosphorylation, binds to and modifies p53 on the p21 promoter, incapacitating p53's transcription activation function, and priming the cell to induce apoptosis by the ablation of p21 expression. Although additional details will need to be worked out (e.g. the specific and sequential involvement of kinases and phosphatases, acetylases and deacetylases, and other p53 modifying enzymes etc.), the proposed model represents a general framework whereby ATM/ATR and DNA-PK are portrayed as key opposing players that dictate cell fate through the manipulation of a common switch, that of p53-mediated p21 transcription.
Our results lend credence to the intriguing notion that DNA-PK has dual roles, the first being its classically defined DNA repair role of mediating non-homologous end joining (NHEJ) and the second being its emerging role of driving apoptosis. It would seem logical to view these two roles as being mutually exclusive, which would in turn suggest the existence of a switch mechanism capable of exerting opposing biochemical and functional control of DNA-PK. While DNA-PK's DNA repair function necessitates its binding to broken DNA ends, the nature of putative DNA-PK binding to promoter elements is less clear. In this regard, it is noteworthy that signal-dependent activation of gene transcription has been shown to involve topoisomerase IIβ-dependent transient double strand DNA breaks with subsequent activation of poly(ADP-ribose) polymerase-1 (PARP-1) enzymatic function [54, 55], which could involve the recruitment of DNA-PK to the promoter site. While it remains to be seen how common DNA-PK-mediated transcription regulation through promoter binding occurs, the present study shows that the potential role of DNA-PK in apoptosis and cancer control cannot be understated and warrants further investigation.
METHODS
Cell lines and reagents
[p53+/+] HCT116 and [p53−/−] HCT116 human colon carcinoma cells were maintained in McCoy's 5A medium. A549 and Panc-1 human lung and pancreatic carcinoma cell lines were maintained in DMEM. All were supplemented with 10% FBS and antibiotics. Antibodies shown in supplemntal table 1 were used for our immunoblots and visualization of signal was achieved using an Odyssey® Infrared Imaging System (Licor Bioscience, US). Doxorubicin (Sigma, US), gemcitabine hydrochloride (Eli Lilly #VL7502), chromium(VI) (potassium chromate (#03377 Sigma, US) were used at a concentration of 0.5 μM, 10 μM and 30 μM respectively. γ-Radiation (10 Gy) was delivered by a 137Cs gamma radiator (MDS Nordion) at 2.5 Gy min−1. Cells were pre-incubated with either the DNA-PK inhibitor NU-7026 (10 μM) or the ATM/ATR inhibitor CGK-733 (20 μM) (Tocris Bioscience, US) for 6 h prior to DNA damage.
Cell viability (MTS) assay
Cells were seeded in 96-well plates at 1 x 104 cells/well and 24 hr later treated with various DNA damaging agents Doxorubicin (Sigma, US), gemcitabine hydrochloride (Eli Lilly #VL7502), chromium(VI) (potassium chromate (#03377 Sigma, US) or γ-Radiation at the concentrations described. At the time points indicated post-treatment, cell survival was determined by CellTiter 96© Aqueous non-radioactive proliferation assay (MTS assay; Promega, CA, USA) following the manufactures guidelines.
Nuclear and cytoplasmic fractionation
Cells were washed once with ice cold PBS and then 1 ml of hypotonic lysis buffer (20 HEPES, 10 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 20% glycerol, 0.1% triton X-100, 100 mM DTT, 1 mM AEBSF, 1 mM PIC) was added to each pellet. Nuclear fractions were harvested in hypertonic lysis buffer (20 mM HEPES, 500 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 20% glycerol, 0.1% Triton X-100, 10 mM DTT, 1 mM AEBSF, 1 mM PIC).
Co-immunoprecipitation
Co-immunoprecipitations (Co-IP) were performed as described in [34]). Cells were lysed in cold lysis buffer (50 mM Tris-Cl at pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% sodium deoxycholate, protease inhibitor mixture). Cell extracts (500 μg) were incubated with the first antibodies (supplemental table S1) or control normal IgG on a rotator overnight at 4°C, followed by addition of protein G magnetic beads (Invitrogen, US) for 2 h at 4°C. Beads were then washed four times using the lysis buffer. The immune complexes were subjected to SDS-PAGE followed by immunoblotting with the secondary antibody.
Western blot analysis
For the preparation of whole cell lysate, cells were harvested and lysed using RIPA buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.5% Na-deoxychlorate, 150 mM NaCl, 1 mM EDTA, 2 mM NaF, 2 mM NaVO4 and 1x protease inhibitor cocktail (PIC) (Sigma, US)). For SDS–PAGE, protein samples were boiled for 5–10 min in protein sample buffer (50 mM Tris pH 6.8, 1% SDS, 10% glycerol, 0.01% Bromophenol Blue, β mercaptoethanol [50 μL per 950 μL sample buffer]). Following electrophoresis, proteins were transferred onto nitrocellulose membrane (BioRad, US). The membrane was blocked for for 1 hour at room temperature or overnight at 4°C using 1X Odyssey® blocking buffer (Li-Cor, US). Primary antibodies were added to the membrane (supplemental table S1) overnight at 4°C or for 2 hours at room temperature. Secondary antibody was added (Licor, US) at typically 1:10,000 dilution for 1 hour at room temperature. Visualisation of signal was achieved using an Odyssey Infrared Imaging System (Li-Cor, US).
Chromatin immunoprecipitation (ChIP)
Chromatin immunoprecipitation (ChIP) assays were performed essentially the same as described by Kaeser et al and Mattia et al [56, 57]. Briefly, cells were fixed with 1% formaldehyde, and then whole-cell lysates were prepared. Protein lysate was subjected to ChIP with the indicated antibodies (Supplemental table S1), followed by DNA purification. ChIP-enriched DNA was analyzed by PCR with the indicated primer sets (Supplemental Table S3). Visualization of bands was achieved using a Typhoon Phospho-imager (Amersham, UK) and quantified using the Image-Quant software (Amersham, UK).
Quantitative Real time PCR (qRT-PCR)
Total RNA was extracted by using Trizol (Invitrogen). Real Time PCR was performed on a Stratagene MX3000P PCR machine using the Stratagene Sybr® green master mix (Stratagene, Canada). The primer sequences for measuring p21, Bax, PUMA, MDM2 and GAPDH were purchased from Invitrogen and are shown in supplemental table S2. Data analysis was carried out using the 2−∆∆CT method described by [58].
Flow cytometric cell cycle analysis
Cells were grown to 60% confluence. BrdU was added to the medium 2 hours prior to DNA damage. Cells were mock treated/exposed to each DNA damage agent for 4 hours (in the presence of the DNA damage agent or following IR treatment). This corresponded to the 6 hour damage sample. Additional time points investigated were 0 hr (cells incubated in BrdU containing medium for 4 hours prior to sample collection), 2 hr, 12 hr, 24 hr and 48 hr. BrdU labeling was carried out utilizing an anti-BrdU-FITC antibody (Becton Dickinson) following the manufacturer's instructions. Samples were run on a Fluorescence Activated Cell Scanner (FACS) and the percentage with incorporated BrdU, the sub-G1 (non-viable apoptotic) population and the BrdU negative non-subG1 (viable) populations determined. 20,000 total events were scored per study BrdU was added to the medium 2 h prior to DNA damage. BrdU labeling utilizing an anti-BrdU-FITC antibody (#555627 BD Biosciences, US) was conducted and propidium iodide (2.5 mg mL−1) was added to the fixed, stained cells prior to analysis. 20,000 total events were scored per study from triplicate studies. Data was analyzed using FACS-express 3 (De Novo software, US).
Statistical analysis
Statistical significance was assessed by one-way ANOVA or the two-tailed Students t-test. Statistical significance was defined as P < 0.05. Results are expressed as the mean ±SD.
SUPPLEMENTAL INFORMATION
Supplemental information is linked to the online version of the paper on the Oncotarget website and includes supplemental figure legends, 9 supplemental figures (S1-S9) and 3 supplemental tables (T1-T3).
Supplementary Figures and Tables
We thank B. Vogelstein (Johns Hopkins University) for our [p53+/+] and [p53−/−] HCT116 cell lines. This work is funded by the Canadian Institute for Health Research (CIHR). RH and PM performed the experiments; RH and PL designed the experiments; RH, PM and PL analysed the data; RH and PL wrote the paper; PM and DW corrected the paper.
The authors declare that they have no conflict of interest.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22363731PONE-D-11-2200110.1371/journal.pone.0031783Research ArticleBiologyBiochemistryModel OrganismsAnimal ModelsMedicineHematologyHematologic Cancers and Related DisordersNutritionOncology3, 3′-Diindolylmethane Exhibits Antileukemic Activity In Vitro and In Vivo through a Akt-Dependent Process DIM Exhibits Antileukemic ActivityGao Ning
1
2
*
Cheng Senping
2
Budhraja Amit
2
Liu E-Hu
1
Chen Jieping
3
Chen Deying
1
Yang Zailin
3
Luo Jia
4
Shi Xianglin
2
Zhang Zhuo
2
*
1
Department of Pharmacognosy, College of Pharmacy, 3rd Military Medical University, Chongqing, China
2
Graduate Center for Toxicology, College of Medicine, University of Kentucky, Lexington, Kentucky, United States of America
3
Department of Hematology, Southwest Hospital, 3rd Military Medical University, Chongqing, China
4
Department of Internal Medicine, University of Kentucky, Lexington, Kentucky, United States of America
Bunting Kevin D. EditorEmory University, United States of America* E-mail: [email protected] (NG); [email protected] (ZZ)Conceived and designed the experiments: NG SC AB. Performed the experiments: NG SC AB E-HL JC DC ZY. Analyzed the data: NG AB JL XS ZZ. Contributed reagents/materials/analysis tools: NG SC ZZ. Wrote the paper: NG.
2012 21 2 2012 7 2 e317832 11 2011 16 1 2012 Gao et al.2012This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.3,3′-diindolylmethane (DIM), one of the active products derived from Brassica plants, is a promising antitumor agent. The present study indicated that DIM significantly induced apoptosis in U937 human leukemia cells in dose- and time-dependent manners. These events were also noted in other human leukemia cells (Jurkat and HL-60) and primary human leukemia cells (AML) but not in normal bone marrow mononuclear cells. We also found that DIM-induced lethality is associated with caspases activation, myeloid cell leukemia-1 (Mcl-1) down-regulation, p21cip1/waf1 up-regulation, and Akt inactivation accompanied by c-jun NH2-terminal kinase (JNK) activation. Enforced activation of Akt by a constitutively active Akt construct prevented DIM-mediated caspase activation, Mcl-1 down-regulation, JNK activation, and apoptosis. Conversely, DIM lethality was potentiated by the PI3K inhibitor LY294002. Interruption of the JNK pathway by pharmacologic or genetic approaches attenuated DIM-induced caspases activation, Mcl-1 down-regulation, and apoptosis. Lastly, DIM inhibits tumor growth of mouse U937 xenograft, which was related to induction of apoptosis and inactivation of Akt, as well as activation of JNK. Collectively, these findings suggest that DIM induces apoptosis in human leukemia cell lines and primary human leukemia cells, and exhibits antileukemic activity in vivo through Akt inactivation and JNK activation.
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Introduction
Epidemiological studies have revealed an association between high dietary intake of cruciferous vegetables and decreased cancer risk [1]. The dietary indoles have been shown to be protective against several types of cancers [2]. Indole-3-carbinol (I3C) and 3,3′-diindolylmethane (DIM) are naturally occurring plant alkaloids formed by the hydrolysis of indole glucosinolate (glucobrassicin) and are found in abundance in cruciferous vegetables such as broccoli and Brussels sprouts [3]. I3C is chemically unstable in an acidic environment and is rapidly converted to a variety of condensation products in the stomach. Among those products, DIM is a major one that is readily detectable in the liver and feces of rodents fed with I3C [4]. The parent I3C could not be detected in tissues of I3C-treated rodents, whereas DIM is acid stable and detectable in biological tissues suggesting that DIM may mediate the physiologic effects of dietary I3C [5], [6]. It has been shown that I3C and DIM exert anticarcinogenic effects in experimental animals and humans [7], [8]. Several studies have also shown that DIM is a potent inhibitor of cell growth and inducer of apoptotic cell death in breast cancer cells [9], [10]. Since DIM is relatively nontoxic to normal tissues of animals and humans [11], it might play an important role in the chemoprevention and chemotherapy of cancers. It has been reported that consumption of cruciferous vegetables appeared to be associated with a decreased risk of leukemia especially AML [12]. Because DIM is a major component of cruciferous vegetables, DIM would be a particularly effective agent for leukemia.
The PI3K/Akt signaling pathway plays an important role in cell survival and anti-apoptotic decisions [13]. A major activator of Akt is phosphatidylinositol-3, 4, 5-triphosphate (PIP3) which is generated by phosphatidylinositol-3- kinase (PI3K) [14]. Akt is activated by phosphorylation at Thr308 by PDK1 or at Ser473 by mTORC2 [15]. Activated Akt functions to promote cell survival by inhibiting apoptosis through inactivation of several pro-apoptotic factors including Bad, Forkhead transcription factors, and caspase-9 [16], [17], [18]. Akt has been believed to be an attractive target for cancer prevention or treatment.
c-Jun N-terminal kinase (JNK) belongs to the super family of MAP kinases, which can promote apoptosis by different mechanisms [19]. Activation of JNK may result in phosphorylation of c-Jun and activator protein-1 (AP-1) activity, which could drive cells to apoptosis [20]. Previous studies on the role of cell signaling cascades in DIM-mediated lethality have primarily focused on those related to oxidative stress and cell signaling pathways. For example, in MCF-7 human breast cancer cells, DIM induced G1 cell cycle arrest through up-regulation of p21, and the oxidative stress and stress-activated signaling cascades including p38 MAPK and JNK play the key roles in these events [21]. It has also been shown that DIM induced apoptosis through inactivation of NF-κB in human breast cancer cells [22]. In cholangiocarcinoma cells, Fas mediated apoptosis was enhanced by DIM through inhibition of Akt and FLIP [23]. Furthermore, in human prostate cancer cells, DIM inhibited cell proliferation and induced apoptosis through down-regulation of AR, Akt, and NF-κB signaling [24]. However, neither the effects of DIM on apoptosis induction nor the relationships between DIM lethality and cell signaling cascades been examined in depth in human leukemia. Here, for the first time we reported that DIM significantly induced apoptosis in human leukemia cells. Our results indicate that inactivation of Akt and activation of JNK play important roles in DIM-mediated apoptosis in these cells. Our results also indicate that DIM inhibits growth of mouse U937 xenograft tumors, and the interruptions of the Akt and JNK pathways are involved in DIM-mediated antileukemic activity in vivo.
Results
DIM induces apoptosis in U937 human leukemia cells in dose- and time-dependent manners
Dose-dependent study revealed a moderate increase in apoptosis 12 h and 24 h after exposure to DIM at concentration of 60 μM and very extensive apoptosis at concentration of 80 μM (Fig. 1a). Time-course study of cells exposed to 80 μM DIM demonstrated a significant increase in apoptosis at 9 h after drug exposure. These events became apparent after 12 h of drug exposure, and reached near-maximal levels after 24 h (Fig. 1a). Consistent with these findings, the same DIM concentrations and exposure intervals resulted in cleavage/activation of caspase-3, -7, -8 and -9, as well as PARP degradation (Fig. 1b).
10.1371/journal.pone.0031783.g001Figure 1 DIM induces apoptosis, caspase activation, downregulation of Mcl-1, upregulation of p21, inactivation of Akt, and activation of JNK in U937 human leukemia cells in dose- and time-dependent manners.
U937 cells were treated with various concentrations of DIM as indicated for 12 h and 24 h or treated with 80 µM DIM for 1, 3, 6, 9, 12, and 24 h. (a) Cells were washed twice with PBS and stained with Annexin V/propidium iodide (PI), and apoptosis was determined using flow cytometry. Both early apoptotic (Annexin V-positive, PI-negative) and late apoptotic (Annexin V-positive and PI-positive) cells were included in cell death determinations. The values obtained from annexin V/PI assays represent the mean ± SD for three separate experiments. (b–d) Total cellular extracts were prepared and subjected to Western blot assay using antibodies as indicated.
Exposure of U937 cells to DIM results in down-regulation of Mcl-1 and up-regulation of p21
The effects of DIM were then examined in relation to expression of various Bcl-2 family members and cell cycle regulatory protein. A dose-dependent study revealed that exposure of U937 cells to DIM at concentration of 40 μM resulted in decrease in expression of Mcl-1 and increase in expression of p21. These events became very extensive at concentrations ≥60 μM (Fig. 1c). Time-course study demonstrated that exposure of U937 cells to 80 μM DIM resulted in slight decrease in expression of Mcl-1 as early as 1 h after drug exposure. This event became apparent after 3 h of drug exposure, and the expression of Mcl-1 was complete blocked after 12 h (Fig. 1c). On the other hand, exposure of cells to 80 μM DIM resulted in increase in expression of p21 at 9 h after drug exposure, and this event became extensive after 24 h of drug exposure (Fig. 1c). In contrast, exposure of cells to DIM resulted in little or no change in expression of Bcl-2, Bcl-xL, Bax, Bad, and Bim (data not shown).
DIM induces inactivation of Akt and activation of JNK in U937 cells
Effects of DIM in U937 cells were also examined in relation to various signaling pathways implicated in apoptosis regulation. Exposure of cells to DIM for 12 h and 24 h resulted in diminished phosphorylation of Akt in a dose-dependent manner. This event was accompanied by a pronounced increase in phosphorylation of JNK (Fig. 1d). Time-course study demonstrated that exposure of cells to DIM (80 μM) resulted in inhibition of phosphorylation of Akt and activation of JNK as early as 3 h after drug exposure. These events became apparent at 6 h and very extensive at 12 h and 24 h after drug exposure (Fig. 1d). In contrast, DIM had little or no effect on expression of total or phospho-mTOR, ERK, or p38 MAPK (data not shown). These results suggest that inactivation of Akt and activation of JNK may play important roles in DIM-induced apoptosis.
DIM has similar effects on apoptosis in Jurkat and HL-60 cells, and AML primary leukemia cells
To determine whether these events were restricted to myeloid leukemia cells, parallel studies were performed in Jurkat and HL-60 leukemia cells. These cells exhibited apoptotic effects upon DIM exposure similar to those observed in U937 cells although Jurkat and HL-60 cells are less sensitive than U937 cells in DIM-induced apoptosis (p<0.01 versus control), PARP degradation, caspases activation (Fig. 2a and 2b).
10.1371/journal.pone.0031783.g002Figure 2 DIM induces apoptosis in U937, Jurkat, and HL-60 cells, and in AML blast samples, but not in normal bone marrow mononuclear cells.
(a) U937, Jurkat, and HL-60 cells were treated with 80 µM DIM for 24 h, after which apoptosis was determined by annexin V/PI staining and flow cytometry. ** Values for cells treated with DIM were significantly increased compared to values in control cells by Student's t-test, p<0.01. (b) Total cellular extracts were prepared and subjected to Western blot analysis using antibodies as indicated. (c–d) Blasts from 15 patients with AML and normal bone marrow mononuclear cells were treated with 80 µM DIM for 24 hours, apoptosis was determined by annexin V/PI staining and flow cytometry.
The effects of DIM on apoptosis in primary leukemia cells from 15 AML patients (four of those patients are M2, five are M4, and six are M5 according to FAB classification system) were investigated. As shown in Fig. 2c, exposure of AML cells to DIM resulted in pronounced increase in apoptosis. Parallel study was also performed with normal bone marrow mononuclear cells. The DIM regimen induced relatively little apoptosis in normal bone marrow mononuclear cells (Fig. 2d). Together, these findings indicate that DIM selectively kills human leukemia cell lines and primary leukemia cells but not normal hematopoietic cells.
DIM-induced apoptosis is associated with the caspase-independent inactivation of Akt and activation of JNK
To determine whether any of these perturbations mediated by DIM were secondary to caspase-mediated processes, U937 cells were treated with 80 μM DIM in the presence of the broad-spectrum caspase inhibitor Z-VAD-FMK at 20 μM. Addition of Z-VAD-FMK, which abrogated DIM-induced apoptosis, PARP degradation, and caspases activation (Fig. 3a and 3b), failed to protect cells from DIM-mediated down-regulation of Mcl-1 and up-regulation of p21 (Fig. 3c). Similarly, Z-VAD-FMK did not attenuate DIM-mediated Akt inactivation and JNK activation (Fig. 3d). Together, these findings suggest that DIM-mediated Akt inactivation, JNK activation, Mcl-1 down-regulation, and p21 up-regulation do not simply reflect activation of the caspase cascade.
10.1371/journal.pone.0031783.g003Figure 3 Effects of inhibition of caspases by Z-VAD-FMK on apoptosis, expression of Mcl-1 and p21, and phosphorylation of Akt and JNK.
U937 cells were pretreated with the caspase inhibitor Z-VAD-FMK (20 µM) for 1 h, followed by treatment with 80 µM DIM for 12 h and 24 h. (a) Cells were stained with Annexin V/PI, and apoptosis was determined using flow cytometry. **Values for cells treated with DIM and Z-VAD-FMK were significantly reduced compared to values obtained for DIM alone by Student's t-test, p<0.01. (b–d) Total protein extracts were prepared and subjected to Western blot assay using antibodies as indicated.
Akt inactivation plays an important functional role in DIM-mediated apoptosis
To understand the functional role of Akt inactivation in DIM-induced apoptosis, U937 cells were pretreated with PI3K inhibitor LY294002 for 1 h, followed by treatment with DIM for 24 h, and apoptosis was monitored. As shown in Fig. 4a, co-administration of a nontoxic concentration of LY294002 (i.e. 20 μM) with a modestly toxic concentration of DIM (40 μM, ∼25% apoptosis at 24 h) resulted a pronounced increase in apoptosis (∼60% at 24 h). Western blot analysis demonstrated that co-administration of DIM and LY294002 resulted in pronounced increase in caspases activation and PARP degradation (Fig. 4b). Combined treatment with DIM and LY294002 also resulted in potentiation of Mcl-1 down-regulation, Akt inactivation and pronounced increase in JNK activation (Fig. 4c). Together, these findings suggest that inactivation of Akt plays a critical role in DIM-induced apoptosis in human leukemia cells.
10.1371/journal.pone.0031783.g004Figure 4 Effects of PI3K inhibitor, LY294002 (LY) and genetic activation of Akt on apoptosis induced by DIM.
U937 cells were pretreated with 20 µM of LY for 1 h, followed by the addition of 40 µM of DIM for 24 h. (a) Cells were stained with Annexin V/PI, and apoptosis was determined using flow cytometry. **Values for cells treated with DIM and LY were significantly greater than those for cells treated with DIM alone by Student's t-test; p<0.01. (b–c) Total cellular extracts were prepared and subjected to Western blot analysis using antibodies as indicated. (d) U937 cells were stably transfected with an empty vector (pcDNA3.1), Akt-CA, and Akt-DN. Cells were treated with 80 µM of DIM for 24 h, after which apoptosis was analysed using Annexin V/PI assay. **Values for Akt-CA cells treated with DIM were significantly decreased compared to those for pcDNA3.1 cells by Student's t-test, p<0.01. (e–f) Total cellular extracts were prepared and subjected to Western blot analysis using antibodies as indicated.
To further assess the functional significance of Akt inactivation in DIM-mediated lethality, U937 cells ectopically expressing Akt-CA and Akt-DN were employed. As shown in Fig. 4d, Akt-CA cells were markedly less sensitive to DIM-induced apoptosis than pcDNA3.1 vector control cells (p<0.01). However, DIM-induced apoptosis in Akt-DN cells is similar to that in pcDNA3.1 control cells. Consistent with these findings, DIM was considerably less effective in triggering caspases activation and PARP degradation in Akt-CA cells compared to pcDNA3.1 vector control cells and Akt-DN cells (Fig. 4e). In addition, enforced activation of Akt effectively blocked DIM-mediated Mcl-1 down-regulation (Fig. 4f). Western blot analysis displayed marked increase in levels of total and phospho-Akt in Akt-CA cells. DIM failed to induce inactivation of Akt in these Akt-CA cells (Fig. 4f). Interestingly, the ability of DIM to induce JNK activation was essentially abrogated in Akt-CA cells (Fig. 4f), indicating that activation of JNK mediated by DIM depends upon inactivation of Akt.
JNK activation plays an important functional role in DIM-mediated apoptosis
To understand the functional significance of JNK activation in DIM-induced apoptosis, both pharmacologic and genetic approaches were employed. Pretreatment with JNK inhibitor SP600125 (i.e. 10 μM) essentially abrogated DIM-induced apoptosis (Fig. 5a), caspases activation, and PARP degradation (Fig. 5b). Co-administration of SP600125 also blocked Mcl-1 down-regulation and effectively diminished phosphorylation of JNK mediated by DIM (Fig. 5c). To further confirm the functional role of JNK in DIM-induced apoptosis, a genetic approach utilizing JNK1 siRNA was employed. As shown in Fig. 5d, transient transfection of U937 cells with JNK1 siRNA reduced expression of JNK1, and resulted in a significant reduction in DIM-mediated apoptosis. Collectively, these findings indicate that DIM-induced JNK activation plays an important functional role in DIM-related lethality.
10.1371/journal.pone.0031783.g005Figure 5 Inhibition of JNK significantly protect cells from DIM-induced apoptosis.
U937 cells were pretreated with 10 µM of JNK inhibitor, SP600125 (SP), for 1 h, followed by the addition of 80 µM of DIM for 24 h. (a) Cells were stained with Annexin V/PI, and apoptosis was determined using flow cytometry. **Values for cells treated with DIM and SP were significantly less than those obtained for cells treated with DIM alone by Student's t-test, p<0.01. (b–c) Total cellular extracts were prepared and subjected to Western blot assay using antibodies as indicated. (d) U937 cells were transfected with JNK1 siRNA oligonucleotides or controls and incubated for 24 h at 37°C, after which cells were treated with 80 µM of DIM for 24 h. Apoptosis was determined using the Annexin V/PI assay. **Values for cells treated with DIM after transfection with JNK1 siRNA were significantly decreased compared to those for control cells treated with DIM by Student's t-test; p<0.01. Total cellular extracts were prepared and subjected to Western blot analysis using antibodies against JNK1.
DIM inhibits growth of a U937 tumor xenograft in immunodeficient Mice
Finally, we evaluated whether our in vitro observations could be translated into an animal model system in vivo. To address this issue, NOD/SCID mice were inoculated in the flank with U937 human leukemia cells, after which mice were treated daily intraperitoneally with either vehicle or DIM (50 mg/kg) for 20 days and tumors were measured. As shown in Fig. 6a, treatment with DIM resulted in a dramatic suppression of tumor growth of U937 xenograft (p<0.01 compared with vehicle control), whereas 20 days following drug exposure no statistically significant change in body weight was noted comparing vehicle control (Fig. 6b), suggesting that no severe toxicity was observed. Tumors exposed to DIM compared with vehicle control exhibited a reduced number of cells per field under H&E staining, with signs of necrosis with infiltration of inflammatory cells (i.e. phagocytic cells), fibrosis, as well as apoptotic regions (Fig. 6c top panels). Moreover, treatment with DIM resulted in a striking induction of apoptosis in tumor cells determined by TUNEL analysis (Fig. 6c middle panels). Exposure to DIM also caused marked increase in immunoreactivity for the cleaved caspase-3 (Fig. 6c bottom panels).
10.1371/journal.pone.0031783.g006Figure 6
In vivo antileukemic activity of DIM in U937 xenografts.
20 NOD/SCID mice were inoculated with U937 cells (2×106 cells/mouse, i.p.) and randomly divided into two groups (10/group) for treatment with DIM (50 mg/kg, i.p., daily, five times per week) or with vehicle control solvent. (a) Average tumor volume in vehicle control mice and mice treated with 50 mg/kg DIM. P<0.01, significantly different compared with vehicle control by Student's t-test. (b) body weight changes of mice during the 20 days of study. (c) At the 20 days after DIM treatment, the tumors were excised and subjected to H&E staining for determination of pathological evaluation, TUNEL assay for determination of apoptosis, and immunohistochemical staining to determine Cleavage-caspase-3 immunoreactivity. Original magnification ×400. (d) After treatment with DIM, tumor tissues were sectioned and subjected to immunohistochemistry using anbibodies as indicated.
Lastly, we performed the immunohistochemistry analysis to evaluate the expression of Mcl-1, phospho-Akt, and phospho-JNK in tissue sections of U937 xenografts. As expected, tumors from vehicle-treated control mice stained strongly for Mcl-1 and phospho-Akt, which were immunolocalized to the cytoplasm of cancer cells. Treatment with DIM resulted in markedly decrease in expression of Mcl-1 (Fig. 6d top panels) and phospho-Akt in tissue sections of tumors (Fig. 6d middle panels). Furthermore, immunostaining of tumors from mice treated with DIM showed marked increase in phospho-JNK (Fig. 6d bottom panels). Collectively, these findings demonstrated that DIM administration significantly inhibited U937 xenograft growth without causing any side effects to the mice. DIM-mediated antileukemic activity in vivo is associated with inactivation of Akt and activation of JNK.
Discussion
The present study shows that exposure of U937 human leukemia cells resulted in increase in apoptosis in dose- and time-dependent manners. In addition, our results provide mechanistic information, for the first time, how DIM exerts its proapoptotic effects on human leukemia cells (i.e. by inhibiting phosphorylation of Akt and expression of Mcl-1, and by inducing phosphorylation of JNK and the expression of p21). Experimental studies have revealed that DIM induces apoptosis in a variety of cancer cells including breast and prostate cancer cells through different cell signaling pathways including NF-κB, Akt, MAPK, p53, AR, and ER pathways [22], [24], [25]. Presently, no information is available concerning the functional roles of the Akt and JNK pathways in mediating in DIM-induced lethality, particularly in malignant hematopoietic cells. The results of the present study demonstrate that Akt inactivation and JNK activation play key functional contributions in caspase activation and subsequent lethality.
Akt is a serine–threonine kinase, which has been shown to be activated in various cancers [26]. Akt plays a critical role in cell survival through multiple downstream targets [27], [28]. Akt is activated by PI3K, which in turn is regulated by the PTEN phosphatase [29], mutations of which are among the most commonly encountered in human cancers [30]. Akt activation generally involves PTEN inactivation [31], and results in attenuation of lethality [32]. The present findings show that DIM exposure resulted in diminished Akt phosphorylation. While it would be tempting to attribute this phenomenon to PTEN activation, the fact that U937 cells do not express wild-type PTEN argues against this notion [33]. A more likely possibility is that DIM, through a mechanism not yet elucidated blocks the actions of PI3K. The finding that LY294002, an inhibitor of PI3K, enhanced DIM-mediated inhibition of Akt and lethality is potentially consistent with this hypothesis. It should also be noted that various stimuli induce apoptosis by different mechanisms linked between Akt inactivation and caspase activation. The caspase-dependent down-regulation of Akt is a well-described positive feedback regulation. For example, previous study demonstrated that 4-Hydroxynonenal (HNE)-mediated Akt inactivation was caspase-3-dependent [34]. However, our previous results indicate that inactivation of Akt by 2-methoxyestradiol does not simply represent a secondary, caspase-dependent event [35]. In this study, co-treatment of cells with the caspase inhibitor Z-VAD-FMK failed to prevent Akt inactivation. Such findings indicate that inactivation of Akt by DIM does not simply represent a secondary caspase- dependent event.
A number of evidence suggests that in human leukemia cells, DIM-induced Akt inactivation plays a critical role in DIM-mediated lethality. First, co-administration of LY294002 significantly enhanced DIM-mediated caspases activation and apoptosis. Second, enforced activation of Akt largely reversed the lethal consequences of DIM. Particularly, DIM exposure resulted in down-regulation of Mcl-1, an anti-apoptotic protein that may play an important role in regulating apoptosis in malignant hematopoietic cells [36]. Mcl-1 has a short half-life and is a highly regulated protein, induced by a wide range of survival signals and rapidly down regulated during apoptosis [37]. Although the precise mechanism underlying the Mcl-1 reduction upon DIM treatment remains to be elucidated, interventions disabling Mcl-1 may be an optimal way to kill leukemia cells. It has been reported that the anti-apoptotic gene Mcl-1 is up-regulated by the PI3K/Akt signaling pathway [38], and down-regulation of Mcl-1 by inhibition of PI3K/Akt pathway is required for cell death [39]. The finding that enforced activation of Akt largely blocked DIM-mediated down-regulation of Mcl-1 may significantly contribute to DIM-mediated lethality. However, additional mechanistic studies are required to demonstrate the causative role of Mcl-1 in DIM induced lethality in leukemia.
Induction of apoptosis was also associated with activation of JNK pathway. Engagement of the SEK/JNK pathway has been shown to play a key functional role in the lethal effects of diverse cytotoxic stimuli, including inflammation and oxidative stress [40]. In fact, the net balance between cytoprotective (e.g. ERK) and stress-related (e.g. JNK) signaling may play a critical role in cell survival and death decisions [41]. The finding that pharmacologic and genetic interruption of the JNK pathway attenuated DIM-mediated lethality indicates that stress pathway play a critical role in apoptosis induction by this agent. The mechanism by which oxidative stress triggers JNK activation is not known with certainty, but may involve release from GSH-mediated inhibitory effects, or alternatively, perturbations in thioredoxin, leading to activation of ASK-1 (apoptosis signal-regulating kinase-1), of which JNK is a downstream target [42], [43]. Interestingly, ectopic expression of Akt not only blocked DIM-mediated caspases activation and apoptosis but also prevented the striking increase in JNK activation, raising the possibility that one of the mechanisms by which Akt protects cells from DIM lethality is by opposing JNK activation. Evidence that the Akt-mediated inhibition of SEK1 and/or ASK1 may act as a negative regulation of the JNK pathway through Akt-dependent induction of specific JNK-interacting protein (JIP) provides a possible explanation for this phenomenon [44]. Lastly, the observation that pharmacologic or genetic interruption of the JNK pathway attenuated DIM-mediated caspases activation and lethality demonstrates an important functional role for this stress pathway in triggering the cell death program. Such findings are compatible with previous reports suggesting a direct role for JNK activation in promoting cytochrome c release from the mitochondria [45].
Our present study has revealed that DIM causes up-regulation of Cip/p21 expression in human leukemia cells. p21 protein is an inhibitor of cyclin-dependent kinase (CDK) and plays an important role in regulating CDK activity and cell cycle progression in response to a wide variety of stimuli [46]. In addition to normal cell cycle progression, p21 has been postulated to participate in growth suppression and apoptosis through a p53-dependent or –independent pathway following stress and induction of p21 may cause cell cycle arrest [47], [48]. In a recent study, DIM has been shown to inhibit cell growth and induce G1 phase cell cycle arrest in human breast cancer cells through Cip1/p21 up-regulation [21]. Consistent with this result, DIM-mediated apoptosis in human leukemia cells may be associated with Cip/p21 up-regulation and cell cycle arrest. Additional mechanistic studies, however, are required in future to elucidate how Cip1/p21 plays a role in DIM-induced apoptosis in human leukemia cells.
Our study reported that DIM induces apoptosis in diverse human leukemia cell lines (i.e. U937, Jurkat, and HL-60) and primary human AML blast cells, and cell-signaling pathways including Akt inactivation and JNK activation may be involved in these events. However, it is still unclear whether the underlying mechanism of apoptosis in vitro is identical to those in vivo. Our present results showed that treatment with DIM could inhibit tumor growth in U937 tumor xenograft model that could be mechanistically linked with inactivation of Akt and activation of JNK. These results further confirmed that inactivation of Akt and activation of JNK could play an important role in DIM-induced apoptosis in vivo.
In summary, the present study has provided evidence that DIM induces human leukemia cell death with caspases activation and PARP cleavage, and that DIM-induced apoptosis proceeds via inactivation of Akt, activation of JNK, and down-regulation of Mcl-1. The data presented here suggest that Akt and JNK signaling pathways may represent attractive targets for DIM-induced apoptosis in human leukemia cells in vitro and in vivo. The results of the present study could have important implications for the incorporation of agents such as DIM into the chemopreventive or therapeutic intervention against leukemia and possibly other hematologic malignancies.
Materials and Methods
Chemicals and reagents
DIM was purchased from Sigma (St Louis, MO). LY294002, SP600125, and Z-VAD-FMK were from EMD Biosciences (La Jolla, CA). Antibodies against Akt, phospho-JNK, JNK, and β-actin were from Santa Cruz (Santa Cruz, CA). Cleaved-caspase-3, Cleaved-caspase-7, Cleaved-capase-9, and phospho-Akt (Ser473) were from Cell Signaling (Beverly, MA). Mcl-1 and p21 were from PharMingen (San Diego, CA). PARP was from Biomol (Plymouth Meeting, PA). Caspase-8 was from Alexis (Carlsbad, CA).
Cell culture and transfection
U937, HL-60, and Jurkat cells were provided by American Type Culture Collection (ATCC, Manassas, VA) and maintained in RPMI 1640 medium containing 10% fetal bovine serum (FBS). The constitutive active form of Akt (Akt-CA) and the dominant negative Akt mutant (Akt-DN) were kindly provided by Dr. Richard Roth (Stanford University, School of Medicine, Stanford, CA), and were subcloned into the pcDNA3.1. U937 cells were stably transfected with Akt-CA and Akt-DN using the Amaxa nucleofector™ (Koeln, Germany) as recommended by the manufacturer. Stable single cell clones were selected in the presence of 400 μg/ml of geneticin. Thereafter, the expression of Akt from each cell clone was analyzed by Western blot analysis.
Peripheral-blood samples for the in vitro studies were obtained from 15 patients with newly diagnosed or recurrent acute myeloid leukemia (AML) after informed consent. The percentage of AML cells from each patient were greater than 20%. According to French-American-British (FAB) classification system, 4 patients are M2, 5 patients are M4, and 6 patients are M5. Approval was obtained from the Southwest Hospital (Chongqing, China) institutional review board for these studies. AML blasts were isolated by density gradient centrifugation over Histopaque-1077 (Sigma Diagnostics, St Louis, MO) at 400 g for 30 minutes. Isolated mononuclear cells were washed and assayed for total number and viability using trypan blue exclusion. Cells were suspended at 8×105/mL for treatment. Fresh normal bone marrow mononuclear cells were purchased from Allcells (Emeryvill, CA). After washing and enumerating, cells were suspended at 8×105/mL prior to treatment.
RNA interference and transfection
U937cells (1.5×106) were transfected with 1 μg JNK1-annealed dsRNAi oligonucleotide 5′-CGUGGGAUUUAUGGUCUGUGTT-3′/3′-TTGCACCUAAAUACCAGACAC-5′ (Orbigen, San Diego, CA, USA) using the Amaxa nucleofectort (Koeln, Germany) as recommended by the manufacturer. After incubation at 37°C for 24 h, transfected cells were treated with DIM, and subjected to determination of apoptosis and JNK1 expression using Annexin V/PI and flow cytometry and Western blot analysis.
Assessment of apoptosis
The extent of apoptosis in leukemia cells was evaluated by flow cytometric analysis using FITC conjugated Annexin V/propidium iodide (BD PharMingen, San Diego, CA) staining according to the manufacturer's instructions. Both early apoptotic (Annexin V-positive, PI-negative) and late apoptotic (Annexin V-positive and PI-positive) cells were included in cell death determinations.
Western blot analysis
The total cellular samples were washed twice with ice-cold PBS and lysed in 1× NuPAGE LDS sample buffer supplemented with 50 mM dithiothreitol. The protein concentration was determined using Coomassie Protein Assay Reagent (Pierce, Rockford, IL). 30 μg of sample proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane. Membranes were blocked with 5% fat-free dry milk in 1× Tris-buffered saline (TBS) and incubated with antibodies. Protein bands were detected by incubating with horseradish peroxidase-conjugated antibodies (Kirkegaard and Perry Laboratories, Gaithersburg, MD) and visualized with enhanced chemiluminescence reagent (Perkin Elmer, Boston, MA).
Xenograft assay
NOD/SCID (severe combined immunedeficient) mice (5 weeks old) were purchased from Vital River Laboratories (VRL, Beijing, China). All animal studies were conducted according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Third Military Medical University (Approval ID: 2009020014). U937 cells (2×106/0.2 mL/mouse) were suspended in sterile PBS and injected s.c. into the right flank of the mice. Mice were randomized into two groups (10 mice/group). Three days after tumor inoculation, the treatment group received DIM (50 mg/kg, i.p., five times per week). The control group received an equal volume of solvent control. Tumor size and body weight were measured after treatment at various time intervals throughout the study. At the termination of the experiment, mice were sacrificed at 24 h after the last administration. The tumors were excised and weighed. Tumors were collected at selected times and fixed in paraformaldehyde. Paraffin-embedded tissues were sectioned and processed for TUNEL staining and immunohistochemical staining.
TUNEL assay
The apoptotic cells in tissue samples were detected using an In Situ Cell Death Detection kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's manual. After deparaffinization and permeabilization, the tissue sections were incubated in proteinase K for 15 min at room temperature. The sections were then incubated with the TUNEL reaction mixture that contains terminal deoxynucleotidyl transferase (TdT) and fluorescein-dUTP at 37°C for 1 h. After washing three times with PBS, the sections were incubated with the Converter-POD which contains anti-fluorescein antibody conjugated with horse-radish peroxidase (POD) at room temperature for 30 minutes. After washing three times with PBS, the sections were incubated with 0.05% 3-3′-diaminobenzidine tetrahydrochloride (DAB) and analyzed under light microscope.
Histological and immunohistochemical evaluation
At the termination of experiments, tumor tissues from representative mice were sectioned, embedded in paraffin, and stained with hematoxylin and eosin for histopathologic evaluation. For immunohistochemical analysis, tissue sections 4 µm in thickness were dewaxed and rehydrated in xylene and graded alcohols. Antigen retrieval was performed with 0.01 M citrate buffer at pH 6.0 for 20 min in a 95°C water bath. Slides were allowed to cool for another 20 min, followed by sequential rinsing in PBS and TBS-T buffer. Endogenous peroxidase activity was quenched by incubation in TBS-T containing 3% hydrogen peroxide. After washing in TBS-T for three times and blocking with 10% goat serum for 1 h, sections were incubated with primary antibodies, washed three times in PBS, incubated with biotinylated secondary antibody for 1 h, followed by incubation with a streptavidin-peroxidase complex for another 1 h. After three additional washes in PBS, diaminobenzidine working solution was applied. Finally, the slides were counterstained in hematoxylin.
Statistical analysis
Tumor volumes, body weights, and percentage of apoptotic cells were represented as mean ± SD. The statistical significance of the difference between control and UA-treated groups was evaluated using Student's t test. p<0.05 or p<0.01 were considered significant.
Competing Interests: The authors have declared that no competing interests exist.
Funding: The study was supported by NIH Grants RO1 ES015375 (X.Shi), 1R21ES019249 (Zhuo Zhang) and National Natural Science Foundation of China (No. 30971288). The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.
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Mol PainMolecular Pain1744-8069BioMed Central 1744-8069-8-32223646110.1186/1744-8069-8-3ResearchComparison of central versus peripheral delivery of pregabalin in neuropathic pain states Martinez Jose A [email protected] Manami [email protected] Alma [email protected] Leah R [email protected] William H [email protected] Cory C [email protected] Department of Clinical Neurosciences and the University of Calgary, Calgary, AB, Canada2 Alzheimer's Research Center, Regions Hospital, and HealthPartners Research Foundation, St. Paul, MN, USA3 Department of Pharmaceutics, University of Minnesota, Minneapolis, MN, USA2012 11 1 2012 8 3 3 27 5 2011 11 1 2012 Copyright ©2012 Martinez et al; licensee BioMed Central Ltd.2012Martinez et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
Although pregabalin therapy is beneficial for neuropathic pain (NeP) by targeting the CaVα2δ-1 subunit, its site of action is uncertain. Direct targeting of the central nervous system may be beneficial for the avoidance of systemic side effects.
Results
We used intranasal, intrathecal, and near-nerve chamber forms of delivery of varying concentrations of pregabalin or saline delivered over 14 days in rat models of experimental diabetic peripheral neuropathy and spinal nerve ligation. As well, radiolabelled pregabalin was administered to determine localization with different deliveries. We evaluated tactile allodynia and thermal hyperalgesia at multiple time points, and then analyzed harvested nervous system tissues for molecular and immunohistochemical changes in CaVα2δ-1 protein expression. Both intrathecal and intranasal pregabalin administration at high concentrations relieved NeP behaviors, while near-nerve pregabalin delivery had no effect. NeP was associated with upregulation of CACNA2D1 mRNA and CaVα2δ-1 protein within peripheral nerve, dorsal root ganglia (DRG), and dorsal spinal cord, but not brain. Pregabalin's effect was limited to suppression of CaVα2δ-1 protein (but not CACNA2D1 mRNA) expression at the spinal dorsal horn in neuropathic pain states. Dorsal root ligation prevented CaVα2δ-1 protein trafficking anterograde from the dorsal root ganglia to the dorsal horn after neuropathic pain initiation.
Conclusions
Either intranasal or intrathecal pregabalin relieves neuropathic pain behaviours, perhaps due to pregabalin's effect upon anterograde CaVα2δ-1 protein trafficking from the DRG to the dorsal horn. Intranasal delivery of agents such as pregabalin may be an attractive alternative to systemic therapy for management of neuropathic pain states.
neuropathic painpregabalindiabetic peripheral neuropathyspinal nerve ligation
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Background
Neuropathic pain is a consequence of nerve damage or disease in the central and/or peripheral nervous system such as with diabetes and trauma. The clinical presentation of neuropathic pain includes hyperalgesia, allodynia, and spontaneous pain [1]. Its high prevalence in humans [2-4] has led to the development of a number of animal models of neuropathic pain, including diabetic peripheral neuropathy and spinal nerve ligation.
Changes within the nervous system associated with neuropathic pain include critical upregulation of the calcium channel subunit CaVα2δ-1 [5-7], particularly at the dorsal horn [8,9]. This is of importance for the clinical utility and potential mechanism of two different pharmacotherapies, gabapentin and pregabalin, both of which are indicated for the management of neuropathic pain due to multiple etiologies. There is uncertainty about the anatomical location of pregabalin's beneficial effect, as CaVα2δ-1 subunit upregulation is multifocal [10]. In addition to the known expression within the DRG, CACNA2D1 mRNA is also localized to brain regions known to be involved in cortical processing, sensory conduction, and arousal [11]. Also, there is also marked CaVα2δ-1 subunit expression in spinal cord regions where DRG projections occur. As a result, the localization of pregabalin's greatest therapeutic effect is uncertain. In addition to changes in CaVα2δ-1, other changes in voltage gated channels [12-14] and microgliosis with associated elevation in cytokines may occur [15-18].
Pregabalin, as with gabapentin, interacts specifically with the CaVα2δ-1 subunit of voltage-gated calcium channels [19-21] providing an antihyperalgesic and antiallodynic effect specific for its action at the CaVα2δ-1 subunit [22]. Recently, Bauer et al [23] demonstrated the importance of trafficking of the CaVα2δ-1 subunit from the dorsal root ganglia to the dorsal horn in the development of neuropathic pain, with its subsequent alleviation with pregabalin treatment. In the present study, we attempted to determine the central and peripheral contributions of pregabalin for relief from neuropathic pain in two separate models: streptozotocin-induced diabetic peripheral neuropathy and traumatic injury (spinal nerve ligation). We also examined the therapeutic potential for intranasal delivery of pregabalin with comparison to more localized delivery systems using implantable pump systems permitting a continuous delivery of pregabalin to specific anatomical locations [10,24]. Intranasal delivery, first developed to bypass the blood-brain-barrier and directly target therapeutic agents to the central nervous system [25-28] of rodents [29-31] and humans [30,31], occurs along both the olfactory and trigeminal neural pathways using extracellular pathways rather than axonal transport [30]. Such directed targeting of pregabalin to the brain can avoid gastrointestinal uptake of oral therapy and may permit more potent relief of neuropathic pain behavior while limiting systemic side effects. For neuroinflammatory, neurodegenerative, and neurovascular disorders [28], intranasal delivery is an attractive non-invasive method to target molecules to the central nervous system [32,33] and even the peripheral nervous system [34].
Our primary objective was to study the central delivery of pregabalin using either intranasal or intrathecal therapy and compare to near-nerve delivery for impact upon neuropathic pain behaviors. Previously, intranasal delivery targeting the central nervous system has been used as a method of delivery for neuropathic pain states [34-36]. Intranasal delivery is dependent upon the olfactory and trigeminal nerves connecting the nose to the brain, the rostral migratory stream, and less so upon vasculature and lymphatic pathways [28,37]. The benefit of intranasal delivery is the concentration of effect in the central nervous system; systemic delivery to some extent will occur with small, lipophilic molecules delivered intranasally but is otherwise quite limited [28]. A small, lipophilic molecule such as pregabalin would be anticipated to have greater systemic distribution following intranasal delivery than other compounds [38]. Rarely used in human clinical management at present [35], the non-invasive nature of intranasal delivery along with limited systemic exposure make it potentially attractive for use in the clinic with human patients. Intrathecal delivery of compounds with various mechanisms [10,39,40] has also led to amelioration of neuropathic pain states. Already used in the clinic for delivery of opioids, baclofen and conotoxins, intrathecal delivery is an invasive technique used in specific situations of refractoriness or intolerance of oral delivery, but its use depends upon the agent used, its site of action, and its ability to cross the blood-brain barrier. Finally, near nerve delivery has also been used to modulate sensory nerve regeneration and impact upon pain behaviors [24,41,42]. Our secondary objectives of this study were to evaluate changes in expression of the CaVα2δ-1 subunit in the dorsal spinal cord, DRG peripheral nerve and brain, evaluate potential changes in other voltage-gated calcium and sodium channels in the spinal cord, DRG and nerve, evaluate for changes in accumulation of microglia, and to evaluate the safety and tolerability of pregabalin using the various delivery routes in rodents, including the use of intranasal delivery. We also examined the previously demonstrated role of CaVα2δ-1 subunit trafficking [23] in a dynamic and progressive model of neuropathic pain, diabetic peripheral neuropathy. The determination of impact of pregabalin upon the CaVα2δ-1 subunit within nervous system tissues depending upon the mode of delivery would assist in the understanding of the pharmacological action of pregabalin upon the CaVα2δ-1 subunit and its effects upon pathways relevant in the development of neuropathic pain.
Results
Radiolabelled Detection of Pregabalin Localization
At 73 hours of intrathecal or intranasal delivery, pregabalin concentrations were detected throughout the nervous system (Figure 1). Intranasal delivery gave consistently higher elevated pregabalin levels throughout the central nervous system structures at and above the cephalad portion of the cervical spinal cord. In contrast, intrathecal delivery was associated with higher pregabalin penetration into the lumbar spinal cord and much higher levels within lumbar dorsal root ganglia. Systemic presence of pregabalin was much higher at 73 hours with intranasal pregabalin delivery, but was similar to concentrations achieved with intrathecal delivery at 74 and 77 hours.
Figure 1 Radiolabelled pregabalin detection. The harvested tissues are displayed on the X axis for the systemic (A, D) and for nervous system tissues (B, C, E, F). Systemic (A) or nervous system (B, C) pregabalin concentrations are demonstrated for 73-74 hours after intrathecal (white bars) or intranasal (black bars) or near nerve (gray bars) pregabalin delivery. Note that the 73 and 74 hour timepoints were analyzed together for these timepoints early after completion of 72 hours of delivery. At 77 hours, at 5 hours after the most recent intranasal delivery, systemic (D), brain (E), and cord (F) pregabalin concentrations started to fall. Near nerve delivery led to sciatic nerve pregabalin concentrations several fold higher than achieved with either intrathecal or intranasal delivery, but near nerve delivery was not associated with detectable pregabalin elsewhere. Significant differences between concentrations achieved with intervention methods were determined by matched ANOVA testing, with * indicating significant differences (p < 0.05) between the intranasal or intrathecal pregabalin delivery and saline delivery techniques for each tissue and respective delivery method. The doses provided through either intranasal or intrathecal delivery were selected to be similar to doses of pregabalin used in later subexperiments [n = 4-6 rats in each rat cohort for each time point]. Note that data are not shown for locations other than the sciatic nerve for near nerve pregabalin delivery, as other locations had no measureable pregabalin found.
After 74 hours of chronic delivery, concentrations with intranasally delivered pregabalin began to fall while intrathecal levels were remaining stable throughout the nervous system and systemically (Additional File 1). At 77 hours after intranasal delivery, both systemic and nervous system levels of intranasally delivered pregabalin began to fall while intrathecal dosing provided relatively stable pregabalin concentrations.
Near nerve delivery of pregabalin only led to detection at the proximal sciatic nerve, with zero detection of meaningful radioactive pregabalin at other systemic or nervous system sites. The concentrations of pregabalin received at the proximal sciatic nerve were several fold higher with near nerve delivery than could be achieved with intrathecal or intranasal delivery (Figure 1).
Maintenance of Diabetes in the Diabetic Peripheral Neuropathy Model
After streptozotocin injection, rats developed diabetes within 1 week in greater than 80% of animals, and in each case, diabetes was maintained over the length of the study. Diabetic rats were smaller than non-diabetic rats at endpoint (201.8 ± 14.7 grams as compared to 234.5 ± 15.2 grams). Diabetic rat glycemia was increased as compared to non-diabetic rats at endpoint (26.4 ± 3.1 mmol/L as compared to 6.1 ± 2.2 mmol/L).
Neuropathic Pain Behavior
Despite different methods and dosing of pregabalin delivery, there was no impact upon locomotor functioning in rat cohorts studied with Rotarod testing at 72-74 hours post-pregabalin delivery (data not shown). Diabetic rats and rats subjected to spinal nerve ligation both developed tactile allodynia and thermal hyperalgesia during experimentation (Figure 2). Both intrathecal and intranasal pregabalin provision led to improvement in tactile allodynia and thermal hyperalgesia at high doses (2.04 mg/kg/d) in both diabetic peripheral neuropathy and spinal nerve ligation models. Medium (0.51 mg/kg/d) doses of pregabalin decreased thermal hyperalgesia due to diabetic peripheral neuropathy and spinal nerve ligation without impact upon tactile allodynia. Lower doses (0.051 mg/kg/d) of intrathecal or intranasal pregabalin had no impact upon either tactile allodynia or thermal hyperalgesia in either model. Near-nerve delivery of pregabalin had no impact upon tactile allodynia or thermal hyperalgesia with any dose (data not shown) in either model of neuropathic pain.
Figure 2 Behavioral testing results for both models of neuropathic pain. Tactile (A) and thermal (B) sensory testing data for rats with or without (control) diabetes and diabetic peripheral neuropathy (DPN) receiving intranasal pregabalin are presented for multiple doses of pregabalin or saline. Intrathecal pregabalin delivery was also followed by measurements of tactile allodynia (C) and thermal hyperalgesia in diabetic rats (D). High doses of either intranasal or intrathecal pregabalin impacted upon these neuropathic pain behaviours in DPN. For rats with or without spinal nerve ligation receiving intranasal pregabalin, tactile (E) and thermal (F) sensory testing data are presented; intranasal pregabalin at high dose impacted upon both measures of neuropathic pain behaviour. Finally, intrathecal pregabalin also impacted upon both tactile allodynia (G) and thermal hyperalgesia (H) due to spinal nerve ligation when compared to saline delivery. Significant differences were detected between the diabetic rats receiving high dose (2.04 mg/kg/d) (*) and medium dose (0.51 mg/kg/d) (θ) pregabalin as compared to diabetic rats receiving saline delivery (non-matched ANOVA tests, F-values range between 1.98-22.86 for indicated groups and time points, n ≥ 5, p < 0.0125 after Bonferroni correction). Significant differences were detected between rats with spinal nerve ligation receiving high dose (2.04 mg/kg/d) (*) and medium dose (0.51 mg/kg/d) (θ) pregabalin as compared to rats with spinal nerve ligation receiving saline delivery (non-matched ANOVA tests, F-values range between 2.25-8.11 for indicated groups and time points, n ≥ 5, p < 0.0125 after Bonferroni correction) [n = 5-6 rats in each cohort for each time point]. Data for rats receiving low dose pregabalin were similar to data for rats receiving saline and are not shown.
mRNA Expression for CACNA2D1 and Other Voltage Gated Channels in Diabetic Peripheral Neuropathy
Both diabetic peripheral neuropathy and spinal nerve ligation were associated with elevation of CACNA2D1 mRNA expression within both the DRG and dorsal spinal cord (Figure 3). However, there was no observed impact of pregabalin delivery upon CACNA2D1 mRNA expression within either location when compared to saline delivery in either model.
Figure 3 Expression of CACNA2D1 mRNA. Expression of CACNA2D1 mRNA was upregulated in both dorsal spinal cord (A) and lumbar DRGs (B) in the presence of either diabetic peripheral neuropathy (DPN) or spinal nerve ligation (SNL). Dorsal horn mRNA expression was unchanged by presence of high dose pregabalin (2.04 mg/kg/d) provided either through intrathecal or intranasal delivery when compared to saline delivery (A). There was no impact of pregabalin delivered in any manner upon mRNA expression in lumbar DRGs when compared to saline delivery, although the presence of spinal nerve ligation or diabetic peripheral neuropathy was associated with elevated CACNA2D1 mRNA expression when compared to control rat specimens (B), indicated by the * above the Control rat data (non-matched ANOVA tests, F-values range between 0.42-1.00 for indicated groups and time points, n ≥ 3, * p < 0.0125) [n = 6-8 specimens for each cohort].
Protein Expression for α2δ-1 and other Voltage Gated Channels
Immunohistochemical detection of CaVα2δ-1 protein in nerves exposed to diabetes or spinal nerve ligation demonstrated an increased number of CaVα2δ-1 positive profiles near the site of spinal nerve ligation, but without impact of pregabalin delivery, irrespective of site of delivery (Additional File 2). The presence of diabetes or the provision of spinal nerve ligation was also associated with a shift to a greater number of CaVα2δ-1 positive DRG neurons within the DRG, but again there was no impact with delivery of pregabalin regardless of method of delivery or form of neuropathic pain (Additional File 2). Both diabetes and spinal nerve ligation led to greater expression of CaVα2δ-1 protein at the dorsal horn; at this location, however, receipt of high dose pregabalin via intrathecal or intranasal delivery was associated with relatively diminished expression of CaVα2δ-1 protein (Figure 4) at the dorsal horn.
Figure 4 CaVα2δ-1 protein expression. CaVα2δ-1 protein expression was detected by immunohistochemistry at the dorsal horn regions of rats subjected to spinal nerve ligation or diabetes (A). Rats exposed to high dose (2.04 mg/kg/d) pregabalin provided intrathecally or intranasally had less expression of CaVα2δ-1 at the dorsal horn as compared to rats receiving near-nerve delivery of pregabalin or with saline delivery for both models of diabetic peripheral neuropathy (DPN) (B) and spinal nerve ligation (SNL) (C). As compared to control rat specimens, the presence of either spinal nerve ligation or diabetes uniformly led to upregulation of CaVα2δ-1 at the dorsal horn. Unmatched ANOVA tests were performed between cohorts receiving pregabalin and saline for each intervention and condition, and between intervention locations, with * indicating significant difference (p < 0.0125 after Bonferroni corrections) between cohorts [n = 3-5 specimens for each cohort].
Dorsal spinal cord protein expression revealed by Western blotting for CaVα2δ-1 protein was elevated in the presence of diabetes or after spinal nerve ligation (Figure 5). In either model of neuropathic pain, delivery of intranasal or intrathecal high dose pregabalin was associated with less significant elevation of CaVα2δ-1 protein at the dorsal horn; meanwhile, near-nerve pregabalin delivery had no impact upon dorsal horn expression of CaVα2δ-1 protein. Similarly, diabetes and spinal nerve ligation led to elevation of CaVα2δ-1 protein at the dorsal root ganglia, greatest in smaller nociceptive neurons. However, no method of pregabalin delivery impacted upon CaVα2δ-1 cellular or axonal patterns of expression in the dorsal root ganglia or in peripheral nerve (Additional Files 2, 3).
Figure 5 Protein Quantification. Western blotting identified increased levels of CaVα2δ-1 protein in the dorsal horn and lumbar DRGs for rats exposed to either diabetic peripheral neuropathy (DPN) or spinal nerve ligation (SNL) when compared to control rats (A). In the dorsal spinal cord, either intrathecal or intranasal delivery of high dose (2.04 mg/kg/d) pregabalin was associated with less upregulation of CaVα2δ-1 protein than with saline delivery (B). The near nerve delivery of pregabalin failed to impact upon CaVα2δ-1 protein expression in the dorsal spinal cord following SNL or DPN (A, B). Finally, pregabalin delivered in any manner was not associated with impact upon CaVα2δ-1 protein expression in the lumbar DRGs exposed to either DPN or SNL when compared to saline delivery (intrathecal delivery results shown) (A). Multiple unmatched ANOVA tests were performed between cohorts receiving pregabalin and saline for each intervention and condition, with * indicating significant difference (p < 0.0125 after Bonferroni corrections) between cohorts. β-actin protein levels were used for quantification of ratios of CaVα2δ-1/β-actin demonstrated in B.
CaVα2δ-1 protein in the thalamus and primary sensory cortex of the brain from rats exposed to diabetic peripheral neuropathy, as well as in the contralateral thalamus and primary sensory cortex, was unchanged in the presence of neuropathic pain when compared to control rats without diabetes or traumatic nerve injury (Additional File 4).
Development of Increased Microglial Density and Activation in Diabetic Peripheral Neuropathy
Microglial quantification was performed in the dorsal thoracic and lumbar spinal cords in rats subjected to diabetes and spinal nerve ligation. There was an increased density of activated microglia in the dorsal spinal cord regions in rats in either neuropathic pain model when compared to control rat spinal cord specimens (Figure 6). There was no difference in microglial quantification in rats receiving saline or pregabalin with any method of delivery in either model of neuropathic pain.
Figure 6 Microglia Assessment. Microglia accumulation was assessed in the dorsal regions of thoracic and lumbar spinal cord for control rat spinal cord (A), and with either spinal nerve ligation (B) or diabetic peripheral neuropathy (C) in rats. Greater immunohistochemically-identified accumulation and activation of microglia in the dorsal spinal cord of rats was seen with spinal nerve ligation or diabetic peripheral neuropathy (D) without impact of pregabalin delivery.* indicates significant differences between control (sham SNL surgery or citrate-injected non-diabetic rat respectively) and all SNL and DPN rat values using individual unmatched ANOVA testing (p < 0.0125 after Bonferroni corrections). Bar = 50 μm.
Verification of Anterograde Trafficking of CaVα2δ-1 in Dorsal Root Ligation and Spinal Nerve Ligation
For rats undergoing spinal nerve ligation +/- dorsal root ligation, immunohistochemistry identified accumulation of CaVα2δ-1 at sites proximal to the ligatures placed at the dorsal root but not spinal nerve at 7 days after procedures were performed (Figure 7). Dorsal horn expression of CaVα2δ-1 was elevated with immunohistochemistry or with Western blotting with the absence of dorsal root ligation regardless of delivery of intrathecal pregabalin or saline; however, CaVα2δ-1 protein levels at the dorsal horn were suppressed in the presence of a dorsal root ligature. When dorsal root ligature was present, CaVα2δ-1 protein accumulation occurred at the portion of dorsal root distal (but not proximal) to dorsal root ligature, with partial suppression due to intrathecal pregabalin delivery. At the spinal root, the presence or absence of spinal nerve ligation did not influence expression of CaVα2δ-1 protein. These findings demonstrate the anterograde transport of CaVα2δ-1 protein away from the dorsal root ganglia after traumatic nerve injury.
Figure 7 Dorsal and Spinal Root Ligation Experiments. A cartoon diagram of the procedures performed in spinal nerve ligation and dorsal root ligation is provided. Tissues obtained after 7 days of spinal nerve ligation and/or dorsal root ligation (or neither in control rats) were examined using immunohistochemistry for CaVα2δ-1 protein and with Western blotting for CaVα2δ-1 protein and β-actin, used as a loading control. At the level of the dorsal horn (1), the presence of a dorsal root ligature prevented upregulation of CaVα2δ-1 protein regardless of delivery of intrathecal pregabalin or intrathecal saline. At the dorsal root proximal to dorsal root ligature (2), there was a similar lack of upregulation of CaVα2δ-1 protein regardless of delivery of intrathecal pregabalin or intrathecal saline, unless dorsal root ligature was absent. At the level of the dorsal root distal to spinal nerve ligation (3), there was an accumulation of CaVα2δ-1 protein, with less significant CaVα2δ-1 protein expression when intrathecal pregabalin was delivered or if spinal nerve ligation was absent. Distal to the dorsal root ganglia and proximal to spinal nerve ligation (4) or at the spinal root distal to spinal nerve ligation (5), there was no change in expression of CaVα2δ-1 protein irregardless of delivery of intrathecal pregabalin or intrathecal saline, or even if spinal nerve ligation was absent. Multiple unmatched ANOVA tests were performed between cohorts receiving intrathecal pregabalin and saline as compared to control rat samples and samples without placement of either spinal or dorsal root ligatures, with * indicating significant difference (p < 0.017 after Bonferroni corrections) between cohorts. Bars = 10 μm.
Discussion
Sufficient doses of pregabalin provided through intranasal or intrathecal methods ameliorated tactile allodynia and thermal hypersensitivity due to spinal nerve ligation or diabetic peripheral neuropathy, but there was no benefit of providing pregabalin at the level of the peripheral nerve in either condition, suggesting that pregabalin's benefit is localized to the dorsal root ganglia or its central projections. In particular, pregabalin provision intranasally or intrathecally led to partial reversal of upregulation of CaVα2δ-1 at the pre-synaptic nerve terminals in the dorsal horn of the spinal cord [7,8]. This may relate to postulated central trafficking of the CaVα2δ-1 subunit from the dorsal root ganglia [23], and its potential block with pregabalin [23]. Pregabalin's effect appeared to be limited to impact upon trafficking of CaVα2δ-1 protein away from the dorsal root ganglia centrally.
Voltage-gated calcium channels are heterogenous multimeric complexes composed of several different subunits including α1, β, α2δ, and γ [43,44]. The CaVα2δ-1 subunit is an auxiliary subunit which facilitates targeting and assembly of channels at the cell surface [45]. CACNA2D1 is transcribed as a single mRNA from a single gene, with the translated CaVα2δ-1 subunit protein cleaved to yield α2 and δ proteins; these are subsequently disulfide-linked and glycosylated, leading to functional CaVα2δ-1 subunits [46]. There are four different CaVα2δ-1 subunits: the CaVα2δ-1 subunit has been investigated most, due to its ability to serve as a ligand for the gabapentinoids [19]. Expression of CaVα2δ-1 in normal situations is essentially confined to neurons withn the brain, spinal cord, and DRG [11]. In the brain, CACNA2D1 mRNA is localized to regions important for cortical processing, primary sensory transmission, and arousal; however, we discovered no measurable changes in CaVα2δ-1 protein expression in the neuropathic pain states studied, regardless of mode of pregabalin delivery. Instead, the observed changes in CaVα2δ-1 protein expression at the dorsal horn appear to be of greater importance.
Upregulation of CaVα2δ-1 in the spinal cord is essential for both initiation and maintenance of neuropathic pain in many conditions [47]. Although upregulation of CaVα2δ-1 occurred in the DRG and at the dorsal root as well, there was no impact of pregabalin upon CaVα2δ-1 protein levels at these locations. In addition, elevation of CACNA2D1 mRNA in neuropathic pain states at the dorsal horn and dorsal root ganglia was not impacted by forms of pregabalin delivery. The isolated impact of pregabalin with lowering of elevated CaVα2δ-1 protein levels at the dorsal horn suggests a pregabalin-mediated prevention of an anterograde trafficking process [23]. However, no such distal anterograde trafficking of CaVα2δ-1 protein towards the periphery could be detected, and there was no accumulation of CaVα2δ-1 protein at the spinal nerve ligation as occurred at the ligature around the dorsal nerve root. The presence of a dorsal root ligature prevented the upregulation of CaVα2δ-1 protein levels at the dorsal horn, leading to accumulation of CaVα2δ-1 protein proximal to the dorsal root ligature, implicating CaVα2δ-1 protein trafficking away centrally from the dorsal root ganglia as an important factor in establishment of neuropathic pain, confirming the results of Bauer et al [23]. These same unmyelinated DRG sensory neurons important for nociception (unmyelinated C-fibers for transmission of thermal hyperalgesia and large-diameter Aδ afferent fibers for transmission of tactile allodynia [48,49]) synapse in laminae I and II of the dorsal horn or at Lissauer's tract. We determined that CaVα2δ-1 immunoreactivity was elevated unilaterally with spinal nerve ligation and bilaterally with diabetic peripheral neuropathy in the dorsal column at thoracolumbar levels above the spinal nerve ligation injury, as well as in DRG neurons [23]. Most importantly, decreases in CaVα2δ-1 immunoreactivity at the dorsal horn were associated with successful analgesia of neuropathic pain with pregabalin. As a result, we hypothesize that pregabalin acts to slow or prevent the trafficking of the CaVα2δ-1 protein from the dorsal root ganglia to the dorsal horn.
The upregulation of CaVα2δ-1 within the spinal cord leads to increased presynaptic Ca2+ influx as well as neurotransmitter release, contributing to sensitization and neuropathic pain [50]. In models of neuropathic pain, the gabapentinoids gabapentin and pregabalin reduce neurotransmitter release and excitatory synaptic transmission at the spinal cord [51,52]. Controversies regarding the mechanism of action of the gabapentinoids have occurred, with some studies demonstrating no acute effects of gabapentin upon spontaneous synaptic currents in lamina II neurons [53], while other studies have shown gabapentin's ability to reduce both inhibitory and excitatory neurotransmission at the dorsal horn by preferentially blocking only P/Q-type Ca2+ channels [54] and through prevention of intracellular endosome recycling of CaVα2δ-1 protein [55]. There has been no effect upon the levels of CaVα2δ-1 protein or CACNA2D1 mRNA elsewhere, though, such as at the DRG (despite the presence of all three CaVα2δ subunits at the DRG) [11] and in the peripheral nerve. This may signify that trafficking, but not expression or endocytosis, of CaVα2δ-1 from DRG to presynaptic terminals is impacted upon by pregabalin [23]. Other potential mechanisms for gabapentinoids include a more acute inhibitory effect upon neurotransmitter release [56] which may relate to upregulation of protein kinase C activation [57,58].
Chronic, and not acute, pregabalin delivery reduces the neuronal expression of CaVα2δ-1 in vitro without impact upon constitutive endocytosis, leading to high levels of CaVα2δ-1 being present in intracellular vesicles [23]. This effect may be a consequence of calcium channel trafficking through the Von Willebrand factor-A domain in the α2 subunit of CaVα2δ-1 [59], as well as blocked CaVα2δ-1 trafficking [23,60]. Transgenic mutants for CaVα2δ-1 unable to bind gabapentinoids have reduced trafficking, supporting this hypothesis [60,61]. This hypothesized mechanism of disrupted trafficking would be unique, distinct from that of other pharmacotherapies with a direct neuronal surface action, and may relate to CaVα2δ-1 recycling at the endosome [55]. We postulate that the effects of pregabalin upon CaVα2δ-1 trafficking occurs peripherally, either at the dorsal root ganglia or the dorsal root.
There was no measureable impact of pregabalin delivered by any method upon the activation and accumulation of microglia in the spinal cord either. Ectopic activity contributing to sensitization due to excessive activity in voltage-gated calcium channels and voltage-gated sodium channel may occur within both A or C fibres [62-64], relating to neuronal hyperexcitability [14]. Microglia and their activation, leading to cytokine production important in neuropathic pain, are apparent and play a key role in pathophysiological pain due to both nerve injury [15-17] and diabetes [18]. Pregabalin's lack of impact upon these other established mechanisms for induction of neuropathic pain suggest its specificity at the CaVα2δ-1 subunit of the voltage-dependent calcium channel. However, other pathological changes associated with neuropathic pain continue abated despite pregabalin delivery.
There are limitations to the present study that require discussion. There is overlap between the tissues that receive pregabalin from intranasal and intrathecal delivery-it is probable that both forms of delivery impacted upon the dorsal horn region, but there may have been some undefined impact of intranasal pregabalin delivery at supraspinal locations as well (even with the lack of impact upon CaVα2δ-1 expression in brain). The absence of heightened CaVα2δ-1 expression at the supraspinal locations does not exclude the possibility of pregabalin possessing a role in supraspinal pain processing-it is possible that pregabalin may have important effects at the medulla and cortex [65-68]; this may explain the effects of intranasal pregabalin delivery, which only resulted in small concentrations of pregabalin in the lumbar spinal cord and dorsal root ganglia. Pregabalin's effects within the cerebrum may be contributory, especially after demonstrations of high cerebral concentratons of pregabalin following its intranasal delivery. Although we did not demonstrate impact of forms of delivery of pregabalin upon microglia accumulation in the spinal cord, it is possible that a longer duration of pregabalin delivery, or earlier onset of pregabalin delivery, is required before an anti-neuroinflammatory effect may be demonstrated, as shown in prior studies [36,69,70]. For example, in a model of diabetic peripheral neuropathy with daily gabapentin delivery over 5 days followed by immediate tissue harvesting, amelioration of spinal microglial activation occurred [71]. The levels of pregabalin achieved within the lumbar spinal cord and dorsal root ganglia were measured to be low after 74 hours-we hypothesize that pregabalin's effects may be summated over time rather than related to an absolute concentration achieved at one time point, although we cannot verify this. We did not study a systemic application such as with gastric/oral or intravenous forms of delivery due to the more widespread distribution of gabepentinoids with these methods and the presence of prior data assessing these delivery methods [72,73]. The timing of pregabalin/saline delivery in the spinal nerve ligation experiments was initiated 7 days after the spinal nerve ligation occurred in order to provide intervention during the time of greatest neuropathic pain behavior; however, we selected the first 7 days after double ligation to study CACNA2D1 mRNA and CaVα2δ-1 protein expression during the time period of neuropathic pain behavior initiation-this difference in timing may have led to inconsistencies in molecular test results. Although we, and others [23], have hypothesized that pregabalin's mechanism of action is the disruption of CaVα2δ-1 trafficking, it remains unclear why expression of CACNA2D1 mRNA and CaVα2δ-1 protein is unaffected at the dorsal root ganglia. Together, these findings suggest that pregabalin's action is independent of translation of CaVα2δ-1 or transcription of CACNA2D1. As well, it would be anticipated that disrupted trafficking of CaVα2δ-1 would result in CaVα2δ-1 upregulation at the dorsal root ganglia, but this was not identified. However, pregabalin-mediated effects upon the presence of CaVα2δ-1 protein at the more proximal spinal nerve root and dorsal horn is indicative of pregabalin-induced suppression of the central trafficking of CaVα2δ-1 protein, as previously identified [23]. Rats received isoflurane anesthesia prior to intranasal delivery, but were tested for neuropathic pain at times that were many half lives after isoflurane discontinuation to avoid anesthetic effects upon behavioural testing. Finally, we designed this study to compare different modes of delivery of pregabalin upon neuropathic pain behaviours; however, our results cannot be used to interpret the site of action of pregabalin due to significant overlap in pregabalin's anatomical destinations with the intranasal and intrathecal delivery modes. However, we propose that a non-invasive form of pharmacologically targeting the dorsal horn and dorsal root ganglia, such as with intranasal delivery, may be a consideration for the management of human neuropathic pain.
Conclusions
The present data confirm the efficacy of pregabalin in the modulation of acute thermal and tactile hypersensitivity as features of neuropathic pain. The site of provision is critical, as exclusive delivery to the peripheral nerve has no impact; only delivery using intranasal or intrathecal delivery was associated with impact upon neuropathic pain behaviours and molecular outcomes. Furthermore, delivery using either intranasal or intrathecal methods led to diminished CaVα2δ-1 expression at the dorsal horn, suggesting that pregabalin inhibits central trafficking of the CaVα2δ-1 subunit from the dorsal root ganglia as previously shown by Bauer et al [23]. Future studies should examine the in vivo and in vitro trafficking of CaVα2δ-1 and the mechanisms by which pregabalin can influence the cell surface expression of CaVα2δ-1; another gabapentinoid, gabapentin, has been shown to prevent the recycling of the related protein CaVα2δ-2 from endosomes and the subsequent insertion in the plasma membrane without influencing internalization of CaVα2δ-2 [55]. The different impacts of pregabalin upon the central and peripheral axons of the dorsal root ganglia neurons are not yet understood. Finally, the intranasal delivery of agents such as pregabalin in order to directly target the nervous system may be a realistic method in managing neuropathic pain in humans, and may assist in the avoidance of systemic adverse effects.
Methods
Animals
All experiments were carried out using male Sprague Dawley rats (Charles River Laboratories), weighing 200-225 grams at initiation. Protocols were reviewed and approved by the University of Calgary Animal Care Committee using the Canadian Council of Animal Care guidelines. All attempts were made to minimize animal numbers and to maintain ethical standards. Experimental study groups were randomized and behavioural studies were performed by an experimenter who was unaware of treatment groups.
In all cases, rats were housed in plastic sawdust covered, pathogen-free cages with a normal light-dark cycle and free access to chow and water. Rats were anesthetized with pentobarbital (57 mg/kg) prior to all surgeries and terminal endpoints, and with inhaled isoflurane provided for all intranasal delivery points.
Sample size calculations
A sample size calculation for intervention groups was based upon an anticipated difference in neuropathic pain behavioral changes observed in treated diabetic rats to date, with an α of 0.05 and β of 0.5 providing a minimal sample size of n = 5 within each intervention group; this was increased to 6 rats due to anticipated minimal diabetes-related mortality over the three week study.
Radiolabeled Pregabalin Studies to Determine Localization of Delivery
We performed radiolabelled studies to determine the distribution of pregabalin reaching the central and peripheral nervous system after intranasal or intrathecal delivery. We examined the distribution of pregabalin at 1, 2, and 5 h after 72 hours of chronic delivery of intranasal, intrathecal, or near nerve delivery. A combination of 125I labeled pregabalin and unlabeled pregabalin was delivered during studies at the Alzheimer's Research Center at Regions Hospital in St. Paul, MN, USA. This procedure was approved by the Institutional Animal Care and Use Committee at Regions Hospital. Prior to experimentation, 30 diabetic rats (induced via streptozotocin injections, described below) were sedated for intranasal or intrathecal delivery using pentobarbital anesthesia (60 mg/kg). 125I-labelled pregabalin was provided to 10 rats via intrathecal delivery, to 10 rats via intranasal delivery, and to 10 rats via near nerve delivery (see below for procedure descriptions). Pregabalin (Pfizer Global, New York, New York) with an initial concentration 3.125 μg/μl was dissolved in PBS and custom labeled with 125I (GE Healthcare, Piscataway, New Jersey, USA). Synthesized radiolabelled pregabalin solution contained 266.7 μCi/μg. 125I-labeled pregabalin delivery (intranasal or intrathecal) was performed in a fume hood behind a lead impregnated shield, with anesthetized rats placed supine. A mixture of 125I pregabalin (19.2 μCr) and unlabeled pregabalin (30.0 μg) were administered intranasally or intrathecally. 125I pregabalin was intranasally administered over alternating nares as eight 6-μL drops with an Eppendorf pipetter every 2 minutes, for a total volume of 48 μL, provided twice daily for 3 days prior to a single delivery on day 4 as per a prior schedule [30]. For intrathecal delivery, 125I pregabalin was delivered using a placed intrathecal catheter (see below) attached to an Alzet pump (described below) with delivery of 2.04 mg/kg/d over 72 hours continually delivering a mixture of 125I pregabalin (19.2 μCr) and unlabeled pregabalin (30.0 μg) over each 24 hour period for 73, 74 and 75 hours prior to harvesting. For near nerve delivery, 125I pregabalin was delivered using a placed T-chamber around the proximal sciatic nerve (see below) attached to an Alzet pump (described below) with delivery of 2.04 mg/kg/d over 72 hours continually delivering a mixture of 125I pregabalin (19.2 μCr) and unlabeled pregabalin (30.0 μg) over each 24 hour period for 73, 74 and 75 hours prior to harvesting. Each desired dose contained a calculated radioactive dose of 40 μCi per day to best provide concentrations similar to the long-term duration experiments described below.
At each of 1, 2, and 5 hours after 72 hours of initiating either 125I pregabalin intranasal delivery, intrathecal or near-nerve delivery, cardiocentesis was performed for blood extraction. Euthanasia was performed via transcardial perfusion using 60 mL of saline, followed by 500 mL of 4% paraformaldehyde while the rat was maintained under anesthesia. To quantify 125I distribution, blood, urine, lymphatic (superficial perimandibular lymph nodes and cervical lymph nodes) and visceral organ structures (quadriceps muscle, kidney, liver, and lung), as well as portions of the central (olfactory bulb, anterior olfactory cortex, frontal cortex, caudate putamen, parietal cortex, temporal cortex, hippocampus, septum, thalamus, hypothalamus, midbrain, pons, medulla, cerebellum, cephalad cervical spinal cord, caudal spinal cord, mid-thoracic spinal cord, and lumbar spinal cord) and peripheral nervous systems (fifth lumbar dorsal root ganglia and proximal sciatic nerve) and associated tissues (ventral and dorsal cervical dura mater) were harvested. Gamma signal was recorded for each body region with autoradiographic imaging using a COBRA II Auto-Gamma Counter (Perkin-Elmer, Waltham, Mass., USA). Concentrations of 125I pregabalin were calculated based upon the gamma counting data, tissue weight, specific activity of the drug administered and standards measured. Results were studied for penetration into peripheral and central nervous system tissues.
To confirm patency and delivery with near-nerve chamber placement, infusion pumps containing India ink were implanted in four animals with pumps connected to the nerve regeneration chamber (see below). The presence of India ink was detected in all cases only at the catheter site for days 3, 7, and 14. Pump and catheter volume infusion rates were approximately 15 μl per day, or approximately 200 μl over 14 days for this India Ink experiment.
Models of Neuropathic Pain
Diabetes and Diabetic Peripheral Neuropathy
At the age of 1 month, rats intended to be diabetic were injected with streptozotocin diluted in sodium citratre (pH 4.7, Sigma, St. Louis, MO) intraperitoneally once daily for each of three consecutive days with doses of 60 mg/kg, 50 mg/kg, and then 40 mg/kg, while rats intended to be non-diabetic were injected with volume-matched carrier (sodium citrate pH 4.7) for three consecutive days. Streptozotocin ablates pancreatic β cells leading to an insulin deficient diabetic state. No insulin treatment was used at any time during the protocol. In all cases, whole blood glucose measurements were performed using puncture of the tail vein and a blood glucometer (OneTouch Ultra Meter, LifeScan Canada, Burnaby, BC, Canada). Hyperglycemia was verified 1 week after streptozotocin injections, with a fasting whole-blood glucose level of ≥ 16 mmol/l (normal 5-8 mmol/l) being our definition for experimental diabetes. Rats that did not meet these criteria for diagnosis of diabetes were excluded from further assessment. During diabetes, daily inspection of footpads occurred to rule out the presence of wounds or burns. No insulin or other diabetes therapies were provided during these investigations.
Spinal Nerve Ligation
Spinal nerve ligation was performed as described by Kim and Chung [74]. Briefly, the right L5/6 lumbar spinal nerves of male Sprague Dawley rats (200 g) were exposed in isoflurane/oxygen-anesthetised rats followed by tight ligation with 5.0 silk sutures between the DRG and proximal to the junctions forming the sciatic nerve. Sham operations were performed in the same way except that spinal nerves were not ligated. The left spinal nerves for all rats were left untouched.
Study Timelines, Animal Groups and Drug Administration
We conducted studies using the rat diabetic peripheral neuropathy model. A total of 118 male Sprague Dawley rats were induced diabetic while 26 male Sprague Dawley rats were studied as citrate-injected control littermates over the course of 3 weeks of diabetes without delivery of any other therapeutic agents other than pregabalin. Rates of conversion to diabetes were in excess of 95%. These rats were accustomized and studied with sensory behavioral testing every 3 days beginning prior to streptozotocin injections. Of the 118 rats receiving intraperitoneal streptozotocin injections, those with confirmed diabetes status were randomized to one of four varied dose groups within each of the intervention groups: 1) intranasal delivery with low dose (0.051 mg/kg/d) pregabalin (n = 9), medium dose (0.51 mg/kg/d) pregabalin (n = 8), or high dose (2.04 mg/kg/d) pregabalin (n = 8) or equal volumes of saline (n = 8); 2) intrathecal delivery with low dose (0.051 mg/kg/d) pregabalin (n = 9), medium dose (0.51 mg/kg/d) pregabalin (n = 8), or high dose (2.04 mg/kg/d) pregabalin (n = 8) or equal volumes of saline (n = 8); 3) near-nerve delivery with low dose (0.051 mg/kg/d) pregabalin (n = 9), medium dose (0.51 mg/kg/d) pregabalin (n = 8), or high dose (2.04 mg/kg/d) pregabalin (n = 8) or equal volumes of saline (n = 9). Those receiving intraperitoneal citrate injections (non-diabetic rats) were divided into intranasal (n = 7), intrathecal (n = 6) or near-nerve (n = 7) intervention groups. Following 7 days after diabetes confirmation occurred, intranasal, intrathecal, or near-nerve delivery began and occurred for a total of 14 continuous days. Neuropathic pain behaviors typically initiate within the first week after streptozotocin-induced diabetes confirmation [75], with initiation of interventions occurring 2 weeks after completion of streptozotocin injections.
We examined a model of traumatic neuropathy, the rodent spinal nerve ligation model. A total of 93 male Sprague Dawley rats received the spinal nerve ligation procedure while 23 male Sprague Dawley rats were studied as sham-operated control littermates. These rats were studied with sensory behavioral testing every 3 days beginning prior to spinal nerve ligation or sham procedure. The 93 rats receiving spinal nerve ligation were randomized to one of four varied dose groups within each of the intervention groups: 1) intranasal delivery with low dose (0.051 mg/kg/d) pregabalin (n = 7), medium dose (0.51 mg/kg/d) pregabalin (n = 6), or high dose (2.04 mg/kg/d) pregabalin (n = 6) or equal volumes of saline (n = 6); 2) intrathecal delivery with low dose (0.051 mg/kg/d) pregabalin (n = 7), medium dose (0.51 mg/kg/d) pregabalin (n = 6), or high dose (2.04 mg/kg/d) pregabalin (n = 6) or equal volumes of saline (n = 6); 3) near-nerve delivery with low dose (0.051 mg/kg/d) pregabalin (n = 7), medium dose (0.51 mg/kg/d) pregabalin (n = 6), or high dose (2.04 mg/kg/d) pregabalin (n = 6) or equal volumes of saline (n = 6). Those rats receiving sham procedures were divided into intranasal (n = 7), intrathecal (n = 7) or near-nerve (n = 6) intervention groups. Following 7 days after spinal nerve ligation occurred, intranasal, intrathecal, or near-nerve delivery began and occurred for a total of 14 continuous days. It has been demonstrated that spinal nerve ligation results in osnet of neuropathic pain behaviors in the first 1-7 days after spinal nerve injury [76].
Intranasal, Intrathecal and Near-Nerve Delivery Systems
Doses of pregabalin were selected based upon prior delivery of intrathecal gabapentin, a molecule similar to pregabalin but with less potency for the α2δ-1 subunit of voltage dependent calcium channels [52]. Intranasal pregabalin or saline delivery occurred twice daily during periods of therapy. Isoflurane-anesthetized rats were placed on their back with necks held in extension for intranasal administration, administered twice daily as ten 8 μl drops for a total volume of 80 μl for each nare, with delivery to alternating nares with 1-2 minutes between drops (i.e., ten drops per nare), for a total of 320 μl per day. Intrathecal delivery of pregabalin or saline occurred through Alzet mini osmotic pumps providing a continuous delivery of pregabalin/saline at a rate of 0.5 μl per hour. Near-nerve delivery of pregabalin or saline also occurred through Alzet mini osmotic pumps providing a continuous delivery of pregabalin/saline at a rate of 0.5 μl per hour. In each case where pregabalin was provided, there were three different total daily doses (low dose (0.2 μmol or 0.051 mg/kg), medium dose (2.0 μmol or 0.51 mg/kg), or high dose (8.0 μmol or 2.04 mg/kg)) of pregabalin delivered for each of the intervention systems.
For intrathecal delivery, we used the same surgical exposure as performed for spinal nerve ligation/sham, or created a new surgically exposed area over the right L5/6 spinal nerve region for diabetic/non-diabetic rats [10]. After incising the back skin at the lower lumbar region, a subcutaneous pouch was created. A silicone catheter (0.012 inches × 0.025 inches) was positioned into the lumbar intrathecal space in the created pouch between L6 and S1 vertebrae while connected to a two-week Alzet mini-osmotic infusion pump placed in the dorsal subcutaneous space of the rat. Alzet mini-osmotic infusion pumps are easily inserted into the subcutaneous space and require no external connections while providing a continuous, constant level of delivery to be provided over a two week period after placement. Surgical closure with 9-0 silk suture was performed around the subcutaneous pouch at the lumbar region. Surgeons were blinded as to the contents of the infusion pumps placed, which were randomized to either contain pregabalin (low (0.051 mg/kg/d), medium (0.51 mg/kg/d) or high dose (2.04 mg/kg/d)) or saline in a 3:1 ratio.
For near-nerve delivery, we used the same surgical exposure as performed for spinal nerve ligation/sham, or created a new surgically exposed area over the right spinal nerve region for diabetic/non-diabetic rats [10]. After incising the back skin at the lower lumbar region, a subcutaneous pouch was created. A two-week Alzet mini-osmotic infusion pump is placed in the dorsal subcutaneous pouch of the rodent. A nerve regeneration chamber is placed to surround the spinal nerve ligation site-the equipment used is composed of a 6.5 mm length (modified from 10 mm to compensate for the short distance of the spinal nerve) of silastic tubing (size: 1.98 mm inside diameter × 3.18 mm outside diameter; Dow Corning, Michigan). A small porthole cut into the side of the nerve chamber permits access with one end of a silicone catheter, with a small amount of cyanoacrylate cement (Instant Krazy Glue; Advanced Formula Gel; Elmer's Products Canada, Brampton, Ont.) placed at the outside of the end of the access tube before inserting it into the porthole. A second silastic tube (size: 0.76 mm inside diameter × 1.65 mm outside diameter) permits connection of the silicone catheter to the nerve regeneration chamber and Alzet pump on either end. Before their use, the entire apparatus used is autoclaved. Surgical closure with 9-0 silk suture was performed around the subcutaneous pouch and at the spinal nerve ligation exposure site. Surgeons were again blinded as to the contents of the infusion pumps placed, which were randomized to either contain pregabalin (low (0.051 mg/kg/d), medium (0.51 mg/kg/d) or high dose (2.04 mg/kg/d)) or saline in a 3:1 ratio.
During all intervention protocols, all animals were monitored post-operatively for signs of infection or other complications of surgery. For the purposes of data demonstration, Day 1 is considered to be the last of 7 days after either diabetes confirmation (for diabetic peripheral neuropathy studies) or 7 days after spinal nerve ligation occurred-this is one day before the start of interventions provided using intranasal, intrathecal, or near-nerve delivery of doses of pregabalin or saline. Note that after Day 15, no further intervention was provided prior to surgical harvesting on Day 18.
Sensory Behavioral Testing
In all cases, baseline testing was done beginning prior to streptozotocin injections or spinal nerve ligation procedure and ongoing testing continued until after pregabalin or saline delivery was completed, occurring every 3 days. During maintenance therapy, behavioural testing was performed at 2-4 hours after the morning dose of intranasal pregabalin/saline administration was delivered, during the time in which pregabalin concentrations were peaking in the nervous system. This latency period between dosing and behaviour testing was necessary to permit elimination of isoflurane anesthesia delivered during pregabalin/saline delivery and prevent confounding effects of anesthesia. A minimum of 1 hour was provided between forms of neuropathic pain behavior testing, and a minimum of 6 rats underwent behavioural testing at each time point.
Mechanical Sensitivity
Mechanical withdrawal thresholds were tested using a Dynamic Plantar Aesthesiometer (Ugo-Basile, Milan). In brief, rats were placed in clear acrylic boxes (22 × 16.5 × 14 cm) with a metal grid floor in a temperature controlled room (22°C) and were acclimatized for 15 minutes before testing. The stimulus was applied via a metal filament (0.5 mm) which applied a linearly increasing force ramp (2.5 g/s) to the middle of the plantar surface of the hind paw (within the sural nerve territory). A cut-off of 50 g was imposed to prevent any tissue damage. The force necessary to elicit a paw withdrawal was recorded. The paw withdrawal threshold was calculated as the average of three consecutive tests with at least 5 minutes between each test. Mechanical allodynia was defined as reduced threshold after induction of diabetes/spinal nerve ligation compared to a baseline paw withdrawal threshold.
Thermal Hyperalgesia
To quantitatively assess the thermal threshold of the hindpaw, rats were placed on the glass surface of a thermal testing apparatus with acclimatization for 15 minutes before testing. A mobile radiant heat source (Hargreaves apparatus) located under the glass was focused onto the middle of each of both individual hindpaws (within the sural nerve territory) for each rat for up to 60 seconds, with the latency (seconds) to withdrawal measured. Heating rate ramped from 30°C to 58°C over 60 seconds in consistent fashion on each occasion. The cutoff of 60 seconds was used to prevent potential tissue damage. Paws were inspected before and after thermal testing to ensure that no evidence of thermal damage was present. The mean withdrawal latency of both hindpaws from three consecutive trials was calculated as the thermal threshold. There were 5 minute intervals provided between each trial.
Locomotor Behavioral Testing
Potential changes in the locomotor function of the rats related to neuropathic pain states or therapeutic intervention were evaluated using Rotarod testing (Microprocessor Controlled Rota-Rod Treadmill for Rats, Model 57602, Ugo Basile, Italy). Acclimatization for walking on the revolving drum was performed over three training trials on the revolving drums at 10-15 rpm over three consecutive days. A maximum of 150 seconds for each Rotarod trial was used. The Rotarod performance time was measured at 1) 0.5, 1, 1.5, and 2 hours after intranasal pregabalin delivery following 72 hours of twice daily intranasal pregabalin/saline delivery; 2) 72 hours after initiation of intrathecal pregabalin/saline; and 3) 72 hours after near nerve pregabalin/saline delivery, each using varying doses of pregabalin (low (0.051 mg/kg/d), medium (0.51 mg/kg/d) or high dose (2.04 mg/kg/d)) or saline. The timepoint of after 72 hours was selected as occurring at least 5 half lives for pregabalin. Each test was performed at 5-10 minute intervals, and the average values obtained were compared. A total of 6 rats in each group were studied for locomotor activity, each of which had diabetes induced 7 days prior. A control rat cohort was studied 7 days after diabetes induction without any exposure to pregabalin or saline.
Surgical harvesting
Following 3 days after the last behavioral testing (4 days after completion of pregabalin/saline), rats from each cohort were sacrificed using pentobarbital intraperitoneal injections-a delay of four days was selected in order to assess the chronic effects of pregabalin delivery more than five half lives after pregabalin/saline termination, rather than detect any acute effects of pregabalin delivery. The following tissues were harvested from all rats: dorsal lumbar spinal cord from L2-S1 (left/right), bilateral lumbar DRG from L4-L6, bilateral sciatic nerves, bilateral sural nerves, and thalamus and primary sensory cortex (contralateral to injury in rats subjected to spinal nerve ligation). In diabetic/non-diabetic rats, left-sided tissues were placed in 2% Zamboni's fixative for later immunohistochemistry studies, while right-sided tissues were immediately fresh frozen at -80°C (Invitrogen, Burlington, ON) in liquid nitrogen and stored at -80°C for later protein/mRNA identification. In spinal nerve ligation/sham rats, right sided tissues ipsilateral to spinal nerve ligation were divided equally to be placed in 2% Zamboni's fixative or fresh frozen; left sided tissues were treated similarly.
Double Ligation Experiment
In order to examine the potential trafficking of CaVα2δ-1 anterograde from the dorsal root ganglia, an additional experiment was performed using two nerve ligations-spinal nerve ligation was performed along with ligature placed at the dorsal root. Although spinal nerve ligation forms a model of neuropathic pain, nerve ligation is also a well established method for determination of axonal protein trafficking, which when perturbed, leads to protein accumulation at the ligation site.
Twelve male Sprague Dawley rats had spinal nerve ligation performed (as above); another four rats had only dorsal root ligation performed (see below) another 4 rats had sham (used as controls) surgery performed (as above). Again, the right L5/6 lumbar spinal nerves of male Sprague Dawley rats (200 g) were exposed in isoflurane/oxygen-anesthetised rats followed by tight ligation with 5.0 silk sutures between the DRG and junctions forming the sciatic nerve. For the 4 rats receiving sham surgeries and for 8 of the 12 rats receiving spinal nerve ligation, ligation of the right L5/6 dorsal roots was performed as well using an additional laminectomy and dural splitting at the L4-6 intervertebral foramina in order to expose the dorsal roots. Dorsal root ligation was also performed in 4 rats not receiving spinal nerve ligation. Tight ligation with 5.0 silk sutures was performed for the dorsal root at the midpoint between the dorsal root ganglia and the dorsal horn of the spinal cord. Intrathecal delivery (see above) was performed for all 16 mice receiving procedures. Surgeons were blinded as to the contents of the infusion pumps placed, which were randomized to either contain pregabalin (high dose (8.0 μmol or 2.04 mg/kg)) or saline in a 1:1 ratio for those rats receiving both spinal and dorsal root nerve ligation procedures. For the 8 rats receiving one of spinal nerve ligation or dorsal root ligation, only intrathecal saline delivery was provided. Closure of the dura used 9-0 silk sutures, with surgical closure of the back of each rat performed otherwise as described above. Pregabalin or saline delivery began immediately post-surgery, considered day 0. The four rats with only a sham procedure performed were used as controls and did not receive any intrathecal delivery.
After 7 days, the following tissues were harvested from all rats: dorsal lumbar spinal cord from L2-S1 (left/right), dorsal root proximal to dorsal root ligature, dorsal root distal to dorsal root ligature, spinal nerve proximal to spinal nerve ligation, and spinal nerve distal to dorsal root ligature. The tissues from one rat from each group were used for immunohistochemistry studies to identify CaVα2δ-1 (see below), while the tissues from the other 3 rats in each group were placed in liquid nitrogen and stored at -80°C for later protein (CaVα2δ-1 and β-actin) identification. Due to the small amount of tissues obtained, pooling of tissues from the rats within each cohort was required for Western blot analysis (see below).
Immunohistochemistry
After spinal cord/DRG/peripheral nerve specimens were fixed in 2% Zamboni's fixative overnight at 4°C, they were washed in PBS, kept overnight in 25% sucrose PBS solution, and then embedded in optimal cutting temperature embedding solution (Tissue Tek, Sakura Finetek, USA), before storage at -80°C until sectioning. Cryostat transverse and longitudinal nerve sections (10 μm) were placed onto poly-l-lysine-and acetone-coated slides (SuperFrost Plus, Fisher Scientific, USA). Antigen retrieval was performed with slides placed in sodium citrate in an 80°C water bath, a PBS wash for 5 min, blocking with 10% goat serum for 1 h, and further PBS washing. In all cases, slides were incubated with primary antibody overnight at 4°C. After PBS washing, secondary fluorescent antibody was applied with incubation for 1 h at room temperature, followed by PBS washing and slide mounting. All immunohistochemistry was visualized using a Zeiss Imager Z1 (Zeiss, UK) fluorescence microscope. Calculation of the number of immunofluorescent profiles as well as the relative luminosity was performed using Adobe Photoshop (Adobe Photoshop 9.0, Adobe, San Jose, CA, 2005).
Primary antibodies used were to identify CaVα2δ-1 (1:200, produced in rabbit, Lifespan Biosciences, Seattle, WA), glial fibrillary acidic protein (GFAP) for Schwann cell identification (1:200, produced in mouse, Sigma Aldrich Canada, Oakville, ON), neurofilaments (NF) 200 for axon and neuron identification (1:100, produced in mouse, Santa Cruz, Santa Cruz, CA), goat anti-ionized calcium-binding adaptor molecule 1 (Iba-1) (1:1000, produced in goat, Abcam, Cambridge, MA) and microtubule associated protein-2 (MAP-2) (1:500, produced in rabbit, Sigma Aldrich Canada). Secondary antibodies used were either anti-rabbit IgG fluorescein isothiocyanate (FITC) labelled (1:100; Zymed, San Francisco, CA), donkey anti-goat IgG CY3 labeled (1:200, Fitzgerald Industries, Concord, MA), or goat anti-mouse IgG CY3 labeled (1:200, Sigma Aldrich Canada, Oakville, ON). Slides were cover-slipped with Vectashield mounting medium containing DAPI for nuclear identification (Vector Laboratories, CA, USA). Iba-1, MAP-2 and GFAP-positive cells were determined by counting the number of profiles (cell bodies) as described previously [10].
Samples of spinal cord/DRG/spinal nerve prepared for immunohistochemistry were examined for expression of CaVα2δ-1 along with identification of cell types (GFAP, MAP-2, NF200). For identification of microglia, isolated staining with Iba-1 was performed. Calculation of the number of immunofluorescent fibers as well as the luminosity of individual nerve fibers was performed for CaVα2δ-1. From each of the L4-6 DRGs, neurons were counted in six sections through the midportion of the DRG: total neuron numbers per transverse section, total numbers of neurons immunolabeled, and numbers of neurons with intense expression of channel of interest, as defined using two previously defined cutoff values with Adobe Photoshop [10]. Dorsal root ganglia neurons were counted as those with nuclear profiles that were visible in one section, but not in the subsequent section. Sensory neurons were differentiated from satellite and Schwann cells based on size and appearance as well as positive immunoprofiling for NF200. Total neuronal number was then calculated based upon the summation of neurons with newly identified nuclei identified through all sections of an individual dorsal root ganglion. All counting was performed with the microscopist masked to the identity of the experimental group. All of these measures were easily distinguished among immunolabeled neurons by a single examiner blinded to the identity of the groups using a calculated luminosity measurement of fluorescence for individual fibers examined under 400 × magnifications. Luminosity was classified as none-low (luminosity value of 0-150), moderate (150-250) or high (> 250) using Adobe Photoshop software (scale of 0-255 with arbitrary units).
Transverse sections through the lumbar spinal cord were also immunolabeled for channel expression with specific attention directed toward the dorsal horn. The relative fluorescence intensity was measured for each side ipsilateral and contralateral to spinal nerve ligation injury, or bilaterally for diabetic rats, at a pre-determined exposure time with a digital camera (Zeiss Axioscope, Zeiss) which provided an image of the entire spinal cord. Luminosity of each dorsal quadrant of the spinal cord was calculated using Adobe Photoshop [10].
In dorsal spinal cord regions within the thoracic and lumbar regions, the total numbers of microglia per transverse section were identified using Iba-1 immunohistochemistry. The lateral, central and medial dorsal horn regions, representing laminae 1-3, were examined. For cellular densities, a box measuring 104 μm2 was placed onto areas of dorsal horn for regions between T2 and S1. A quantitative estimate (proportional area) of changes in the activation state of microglial cells was performed [77-79] in the dorsal spinal cord based on atlas boundaries and after subtraction of background signal. A resting microglia was classified as having a small, compact soma with long, thin, ramified processes. Activated microglia, in contrast, exhibit marked cellular hypertrophy, and retracted processes with process length less than soma diameter. A total of 25 randomly chosen areas of dorsal spinal cord, from a minimum of 4 animals per cohort group, were examined for activated microglia quantification. All measurements were performed by a single examiner blinded to the group identity.
Western Immunoblotting
In order to determine protein expression for membrane bound channels, we performed Western blot with a specialized protocol to protect membrane structure. Tissue portions from the dorsal spinal cord, DRG, thalamus and primary sensory cortex were placed in chilled phosphate buffer solution (PBS), followed by centrifugation at 400 rpm. Tissue was ground down with a pestle in ice-cold lysis buffer (HEPES 15 mM at pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM 2Na-EDTA, 5% glycerol, 0.5% Nonidet 40 (NP-40), 1 mM Phenylmethylsulfonyl Fluoride, Roche Mini-Complete Protease Inhibitors, and double distilled H20) at a ratio of 1 g of tissue: 10 ml of lysis buffer. Centrifugation of samples at 5,000 rpm followed for 15 minutes at 4°C. Supernatant was kept in tubes, and the pellet was homogenized again with half amounts of lysis buffer, followed by repeat centrifugation at 5,000 rpm for 15 minutes at 4°C. Both supernatants were transferred to ultracentrifuge rubes for repeat centrifuge at 26,000 rpm for 15 minutes at 4°C. Pellets were taken and resuspended in 25-50 μl of resuspension buffer (75 mM Tris at pH 7.4, 12.5 mM MgCl2, 5 mM 2Na-EDTA, 1.5% SDS, 0.1% Triton X-100, and double distilled H20) for protein quantification. These protein samples were then separated by SDS-PAGE techniques under conditions previously described [34]. Blots were probed for CaVα2δ-1 (1:4000, produced in rabbit, Lifespan Biosciences, Seattle, WA). For a loading control, anti-β-actin (1:100, Biogenesis Ltd. Poole, UK) was analyzed as well. Signal detection was performed by exposing of the blot to enhanced chemi-luminescent reagents (Amersham, GE Healthcare, USA) and captured on Kodac X-OMAT K film. In each case, 4-6 blots were performed for each protein of interest from different rats in each cohort. Blots were analyzed with Adobe Photoshop (Adobe Photoshop 9.0, Adobe, San Jose, CA, 2005) for semi-quantification of blotting density [34].
Quantitative Reverse-Transcriptase Polymerase Chain Reactions
Total RNA was extracted from peripheral nerve and spinal cord regions using Trizol reagent (Invitrogen). Total RNA (1 μg) was processed directly to cDNA synthesis using the Superscript II Reverse Transcriptase® system (Invitrogen). CACNA2D1 primers were: Forward 5'-GAAAGGCTTTAGCTTCGCGTTT-3', Reverse 5'-TCTCTCTTCTCCTCCATCCGTG-3' [GenBank: NM_001110847.1]. For a housekeeping gene, ribosomal protein (large P0) (RPLP0) was used, with primers: Forward 5'-TACCTGCTCAGAACACCGGTCT-3', Reverse 5'-GCACATCGCTCAGGATTTCAA-3' [GenBank: NM_022402.2]. qRT-PCR was done using SYBR Green dye. All reactions were performed in duplicate in an ABI PRISM 7000 Sequence Detection System. Data were calculated by the 2-ΔΔCT method and are presented as the fold induction of mRNA for the specific target in diabetic tissues normalized to RPLP0 and compared to control tissues (defined as 1.0-fold). The 2-ΔΔCT method is a standard technique to permit analysis of quantitiative real-time polymerase chain reaction results; this is an approximation technique which assumes similar efficiencies of reactions for both standard and target genes, permitting comparisons of differences in messenger RNA quantities following polymerase chain reactions after baseline measurements.
Data analysis
The presence of multiple doses of pregabalin within each intervention led to the use of two-way repeated unmatched ANOVA measurements followed by Tukey's test were performed for analysis of behavioural studies. We chose to analyze behavioral data based upon comparison of either low (0.051 mg/kg/d), medium (0.51 mg/kg/d) or high dose (2.04 mg/kg/d) pregabalin intervention with either the sham group (for spinal nerve ligation) or non-diabetic cohort group as appropriate. For immunohistochemistry, we chose to analyze the tissues from rodents receiving the highest doses for each intervention with those tissues from rats receiving placebo using mean values-therefore, one-way unmatched ANOVA followed by Tukey's test was used for immunohistochemistry analysis as well. Data collected in the groups were expressed as mean ± standard error in all cases. For immunohistochemistry comparisons demonstrated as low/medium/high intensity, the individual values were compared using unmatched ANOVA testing. Bonferroni corrections were applied as appropriate in all cases.
List of abbreviations
ANOVA: Analysis of variance; CaVα2δ-1: The Alpha-2 Delta-1 protein subunit of the calcium channel; CACNA2D1: The gene and mRNA for the Alpha-2 Delta-1 subunit of the calcium channel; DRG: Dorsal root ganglia; EDTA: Ethylenediaminetetraacetic acid; FITC: Fluorescein isothiocyanate; GFAP: Glial fibrillary acidic protein neurofilaments; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HRP: Horseradish peroxidase; Iba-1: Ionized calcium binding adaptor molecule 1; MAP-2: Microtubule associated protein-2; NF200: Neurofilament 200; PBS: Phosphate Buffered Solution; PMSF: Phenylmethanesulphonylfluoride; qRT-PCR: Quantitative real-time polymerase chain reaction; SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis.
Competing interests
CT receives salary support from the Alberta Heritage Foundation for Medical Research, and also receives research operating funds from the Juvenile Diabetes Research Foundation, The University of Calgary, The University of Calgary Department of Clinical Neurosciences, Pfizer Global, Valeant Canada, and Talecris Canada. CT has received honoraria for medical education seminars and consultancy from Pfizer Global, Valeant Canada, and Talecris Canada.
Authors' contributions
CT conceived of the study, participated in its design and coordination and drafted the manuscript. CT also performed the surgeries and molecular studies related to the project. JM and MK carried out the delivery of agents and performed behavioral testing in all cases, and performed surgeries for harvesting of tissues. LH and WF participated in study design, assisted in performance of radiolabelled studies, and helped to edit the manuscript. All authors read and approved the final manuscript.
Authors' information
CT is the Director of the Neuropathic Pain Clinic, University of Calgary and is a Clinician-Scientist. WHF is the Director of the Alzheimer's Research Centre at the University of Minnesota and is the patent owner for delivery of intranasal compounds for neurodegenerative conditions.
Supplementary Material
Additional file 1
Supplementary Figure 1- Pregabalin Detection. Concentrations of pregabalin within specific regions of the brain and spinal cord in the hours after delivery of either intranasal or intrathecal pregabalin were measured beginning at 72 hours after initiation of either therapy. Intranasal (A) pregabalin delivery led to peak tissue concentrations of pregabalin at about the 74 hour timepoint, 2 hours after most recent intranasal delivery before falling at the 77 hour timepoint; intranasal delivery led to highest concentrations in the cerebral structures. Intrathecal pregabalin delivery (B) led to concentrations that were essentially stable over time and greatest in the lumbar cord region. Doses provided through either intranasal or intrathecal delivery were comparable to medium doses (0.51 mg/kg/d) of pregabalin in later subexperiments. [n = 3-4 rats in each rat cohort for each time point].
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Additional file 2
Supplementary Figure 2-CaVα2δ-1 nerve immunohistochemistry. Co-localized immunohistochemistry pictures for CaVα2δ-1 demonstrated for sciatic peripheral nerve of control (non-diabetic) rat (A) or diabetic rat (B), and spinal nerve of control (no injury) rat (C) or spinal nerve nerve distal to spinal nerve ligation (D). Axonal fibers are demonstrated with neurofilament 200 (NF200) (red), Schwann cells with glial fibrillary acidic protein (GFAP) (green), and CaVα2δ-1 presence is also demonstrated (blue). Axons expressing CaVα2δ-1 appear purple-blue after co-localization of images. A bar graph demonstrates a greater number of CaVα2δ-1 positive sciatic fibers in diabetic nerve with diabetic peripheral neuropathy (DPN) (E) and in spinal nerve at or distal to spinal nerve ligation (SNL) when compared to control rat nerve (F). There was no impact of pregabalin interventions upon expression of CaVα2δ-1 within the peripheral nerve itself distal to the site of traumatic injury when compared to saline delivery (values are mean ± S.E.M., unmatched ANOVA testing between intervention groups, * indicates a significant difference between the control rat group as compared to each of the intervention groups receiving either pregabalin or saline (p < 0.0125 after Bonferroni corrections). Bar = 10 μm.
Click here for file
Additional file 3
Supplementary Figure 3-CaVα2δ-1 dorsal root ganglia immunohistochemistry. Co-localized immunohistochemistry pictures demonstrating DRG neurons of control (non-diabetic) rat (A), diabetic rat (B), control (no injury) rat (C), and DRG ipsilateral to spinal nerve ligation (D). Neurons are demonstrated with neurofilament 200 (NF200) (red), while CaVα2δ-1 presence is also demonstrated (green). Neurons expressing CaVα2δ-1 appear yellow-green after co-localization of images. A bar graph demonstrates a greater number of medium-high CaVα2δ-1 expressing DRG neurons ipsilateral to spinal nerve ligation (SNL) (E) and in diabetic DRG neurons with diabetic peripheral neuropathy (DPN) (F). There was no impact of pregabalin interventions upon expression of CaVα2δ-1 within the DRG neurons themselves when compared with saline interventions (values are mean ± S.E.M., unmatched ANOVA testing between intervention groups, * indicates a significant difference between the control rat group as compared to each of the intervention groups receiving pregabalin or saline (p < 0.0125 after Bonferroni corrections). Bar = 10 μm.
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Additional file 4
Supplementary Figure 4-Central nervous system CaVα2δ-1 protein detection. Western blotting identified no increase in levels of CaVα2δ-1 protein in either the thalamus or sensory cortex for rats exposed to either diabetic peripheral neuropathy (DPN) or spinal nerve ligation (SNL) when compared to control rats. Levels of CaVα2δ-1 protein in the thalamus and sensory cortex were also unchanged with either intrathecal or intranasal delivery of high dose (2.04 mg/kg/d) pregabalin or with saline delivery. β-actin was used as a loading control as displayed.
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Acknowledgements
This project was supported by an unrestricted grant from Pfizer Global. Pregabalin used in this work was generously donated by Pfizer Global.
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1
Institut de Recherche pour le Développement, Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes (URMITE) UMR 198, Campus commun UCAD-IRD de Hann, BP 1386, CP 18524, Dakar, Sénégal
2
Laboratoire Ecologie Vectorielle et Parasitaire, UCAD, Fann Dakar, Sénégal
Paul Rick Edward EditorInstitut Pasteur, France* E-mail: [email protected] and designed the experiments: MON. Performed the experiments: MON SS AG. Analyzed the data: CM. Contributed reagents/materials/analysis tools: LK. Wrote the paper: CM. Substantial improvement of the manuscript: OF JFT. Scientific supervision of the study: CS JFT.
2012 22 2 2012 7 2 e319431 9 2011 20 1 2012 Ndiath et al.2012This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Introduction
The aim of this study was to evaluate the susceptibility to insecticides of An. gambiae mosquitoes sampled in Dielmo (Senegal), in 2010, 2 years after the implementation of Long Lasting Insecticide-treated Nets (LLINs) and to report the evolution of kdr mutation frequency from 2006 to 2010.
Methods
WHO bioassay susceptibility tests to 6 insecticides were performed on adults F0, issuing from immature stages of An. gambiae s.l., sampled in August 2010. Species and molecular forms as well as the presence of L1014F and L1014S kdr mutations were assessed by PCR. Longitudinal study of kdr mutations was performed on adult mosquitoes sampled monthly by night landing catches from 2006 to 2010.
Findings
No specimen studied presented the L1014S mutation. During the longitudinal study, L1014F allelic frequency rose from 2.4% in year before the implementation of LLINs to 4.6% 0–12 months after and 18.7% 13–30 months after. In 2010, An. gambiae were resistant to DDT, Lambda-cyhalothrin, Deltamethrin and Permethrin (mortality rates ranging from 46 to 63%) but highly susceptible to Fenitrothion and Bendiocarb (100% mortality). There was significantly more RR genotype among An. gambiae surviving exposure to DDT or Pyrethroids. An. arabiensis represented 3.7% of the sampled mosquitoes (11/300) with no kdr resistance allele detected. An. gambiae molecular form M represented 29.7% of the mosquitoes with, among them, kdr genotypes SR (18%) and SS (82%). An. gambiae molecular form S represented 66% of the population with, among them, kdr genotype SS (33.3%), SR (55.6%) and RR (11.1%). Only 2 MS hybrid mosquitoes were sampled and presented SS kdr genotype.
Conclusion
Biological evidence of resistance to DDT and pyrethroids was detected among An. gambiae mosquitoes in Dielmo (Senegal) within 24 months of community use of LLINs. Molecular identification of L1014F mutation indicated that target site resistance increased after the implementation of LLINs.
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Introduction
Recently, huge progress has been made in the control of malaria in Sub Saharan African countries [1], [2]. In Senegal, between 2006 and 2009, malaria proportional morbidity fell from 33.57% to 3.1%. During the same period, proportional mortality decreased from 18.17% to 4.4% [3]. These changes followed the introduction of new prevention, diagnostic and treatment polices [4]. As recommended by WHO, control strategy included actions to targeting malaria parasite vectors including indoor residual spraying (IRS), the use of long-lasting insecticide-treated bed nets (LLINs) and the destruction of larvae breeding sites [5]. The major challenge faced by vector control programs is the development of resistance to insecticides [6]. In recent years, the widespread use of insecticides in agriculture [7] but also for bed net treatment [8], [9] contributed to the selection of resistant mosquito strains. Resistance to pyrethroids is a particular threat for malaria control, since they are currently the only recommended and approved insecticides for treating bed nets, primarily because of their low toxicity for humans compared to other pesticides [10].
Mosquitoes' resistance to insecticide has been demonstrated by both in vivo biological test and by the identification of resistance alleles in a vast number of sites across Africa. Especially, kdr mutation genotype has been recognized to be related to DDT and pyrethroid resistance [11]. An. gambiae s.l. and An. funestus are the two major malaria vectors in Dielmo (Senegal) [12]; both have previously been found to be potentially resistant to pyrethroids [13], [14]. The resistance to insecticide has been shown to be locally highly variable even inside a country or a region [15]–[19]. An early detection of resistance is necessary for the implementation of rational vector control programs [20]. It will not be possible to have reliable information without a regular and tight mapping of the resistance status of mosquitoes.
Since 1990, an epidemiological study is ongoing in Dielmo (Senegal) that involves long-term investigations on host-parasite relationships and mechanisms of protective immunity in residents of this Senegalese village [21]. For the first time in Senegal, universal coverage with LLINs (Permanet® 2.0) was implemented in Dielmo in July 2008. After a dramatic decrease in malaria morbidity observed after the implementation of LLINs, a rebound was observed in this village 2 years later [22]. In order to identify the causes for increased morbidity, a study of mosquito susceptibility to insecticide was needed. This paper reports the evolution of the presence of kdr mutation, in Anopheles gambiae s.l., 2 years before and after the implementation of LLINs and the results of resistance tests to 6 frequently used insecticides performed in 2010, 2 years after the implementation of LLINs.
Methods
Mosquito sampling
This study is part of the Dielmo Project that has been described in detail elsewhere [21]. Briefly, the village of Dielmo (13°43N, 16°24W) is located 280 km Southeast of Dakar and about 15 km north of the Gambian border in an area of Sudan-type savannah. About 400 inhabitants are living in the village. Rainfall occurs during a four-month period, from June to October. Dielmo is situated on the marshy bank of a small permanent stream, with anopheles larval sites present all year round.
Adult mosquitoes were collected by human landing catches (HLC) monthly from July 2006 to December 2010. Night captures (7:00 PM–7:00 AM) were conducted once or more times each month in two indoor and two outdoor sites. In each site, two trained collectors (adult male volunteers) worked alternatively for one hour and rested for one hour. Anopheline identification was performed following the Gillies and DeMeillon morphologic identification keys [23]. Mosquitoes belonging to the Anopheles gambiae sensus lato (s.l.) group were stored for following steps.
In August 2010, during the rainy season, immature stages of An. gambiae s.l. were collected from 10 breeding sites situated in and around the village (river, rain pools and cattle watering places). Larvae were pooled and fed with Tetramin® baby fish food locally until emergence. Unfed 2–3 days female An. gambiae s.l. mosquitoes were used for insecticide susceptibility tests.
Susceptibility test
Bioassays were carried out using WHO test kits for adults mosquitoes [24] with six insecticides of technical grade quality: one belonging to the Carbamate group (0.1% Bendiocarb), one Organophosphate (1% Fenitrothion), 3 pyrethroids (0,05% Lambda-cyhalothrin, 0.05% Deltamethrin, 0,75% Permethrin) and one Organochlorine (4% DDT). Impregnated papers were obtained from the WHO reference center (Vector Control Research Unit, University Sains Malaysia, Penang, Malaysia). Tests were performed with batches of 25 An. gambiae s.l., with four batches tested against each insecticide. Mosquitoes were exposed to insecticide-impregnated filter paper for 1 hour at 25–27°C and 80% relative humidity. The number of knockdown mosquitoes was recorded at 10, 15, 20, 30, 40, 50, 60 and 80 min. After exposure, mosquitoes were kept in observation tubes and supplied with a 10% sugar solution. Mortality was recorded after 24 hours. The mortality of a control stain of An. gambiae (Yaoundé known to be 100% susceptible to all tested insecticides [25], [26]) was studied as a positive control. Batches exposed to untreated papers were used as negative control. Since mortality in negative controls was always <5%, no adjustment was performed for treated batches. For each insecticide, a sample of 50 An. gambiae s.l. specimens was randomly selected, including equal numbers of dead and surviving specimens (when available) and used for molecular tests.
Molecular identification and kdr genotyping
In the subsample of mosquitoes used for bioassay and in adults sampled by HLC during the longitudinal study, detection of L1014F and L1014S kdr mutations (thereafter identified as kdr-w and kdr-e respectively) was performed by PCR [27], [28]. Mosquitoes used for bioassay were identified down to their species and molecular form with the PCR RFLP method [29].
Data analysis
WHO (1998) criteria were used to evaluate the resistance/susceptibility status of the tested mosquito populations (<80% mortality: resistance, 80–98% mortality: increased tolerance, >98% mortality: susceptibility) [24]. Fifty and 95 percent knockdown times (respectively KDT50 and KDT95) were computed with probit regression models. Rates were compared using Fisher exact and Pearson Chi2 tests. Statistical analyses were performed using Stata 10.1 software. A P value of 0.05 or less was considered as significant.
Ethics approval
The Dielmo project was initially approved by the Ministry of Health of Senegal and the assembled village population. Approval was then renewed on a yearly basis. Audits were regularly conducted by the National Ethics Committee of Senegal and ad-hoc committees of the Ministry of Health, the Pasteur Institute and the Institut de Recherche pour le Développement.
Results
Kdr genotype dynamic in adult An. gambiae s.l.
From July 2006 to December 2010, no specimen with L1014S (kdr-e) mutation was identified.
The repartition of kdr genotype during the study period is presented in Figure 1. Before the implementation of LLINs, L1014F allelic frequency was low and not different when comparing 24-13 months and 12-0 months before periods (2.0 and 3.5% respectively, Chi2 = 1.4, p = 0.24). This rate significantly increased to 4.6% within the first 12 months that followed the distribution of LLINs (Chi2 = 4.4, p<0.05 vs. pre-implementation) and again 13–30 months after to 18.7% (Chi2 = 70, p<0.001 vs. preceding period) (Figure 1).
10.1371/journal.pone.0031943.g001Figure 1 kdr mutation in An. gambiae before and after the implementation of LLINs.
Proportion (and 95% CI) of An. gambiae with L1014F homozygote mutation (RR), heterozygote mutation (RS) or wild type (SS) sampled 24-13 months (n = 228) and 12-0 months (n = 99) before the implementation of long lasting insecticide-treated nets (LLINs) in July 2008, 0–12 months (n = 327) and 13–30 months after (n = 582).
Sensitivity to insecticides in 2010
Mortality data indicated that mosquitoes were highly resistant to 4 of the 6 insecticides tested including DDT and all Pyrethroids (Deltamethrin, Lambda-cyhalothrin and Permethrin). Mortality rates ranged from 46 to 63%, far below the susceptibility limit of 80% (Table 1). Mosquitoes were totally susceptible to Fenitrothion (Organophosphate) and Bendiocarb (Carbamate) with a 100% mortality observed for both insecticides.
10.1371/journal.pone.0031943.t001Table 1 Bioassay susceptibility tests in 2010.
Mortality % 95% CI KDT50
95% CI KDT95
95% CI
Deltamethrin 0,05%
63 [53.5–72.5] 28.0 [25.3–30.7] na -
Lambda-cyhalothrin
60 [50.3–69.7] 43.6 [40.9–46.3] na -
Permethrin 0,75%
46 [36.2–55.8] 48.7 [45.5–51.9] na -
DDT 4%
61 [51.4–70.6] 64.6 [58.1–71.0] na -
Fenitrothion
100 - 70.4 [67.1–73.7] na -
Bendiocarb 0,1%
100 - 17.5 [16.4–18.5] 39.0 [37.9–40.0]
Mortality rate (%) 24 hours after exposition, 50 and 95% knockdown (KDT50 KDT95) time (min) with 95% confidence interval (CI), obtained on 100 An. gambiae for each insecticide tested. na: not applicable, 95% knock down time exceeded 80 min.
Knockdown time 50 (KDT50) was higher than 40 minutes for Lambda-cyhalothrin, Permethrin, DDT and Fentrothion and KDT95 exceeded the 80-min observation period (Table 1). Knockdown time was shorter for Deltamethrin (KDT50 = 28.0 min) and even shorter for Bendiocarb (KDT50 = 17.5 and KDT95 = 39.0 min).
In the 300-specimens sample selected for molecular analysis among dead and surviving mosquitoes, 11 (3.7%) were An. arabiensis, 89 (29.7%) An. gambiae s.s. molecular form M, 2 (0.7%) MS hybrids and 198 (66.0%) form S. When comparing the species and molecular forms of An. gambiae s.l. among dead and surviving mosquitoes, no association could be identified for Deltamethrin, Lambda-cyhalothrin, Permethrin and DDT (Fisher exact test p>0.2, Table 2). This analysis could not be performed for Fenitrothion and Bendiocarb since all mosquitoes died.
10.1371/journal.pone.0031943.t002Table 2 Molecular forms of An. gambiae s.l. among dead and surviving mosquitoes after insecticide exposure.
Molecular form of An. gambiae s.l.
Fisher exact test p
An. arabiensis
M MS S
Deltamethrin 0,05%
Dead n = 25 4% (1) 36%(9) 0%(0) 60%(15) 0.551
Survivors n = 25 0%(0) 28%(7) 0%(0) 72%(18)
Lambda-cyhalothrin
Dead n = 25 4%(1) 12%(3) 0%(0) 84%(21) 0.289
Survivors n = 25 0%(0) 28%(7) 0%(0) 72%(18)
Permethrin 0,75%
Dead n = 25 0%(0) 20%(5) 0%(0) 80%(20) 0.702
Survivors n = 25 0%(0) 12%(3) 0%(0) 88%(22)
DDT 4%
Dead n = 25 12%(3) 44%(11) 0%(0) 44%(11) 0.385
Survivors n = 25 4%(1) 32%(8) 4%(1) 60%(15)
Fenitrothion
Dead n = 50 4%(2) 42%(21) 2%(1) 52%(26) -
Bendiocarb 0,1%
Dead n = 50 6%(3) 30%(15) 0%(0) 64%(32) -
Total
n = 300 3.7% (11) 29.7% (89) 0.7% (2) 66.0% (198)
Proportion and number of mosquitoes belonging to An. arabiensis specie and An. gambiae s.s. molecular form M, MS and S assessed after insecticide sensitivity in both dead and surviving (when available) mosquitoes.
Kdr mutations and resistance phenotype
Among the 300 surviving and dead specimens selected for kdr-w identification, 152 (50.7%) were SS, 126 (42%) SR and 22 (7.3%) RR kdr-w genotype (Table 3). No specimen presented the kdr-e mutation. There was a significant difference in kdr-w genotype between dead and surviving mosquitoes for DDT and all Pyrethroids (Fisher exact p ranging from 0.041 to 0.002). R allelic frequency was significantly higher in survivors for each insecticide (Fisher exact p≤0.001). No RR genotype was identified among dead mosquitoes after DDT or pyrethroids exposure (Table 3). Among survivors, 70% of specimens presented a mutated allele; 30% had a resistant phenotype although they did not present kdr mutation.
10.1371/journal.pone.0031943.t003Table 3 kdr-w mutation genotypes and allelic frequencies among dead and surviving mosquitoes after insecticide exposure.
Genotype Fisher exact test p Allelic frequency Fisher exact test p
SS SR RR S R
Deltamethrin 0,05%
Dead n = 25 64% (16) 36%(9) 0% (0) 0.002 82% (41) 18% (9) <0.001
Survivors n = 25 28% (7) 40% (10) 32% (8) 48% (24) 52% (26)
Lambda-cyhalothrin
Dead n = 25 60% (15) 40% (10) 0% (0) 0.013 80% (40) 20% (10) 0.001
Survivors n = 25 24% (6) 68% (17) 8% (2) 58% (29) 42% (21)
Permethrin 0,75%
Dead n = 25 52% (13) 48% (12) 0% (0) 0.002 76% (38) 24% (12) <0.001
Survivors n = 25 20% (5) 52% (13) 28% (7) 46% (23) 54% (27)
DDT 4%
Dead n = 25 76% (19) 24% (6) 0% (0) 0.041 88% (44) 12% (6) <0.001
Survivors n = 25 48% (12) 36% (9) 16% (4) 66% (33) 34% (17)
Fenitrothion
Dead n = 50 50% (25) 50% (25) 0 - 75% (75) 25% (25) -
Bendiocarb 0,1%
Dead n = 50 68% (34) 30% (15) 2% (1) - 82% (83) 17% (17) -
Total
n = 300 50.7% (152) 42% (126) 7.3% (22) - 71.7% (430) 28.3% (170) -
Proportion and number of mosquitoes with kdr-w genotype SS (sensitive, sensitive), SR (resistant, sensitive) and RR (resistant, resistant) and corresponding allelic frequency assessed after insecticide sensibility in both dead and surviving (when available) mosquitoes.
The frequency of kdr-w mutation was significantly different according to the molecular form of An. gambiae (Fisher exact test p<0.001, Table 4). Molecular form S had a specific kdr-w genotype compared to An. arabiensis and An. gambiae s.s. M form (Fisher exact test p<0.001 in both cases). Allelic form R was totally absent in An. arabiensis and in An. gambiae MS form. In An. gambiae M form, SR genotype was present (18%) and RR genotype was absent. Allele R frequency was 38.9% for molecular form S vs. 7.8% in the other groups (Fisher exact test p<0.001).
10.1371/journal.pone.0031943.t004Table 4 kdr-w genotypes and allelic frequencies among the different molecular forms of An. gambiae s.l.
Molecular form of An. gambiae
An. arabiensis
M MS S
Kdr-w mutation
SS
100% (11) 82.0% (73) 100% (2) 33.3% (66)
SR
0% (0) 18.0% (16) 0% (0) 55.6% (110)
RR
0% (0) 0% (0) 0% (0) 11.1% (22)
Allelic frequency
S
100% (22) 91.0% (162) 100% (4) 61.1% (242)
R
0% (0) 9.0% (16) 0% (0) 38.9% (154)
Proportion and number of mosquitoes belonging to An. arabiensis species and An. gambiae s.s. molecular form M, MS and S and presenting kdr-w mutation genotypes SS, SR, and RR (Fisher exact test p<0.001) and corresponding allelic frequency.
Discussion
The results of this study demonstrated that field population of An. gambiae s.l. display a high biological level of resistance to DDT and pyrethroids (Deltamethrin, Lambda-cyhalothrin and Permethrin). Similar resistance has been observed all around Africa but little information was previously published about Senegal. Investigations on the biological susceptibility to DDT performed in sentinel sites of Senegal reported a resistance to DDT in 4/10 sites [26] in 2008 and in 11/15 sites in 2010 [25]. In Africa, the resistance to DDT is widespread [15]–[18], [30] with mortality rate as low as 0% in RDC [31]. On the other hand, total susceptibility to DDT was observed in other countries [19], [32] or even in other regions of the same countries [30]. In regions where mosquitoes are still relatively susceptible to DDT, KDT50 is short (6–26 min) [19], whereas in regions where specimens are highly resistant to DDT, KDT50 is longer (more than the 80-min observation period) [18]. In our study, although resistance was detected, according to the WHO criteria, mortality rates as well as KDT50 were at an intermediate level.
While the resistance to pyrethroids was limited in 2008 in Senegal (detected in 0/10 sentinel sites for Deltamethrin, 2/11 for Lambda-cyhalothrin and 4/10 for Permethrin [26]), it was found to be widespread in 2010 (detected in 9/15 sites for Deltamethrin, 10/15 for Lambda-cyhalothrin and 12/15 for Permethrin [25]). Resistance to pyrethroids has been reported in various African countries [16]–[18], [30], [33]. Whilst full susceptibility to pyrethroids is still reported in other countries [19] or even in other areas of the same countries [18], [30], [34]. KDT50 was 49 min in our study, slightly longer than that observed in Dakar in 1995 when susceptibility was higher (77% mortality vs. 46% in our study) [14]. In studies where various pyrethroids were tested, cross-resistance or increased tolerance to all pyrethroids was confirmed [16], [17]. In our study, a cross-resistance to all pyrethroids tested was observed with low mortality rates.
In this study, the presence of kdr-w mutation was detected in An. gambiae s.s.; kdr-e mutation was not identified in any tested taxa. The presence of kdr mutations have been studied all around Africa [33]. While, kdr-w mutation that was initially described in Cote d'Ivoire, has been detected as far East as Uganda, kdr-e that originated in Kenya have spread into Central Africa (for review see [33]) and have recently been found in Benin [35]. Until now, kdr-e mutation has never been detected in Senegal. On the other hand, kdr-w mutation has already been observed in 2005–2006 in Senegal at a rate of 9–12% in Dakar [36] and 19% in Kedougou (Western Senegal) [37] that was lower than that observed in our study (28%). In recent studies, the allelic R frequency was found to be higher in Ghana [17], similar in RDC [31] and lower in Guinea Conakry [15]. In this study, the presence of kdr-w mutation has been shown to precede the implementation of LLINs but their rate significantly increased after.
Resistance to pyrethroids and DDT in An. gambiae is known to associate closely with kdr-w [11], [14], [27]. In our study, the frequency of the kdr-w allele was significantly higher in resistant-selected samples confirming the association between kdr-w mutation and the resistance phenotype to DDT and all pyrethroids tested. Moreover, a similar level of resistance was observed with DDT and all pyrethroids. Therefore a mutation of the sodium channel, that is the common target of both DDT and Pyrethroids, is likely to be involved in the observed resistance. However, 30% of specimens found among survivors presented the wild homozygote genotype. These findings support the hypothesis that target mutation is only one of the mechanisms implicated in insecticide resistance [11] and that metabolic resistance likely occurs in the An. gambiae population of Dielmo.
In our study, the presence of kdr-w mutation was mainly found in S molecular form of An. gambiae. It was absent in An. arabiensis and in the small sample of MS hybrids (4 specimens). Interestingly, kdr-w mutation was identified at a low rate (9%) in the M molecular form. Many studies reported the high frequency of kdr-w mutation in molecular form S in Western and Central Africa and its low frequency or absence in molecular S form (see [37] for review or recent studies [17], [31]). The kdr-w mutation, in molecular form S, has therefore spread or occurred west of the 5°W limit identified by Santolamazza et al.
[37]. It has been hypothesized that the difference in kdr-w mutation frequency in both molecular forms was related to a different origin of the mutation in the two populations or linked to different ecological or behavioral characters between M and S forms [37].
The use of pyrethroids as pesticides for agriculture and for net treatment have both been recognized as factors responsible for the selection of resistant mosquitoes in Sub-Saharan Africa [7]–[9]. Near Dielmo, although some gardening and rice culture are performed, the use of pesticide is limited. Therefore, agriculture probably had a limited role in the emergence of the resistance to insecticides in this area. In Kenya, a lower susceptibility of An. gambiae to Permethrin was found in villages where Permethrin impregnated nets were implemented for one year as compared to villages without nets. The mechanism involved was postulated to include kdr mutation together with metabolic resistance [38]. In another study, an increased frequency of kdr was observed in villages with nets [39]. Insecticide treated nets were implemented in Dielmo in July 2008 as a part of the vector control study. We speculate that this may have contributed to the selection of pyrethroids resistant strains of An. gambiae.
In Dielmo, mosquitoes have been found to be totally susceptible to Bendiocarb, an insecticide belonging to the Carbamate class and to Fenitrothion (Organophosphate class) since we observed 100% mortality. Susceptibility of An. gambiae s.l. populations to Fenithrothion was also observed in all the sentinel sites in Senegal in 2008 [26] and 2010 [25]. On the other hand, a resistance to Bendiocarb appeared between 2008 and 2010 in 2/15 sentinel sites [25], [26]. In other areas, An. gambiae s.l. presented a resistance [15]–[17] or an increased tolerance [31] to Carbamates and increased tolerance to Malathion, another member of the Oraganophosphate class [15], [17]. In addition to pyrethroids, Carbamates (Bendiocarb and Propoxur) or Organophosphates (Fenitrothion, Malathion and Pirimiphos-methyl) are recommended by the WHO for IRS [40]. Until now, no IRS has been performed in Dielmo as part of the vector control strategy. Since complete susceptibility to Carbamates and Organophosphates has been detected in Dielmo, these insecticides should be used in priority if IRS were to be performed in this area. The reduced effectiveness of insecticides coincides with an important international effort to increase bed net coverage in African Countries in order to control malaria transmission. Notably, in Senegal 6 millions insecticide-treated nets were freely distributed to the populations between 2005 and 2010 [41], [42]. Resistance to pyrethroids is worrying, since it is the only class of insecticide safe enough to be recommended for treatment of bed nets. It appears to have a significant impact on net or IRS efficacy [43]. In Dielmo, we have recently demonstrated a rebound and age shift in malaria cases two years after the implementation of nets [22]. Since resistance of mosquitoes to several insecticides is reported in various sentinel sites in Senegal, planning alternative strategies for vector control should begin. Indeed, in Senegal, the insecticide vector resistance management started in 2011 by shifting from Deltamethrin to Bendiocarb for IRS in six selected districts. This study underlines the need to carefully document resistance and its impact on the efficacy of interventions.
In conclusion, this study demonstrated an increased frequency of kdr mutation in An. gambiae after the implementation of LLINs in Dielmo (Senegal). This coincided with a cross-resistance to DDT and all pyrethroids observed in 2010. Resistance was associated with a higher kdr-w allele frequency in surviving specimens. Moreover, kdr-w mutation was detected in both M and S molecular forms of An. gambiae and significantly more frequently in molecular form S. On the other hand, mosquitoes were fully sensitive to Bendiocarb and Fenitrothion.
We thank Charles Bouganali and Babacar Ndiouck for their technical assistance, Dr S. Clarke for English correction and helpful comments, the villagers in Dielmo for their participation in the study for 20 years.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by the French Ministry of Research and the Department Support and Formation of the south communities of the Research Institute for the Development (IRD). The funders had no role in study design, data collocation and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22396731PONE-D-11-1844010.1371/journal.pone.0031256Research ArticleBiologyPopulation BiologyMedicineClinical Research DesignGastroenterology and HepatologySurgeryPretransplant Prediction of Posttransplant Survival for Liver Recipients with Benign End-Stage Liver Diseases: A Nonlinear Model A Predictive Model for Liver TransplantsZhang Ming
1
2
Yin Fei
3
Chen Bo
4
Li You Ping
2
Yan Lu Nan
1
Wen Tian Fu
1
Li Bo
1
*
1
Liver Transplantation Center, West China Hospital, Sichuan University Medical School, Chengdu, People's Republic of China
2
Chinese Cochrane Center and Chinese Evidence-Based Medicine Center, West China Hospital, Sichuan University Medical School, Chengdu, People's Republic of China
3
Department of Biostatistics, West China School of Public Health, Sichuan University, Chengdu, People's Republic of China
4
Department of Medical Informatics, West China Hospital, Sichuan University Medical School, Chengdu, People's Republic of China
Man Kwan EditorThe University of Hong Kong, Hong Kong* E-mail: [email protected] and designed the experiments: MZ FY YPL BL. Performed the experiments: MZ FY BC LNY TFW BL. Analyzed the data: MZ FY BC BL. Contributed reagents/materials/analysis tools: MZ BC LNY TFW BL. Wrote the paper: MZ FY.
2012 1 3 2012 7 3 e3125620 9 2011 5 1 2012 Zhang et al.2012This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
The scarcity of grafts available necessitates a system that considers expected posttransplant survival, in addition to pretransplant mortality as estimated by the MELD. So far, however, conventional linear techniques have failed to achieve sufficient accuracy in posttransplant outcome prediction. In this study, we aim to develop a pretransplant predictive model for liver recipients' survival with benign end-stage liver diseases (BESLD) by a nonlinear method based on pretransplant characteristics, and compare its performance with a BESLD-specific prognostic model (MELD) and a general-illness severity model (the sequential organ failure assessment score, or SOFA score).
Methodology/Principal Findings
With retrospectively collected data on 360 recipients receiving deceased-donor transplantation for BESLD between February 1999 and August 2009 in the west China hospital of Sichuan university, we developed a multi-layer perceptron (MLP) network to predict one-year and two-year survival probability after transplantation. The performances of the MLP, SOFA, and MELD were assessed by measuring both calibration ability and discriminative power, with Hosmer-Lemeshow test and receiver operating characteristic analysis, respectively. By the forward stepwise selection, donor age and BMI; serum concentration of HB, Crea, ALB, TB, ALT, INR, Na+; presence of pretransplant diabetes; dialysis prior to transplantation, and microbiologically proven sepsis were identified to be the optimal input features. The MLP, employing 18 input neurons and 12 hidden neurons, yielded high predictive accuracy, with c-statistic of 0.91 (P<0.001) in one-year and 0.88 (P<0.001) in two-year prediction. The performances of SOFA and MELD were fairly poor in prognostic assessment, with c-statistics of 0.70 and 0.66, respectively, in one-year prediction, and 0.67 and 0.65 in two-year prediction.
Conclusions/Significance
The posttransplant prognosis is a multidimensional nonlinear problem, and the MLP can achieve significantly high accuracy than SOFA and MELD scores in posttransplant survival prediction. The pattern recognition methodologies like MLP hold promise for solving posttransplant outcome prediction.
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Introduction
Orthotopic Liver transplantation (OLT) has become an established treatment approach for patients with benign end-stage liver diseases (BESLD, i.e. non-neoplastic diseases), but the growing scarcity of grafts compared to numbers of waiting patients, coupled with the high cost of this procedure, make it imperative to make difficult decisions about how to distribute such scarce organs [1]–[3], and highlight the need to identify patients likely to have relatively good outcomes after transplantation [4]–[6]. This need is particularly acute in the Asia-Pacific region, where the carrier rate of hepatitis B virus (HBV) is estimated at 20%–30% [7], [8] and large numbers of BESLD patients with HBV-related cirrhosis and severe hepatitis B need OLT. Under such circumstances, the ideal allocation system would allocate livers to candidates who are most likely to die without a transplant, but who also have a high probability of survival after OLT. The balanced application of a model for liver transplant outcome estimation, in concert with a model for end-stage liver disease (MELD) estimating disease severity, would improve transplant outcomes and maximize patients' benefit from OLT [9].
In order to incorporate likely posttransplant prognosis into decisions about grafts allocation, and to facilitate informed decision-making by potential transplant recipients and their relatives [10]–[12], it is necessary to accurately assess the likelihood of posttransplant survival based on information that is available before transplantation.
Although there have been some attempts to develop a model that meets this requirement, most lacked sufficient discriminating accuracy or simply stratified the prognostic risk [4], [6], [9], [11]–[14]. One major reason for this is inappropriate choice of modeling method [13]. Survival prognosis is a complex nonlinear relationship affected by many interactive factors, especially for a complicated organ transplantation procedure; however, most current models were developed by linear methods, such as multiple regression.
Artificial neural network (ANN) is a computer-based nonlinear data mining mode that can recognize relationships between a series of independent variables and the corresponding dependent variable. It is more successful than traditional linear methods when the prognostic effect of a variable is influenced by other variables in a complex multidimensional nonlinear function, or when the importance of a given prognostic variable is expressed as a complex unknown function of the value of the variable [15], [16]. Thus, ANN is particularly suited to modeling complex multidimensional patterns [17], [18], and has had remarkable success in many medical problems that are too complicated for linear models [15], [19], [20]. To date, there have been a few attempts to use ANN for outcome prediction after organ transplantation [17], [21], [22], but no reliable ANN model had been developed specifically for BESLD recipients.
We investigated the feasibility of using multi-layer perceptron (MLP), arguably one of the most efficient ANN for prognostic research [22], [23], to develop a prognostic model to predict individualized survival probability after deceased donor OLT in recipients with BESLD, employing typically available, objective preoperative characteristics. Furthermore, we evaluated and compared the predictive accuracy of this MLP network with a BESLD-specific prognostic model (MELD) and a general-illness severity prognostic model (the sequential organ failure assessment score, or SOFA score).
Methods
Data source
Between February 1999 and August 2009, 386 adults with BESLD received deceased-donor (either no heartbeat or brain dead) liver transplants at the 4300-bed West China Hospital of Sichuan University. We excluded 15 recipients with combined organ transplants or partial organs and 11 recipients with incomplete follow-up records. The remaining 360 transplants were involved in this study and followed up by August 31, 2010. Maintenance immunosuppression initially consisted of a triple-drug regimen that included either tacrolimus or cyclosporine, mycophenolate, and prednisone; and that recipients were eventually weaned to dual or single agent.
We extracted demographic characteristics of donors and recipients, pretransplant clinical records (Tables 1 and Table S1), and recipients' follow-up information form the electronic database of the liver transplantation center at West China Hospital. Surgical and some donor factors were not included in the model development, since they could not have been known when recipients decided whether to undergo OLT and were ranked on the waiting list. All included data were taken from the most recent examinations prior to transplantation, since they reflected the current medical condition of the candidate at time of transplantation.
10.1371/journal.pone.0031256.t001Table 1 Baseline quantitative characteristics of the training set and validation set.
Variables Training set (N = 290) Validation set (N = 70)
P-Value
Donor characteristics
D-Age (yr) 38.12±12.33 37.88±11.99 0.883
D-BMI 23.95±5.84 24.89±5.16 0.218
cold ischemia times (hour)*
8.65±4.16 8.92±4.03 0.624
warm ischemia times (min)*
9.67±2.78 9.26±2.65 0.265
Recipient characteristics
Age (yr) 45.25±10.32 44.88±10.18 0.787
BMI 20.06±4.41 20.18±4.35 0.838
HB (g/dl) 9.58±5.36 9.03±5.11 0.437
WBC (×109) 6.36±4.66 7.05±4.69 0.268
PLT (×109) 83.86±85.58 79.99±81.56 0.732
ALB (g/dl) 3.06±0.65 2.89±0.55
0.044
TB (mg/dl) 13.81±14.66 12.39±13.46 0.461
ALK (u/l) 161.46±186.61 172.11±185.32 0.668
GGT (u/l) 102.87±184.35 97.56±173.23 0.827
AST (u/l) 124.62±176.05 149.76±175.69 0.284
ALT (u/l) 113.06±191.98 143.25±183.21 0.234
BUN (mmol/l) 20.55±16.86 21.02±16.36 0.833
Crea (mg/dl) 1.19±1.16 1.26±1.15 0.650
APTT (s) 57.93±25.69 62.57±25.43 0.175
INR 2.15±1.28 2.49±1.26
0.046
Na+ (mmol/l) 130.97±9.65 129.56±9.47 0.272
SOFA scores*
8.69±1.76 8.72±1.68 0.897
* Ischemia times and recipients' SOFA scores were not used as candidate factors. BMI = body mass index; HB = hemoglobin; WBC = white blood cell; PLT = platelet; BUN = blood urea nitrogen; Crea = creatinine; ALB = albumin; TB = total bilirubin; ALK = alkaline phosphatase; GGT = γ-glutamyltransferase; AST = aspertate aminotransferase; ALT = alanine aminotransferase; INR = international normalized ratio of prothrombin time; APTT = activated partial thromboplastin time; Na+ = serum sodium; N/A = not applicable.
All organ donations recorded in the electronic database were contributed voluntarily, and no grafts were obtained from executed prisoners or other institutionalized persons. All of the donors or their families had provided written, valid informed consent for donation before the organs were procured. Each liver donation and transplantation in our center was approved by the Medical Ethics Committee of West China Hospital, Sichuan University, and the study protocol was carried out in accordance with the Declaration of Helsinki.
Dataset division
A data-splitting approach was used in this study. The recipients were randomly divided into a modeling set (80% of the total sample, 290 recipients) used to construct the MLP network, and a validation set (20% of the total sample, 70 recipients) used to assess the models' predictive accuracy; the validation samples would not be involved in the model development. The modeling set was randomly re-divided into a general training set (80% of the modeling set, 232 recipients) and a cross validation set (20% of the modeling set, 58 recipients) to perform the internal cross validation in MLP training.
Statistical analysis
Continuous variables were reported as mean ± standard deviation and compared using Student's t test; categorical variables were reported as numbers and percentages, modeled as dummy variables, and compared using the chi-square test. A value of P<0.05 was considered significant in all the analyses. All analyses, except the MLP development, were carried out using SAS 8.0.
MELD and SOFA scores calculation
The BESLD-specific illness severity was evaluated by the MELD and MELD-Na+ scores, which were calculated according to the following formulas: MELD = 3.78×loge TB (mg/dl)+11.20×loge INR+9.57×loge Crea (mg/dl)+6.4 [24], MELD-Na+ = MELD - Na+−(0.025×MELD×(140−Na+))+140 [25].
The general illness severity was assessed by the SOFA score, which is composed of scores from six organ systems (respiratory, coagulation, liver, cardiovascular, renal, and neurological) graded from 0 to 4 points according to normal function or the degree of dysfunction [26] (Table 2).
10.1371/journal.pone.0031256.t002Table 2 Sequential Organ Failure Assessment (SOFA) score.
Variables/score 0 1 2 3 4
PaO2/FiO2 (mmHg) >400 ≤400 ≤300 ≤200 ≤100
Platelets (×103/uL) >150 ≤150 ≤100 ≤50 ≤20
Bilirubin (mg/dL) <1.2 1.2–1.9 2–5.9 6–11.9 >12
Cardiovascular (Hg/kg/min) – MAP<70 Dop≤5 Dop>5(Epi≤0.1) Epi>0.1
Glasgow Coma Scale 15 13–14 10–12 6–9 <6
Creatinine (mg/dL) <1.2 1.2–1.9 2–3.4 3.5–4.9 >5
MAP = mean arterial pressure; Dop = dopamine; Epi = epinephrine.
The MLP network development
An MLP consists of a densely interconnected set of units. In this study, we developed a three-layer network which not only can approximate any reasonable function to any degree of required precision as long as the hidden layer is large enough, but also has an advantage in computing speed compared to multiple hidden layer networks [27]. The concept of a neuron is a high-level abstraction that encompasses both certain values and a set of operations that are performed on those values, and neurons are tied together with weighted connections. The MLP was developed using STATISTICA 8.0.
Determination of input neurons
We performed the forwards stepwise selection algorithm to screen and identify the input feature variables from the candidate variables (Table 1 and Table S1), in which quantitative variables were assigned one-to-one to the neurons and each sub-category of every categorical variable was defined as an input neuron. All input quantitative variables were scaled linearly between 0 and 1.0 using the following transformation formula, where min{xij} and max{xij} were the minimum and maximum values of the variable. The input categorical variables were entered as dummy variables.
Determination of output neuron
The probability of survival at posttransplant one year and two years was entered as continuous output on the interval 0–1, in which 0 represents death and 1 represents survival, so the MLP output values represent the probability of posttransplant recipient survival. Survival was chosen as the outcome endpoint because it is the most reliable and unbiased variable in the prognostic research [28].
Determination of hidden neurons and network transfer function
The hidden neurons calculate the weighted sum of inputs from the input neurons and produce the output result through an activation algorithm (i.e. transfer function). The weights are adjusted based on the training data in order to minimize the error estimate function [29]. Therefore, the approximate number of hidden neurons and the corresponding transfer function are closely related to the predictive accuracy of the network. In this study, the number of hidden neurons varied from two to 35, and the alternative transfer functions included identity, logistic, tanh, exponential, gaussian and softmax. We applied the enumerative combinatory method to exhaustively evaluate all possible combinations of hidden neuron numbers and transfer functions, then identified the combination with the best predictive accuracy.
Cross-validation
Experiments have verified that the predictive accuracy of an MLP initially increases with the number of training iterations, but starts deteriorating after a critical point, because the network becomes over-fitted to recognize specific cases rather than learning general characteristics [27]. One effective and widely-accepted way to prevent this over-fitting is to use cross-validation to stop the training at the point of maximum generalization.
Network training process
The training rule used in this MLP was supervised, feedforward, back-propagation of error, which could adjust the internal parameters of the network over repeated training iterations to improve the overall accuracy, by modifying the weight of the connections between neurons. In detail, once an input variable is applied as a stimulus to the input layer, it is propagated through hidden layer until an output is generated; this output is then compared with the desired output and an error signal is calculated; this error signal is then transmitted backwards across the net and the weight of the connections between neurons is updated to decrease the overall error of the network; as training proceeds, the difference between the network output and the desired output decreases to a minimum [30].
Model Validation
The performances of the MLP, SOFA score, and MELD score in predicting survival at posttransplant one year and two years were assessed in a validation set by measuring both calibration and discrimination ability [31]. We chose these two intervals because outcome at posttransplant one year could reflect surgical and perioperative risk [4], and outcome at two years could also capture mortality associated with most transplant complications, such as rejection and biliary stricture. Calibration refers to the degree of correspondence between predicted and actual survival probabilities. In this study, we used goodness-of-fit testing to evaluate calibration by the Hosmer-Lemeshow test [32], in which the χ
2 statistic is the sum of the squared differences between actual and predicted survival probability. Discrimination is usually assessed by the area under a receiver operating characteristic (ROC) curve [33], which is equal to the index of concordance (i.e., c-statistic). The ROC analysis was also performed to measure the sensitivity, specificity, positive predictive value, negative predictive value, and the total accuracy of these three predictive models.
Results
Outcomes of the entire series of recipients
Of the 360 DDLT recipients, the mean time on the waiting list was 9.16±3.56 months, and the median follow-up period was 56.23±26.46 months. The overall 6-month, 1-, 2-, 3- and 5- year survival rates were 89.6%, 86.1%, 82.9%, 78.2% and 73.1%, respectively. Of the 360 recipients, 89 recipients (24.7%) died during the 5-year follow-up period. Of these, 23 (6.4%) died within the first 3 months after transplantation of various perioperative causes, including severe fungal infection or sepsis (n = 6), multiple organ failure (n = 4), hepatic artery thrombosis (n = 3), acute rejection (n = 3), primary graft dysfunction (n = 2), upper gastrointestinal bleeding (n = 2), graft versus host disease (n = 2), and subarachnoid hemorrhage (n = 1). 57 (15.8%) recipients died for chronic graft dysfunction with different causes, such as the HBV or HCV recurrence, biliary complications, pathologically-proven chronic rejection, and hepatic vein stenosis, etc. The remaining 9 recipients (2.5%) died of other causes in long-term follow-up, including severe fungus infection or sepsis (n = 3), de novo cancers (n = 2), multi-organ failure (n = 2), respiratory failure (n = 1), cerebral hemorrhage (n = 1).
Recipients' baseline characteristics
Table 1 and table S1 showed the baseline characteristics of the modeling set and validation set. Most of the characteristics between the two sets have no differences, but we also observed significant differences in the percentage of HBV-DNA level, as well as in the mean values of ALB and INR between the modeling and validation set.
MLP input features selection
Two donor factors and ten recipient factors were identified as optimal input features by the forwards stepwise selection algorithm: donor age and BMI; serum concentration of HB, Crea, ALB, TB, ALT, INR, Na+; presence of pretransplant diabetes; dialysis prior to transplantation, and microbiologically-proven sepsis. As each sub-category of every categorical variable is an input neuron, there are 18 input neurons in the MLP network.
Training and development of the MLP network
By enumerative combinatory method and making many iterations of training and cross-validation in each combination, we identified 12 hidden neurons that optimally delineated the network and produced the best performance in both one- and two-year intervals. The most appropriate transfer functions were Logistic, Gaussian for one-year network, and Exponential, Identity for two-year network (Fig. 1.).
10.1371/journal.pone.0031256.g001Figure 1 Topological architecture of the MLP network constructed in this study.
The network consisted of 18 input neurons, 12 hidden neurons, and 1 output neuron.
Taking one input variable, HB as an example, Figure 2 represents the relationships between HB and other variables, and the output prognosis of the trained MLP network. In every subgraph, HB, another variable, and the output prognosis (ie., the MLP target) composed a simulated 3-D rendering; the output prognosis of the network is plotted versus HB and another variable, and the curved surface represents the relationship between HB, the other variable, and the output prognosis. In such a simulated 3-D rendering composed of only two input variables (HB and another variable) and the output prognosis, there is a nonlinear relationship between HB, other variables, and the output prognosis. The relationships between multi-variables and the output prognosis would undoubtedly be even much more complex in corresponding multidimensional space.
10.1371/journal.pone.0031256.g002Figure 2 Curved surface diagram of outcome prediction in the MLP network (taking HB as an example).
(2A): The one-year network. The x-axis represents input variable HB (x1), while the y-axis represents another variable: donor BMI (x2), TB (x3), or ALB (x4). The z-axis represents the output prognosis (ie., the MLP target). (2B): The two-year network. The x-axis represents HB (x1), and the y-axis represents another variable: Crea (x5), INR (x6), or Na+ (x7). The z-axis represents the output prognosis.
Model validation
With the Hosmer-Lemeshow test, a P-value greater than 0.05 and close to 1.0 is considered to indicate better calibration, and the smaller the χ2 value, the better the calibration ability of a model [34]. The MLP's calibration ability (χ2 = 1.56, P = 0.82 in one-year prediction; χ2 = 1.74, P = 0.78 in two-year prediction) was higher than that of the SOFA and MELD in both intervals' prediction (Table 3).
10.1371/journal.pone.0031256.t003Table 3 Calibration for MLP, SOFA, and MELD in posttransplant survival prediction.
Goodness-of-fit (χ2)
P-Value
Postransplant one-year survival prediction
MLP 1.56 0.82
SOFA 5.26 0.26
MELD 6.48 0.17
Postransplant two-year survival prediction
MLP 1.74 0.78
SOFA 5.64 0.23
MELD 6.98 0.14
Table 4 and Figure 3 show the discrimination of the MLP, SOFA score, and MELD score for predicting posttransplant 1-year and 2-year survival probability. The c-statistic values range from 0 to 1, with 0.5 corresponding to what is expected by chance alone and 1.0 to perfect discrimination. For a prognostic model, a c-statistic below 0.7 generally suggests poor prediction, while a c-statistic above 0.7 indicates a useful model, and a c-statistic greater than 0.8 indicates excellent predictive accuracy [24]. The MLP had c-statistics of 0.91 (P<0.001) and 0.88 (P<0.001) in one-year and two-year prediction, respectively (Table 4 and Fig. 3). The c-statistics of the SOFA were 0.70 (one-year) and 0.67 (two-year). MELD yielded the least accurate predictions (Table 4 and Fig. 3).
10.1371/journal.pone.0031256.g003Figure 3 ROC curves for MLP, SOFA score, and MELD score in posttransplant survival prediction.
(3A): Posttransplant one-year prediction. (3B): Posttransplant two-year prediction.
10.1371/journal.pone.0031256.t004Table 4 Discrimination of MLP, SOFA, and MELD in posttransplant survival prediction.
C-statistic ± SE 95% CI Sensitivity (%) Specificity (%) PPV (%) NPV (%) Accuracy (%)
P-Value
Posttransplant one-year survival prediction
MLP 0.91±0.05 0.80–0.97 91.3 88.6 84.0 93.9 89.7
<0.001
SOFA 0.70±0.08 0.54–0.86 72.0 66.7 62.1 75.9 69.0
0.04
MELD 0.66±0.10 0.47–0.84 68.0 63.6 58.6 72.4 65.5 0.10
Posttransplant two-year survival prediction
MLP 0.88±0.07 0.74–0.96 88.0 84.8 81.5 90.3 86.2
<0.001
SOFA 0.67±0.09 0.50–0.84 68.0 66.7 60.7 73.3 67.2 0.07
MELD 0.65±0.10 0.47–0.84 64.0 63.6 57.1 70.0 63.8 0.11
PPV = positive predictive value; NPV = negative predictive value.
Discussion
The large disparity between patient demand and donated organs is a pressing problem for all transplant surgeons, especially in the Asia-Pacific region. The best solution to this problem is still in dispute, as there are two sometimes-contradictory principles of organ allocation: urgency of patient need, and efficiency of organ use [35]. Unfortunately, prioritizing extremely sick patients make it likely that patients who are not as sick “will be forced to wait until their condition worsens and their chances for success are also diminished” [36], and patients who are very sick may have worse posttransplant outcomes than healthier patients [37]. Thus, the optimal system would offer grafts to those who are sufficiently sick to justify the transplantation but not too sick to benefit from it [38], that is, the urgency of need should be jointly optimized with the likelihood of satisfactory outcomes so as to avoid “futile transplantation”.
Furthermore, OLT ranks among the most expensive medical interventions [39], so the urgency-based principle has contributed to rising healthcare costs [37], [40]. An accurate prognostic model could also help potential transplant recipients and their families make informed decisions by providing them with information on the patient's posttransplant survival probability [11], [13].
With the aforementioned goals, a newly-adopted lung allocation score in the United States has incorporated likelihood of posttransplant survival in addition to lung disease severity [41]. The liver transplantation field would also benefit from a continuously optimized allocation system that prioritizes patients who need grafts most, without sacrificing the overall utility of this scarce resource. Such a system necessitates a strong prognostic model that can identify potential recipients with satisfactory survival prospects.
Over the past decade, MELD [42] has proved to be an excellent marker of BESLD-specific illness severity and corresponding pretransplant mortality risk, but many studies have also shown its poor accuracy in predicting posttransplant survival [43], [44], which is consistent with our results. The SOFA score was originally developed to quantitatively describe the degree of organ dysfunction in six organ systems and to evaluate morbidity in intensive care unit septic patients [26], but later studies found that it could be applied equally well in non-septic critically ill patients to measure individual or aggregate organ dysfunction and to describe morbidity risk [45]. Since its introduction, the SOFA score has also been widely applied to prognostic mortality assessment in critically ill patients with good results [46], although it was not developed for this purpose. In recent years, some investigations have applied the SOFA to critically ill cirrhotic patients and have also proven its validity in mortality risk assessment for BESLD patients [47]–[49]. We believe that because BESLD patients usually display multiple-organ damage or dysfunction, such as the renal failure, coagulopathy, and encephalopathy, the SOFA is an excellent scoring model for assessing BESLD patients' illness severity and mortality risk. Additionally, several studies have analyzed the predictive power of SOFA on post-liver transplant mortality; although these achieved some encouraging results in short-term prognosis assessment [50], [51], its value in long-term outcome prediction still requires study. In this study, SOFA achieved good calibration abilities in both intervals and satisfactory discrimination power in one-year prediction, which is consistent with other studies [50], [51], but its accuracy was poor in two-year prediction. Although SOFA encopasses the functions of multiple systems including respiratory, hemostastics, hepatic, circulatory, and brain and kidney, it is not specific enough to BESLD patients and is not tailored to posttransplant outcome prediction. Lack of these specificities may account for its discriminative and calibration inferiority to the MLP network.
Although there have been many attempts to develop a specific model to assess posttransplant prognosis, to date, they have not achieved sufficient accuracy, or have simply categorized the patients into various risk groups [4], [11]; even with some of the most comprehensive efforts, the predictive accuracy of these models has always been reported in the 60–70% range [4], [9], [11]–[14] with no single model being more accurate than any other. We believe there are several possible explanations for this. First, the effect of prognostic factors depends on the underlying liver disease [11]–[13]. Thus, effort would be better spent developing disease-specific models targeted to BESLD patients or cancer patients. Second, Existing studies rely heavily on a few specific variables derived from linear regression analyses, rather than from data mining. The omission of many variables may hinder the discovery of underlying relationships between prognosis and related factors, and the interactions among factors. Third, transplant recipients represent a very complex biological system where the relationship between pretransplant variables and posttransplant prognosis is multidimensional and nonlinear (as shown in Fig. 2) [17], [23], so linear methods are inadequate in predicting regression coefficients and constructing risk factor models.
With the development of artificial intelligence in recent years, ANN has been a superior data-mining solution for complex prognostic problems [17], [20], and MLP has been proven to perform better than other architectures such as radial basis function, recurrent neural network, and self-organizing map [22]. MLP is a computation system that uses a large number of simple units to process information in parallel, so it is capable of learning arbitrarily complex nonlinear functions to arbitrary accuracy levels [22]. Furthermore, MLP allows a certain degree of flexibility when it comes to handling noise [18]. Most importantly, MLP is a nonparametric dynamic model, which can automatically self-training and readjust the internal parameters by back-propagation when more transplants enter the network [52], thus yielding more accurate responses and becoming progressively more dependable over time; this is what the linear models could not achieve.
In this study, although three characteristics of the recipients in the validation set differed from the training set, the MLP still achieved good calibration ability and high discrimination power in posttransplant survival prediction, with c-statistics around 0.9 and satisfactory sensitivity and specificity in both intervals, as well as the small χ2 statistics and associated P-values around 0.8 in both intervals. These results were not only superior to that of the linear regression models reported in previous studies [4], [9], [12], [13], but also outstripped the performances of SOFA and MELD in this study. We believe that several factors may account for the MLP's outstanding performance. First, the MLP network, employing 12 variables to make predictions, included more comprehensive information associated with the posttransplant prognosis. Second, the input features of our MLP included not only donor factors and measurements of disease severity, but also some well-recognized variables reflecting the complications and comorbidities (such as sepsis and diabetes) in BESLD patients. Meanwhile, it should be noted that we decided not to include some subjective variables (such as encephalopathy or ascites) in our model development because their classifications are subjective and could therefore be arbitrary. Third, being computer-based, the MLP can process more information about the survival process and model much more complex nonlinear multidimensional relationship, thus yielding more accurate prognostic estimations.
In this study, donor age and BMI were identified as input features. These two factors could be obtained before transplantation, and have been proved to be associated with graft quality [53], [54] and recipient outcomes [9], [14]. Although some other donor factors (such as the graft steatosis and ischemia times) may directly reflect graft quality and contribute to posttransplant prognosis, they would have been difficult or impossible to know when clinicians and patients make transplant acceptance decisions and when candidates are ranked on a waiting list. This problem would seem to be an inherent difficulty in pretransplant prediction. Therefore, in order to maximize the practical applicability of a pretransplant model, we believe that it must be constructed in accordance with actual clinical conditions, and enhancing the model's performance based on the variables available is the most important goal. Thus, we decided not to include this kind of characteristics in our pretransplant model development.
Meanwhile, we chose posttransplant one-year and two-year as the study endpoints in this study because outcomes within this timeframe could reflect surgical and perioperative risk [4] and mortality associated with most early complications. However, as we know, the recipient's long-term survival would be affected by not only the pretransplant characteristics, but also many intraoperative and posttransplant factors, such as the graft cold-ischemia time and biliary complications. Thus, in our view, once the appropriate modeling method is identified, development of sequential correction models according to the different variable acquisition phases may be a reasonable way to meet the evaluation requirement in different phases. When certain donor characteristics, operative parameters, and even some posttransplant variables could be available after operation, another posttransplant predictive model that incorporated above features should be developed and used to perform a further corrective assessment. We believe the two kinds of model can provide more comprehensive perioperative evaluation information at different variable acquisition phases, and, most importantly, they are consistent with actual clinical conditions.
In this study, we clarified the complex multidimensional and nonlinear relationship between transplant variables and posttransplant outcomes, and identified the value of MLP in solving this complex prognostic problem. We believe this methodological result is the key point of this study, and is more important than the specific factors and specific study intervals included in the presented model.
We believe that this kind of pretransplant model would provide patients and clinicians with important reference information about their early posttransplant prospects during the initial counseling and evaluation phases of referral [4], [11], [13]. If used alongside the MELD system, the pretransplant model can also help predict early outcome with and without transplantation. This provides clinicians with a combined tool to identify patients likely to benefit most from transplantation [9].
Meanwhile, how to ethically balance medical urgency with posttransplant survival prospects is an important issue. For instance, it could be argued that the patient with the highest combined MELD score and survival prospects should be given priority. But we expect that in practice, scientifically combining the two conflicting determinants would not be so simple, just as the use of MELD to guide graft allocation has sparked a wealth of studies and discussion. Therefore, we believe that comprehensively considering and weighing urgency and survival prospects will require further evidence-based research. Whatever shape the final system takes, however, it will undoubtedly include a prognostic model with high predictive accuracy as an important component.
Although this MLP model was more sophisticated than conventional linear models, in practical application, its software implementation allowed the creation of a new interface that can be incorporated into a website and be easily used by everyone, as in the UNOS website, where an interface was created for MELD calculation. Thus, we believe the model's complexity should not present a problem in clinical practice.
Despite our encouraging results, our study has some potential limitations. First, it was developed using data from a single center; we did not validate our model externally with data from different sources. Indeed, we divided our dataset into training and validation sets, and the validation samples were not used in model development. Thus, the proposed MLP network should be further verified with data at other major centers. Fortunately, the dynamic nature of the MLP makes it capable of continuously and automatically adjusting its internal parameters and improving as more transplant data from other centers enter the network [52]. Second, the patient population had a high proportion of HBV infection; therefore, this MLP network may have limited applicability to typical North American and European patients, who tend to have a lower rates of HBV but higher rates of hepatitis C and alcoholism than do Chinese BESLD patients.
In summary, artificial intelligence methodologies such as MLP offer significant advantages over conventional statistical techniques in variable selection and dealing with restrictive assumptions of normality and linearity, and thus hold promise for solving posttransplant outcome prediction. Therefore, in future research we plan to use MLP to develop a posttransplant multi-interval sequential correction model, a step toward establishing a balanced system that considers both pretransplant mortality and expected posttransplant survival.
Supporting Information
Table S1
Baseline categorical characteristics of the training set and validation set.
(DOC)
Click here for additional data file.
The authors thank Shawna Williams for her editing assistance in the preparation of this manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by grants from the National Basic Research Program of China (also called the 973 Program) (No. 2009CB522401), the Ph.D. Programs Foundation of the Ministry of Education of China (No. 20110181120037), and the China Postdoctoral Science Foundation (No. 20090461343). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Case Rep MedCase Rep MedCRIMCase Reports in Medicine1687-96271687-9635Hindawi Publishing Corporation 2243192910.1155/2012/347939Case ReportOral Haemangioma Gill Jaspreet Singh
1
*Gill Sharanjeet
2
Bhardwaj Amit
1
Grover Harpreet Singh
1
1Department of Periodontics and Oral Implantology, SGT Dental College, Hospital and Research Institute, Gurgaon, Budhera, Haryana 122505, India2Department of Oral Pathology, Manav Rachna Dental College, Haryana, Faridabad 121004, India*Jaspreet Singh Gill: [email protected] Editor: Jahn M. Nesland
2012 6 2 2012 2012 34793927 7 2011 13 11 2011 Copyright © 2012 Jaspreet Singh Gill et al.2012This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Vascular anomalies comprise a widely heterogeneous group of tumours and malformations. Haemangioma is the most common benign tumour of vascular origin of the head and neck region. The possible sites of occurrence in oral cavity are lips, tongue, buccal mucosa, and palate. Despite its benign origin and behaviour, it is always of clinical importance to the dental profession and requires appropriate management. This case study reports a rare case of capillary haemangioma on the palatal gingiva in a 14-year-old female.
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1. Introduction
Haemangioma are the most common benign vasoformative tumours of infancy and childhood [1, 2]. They usually are manifested within the first month of life, exhibit a rapid proliferative phase, and slowly involute to near complete resolution. There are many ways to classify haemangiomas. According to Enzinger and Weiss, haemangiomas are broadly classified into capillary, cavernous, and miscellaneous forms like verrucous, venous, arteriovenous haemangiomas, and so forth [3]. Capillary haemangiomas further include juvenile, pyogenic granuloma, and epitheliod haemangioma [3]. The term haemangioma has been commonly misused to describe a large number of vasoformative tumours [4]. However, the International Society for the Study of Vascular Anomalies (ISSVA) has recently provided guidelines to differentiate these two conditions, according to the novel classification first published by Mulliken et al. in 1982 [5]. Vasoformative tumours are broadly classified into two groups: haemangioma and vascular malformation [5]. Haemangioma is histologically further classified into capillary and cavernous forms [6, 7]. Capillary haemangioma is composed of many small capillaries lines by a single layer of endothelial cells supported in a connective tissue stroma of varying density, while cavernous haemangioma is formed by large, thin walled vessels, or sinusoids lined by epithelial cells separated by thin layer of connective tissue septa [8].
The majority of haemangioma involve the head and neck. However, they are rare in the oral cavity but may occur on tongue, lips, buccal mucosa, gingiva, palatal mucosa, salivary glands, alveolar ridge, and jaw bones [3, 4, 6, 9–12]. Clinically, haemangioma appears as soft mass, smooth or lobulated, and sessile or pedunculated and may vary in size from a few mms to several cms [8]. They are usually deep red and may blanch on the application of pressure and if large in size, might interfere with mastication [9]. In the present case study, we report a rare and an unusual case of capillary haemangioma of the palatal mucosa.
2. Case Report
A female patient aged 14 years, reported with a chief complaint of a swelling and growth on the inner side of her upper front teeth since 4-5 months. She also complains of localized bleeding in that area on brushing, and there was slight pain and discomfort on eating.
Dental history revealed that she had a history of gingival enlargement, one year back, which she had got excised. However, the growth recurred within two months after surgical excision. It was initially small in size, gradually increased and stabilized after 3-4 weeks till the present size. On general physical examination, it was found that the patient was normally built for her age with no defect in gait or stature, and there was no relevant medical history. Family history was also noncontributory.
A comprehensive intraoral examination revealed a localized gingival mass between maxillary right central incisor and lateral incisor (11, 12) on the palatal aspect (Figure 1). It was firm, pedunculated with a distinct stalk arising from the interdental papillary region. The mass was bright red, erythematous and bilobulated with well-defined margins. The two distinct lobes measured about 5 cm × 4 cm and 3 cm × 2.5 cm in diameter. They were firm and rubbery in texture. No surface ulceration or secondary infection was noted. Periodontal examination revealed no clinical attachment loss. Panoramic examination confirmed no alveolar bone loss, and a provisional diagnosis of pyogenic granuloma was made on the basis of history and clinical features.
The other pathologic entities that were included in the differential diagnosis were malignancies, trauma, and or infection (bacterial, viral, and fungal), enlargement due to drugs. Complete blood examination, urine analysis, and an intraoral periapical radiograph with respect to 11, 12 were done. The laboratory investigations of blood and urine were within normal limits that ruled out any leukemic enlargement and diabetes mellitus. HIV, HBs, VDRL, and Mantoux test were negative, ruling out any possibility of infectious involvement. Radiographically, there was no evidence of crestal bone loss, and lamina dura was intact around the roots of both maxillary right central and lateral incisor. Scaling was carried out under universal precautions, and later surgical excision of the lesion was done under local anaesthesia as a part of excisional biopsy. A thread was tied around the stalk of the pedunculated lesion and was stretched tightly so as to reduce blood circulation to the lesion. The growth was then surgically excised along with the stalk, and thorough curettage of the area was performed. The excised lesion was stored in 10% formalin and sent for histopathological examination. Periodontal dressing was applied on the operated area, and the patient was given postoperative instructions. Histopathological examination revealed stratified squamous epithelium which showed atrophy, and in some areas hyperkeratosis was seen. Beneath this, many small and large capillaries filled with blood were present. These vessels were lined by a single layer of endothelial cells and were supported by a connective tissue stroma of varying density with no inflammatory component (Figure 2). On the basis of clinical examination and histopathology, a diagnosis of capillary haemangioma was made. The patient was recalled after a week with normal healing and various plaque control measures were reinforced (Figure 3). The patient was also reviewed 1, 3, and 6 months after the biopsy, and there was no recurrence of the lesion.
3. Discussion
The confusing and misleading terminology has led to inappropriate grouping and classification of vasoformative tumours [4]. The differentiation between haemangioma and vascular malformations is made on the basis of clinical appearance, histopathology, and biological behaviour [4].
Pathogenesis and origin of haemangioma remain incompletely understood. However, various theories have been proposed to elucidate the mechanism and pathogenesis of haemangioma. Aberrant and focal proliferation of endothelial cells results in haemangioma, although the cause behind this remains unclear [13]. The placental theory of haemangioma origin has been described by North et al. [14], who studied various histology and molecular markers such as GLUTI, Lewis Y Antigen, Merosin, CCR6, CD15, IDO, FC, and gamma Receptor II. Positive staining for GLUTI is considered highly specific and diagnostic for haemangioma, and it is useful for making differential diagnosis between haemangioma and other vascular lesions clinically related to it [13]. More recently, somatic mutational events in gene involved in angiogenesis are related to haemangioma growth [4]. Growth factors specifically involved in angiogenesis such as VEGF, b-TGF, and IGF are often increased during the proliferation phases of haemangioma growth [15, 16]. Moreover, it has been noted that during the involution phase of haemangioma, there is a decrease in angiogenic molecules (VEGF, PCNA, Type IV collagenase, Lewis V antigen, CD 31), while there is increase in concentration of marker for apoptosis (T4, TUNNEL, INF, Mast cells, and TGF) [4, 17, 18]. Thus, role of molecular signalling is now clear in haemangioma development.
A variety of other lesions can resemble haemangioma in the oral cavity. The differential diagnosis includes pyogenic granuloma, chronic inflammatory gingival hyperplasia, epulis granulomatosa, telangiectasia, angiosarcoma, squamous cell carcinoma, and other vascular appearing lesions of face or oral cavity such as Sturge Weber Syndrome [9].
In the present case, clinical features resemble that of pyogenic granuloma. However, the lesion did not show microscopic appearance of a pyogenic granuloma. It contained blood-filled capillaries lined by layer of endothelial cells in a connective tissue stroma without any evidence of inflammation.
Angiosarcoma is a rare malignant tumour of vascular endothelium, and it resembles haemangioma. However, it can be differentiated from it on the basis of histopathologic findings as it is characterized by infiltrative proliferation of endothelium-lined blood vessels that form an anastomosing network. The endothelial cells appear hyperchromatic and atypical and often tend to pile up within vascular lamina [8].
Management of haemangioma depends on a variety of factors, and most true haemangioma requires no intervention. However, 10–20% requires treatment because of the size, exact location, stages of growth or regeneration, functional compromise, and behaviour. The range of treatment includes surgery, flash lamp pulsed laser, intralesional injection of fibrosing agent, interferon alpha-2b, and electrocoagulation while cryosurgery, compression and radiation were used in the past [11–13, 19–22]. Each treatment modality has its own risk and benefits. In the present case, surgery was carried out on the basis of size and location. Moreover, the difficulty in swallowing was another factor that was taken in consideration, and surgical approach was preferred as to remove excess residual fibrofatty and redundant tissue after involution.
4. Conclusion
Haemangioma is of benign origin and behaviour, but haemangioma in the oral cavity is of clinical importance. It often mimics other lesion clinically and requires appropriate clinical diagnosis and proper management.
Figure 1 Preoperative intraoral view.
Figure 2 H and E stain section at 10x magnification showing numerous blood-filled capillaries in connective tissue stroma.
Figure 3 Postoperative view after one week.
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De Cicco Paola
1
Troncone Edoardo
1
Ciacci Carolina
3
*
1
Department of Clinical and Experimental Medicine, Federico II University of Naples, Napoli, Italy
2
Department of Chemical Engineering, University of Naples “Federico II”, Naples, Italy
3
Chair of Gastroenterology, University of Salerno Medical School, Baronissi, Italy
Bereswill Stefan EditorCharité-University Medicine Berlin, Germany* E-mail: [email protected] and designed the experiments: IR AL. Performed the experiments: PDC ET. Analyzed the data: IR AL CC. Contributed reagents/materials/analysis tools: IR AL. Wrote the paper: IR AL CC.
2012 6 3 2012 7 3 e3284110 11 2011 31 1 2012 Russo et al.2012This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Oxidative stress plays an important role in the pathogenesis of inflammatory bowel disease (IBD), including Crohn's disease (CrD). High levels of Reactive Oxygen Species (ROS) induce the activation of the redox-sensitive nuclear transcription factor kappa-B (NF-κB), which in turn triggers the inflammatory mediators. Butyrate decreases pro-inflammatory cytokine expression by the lamina propria mononuclear cells in CrD patients via inhibition of NF-κB activation, but how it reduces inflammation is still unclear. We suggest that butyrate controls ROS mediated NF-κB activation and thus mucosal inflammation in intestinal epithelial cells and in CrD colonic mucosa by triggering intracellular antioxidant defense systems. Intestinal epithelial Caco-2 cells and colonic mucosa from 14 patients with CrD and 12 controls were challenged with or without lipopolysaccaride from Escherichia Coli (EC-LPS) in presence or absence of butyrate for 4 and 24 h. The effects of butyrate on oxidative stress, p42/44 MAP kinase phosphorylation, p65-NF-κB activation and mucosal inflammation were investigated by real time PCR, western blot and confocal microscopy. Our results suggest that EC-LPS challenge induces a decrease in Gluthation-S-Transferase-alpha (GSTA1/A2) mRNA levels, protein expression and catalytic activity; enhanced levels of ROS induced by EC-LPS challenge mediates p65-NF-κB activation and inflammatory response in Caco-2 cells and in CrD colonic mucosa. Furthermore butyrate treatment was seen to restore GSTA1/A2 mRNA levels, protein expression and catalytic activity and to control NF-κB activation, COX-2, ICAM-1 and the release of pro-inflammatory cytokine. In conclusion, butyrate rescues the redox machinery and controls the intracellular ROS balance thus switching off EC-LPS induced inflammatory response in intestinal epithelial cells and in CrD colonic mucosa.
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Introduction
Intestinal epithelial cells constitute the interface between the gut lumen and the innate and adaptive immune system [1]. Previous studies show that a loss of immunologic tolerance is the primary cause for the development of inflammatory bowel disease (IBD) in genetically susceptible hosts [1], [2].
IBD is characterized by the loss of tolerance in the intestinal immune system towards the intestinal microbiota resulting in constant immune activation which leads to mucosal tissue damage and chronic inflammation [3]. These spontaneously relapsing chronic intestinal inflammations are subdivided into two main idiopathic pathologies ulcerative colitis (UC) and Crohn's Disease (CrD).
CrD is characterized histologically by transmural inflammation, epithelial ulceration, fissure formation, and stenosis of segments throughout the gastrointestinal tract [4]. Increased ROS production and decreased antioxidant enzyme levels have been found in the intestinal mucosa of CrD patients [5], [6], causing increased oxidative stress, lipid peroxidation and inflammation [7], [8]. Moreover high ROS levels have been reported to promote activation and translocation of NF-κB [9] into the nucleus through alternative phosphorylation of Iκ-B-α which leads to its ubiquitynation and degradation [9]. This ROS/NF-κB self-sustaining regulatory loop may contribute to the perpetuation and exacerbation of chronic inflammation [10].
CrD therapy is presently based on anti-inflammatory non-steroid drugs such as mesalazine, steroid analogues, and/or immuno-suppressive molecules that often produce severe side-effects [11]. An emerging therapeutic approach is the use of specific dietary fibre and/or prebiotics able to enhance butyrate production in the colon of CrD patients [12]. These functional foods have proven effective in delivering butyrate to the colonic mucosa, a process that is difficult to achieve by direct administration of butyrate, either orally or rectally [13]. Butyrate is a four-carbon short-chain fatty acid produced by bacterial fermentation of mainly undigested dietary carbohydrates within the colonic lumen. Although butyrate has been the favoured energy source for colonic epithelial cells and induces changes in gene expression influencing colonic function [14],[15], it has recently been demonstrated to have an anti-inflammatory effect [16]. In two in vitro studies, butyrate was shown to modulate inflammation through NF-κB inhibition [17] and up-regulation of PPAR-γ [18]. Several in vivo studies report a decreased inflammation after rectal administration of butyrate or mixtures of SCFA (short chain fatty acids) in patients with active ulcerative colitis [19],[20] and diversion colitis [21],[22]. However, the detailed biological regulatory mechanisms of butyrate's activity remain unclear.
Since the impaired mucosal anti-oxidative capacity may further promote intestinal inflammation in patients with IBD [23], this study aimed to investigate whether butyrate could modulate GST-A1/A2 mRNA levels, protein expression and catalytic activity and readjust ROS levels, thus switching off ROS mediated NF-κB activation and the inflammatory response in intestinal epithelial Caco-2 cells and CrD colonic mucosa.
Materials and Methods
Patients and ex-vivo organ cultures
Biopsy specimens were taken from uninflamed mucosal areas immediately next to inflamed tissues of fourteen patients with CrD (n = 14, mean age 24 years, range 18–41). The primary site of involvement was ileal in four patients, ileocolonic in four and colonic in five. Disease was active in all patients, as defined by a Crohn's Disease Activity Index (CDAI) of >150 [24]. Normal controls (n = 12, mean age 22.4 years, range 18–29) included mucosal samples taken from eight patients with uncomplicated diverticular disease and four patients with rectal bleeding due to haemorrhoid. Informed written consent was obtained from all subjects, and the protocol of the study was approved by the Ethics Committee of Regione Campania Health Authority. One specimen from each patient was used for diagnosis; the other samples were cultured in vitro for 4 and 24 h [25] with medium alone, EC-LPS (1 µg/ml E. Coli Serotype O127: B8 Lipopolysaccharide, Sigma-Aldrich, Milan, Italy) in the presence or absence of the butyrate (10 mM).
Cell lines
Caco-2 cells, a human colonic epithelial cell line, were cultured as recommended by the American Type Culture Collection (ATCC). Experiments were initiated on day 14 or 15 after seeding and continued for 24–72 h, as the cells progressed through more mature stages of differentiation.
Cell cultures
Cells were seeded onto 12-well plates at a density of 2–3×105cells/cm2 and were pre-treated with butyrate (10 mM) for 24 h and finally stimulated with EC-LPS (1 µg/ml−1) for another 4 h and 24 h.
RNA interference
Cells were seeded onto 12-well plates (Costar®, Corning) at a density of 2–3×105cells/cm2. The cells were transfected with human GSTA1/A2 or scrambled small interfering RNA (50 nmol/L, siRNA) duplex using Lipofectamine RNAiMAX at 37°C for 72 h. Cells were then stimulated in the presence of butyrate with EC-LPS for 4 h and 24 h. The GSTA1/A2 duplex siRNA was a pool of two sequences: siRNA no. 1, GSTA1 (catalog number HSS142318, Invitrogen, Milan, Italy) and siRNA no. 2, GSTA2 (catalog number HSS142323, Invitrogen, Milan, Italy)
Quantitative RT–PCR
Total RNA was extracted using the RNA-easy Mini Kit (Qiagen). The mRNA was reverse transcribed with a SuperScriptTM III First Strand Synthesis System (Invitrogen). Quantitative RT–PCR was performed with an iCycler iQ Multicolour Real-Time PCR Detector (Bio-Rad) with iQ TM SYBR Green supermix (Bio-Rad). Expression levels of genes were normalized to β-actin levels in the same sample. Primer sequences were as follows (5′ to 3′, sense, antisense): GSTA1, Forward primer CCT GCC CAC AGT GAA GAA GT Reverse primer GCC TCC ATG ACT GCG TTA TT; GSTA2, Forward primer GGC TGC AGC TGG AGT AGA GT Reverse primer ATTGGCACTTGCTGGAACAT; β-Actin, Forward primer TGACCCAGATCATGTTTGAG Reverse primer TAATCTCCTTCTGCATCCTG. The relative amounts of mRNA were calculated by using the comparative Ct method.
Nuclear protein extraction
The cells were collected in cold buffer A (10 mmol/L HEPES (pH 7.9), 1.5 mmol/L MgCl2, 10 mmol/L KCl, and 1 mmol/L dithiothreitol (DTT) and protease inhibitor cocktail), homogenized in Potter-Elvehjem pestle and glass tube (Sigma-Aldrich), and centrifuged at 11,000× g for 20 min at 4°C to obtain nuclear pellets. Supernatants were collected as cytoplasmic fractions. Nuclear pellets were washed with buffer A and resuspended in buffer B (20 mmol/L HEPES (pH 7.9), 1.5 mmol/L MgCl2, 0.42 mol/L NaCl, 0.2 mmol/L EDTA, 25% (v/v) glycerol, 1 mmol/L DTT, and protease inhibitor cocktail) and incubated on ice for 50 min with occasional mixing to extract nuclear proteins. Nuclear extracts were cleared by centrifugation (11,000× g, 15 min, 4°C), and supernatants were collected as nuclear fraction. Then, cytoplasmic and nuclear whole-cell fractions were analyzed by immunoblotting.
Immunoblot
Experiments were carried out as previously described [26]. Briefly, cells were washed in ice-cold phosphate-buffered saline (PBS) and lysed with NP-40 lysis buffer (1% NP-40, 150 mmol/L NaCl, 50 mmol/L Tris, pH 7.4, 10 mmol/L NaMoO4) at 4°C for 30 min. Protease inhibitors were added to NP-40 lysis buffer to a final concentration of 1 µg.ml−1 leupeptin, 2 µg.ml−1 aprotinin, 50 µg.ml−1 Pefabloc, 121 µg.ml−1 benzamidine, 3.5 µg.ml−1 E64. Cell lysates were centrifuged at maximal speed in an eppifuge at 4°C, and supernatants were collected. Cell lysates (50 µg) were loaded, separated on 10% SDS-PAGE and transferred to nitrocellulose. After blocking for 2 h (TBS/Tween supplemented with 5% nonfat dry milk), blots were incubated with anti-phospho-p65NF-κB(Cell Signaling Technology, Danvers, Massachusetts, USA), Gluthatione-S-transferase α (goat polyclonal IgG, Abcam, Milan, Italy), phospho-p42/p44 MAP kinases (rabbit polyclonal IgG, Cell Signaling Technology), COX-2 (Cell Signaling Technology, Danvers, Massachusetts, USA), ICAM-1 (SantaCruz Biotechnology, Santa Cruz, California, USA) and β-actin (rabbit polyclonal IgG; Santa Cruz, CA, USA) and αβ-tubulin (rabbit polyclonal IgG; Santa Cruz, CA, USA). The primary antibodies were counterstained using a horseradish peroxidase-conjugated anti-IgG antibody (Amersham, Little Chalfont, UK) for 60 min at room temperature. Proteins were visualized by chemiluminescence (ECL Plus, Amersham) and exposed to X-OMAT film (Eastman Kodak, Rochester, NY). Protein concentrations were determined using a Bio-Rad protein assay to ensure equal protein loading prior to Western blot analysis.
Immunolocalization
Human tissue sections
Five µm thick cryostat sections were fixed in acetone for 10 min. The sections were individually incubated for 2 hours at room temperature with the following antibodies: phopsho-p65NF-κB(Ser536) (rabbit polyclonal; Cell Signaling Technology), p42/p44 MAPK (rabbit polyclonal IgG ; Cell Signaling Technology), COX-2 (rabbit polyclonal; Cell Signaling Technology, Danvers, Massachusetts, USA) and ICAM-1 (rabbit polyclonal; SantaCruz Biotechnology, Santa Cruz, California, USA). Antigen expression and distribution was visualized using a donkey anti-rabbit IgGs conjugated to Alexa Fluor 488 for 60 min at room temperature. Isotype control antibodies (IgG1 or IgG2), isotype-matched non immune Igs, or isotype-matched antibodies against inappropriate blood group antigens were used as control of specificity. Data were analyzed under fluorescence examination using a LSM510 Zeiss confocal laser scanning unit (Carl Zeiss, Germany). COX-2 or ICAM-1 positive mononuclear cells (MNC) were counted per mm2 of mucosa. Epithelial cells with nuclear p65 localization were counted per 100 epithelial cells. Data were examined in a blind fashion by two independent reviewers totally unaware of all the culture conditions to prevent bias in their observation [27],[28].
Measurement of intracellular reduced glutathione (GSH)
Intracellular GSH levels were measured in Caco-2 cells using a fluorometric method as described by Ranganna et al.
[29]. Briefly, cells were collected in ice-cold NaCl/Pi were pelleted by centrifugation (750 g, 5 min, 4°C). The pellets were suspended in buffer A (50 mL of 25% (w/v) metaphosphoric acid and 188 mL of 0.1 mmol/L sodium phosphate buffer supplemented with 5 mmol/L EDTA, pH 8.0) and homogenized on ice. The homogenates were centrifuged (15,000 g, 20 min, 4°C) and diluted 10-fold with 0.1 mL sodium phosphate buffer B (5 mmol/L EDTA, pH = 8.0) incubated with buffer C (1.8 mL buffer B and 0.1 mL 0.1% o phthalaldehyde solution in methanol ) for 15 min at room temperature. Changes in fluorescence were analyzed with a Wallac 1420 multilabel Counter (PerkinElmer Waltham, MA, USA) at an activation wavelength of 350 nm and an emission wavelength of 420 nm. Cellular GSH levels were calculated using standard curve measurements performed simultaneously with the samples and expressed as pmole of GSH/cell.
ROS detection
The cells and five-micrometer cryostat sections were pulsed with 10 µmol/L 5-(and-6)-chloromethyl-2′7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) (Molecular Probes, Invitrogen) [26]. CM-H2DCFDA, a ROS-sensitive probe, was used to track changes in the cellular redox state. The cells were analyzed with a Wallac 1420 multilabel Counter (PerkinElmer Waltham, MA,USA) and detected by a LSM510 Zeiss confocal laser scanning unit (Carl Zeiss, Germany).
ELISA
TNF-α secretion was measured using the BD OptEIATM ELISA kit II (BD Biosciences) according to the manufacturer's instructions. Protein concentrations of whole-cell lysates were measured using the BioRad Dc protein Assay (BioRad). TNF-α levels were normalized to standard protein concentrations [30].
Statistical analysis
All of the experiments were performed in duplicate and repeated at least three times. Group data from all experiments are presented as means ± s.d. . One-way ANOVA was used for all of the statistical analyses among multiple groups. In another set of data the paired two tailed Student's test was used for statistical analyses. Groups were compared by post hoc Tukey-Kramer test. A probability of P-value<0.05 was considered significant.
Results
Effect of butyrate on GSTA1/A2 mRNA levels, protein expression and catalytic activity and ROS levels in intestinal epithelial cells upon challenge with LPS from Escherichia Coli
We demonstrated that butyrate prevents LPS-induced decrease of GSTA1/A2 mRNA, protein and activity in LPS-stimulated intestinal epithelial cells (Figure 1A and B and Figure S1A). To establish that butyrate induces GST expression in lipopolysaccharide stimulated Caco-2 cells, the influence of an siRNA construct specific to GSTA1/A2 was evaluated. Results showed that butyrate-induced GST-α protein (Figure S1B and Figure 1D) was almost completely silenced when Caco-2 cells were transfected with GST A1/A2 siRNA prior to stimulation with butyrate whereas negative siRNA transfection had no relevant effect on butyrate-induced GST expression (Figure S1B).
10.1371/journal.pone.0032841.g001Figure 1 Effect of butyrate on GSTA1/A2 mRNA levels and protein expression in intestinal epithelial cells and CrD mucosal epithelial cells challenged with LPS from Escherichia Coli.
(A–B) Caco-2 cells were treated for 24 hours with butyrate and then were stimulated with EC-LPS for 4 h. (A) Real time PCR of GSTA1/A2 mRNA. Values are means ± s.d., n = 6. Asterisks indicate that means differ from samples cultured with medium alone and from samples cultured with EC-LPS. *P<0.05. (B, top line) Immunoblot of GST-α. β-actin was used as loading control for blot. (B, bottom line) Densitometric analysis of the band intensity. Values are means ± s.d., n = 6. (C–D) CrD colonic mucosa were cultured for 4 h in the presence of medium alone, medium with EC-LPS or EC-LPS with butyrate. (C) Real time PCR of GSTA1/A2 mRNA. Values are means ± s.d., n = 14. Asterisks indicate that means differ from samples cultured with medium alone and from samples cultured with EC-LPS. *P<0.05. (D) confocal microscopy of GST-α protein (green) in CrD colonic mucosa (n = 14). Nuclei counterstained with DAPI(blue). Scale bar, 10 µm.
Using a well established tissue culture model for biopsy of human CrD colonic mucosa [28], we showed that butyrate prevents LPS-induced decrease of GSTA1/A2 mRNA, protein and activity in LPS-treated colonic mucosa in CrD patients (Figure 1C and D and Figure S1C).
Since glutathione-S-transferases (GSTs) play an important role in protection mechanisms against oxidative stress, we investigated the capacity of butyrate-induced GST-α expression to attenuate lipopolysaccharide-mediated oxidative stress in intestinal epithelial cells and CrD colonic mucosa.
We demonstrated that butyrate was effective in controlling the increase of ROS levels and attenuating the concomitant decline in reduced glutathione (GSH) levels generated in response to lipopolysaccharide in intestinal epithelial cells (Figure 2A and Figure S2A). GSTA1/A2 siRNA antagonized the down-regulatory effect of the butyrate on ROS levels and decline in GSH levels induced by EC-LPS (Figure 2A and Figure S2A). We investigated the effect of butyrate in controlling oxidative stress in CrD epithelia. Before challenge, higher ROS levels were observed in CrD colonic mucosa compared with controls. EC-LPS challenge led to an increase of ROS levels at 4 hours of incubation in CrD colonic mucosa but not in controls (Figure 2B). Moreover treatment of cultured biopsies with butyrate was highly effective in preventing LPS-induced ROS levels (Figure 2B).
10.1371/journal.pone.0032841.g002Figure 2 Effect of butyrate on oxidative stress in intestinal epithelial cells and CrD mucosal epithelial cells challenged with LPS from Escherichia Coli.
Caco-2 cells were treated for 24 h with butyrate or transfected with either 50 nM human GST-α siRNA or scrambled oligonucleotides and then challenged with EC-LPS for 4 h. (A) Intracellular ROS levels. Values are means ± s.d., n = 6. Asterisks indicate that means differ from samples cultured with medium alone and from samples cultured with EC-LPS. *P<0.05. [DCF: 2′,7′-dichlorodihydrofluorescein]. (B) ROS levels in control (n = 10) and CrD colonic mucosa (n = 14) before challenge and after challenge with medium alone, medium with EC-LPS or EC-LPS with butyrate following 4 h of incubation. Values are means ± s.d. . Asterisks indicate that means differ from control samples, CrD samples cultured with medium alone and from CrD samples cultured with EC-LPS. *P<0.05. 2′,7′-dichlorodihydrofluorescein (DCF). (C) Caco-2 cells were treated for 24 h with butyrate and then were stimulated with EC-LPS for 4 h. (C, left panel) Immunoblot of p42/p44 phosphorylation. (C, right panel) Densitometric analysis of the band intensity. Values are means ± s.d., n = 6. β-actin was used as loading control for blot. (D–E) Confocal microscopy of phosphorylated p42/p44 (green) and number (F) of epithelial cells with phosphorylated p42/p44 per 100 epithelial in control (n = 10) and in CrD colonic mucosa (n = 14) before challenge (D) and after challenge with medium alone, medium with EC-LPS or EC-LPS with butyrate following 4 h of incubation(E–F). Nuclei counterstained with DAPI (blue). Scale bar, 10 µm. Values are means ± s.d.. Asterisks indicate that means differ from control samples, CrD samples cultured with medium alone and from CrD samples cultured with EC-LPS. *P<0.05.
A pro-oxidative environment induces activation of different stress sensitive signalling pathways [31],[32],[33]. We demonstrated that butyrate prevented ROS-induced p42/p44 MAPK phosphorylation in LPS-stimulated intestinal epithelial cells. (Figure 2C). GSTA1/A2 siRNA antagonized the effect of butyrate in decreasing p42/p44 MAPK phosphorylation (Figure S2B).
We investigated the effect of butyrate in controlling ROS-induced p42p/44 MAPK phosphorylation in epithelia of CrD patients. Before challenge, higher p42/p44 MAPK phosphorylation was observed in CrD colonic mucosa compared with controls (Figure 2D). EC-LPS challenge led to an increase of p42/p44 MAPK phosphorylation at 4 h of incubation in CrD colonic mucosa but not in controls (Figure 2E and F). The increased expression of p42/p44 MAPK phosphorylation following EC-LPS challenge was efficiently controlled by butyrate (Figure 2E and F), which also reduced basal p42/p44 MAPK phosphorylation observed in the absence of any EC-LPS stimulation (Figure S3A and S3B).
Butyrate decreases ROS mediated NF-κB activation and inflammatory response in intestinal epithelial Caco-2 cells upon challenge with LPS from Escherichia Coli
Since ROS levels enhance the signal transduction pathways for NF-κB activation in the cytoplasm and translocation into the nucleus, we examined whether butyrate is able to attenuate ROS-mediated NF-κB activation in Caco-2 cells challenged with EC-LPS. We demonstrated that butyrate was effective in controlling the translocation of phosphorylated p-65- NF-κB into nuclear extracts (Figure 3A) after 4 h and up to 24 h (data not shown) of challenge with EC-LPS. GSTA1/A2 siRNA antagonized the down-regulatory effect of the butyrate on phosphorylated p-65- NF-κB induced by EC-LPS (data not shown).
10.1371/journal.pone.0032841.g003Figure 3 Effect of butyrate on NF-κB activation and inflammatory response in intestinal epithelial cells challenged with LPS from Escherichia Coli.
Caco-2 cells were treated for 24 h with butyrate and then were stimulated with EC-LPS for 4 h. (A) Immunoblot analysis of phospho-p65(Ser536) in cytoplasmic (C) and nuclear (N) cell fractions. αβ-tubulin and laminB were used as protein loading respectively for cytoplasmic and nuclear extract. (B–C) Caco-2 cells were treated for 24 h with butyrate and then were stimulated with EC-LPS for 24 h. (B, left panel) Immunoblot of COX-2 and ICAM-1. β-actin was used as loading control. (B, right panel) Densitometric analysis of the band intensity Values are means ± s.d., n = 6. Asterisks indicate that means differ from samples cultured with EC-LPS. *P<0.05. (C) TNF-α protein. Values are means ± s.d., n = 6. Asterisks indicate that means differ from samples cultured with EC-LPS. *P<0.05.
Since activation of the NF-κB/Rel transcription family plays a central role in inflammation through its ability to induce transcription of pro-inflammatory genes, we investigated whether butyrate is able to dampen down inflammatory response in Caco-2 cells challenged with EC-LPS. We showed that butyrate was effective in controlling COX-2, ICAM1 protein expression and TNF-α release induced by EC-LPS (Figure 3B and C). GSTA1/A2 siRNA antagonized the down-regulatory effect of the butyrate on COX-2, ICAM-1 protein levels and TNF-α release induced by EC-LPS (data not shown).
Butyrate decreases ROS mediated NF-κB activation and mucosal inflammation in CrD mucosal epithelial cells upon challenge with LPS from Escherichia Coli
We investigated the effect of butyrate in controlling ROS mediated mucosal inflammation in CrD colonic mucosa. Before challenge, higher numbers of epithelial cells with p-65-nuclear localisation were observed in CrD colonic mucosa compared with controls (Figure 4 A). EC-LPS challenge led to an increase in numbers of epithelial cells with p-65-nuclear localisation at 4 h of incubation in CrD colonic mucosa but not in controls (Figure 4D). Moreover treatment of cultured biopsies with butyrate was highly effective in controlling EC-LPS induced p-65-nuclear localisation (Figure 4A).
10.1371/journal.pone.0032841.g004Figure 4 Effect of butyrate on mucosal inflammation in CrD mucosal epithelial cells challenged with LPS from Escherichia Coli.
(A) Number of epithelial cells with p65 nuclear localisation per 100 epithelial cells in control (n = 10) and CrD colonic mucosa (n = 14) before challenge and after challenge with medium alone, medium with EC-LPS or EC-LPS with butyrate following 4 h of incubation. Nuclei counterstained with DAPI (blue). Scale bar, 10 µm. Values are means ± s.d.. Asterisks indicate that means differ from control samples, CrD samples cultured with medium alone and from CrD samples cultured with EC-LPS. *P<0.05. (B) Confocal microscopy of COX-2 (green) and ICAM-1(green) and number (C) of COX-2 and ICAM-1 positive lamina propria cells per mm2 of mucosa in CrD colonic mucosa (n = 14) after challenge with medium alone, medium with EC-LPS or EC-LPS with butyrate following 24 h of incubation. Nuclei counterstained with DAPI (blue). Scale bar, 10 µm. Values are means ± s.d., n = 14. Asterisks indicate that means differ from samples cultured with medium alone and from samples cultured with EC-LPS. *P<0.05. (D) TNF-α protein after challenge with medium alone, medium with EC-LPS or EC-LPS with butyrate following 24 h of incubation. Values are means ± s.d., n = 14. Asterisks indicate that means differ from samples cultured with medium alone and from samples cultured with EC-LPS. *P<0.05.
Butyrate also reduced the residual p65 localisation observed in CrD biopsies cultured in the absence of any EC- LPS stimulation (Figure S3C). To determine whether butyrate is able to switch off the mucosal inflammation in human EC-LPS stimulated CrD colonic mucosa, we also analysed the release of pro-inflammatory cytokines, such as TNF-α and COX-2 or ICAM-1 by mucosal mononuclear cells. This latter marker was studied as a broad factor of inflammation as described in previous study [28]. After 24 h of incubation, EC-LPS also induced an increase of ICAM-1 and COX-2 positive mononuclear cells and TNF-α release (Figure 4B, C and D) in CrD colonic mucosa compared with samples cultured in medium alone. The increased expression of ICAM-1, COX-2 and pro-inflammatory cytokine, as TNF-α release, following challenge with EC-LPS was efficiently controlled by butyrate (Figure 4B, C and D). Minimal TNF-α up-regulation and release, ICAM-1 and COX-2 were observed in controls after challenge with EC-LPS (data not shown).
Discussion
This report describes the relationships among a bacterial product, oxidative stress and mucosal inflammation in the mucosa of patients with CrD. We have identified butyrate's role in intestinal epithelial homeostasis by promoting anti-oxidative responses and inhibiting mucosal inflammation in the colon of CrD patients.
The short-chain fatty acid butyrate, which is mainly produced in the lumen of the large intestine by the fermentation of dietary fibers, plays a major role in the physiology of the colonic mucosa. It is also the major oxidative substrate for the colonocyte [34]. Impairment of IEC energy homeostasis is a typical feature of inflamed tissue in CrD. Constitutive energy expenditure mediated by persistent IEC activation and reduced energy supply may be the main causes of failure of IEC to preserve energy homeostasis [35].
Several studies report decreased butyrate oxidation in the inflamed mucosa of patients suffering from UC [36] or CrD [37] and in animal models of experimental colitis [38]. Although other studies found no defect in butyrate oxidation during IBD [39],[40],[41].
Previous studies in active IBD and in experimental DSS-colitis have shown that intestinal inflammation specifically affects butyrate metabolism [42],[43],[44]. Moreover, down-regulation of the Monocarboxylate Transporter 1 (MCT-1) is involved in butyrate deficiency in inflamed colonic mucosa of patients with IBD and of rats [45]. Thus, a decrease in MCT1 expression, which reduces the intracellular availability of butyrate [46] could affect not only its oxidation but also its cell regulatory effects.
Impaired energy availability as well as reduced tissue oxygen supply and the generation of intra and extracellular free radicals have also been to induce oxidative stress [47].
ROS are highly toxic to cells and oxygen radical formation in excess of physiological amounts may overtax the limited intestinal antioxidant defense system initiating oxidative injury to the gut [48] inducing damage to lipids, proteins and/or DNA. Moreover, increased oxidative stress has been seen to destroy the mucosal barrier of intestinal epithelial cells, increasing permeability. Different antioxidant defense mechanisms, including enzymatic antioxidant molecules, such as glutathione-S-transferase (GST) and non-enzymatic antioxidant molecules such as glutathione (GSH) are involved in protection against ROS. Deterioration of anti-oxidative glutathione metabolism [49] and increased colonic oxidative damage to proteins and DNA in association with impaired enzyme activity of Cu-Zn superoxide dismutase has been reported previously in patients with CrD [50]. Our studies demonstrate that butyrate was effective in controlling the increase of GSH reduced by EC-LPS in intestinal epithelial Caco-2 cell and in mucosal biopsies of CrD patients.
There is limited evidence of butyrate's role in controlling oxidative stress in the colonic mucosa. In two in vitro studies, pre-treatment of isolated rat [51] or human [51] colonocytes with butyrate reduced H2O2-mediated DNA damage. Since the butyrate's antioxidant role is not primary, it may be secondary, influencing DNA repair systems and levels of enzymatic or non-enzymatic antioxidants. Fermentable fiber uptake in a rat model of TNBS-induced colitis [52] is reported to increase colonic concentrations of butyrate, to decrease colonic myeloperoxidase (MPO) activity and to restore colonic GSH concentration [53]. We demonstrated that butyrate was effective in controlling the decrease of GST-α protein levels and activity induced by LPS in intestinal epithelial Caco-2 cells and in mucosal biopsies of CrD patients. GST is a detoxifying enzyme system that provides defense against oxidative stress compounds [54]. Since oxidative stress induces the impairment of the intracellular ROS balance, we evaluated whether butyrate reduces ROS levels and ROS-mediated stress sensitive signalling pathways induced by EC-LPS in Caco-2 cells and in mucosal biopsies of CrD patients. We demonstrated that butyrate was effective in controlling the increase of ROS levels and reduces ROS-mediated p42/44 MAPK phosphorylation.
The most extensively studied intracellular pathway that is a target of ROS and oxidative stress is the transcription factor NF-κB [55]. NF-κB is found in cytoplasm and is bound to Iκ-Bα, which prevents it from entering the nuclei [56]. When these cells are stimulated, specific kinases phosphorylate Iκ-Bα, causing its rapid degradation by proteasomes [56]. Activation of NF-κB acts on genes for proinflammatory cytokines, chemokines (chemotactic cytokines that attract inflammatory cells to sites of inflammation) [56], enzymes that generate mediators of inflammation, immune receptors, and adhesion molecules that play a key role in the initial recruitment of leukocytes to sites of inflammation. Moreover, infiltrating macrophages and neutrophils that are abundantly present in inflamed gut expose the inflamed intestine to substantial oxidative stress by production of ROS [8] sustaining a vicious circle that leads to a progressive and uncontrolled inflammatory response. Our results demonstrate that butyrate controlled ROS-mediated p65 NF-κB activation in intestinal epithelial Caco-2 cells after challenge with LPS from Escherichia Coli. Since activation of the NF-κB/Rel transcription family plays a central role in inflammation through its ability to induce transcription of pro-inflammatory genes, we showed that butyrate decreases COX-2, ICAM1 protein expression and TNF-α release induced by EC-LPS.
To provide the rationale and the proof-of-principle for using butyrate in CrD patients, we checked whether the mechanisms we observed in cell lines also take place in human CrD colons. Our approach to testing potential anti-inflammatory strategies in CrD, which we have used to study other inflammatory conditions and potential strategies to modulate the inflammatory response [12], is based on an ex vivo organ tissue culture model. This model represents a good approximation to in vivo studies since, all the anatomical connections in cultured biopsy tissues, are retained and all cell types (epithelial, myeloid, lymphoid) interact with neighboring cells within their natural environment. For this study we have used colonic mucosal biopsies which are routinely removed surgically.
We demonstrate that, in all CrD colonic tissues, butyrate reduced p65 phosphorylation and release of pro-inflammatory cytokines, such as TNF-α and COX-2 or ICAM-1 from mucosal mononuclear cells, thus restoring the pattern observed in controls after challenge with LPS from Escherichia Coli. Our study suggests that the restoration of intracellular ROS balance through appropriate control of the redox machinery may be a novel approach to treatment of CrD and may pave the way for the development of a new class of functional foods that, by enhancing butyrate production, could be effective treatments for CrD.
Supporting Information
Figure S1
Effect of butyrate on GST-α activity in EC-LPS stimulated intestinal epithelial cells and Crohn's mucosa. (A) Intracellular GST catalytic enzyme activity was assessed by conjugation of chloro-2,4-dinitrobenzyne with reduced GSH. Asterisks indicate that means differ from samples cultured with medium alone and from samples cultured with EC-LPS. *P<0.05. (B) Caco-2 cells were transfected with either 50 nM human GSTA1/A2 siRNA or scrambled oligonucleotides and then challenged with butyrate for 24 hours. (B, left panel) Immunoblot of GST-α. β-actin was used as loading control. (B, right panel) Densitometric analysis of the band intensity. Values are means ± s.d., n = 6. (C) Intracellular GSH activity in butyrate treated CrD mucosal epithelial cells stimulated with EC-LPS. Asterisks indicate that means differ from samples cultured with medium alone and from samples cultured with EC-LPS. *P<0.05.
(EPS)
Click here for additional data file.
Figure S2
Effect of butyrate on stress sensitive signalling pathways in EC-LPS stimulated intestinal epithelial cells. Caco-2 cells were cultured with butyrate or transfected with either 50 nM human GST-A1/A2 siRNA or scrambled oligonucleotides and then challenged with EC-LPS. (A) Intracellular GSH levels. Asterisks indicate that means differ from samples cultured with medium alone and from samples cultured with EC-LPS. *P<0.05. (B, left panel) Immunoblot of p42/p44 phosphorylation in Caco-2 cells challenged with EC-LPS. β-actin was used as loading control. (B, right panel) Densitometric analysis of the band intensity. Values are means± s.d., n = 6. β-actin was used as loading control for blot.
(EPS)
Click here for additional data file.
Figure S3
Expression of phosphorylated p42/p44-MAPk and p65-NF-κB in Crohn's mucosa. Confocal microscopy of phosphorylated p42/p44 (green) (A) and number of epithelial cells (B) with phosphorylated p42/p44 per 100 epithelial in CrD colonic mucosa (n = 14) Nuclei counterstained with DAPI (blue). Scale bar, 10 µm. Values are means± s.d.. Asterisks indicate that means differ from CrD samples cultured with medium alone. *P<0.05. (C) Number of epithelial cells with p65 nuclear localisation per 100 epithelial in CrD colonic mucosa (n = 14). Nuclei counterstained with DAPI (blue). Scale bar, 10 µm. Values are means± s.d.. Asterisks indicate that means differ from CrD samples cultured with medium. *P<0.05.
(EPS)
Click here for additional data file.
Competing Interests: The authors have declared that no competing interests exist.
Funding: These authors have no support or funding to report.
==== Refs
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22438885PONE-D-11-2129110.1371/journal.pone.0032853Research ArticleBiologyBiochemistryNucleic AcidsBiophysicsNucleic AcidsMicrobiologyVirologyViral ClassificationIdentification of XMRV Infection-Associated microRNAs in Four Cell Types in Culture Cellular microRNA Profiles of XMRV InfectionMohan Ketha V. K.
1
Devadas Krishnakumar
2
Sainath Rao Shilpakala
1
Hewlett Indira
2
Atreya Chintamani
1
*
1
Section of Cell Biology, Laboratory of Cellular Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, United States of America
2
Laboratory of Molecular Virology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, United States of America
Jeang K.T. EditorNational Institute of Health, United States of America* E-mail: [email protected] and designed the experiments: KVKM KD IH CA. Performed the experiments: KVKM KD SSR. Analyzed the data: KVKM. Wrote the paper: KVKM KD SSR IH CA.
2012 16 3 2012 7 3 e3285328 10 2011 31 1 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.2012This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.Introduction
XMRV is a gammaretrovirus that was thought to be associated with prostate cancer (PC) and chronic fatigue syndrome (CFS) in humans until recently. The virus is culturable in various cells of human origin like the lymphocytes, NK cells, neuronal cells, and prostate cell lines. MicroRNAs (miRNA), which regulate gene expression, were so far not identified in cells infected with XMRV in culture.
Methods
Two prostate cell lines (LNCaP and DU145) and two primary cells, Peripheral Blood Lymphocytes [PBL] and Monocyte-derived Macrophages [MDM] were infected with XMRV. Total mRNA was extracted from mock- and virus-infected cells at 6, 24 and 48 hours post infection and evaluated for microRNA profile in a microarray.
Results
MicroRNA expression profiles of XMRV-infected continuous prostate cancer cell lines differ from that of virus-infected primary cells (PBL and MDMs). miR-193a-3p and miRPlus-E1245 observed to be specific to XMRV infection in all 4 cell types. While miR-193a-3p levels were down regulated miRPlus-E1245 on the other hand exhibited varied expression profile between the 4 cell types.
Discussion
The present study clearly demonstrates that cellular microRNAs are expressed during XMRV infection of human cells and this is the first report demonstrating the regulation of miR193a-3p and miRPlus-E1245 during XMRV infection in four different human cell types.
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Introduction
XMRV is a recently identified gammaretrovirus, closely related to xenotropic murine leukemia viruses (MLVs), that was initially detected in familial cases of prostate cancer tissue using a virus gene array [1]. XMRV was also detected in blood cells of patients with Chronic Fatigue Syndrome (CFS) and normal healthy controls [2], [3]. Subsequently, a number of additional studies have failed to confirm any association of XMRV with CFS or prostate cancer [4]–[11]. Indeed, recent reports suggest that XMRV likely originated as a laboratory contaminant in prostate xenografts serially passaged through nude mice by the recombination of endogenous MLVs. Though the XMRV is of murine origin, it is known to infect different human cell types like T and B lymphocytes, NK cells, prostate cancer cell lines, and neuronal cells [12]–[15]. Various detection methods like serology, cell culture, and nucleic-acid based assays have already been used for detecting XMRV infection [4], [12], [16]–[19]. However, use of microRNAs (miRNAs) as biomarkers of XMRV infection has not been reported so far.
MicroRNAs have known to play a critical role in the life cycle of retroviruses and a few oncogenic viruses such as reticuloendotheliosis virus strain T (REV-T), Epstein-Barr virus and Hepatitis C virus (HCV) wherein the viruses regulate host cells and viral replication through specific microRNAs [20]–[23]. MicroRNAs are a class of evolutionarily conserved, endogenous, small non-coding RNAs that regulate gene expression and play a role in diverse cellular processes, including proliferation, differentiation and cell death [24]. As an abundant class of regulatory molecules, there are hundreds of distinct miRNAs identified in the human genome to date and hundreds more predicted. A single miRNA can regulate expression of multiple genes, and expression of a single gene may be regulated by several distinct miRNAs, creating complicated regulatory networks. It is estimated that roughly 60% of human protein-coding genes are regulated by miRNAs [25]–[28].
In this study, we evaluated whether miRNAs are modulated by XMRV in cultured cells and if so, can they be identified to see whether a single or a set of miRNAs specific to the infection can be detected early that could serve as biomarker(s) of XMRV infection. Our results demonstrate that a) two miRNAs, miR-193a-3p and miRPlus-E1245 (a proprietary sequence of Exiqon Inc, Denmark and named as such to differentiate from miR-1245) were commonly regulated among all 4 cell types infected with XMRV used in the study, and b) while miR-193a-3p is down regulated, miRPlus-E1245 exhibited varied expression profile in the four cell types infected with XMRV.
Materials and Methods
Cell culture and isolation and culture of Peripheral Blood Lymphocytes (PBL)
LNCaP (ATCC, Manassas, VA) and DU145 cells (kind gift from Robert Silverman, Cleveland Clinic, Ohio to Indira Hewlett) were cultured in RPMI 1640 supplemented with 10% FBS, 2 mM glutamine, 100 units/ml of penicillin, and 100 units/ml streptomycin [29], [30]. PBMC were isolated from the peripheral blood of HIV seronegative donors (NIH Blood Bank) by Ficoll/Hypaque density gradient centrifugation. Monocytes were removed by adherence to the culture flasks and the remaining Peripheral Blood Lymphocytes (PBL) were stimulated with 2 µg/ml PHA for 3 days to activate T cells before infection. The PBL were cultured in RPMI 1640 supplemented with 10% FBS, 2 mM glutamine, 100 units/ml of penicillin, 100 units/ml of streptomycin, and 5 units/ml of human Interleukin-2 (Roche, NJ) until further use.
Isolation and culture of Monocyte-derived Macrophages (MDMs)
Monocytes were isolated from PBMC of donors seronegative for HIV-1 and hepatitis B after leukopheresis and purified by countercurrent centrifugal elutriation [31]. Cell suspensions contained >95% monocytes by criteria of cell morphology on Wright-stained cytosmears, by granular peroxidase and by non-specific esterase. The cells were cultured for 5 days in DMEM supplemented with 10% FBS, 2 mM glutamine, 100 units/ml of penicillin, and 100 units/ml streptomycin and 1000 U/ml macrophage colony stimulation factor (M-CSF) before infection with XMRV. All cell culture reagents were tested by Limulus Lysate assay (Associates of Cape Cod, Cape Cod, MA) for endotoxin contamination and the levels were found to be <0.06 EU/ml.
Infection with XMRV
Prostate cell lines LNCaP and DU145, Peripheral Blood Lymphocytes (PBL) and Primary monocyte-derived macrophages (MDM) were infected with 1×107 XMRV copies/mL. After a three-hour exposure, virus particles were removed, and fresh medium was added and cultured at 37°C. Infected cells were isolated at the indicated times, washed twice in 1×PBS and stored at –80°C until further use.
Cellular total mRNA extraction
Total RNA from the four cell types stored as above was extracted by using miRCURY™ RNA isolation kit as per the manufacturer's instructions (Exiqon, Denmark). The final volume of RNA extracted from each column was approximately 75–100 µl which was quantified by Nanovue® Plus spectrophotometer (GE Life Sciences, Piscataway, NJ). All the above experiments were conducted twice and RNAs extracted from duplicate experiments were analyzed for microRNA (miR) expression profile.
10.1371/journal.pone.0032853.g001Figure 1 Quantitative-PCR analysis of XMRV infection.
Total mRNA from (A) LNCaP, (B) DU145, (C) PBLs and (D) MDM cells either mock- or virus-treated were subjected to RT-PCR using the gag primers for detecting XMRV infection. GAPDH primers were used as endogenous controls. As evident, XMRV was able to infect all 4 cell types with substantial increase in infection by the 48 h time point.
Quantitative RT-PCR
The endogenous human Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) control real-time PCR primers and probes (TaqMan endogenous human GAPDH control, Cat# 4352934E) and the XMRV gag gene specific real-time PCR primers and probes were obtained from Applied Biosystems, Foster City, CA. Thermal cycling was performed for XMRV and GAPDH in triplicate on RNA samples in a Micro-Amp Optical 96-well reaction plate (Applied Biosystems, Foster City, CA). Briefly, an equal amount of total RNA was used to quantify XMRV levels using QuantiTect Probe RT-PCR kit (Qiagen Inc., Valencia, CA). Real-time PCR Master Mix (Quantitect Probe RT-PCR, Qiagen Inc., Valencia, CA) was added to the RNA, forward (CGAGAGGCAGCCATGAAGG) and reverse (CCCAGTTCCCGTAGTCTTTTGAG) gag primers, probe (6FAM-AGTTCTAGAAACCTCTACACTC-MGBNFQ) and primer for XMRV viral RNA first strand synthesis (reverse-transcription) (GAGATCTGTTTCGGTGTAATGGAAA) in a total volume of 25 µL. The mixture was incubated at 50°C for 2 min (for RNA, 20 min), at 95°C for 10 min, and then cycled at 95°C for 15 sec and 60°C for 60 sec 40 times, in an Applied Biosystems 7500 sequence detection system. XMRV levels were quantified using XMRV clone VP62-pcDNA3.1 (GenBank accession no. EF185282; obtained through NIH AIDS Research and Reference Reagent Program) as a standard. Each experimental sample was normalized relative to the GAPDH endogenous control and the relative amount of target gene quantified.
Assessment of miRNA Quality and Array analysis
MicroRNA array services were contracted out to Exiqon Services, Denmark. The quality of the total RNA was verified by an Agilent 2100 Bioanalyzer profile. 1000 ng total RNA from sample and reference was labeled with Hy3™ and Hy5™ fluorescent label, respectively, using the miRCURY LNA™ microRNA Power Labeling Kit, Hy3™/Hy5™ (Exiqon, Denmark) following the procedure described by the manufacturer. The Hy3™-labeled samples and a Hy5™-labeled reference RNA sample were mixed pair-wise and hybridized to the miRCURY LNA™ microRNA Array (5th gen-hsa, mmu & rno) (Exiqon, Denmark), which contains capture probes targeting all miRNAs for human, mouse or rat registered in the miRBASE 16.0. The hybridization was performed according to the miRCURY LNA™ microRNA Array instruction manual using a Tecan HS 4800™ hybridization station (Tecan, Austria). After hybridization the microarray slides were scanned and stored in an ozone free environment (ozone level below 2.0 ppb) in order to prevent potential bleaching of the fluorescent dyes. The miRCURY™ LNA array microarray slides were scanned using the Agilent G2565BA Microarray Scanner System (Agilent Technologies, Inc., USA) and the image analysis was carried out using the ImaGene 9.0 software (BioDiscovery, Inc., USA). The quantified signals were background corrected (normal exposure with offset value 10) and normalized using the global LOWESS (LOcally WEighted Scatterplot Smoothing) regression algorithm [32].
10.1371/journal.pone.0032853.g002Figure 2 Principal Component Analysis (PCA) Plot.
The principal component analysis is performed on all samples and on all microRNAs with standard deviation over 1. The normalized log ratio values have been used for the analysis. Abbreviations: LM (LNCaP Mock); LV (LNCaP Virus-treated); DM (DU145 Mock); DV (DU145 Virus-treated); PM (PBL Mock); PV (PBL Virus-treated); and MM (MDM Mock); MV (MDM Virus-treated). The LNCaP and DU145 cell lines form tight cluster indicating minimal variation with regard to infection status and time. PBLs and MDMs are more spread suggesting variation due to both and time and infection status.
Results
Quantitative RT-PCR demonstrates XMRV infection in all 4 cell types studied
XMRV infection in the 4 cell types was determined by performing a quantitative RT-PCR (qPCR) on the total RNAs extracted at various time points. Endogenous GAPDH levels were used for normalizing the expression levels of the XMRV gag target gene. Results demonstrated that XMRV was able to infect all 4 cells types studied, very robustly in the prostate cell lines (LNCaP and DU145) and moderately in PBLs and MDMs (Fig. 1). A 3–10 fold increase in XMRV infection by 48 hours was evident among all 4 cell types (Fig. 1).
10.1371/journal.pone.0032853.g003Figure 3 Heat map and unsupervised Hierarchical Clustering.
The clustering is performed on all samples and on all microRNAs with standard deviation over 1. Each row represents one microRNA and each column represents one sample. The microRNA clustering tree is shown on the left. The color scale shown at the bottom illustrates the relative expression level of a microRNA across all samples: red color represents an expression level above mean, green color represents expression level lower than the mean.
MicroRNA expression profiles of XMRV infected continuous cell lines differ from that of infected primary cells (PBL and MDMs)
Principal Component Analysis (PCA), a method used for reducing the dimension of large data sets and thereby useful to explore naturally arising sample classes/groups based on expression profile was performed. By including the microRNAs that have the largest variation across all samples (SD>1) an overview of how the samples cluster based on this variance is obtained. Based on this method, if the biological differences between various samples are pronounced, then this would become a primary component of the variation leading to segregation of samples in different regions of a PCA plot corresponding to their biology. As demonstrated in Fig. 2, it is observed that the overall miRNA expression profile in LNCaP and DU145 cell lines form a tight cluster suggesting minimal variation due to time and infection status of these two cell types, while the PBLs and MDM profiles are more spread, indicating the global miR levels being affected both due to time and infection status.
10.1371/journal.pone.0032853.g004Figure 4 Identification of top 25 commonly regulated microRNAs in the four cell types tested.
As seen in this venn diagram two miRs are commonly regulated in all four cell types. While 6 miRs were commonly regulated in 3 of the 4 cell types tested, 11 miRs were found to be common in at least 2 of the 4 cell types. Cell-type specific miR regulation was found to be 11 miRs each for PBL and DU145 cell lines and 16 miRs each were regulated in LNCaP and MDM cells.
Heat map analysis and unsupervised hierarchical clustering
MicroRNA expression profiles from four different XMRV-infected cell types at 3 different time points were plotted on a heat map. As observed in Fig. 3, the heat map diagram depicts results of the two-way hierarchical clustering of microRNAs and samples. Each row representing one miRNA and each column representing one sample confirms the variation in miRNA expression profile between the continuous prostate cancer cell lines (LNCaP and DU145) and the PBLs and MDMs. A more distinct pattern of demarcation in miRNA profiles between these two set of cell types is observed in the region of the heat map depicting miR-1275 to miR-765 (Fig. 3). The microRNA clustering tree is shown on left of the figure.
10.1371/journal.pone.0032853.g005Figure 5 Tabular representation of top 25 most differentially expressed miRs in the 4 cell types D-DU145; L-LNCaP; P-PBL, and M-MDMs).
(Yellow represents miRNAs present in all 4 lists (hsa-miR-193a-3p and hsa-miRPlus-E1245), blue represents miRNA present in 3 out of 4 lists and dark-red represents miRNAs present in 2 out of 4 lists.
miR-193a-3p and miRPlus-E1245 are specific to XMRV infection in all 4 cell types
The main objective of the current study was to identify common miRNAs that are uniformly regulated in all 4 cells types due to XMRV infection and hence an overall comparative analysis was performed. While comparing the two sample groups (mock versus infected) using a paired t-test, no microRNA was found to be differentially expressed using a cut-off of p-value<0.05. Since the experiment involved 4 cell lines that behave differently, with two treatments (mock- and virus-treated) and 3 time-points (6 h, 24 h and 48 h), a more logical approach was adopted to simply look into the differences between the two treatments over the three time-points, by subtracting the control (mock) from the virus treatment. Subsequently, the most differentially expressed genes between the 3 time points (6 h to 24 h and 48 h) was estimated. As represented in a venn diagram (Fig. 4) a total of 72 differentially expressed miRs were observed between the 4 four cell types. Among these, 2 miRs were common in all 4 cell types, while 6 miRs were found to be common in 3 of the 4 cell types and 11 miRs were common in at least 2 of the 4 cell types.
10.1371/journal.pone.0032853.g006Figure 6 Differential expression of miR-193a-3p and miRPlus-E1245.
By subtracting the miR expression values from the virus-infected and mock-infected (M-V) and further subtracting the values between the time points (▵T6▵T24 and ▵T6-▵T48) an expression profile was generated for the two miRs as mentioned above. It can be observed that miR-193a-3p is down regulated over time due to virus infection in all 4 cell types, whereas miRPlus-E1245 exhibits varied levels of expression profile between the 4 cell types.
Further analysis of the top 25 differentially expressed miRs between the 4 cell types revealed that the two miRNAs common to all 4 cell types were miR-193a-3p and miRPlus-E1245 (Fig. 5). The miRPlus-E1245 is a recently discovered miRNA and proprietary sequence of Exiqon Inc, Denmark and named as such to differentiate it from miR-1245. The sequence is not yet annotated and hence not been submitted to the miRNA database yet. More significantly, though the miRPlus-E1245 levels were in the top 25 list in one experiment and moderately regulated in the second experiment, the miR-193a-3p expression profile was among the top 25 list in both the microarray experiments. The reason for the observed differences in the relative abundance of the differentially expressed miRNAs could be due to donor-to-donor variations. MicroRNAs that were common in 3 of the 4 cell types were miR-15a, miR-19a, miR-29b, miR-32, miR-33a, and miR-101. Eleven microRNAs that were differentially expressed and common in 2 of the 4 cell types were found to be miR-17, miR-21, miR-29c, miR-141, miR-142-3p, miR-215, miR-494, miRPlus-E1072, miRPlus-E1192, miR-1248, and miR-1973 (Fig. 5).
miR-193a-3p is down regulated and miRPlus-E1245 exhibits varied expression profile
Following the identification of 2 miRs (miR-193a-3p and miRPlus-E1245) that were specific to all 4 cell types infected with XMRV, it was logical to deduce the expression profile of these two miRNAs in all 4 cell types over time in virus-infected cells. By subtracting the miRNA expression values from the virus-infected and mock-infected (M-V) and further subtracting the values thus generated between the time points (▵T6-▵T24 and ▵T6-▵T48), an expression profile was generated for the two miRNAs as mentioned above. It can be observed in figure 5 that miR-193a-3p is down regulated over time due to the virus infection in all 4 cell types. The miRPlus-E1245 however exhibits varied levels of expression profile between the 4 cell types with up regulation in MDM and PBL cell types and down regulation in LNCaP and DU145 cell types (Fig. 6).
10.1371/journal.pone.0032853.t001Table 1 Target prediction for miR-193a-3p using 3 different programs.
S.No Gene description Gene symbol miRDB TargetScan microRNA.org
1 SON DNA binding domain SON + + +
2 Friend Leukemia virus Integration 1 FLI1 + +
3 Abl Interactor 2 ABI2 + +
4 v-erb-erythroblastic leukemia viral oncogene homolog 4 (avian) ERBB4 + +
5 Solute carrier family 10 (Na/bile acid cotransporter family), member 6 SLC10A6 + +
6 FH2 domain containing 1 FHDC1 + +
Top 10 genes commonly picked by at least two different prediction programs are indicated.
Discussion
The discovery of XMRV and its potential association with PC and CFS aroused considerable excitement and promise within the research and clinical community regarding a possible infectious etiology for at least some cases of these disease or conditions. [2], [3], [33]. However, recent research findings have not supported any association between the virus and CFS or prostate cancer [7], [9], [10], [34]–[37]. In fact, the virus itself may have originated as a result of recombination in a laboratory setting [38], [39]. Specifically, it has been postulated that XMRV originated as a result of recombination between two MLV proviruses in laboratory mice [40]. These findings appear to raise doubts about the significance and involvement of XMRV in any human disease or condition [38]–[41]. Nonetheless, because at least some studies have demonstrated that XMRV is a culturable virus and that it can readily infect cells of human origin [12]–[15], additional research efforts will help to further our understanding of XMRV pathogenesis and provide insights into the modes of transmission involved in XMRV infection. It also remains to be seen whether XMRV demonstrates potential to be transmitted across species [12], [37], [41].
The present study further emphasizes that XMRV can infect human prostate and hematopoietic cells and the study clearly demonstrates that microRNAs are regulated during XMRV infection of these culturable human cells. In fact, the qPCR results indicate that while all the 4 cell types were susceptible to XMRV infection with significant increase in viral titers by 48 h time point it was evident that there was a distinct difference in infection levels between the 4 cell types (Fig. 1). The prostate cell lines (LNCaP and DU145) supported robust XMRV infection, while the PBLs and MDMs were moderately infected. It is interesting to note that the variability in infection status of the 4 cell types may potentially be dependent on individual APOBEC levels in each cell type [42]. It has been shown earlier that XMRV is resistant to human APOBEC 3G (hA3G) and that the levels of hA3G are down-regulated by XMRV in LNCaP and DU145 cells thereby supporting efficient viral infection in these cell types [42], [43]. The hA3G is down regulated by the human immunodeficiency virus-1 (HIV-1) vif protein during infection. However, since XMRV lacks vif, an alternate mechanism of hA3G down regulation has been suggested [43]. PBMCs on the other hand, seemingly possess significantly higher levels of h3AG and hence are relatively resistant to XMRV infection [42].
The two microRNAs (miR-193a-3p and miRPlus-E1245) are moderately regulated in the four cell types. However, it is interesting to note that within the four cell types, miR-193a-3p is down regulated over time, while miRPlus-E1245 however exhibited varied levels of expression profile between the 4 cell types: up regulation in MDMs and PBL cell types and down regulation in LNCaP and DU156 cell types. Since the miRPlus-E1245 has not been annotated and not submitted in the miRNA database yet by its discoverer, the Exiqon Inc., Denmark, it is not feasible at this time to identify its potential targets. Therefore, we only analyzed the miR-193a-3p for its tentative mRNA targets by 3 different online programs as indicated in Table 1. Target Prediction by miRDB, TargetScan and microRNA.org programs revealed that out of the top 10 mRNA targets that were identified individually by these 3 different softwares, 1 target mRNA was picked by all three programs and 5 mRNA targets were commonly flagged at least by two different programs. Of the six predicted mRNA targets for miR-193a-3p, five mRNA targets were related to tumorogenesis or suppression. Interestingly 3 mRNA targets, namely SON DNA binding domain (SON), Friend Leukemia Virus Integration 1 (FLI1) and v-erb-erythroblastic leukemia viral oncogene homolog 4 (ERBB4) have been implicated with virus/virus infections. Of the 3, the FLI1 protein (or its homolog) may have a potential role in XMRV infection as this protein has already been implicated in Friend Leukemia Virus which also is a retrovirus causing tumorigenesis [44], [45]. The FLI1 is a protein responsible for the integration of the viral gene into the host DNA thereby leading to carcinogenesis [44], [45]. The human genome was recently analyzed for potential XMRV genome integration sites and results revealed that the virus had integration sites in at least 11 of the 23 chromosomes [46]. Hence it is to be seen whether this particular host mRNA target is being modulated by miR-193a-3p during XMRV infection. Of the other two, while the SON protein binds to hepatitis B virus (HBV) DNA and exhibits sequence similarity to other oncoproteins, the ERBB4 protein affects mitogenesis and cell differentiation and furthermore it is known that mutations within this gene are associated with cancer [47]–[49].
More pertinently, while the qPCR results revealed robust infection in two cell types (LNCaP and DU145 cells) and moderate infection in the other two tested cell types (PBLs and MDMs), what is common to all 4 cell types is the regulation of the two miRNAs (miR-193a-3p and miRPlus-E1245) during XMRV infection regardless of the level of infectivity, virus titer or dose of the infection. This is the first report indicating the expression and regulation of miRs during XMRV infection of human cells. It remains to be seen whether the same set of miRNAs are up regulated during infection of murine cells or cell lines.
The current findings reported here certainly demonstrate that XMRV infection modulates miRNAs in the host cells as is the case with many other viruses that are pathogenic to humans [20]–[23]. In human retroviruses such as HIV-1 and HTLV-1, the role of microRNAs has already been demonstrated [50]–[54]. Many of these exquisite studies have clearly shown how certain miRs up regulate or down regulate certain host genes/proteins to promote viral infection or disease pathogenesis [50]–[52], [54], [55]. In fact, it is now known that HIV-1 and other viruses themselves code for microRNAs, which play a critical regulatory role during virus infection [50], [56]. Our studies also demonstrate that miRNA profiles are different in XMRV-infected prostate cancer cell lines compared to primary hematopoietic cells, suggesting that miRNAs could play a role in XMRV infection, and serve as markers of XMRV infection in cultured cells.
Review of the manuscript by Drs. Xuan Chi and Krishnamurthy Konduru, CBER, is highly appreciated.
Competing Interests: The authors have declared that no competing interests exist.
Funding: SSR is a recipient of a postdoctoral fellowship at the Center for Biologics Evaluation and Research administered by the Oak Ridge Institute for Science and Education through an intra-agency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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J PregnancyJ PregnancyJPJournal of Pregnancy2090-27272090-2735Hindawi Publishing Corporation 2251831410.1155/2012/874290Research ArticleThe Effects of Fetal Gender on Serum Human Chorionic Gonadotropin and Testosterone in Normotensive and Preeclamptic Pregnancies Lorzadeh Nahid
*Kazemirad Sirous
Department of Obstetrics and Gynecology, Asali Hospital, Lorestan University of Medical Sciences, Lorestan, Iran*Nahid Lorzadeh: [email protected] Editor: Albert Fortuny
2012 15 2 2012 2012 8742907 7 2011 6 11 2011 30 11 2011 Copyright © 2012 N. Lorzadeh and S. Kazemirad.2012This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Introduction. The aim of the present study was to evaluate the effects of fetal sex on serum human
chorionic gonadotropin (hCG) and testosterone in normotensive and preeclamptic pregnancies. Materials and Methods. This is a cross-sectional study and 139 women with singleton pregnancies in the third trimester were studied. Seventy-one pregnancies were uncomplicated; among those were 35 male and 36 female fetuses. Sixty-eight pregnancies were complicated by preeclampsia; among those were 35 male and 33 female fetuses. Human chorionic gonadotropin and total testosterone were
measured in maternal peripheral blood. Data analyzed by SPSS software. Results. In male-bearing pregnancies, maternal hCG and testosterone serum levels were significantly higher in preeclamptic than normotensive mothers (P < 0.001 and P < 0.001, resp.) in female-bearing pregnancies testosterone levels were significantly higher in preeclamptic than normotensive mothers (P < 0.001). Total testosterone levels were significantly higher in pregnancies with either
gender and significantly higher in mlae-bearing than in female-bearing pregnancies. Conclusion. According to our results, there is a correlation between maternal serum hCG and testosterone levels and preeclampsia. Therefore these tests can be used as routine during 30–38 weeks of gestation. High maternal serum concentrations of these markers can predict preeclampsia.
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1. Introduction
Following the inflammation cascade, placenta produces some peptides that might be valuable as predictive markers of preeclampsia. Some studied peptides in this field include corticotrophin-releasing hormone (CRH, hCG), human chorionic gonadotropin, activin A, and inhibin A. Since the disease is resolved soon after delivery, most attention is focused on fetus, placenta, and fetal membranes. hCG concentration in maternal blood is 100 U/L during miss period. At 8–10 weeks of gestation, hCG level rises to a peak of 100,000 U/L, then drops down to 10000–20000 U/L at 18–20 weeks and reaches a plateau for the rest of the pregnancy [1]. During the first and second trimesters of normal pregnancy, there are no gender differences in maternal hCG levels. However, from the second to the third trimesters, there is a marked shift in maternal hCG serum concentrations. The hCG levels in female-bearing pregnancies increase significantly, whereas they decrease in male-bearing pregnancies [1–3]. Maternal serum concentration of total testosterone (TT) increases gradually throughout pregnancy, whereas free testosterone (FT) levels change very little until the third trimester and then become double. The sources for the increased testosterone levels in maternal serum are unknown but could be the ovarian theca-interstitial cells and the maternal cortex, which might be stimulated by hCG throughout pregnancy [4].
Gurbuz et al. in a study in 2004 found that the maternal serum hCG level is a useful laboratory tool when managing and treating hypertensive disorders that complicate pregnancy [5]. An increased level of serum hCG is found in mothers with pregnancies complicated by preeclampsia. On the other hand, testosterone levels have been shown to be significantly higher in preeclamptic pregnancies than normal pregnancies [6–8]. Steier et al. in a study in 2002 found that in male-bearing pregnancies maternal serum hCG and testosterone levels are significantly higher in preeclamptic mothers than normotensive mothers [2, 9–11]. Gol et al. in 2004 found that hCG concentration in maternal serum and cord blood is significantly higher in normal pregnancies with female than male fetuses [3, 12]. Elsmén et al. concluded that mean serum hCG level in male-carrying pregnancies was significantly higher in preeclamptic pregnancies than in normal pregnancies [13]. Due to these limited data about the relationship between fetal gender and preeclampsia, this study was undertaken to investigate the effects of fetal gender on maternal serum concentration of hCG and testosterone in normotensive and preeclamptic pregnancies.
2. Materials and Methods
All pregnant women admitted to our outpatient obstetric clinic of Haj Asali Hospital for pregnancy visits from 23 October 2008 to 22 September 2009 were enrolled in this cross-sectional study. All women underwent routine pregnancy examinations, and if they had a blood pressure equal to or higher than 140/90 mmHg, urine analyses were requested to detect proteinuria. Women who had a proteinuria between trace to 2+ and without any signs and symptoms of severe preeclampsia were considered as mild-to-moderate preeclampsia. Pregnancies with severe preeclampsia did not enter the study. Pregnancies with gestational diabetes and fetal anomalies were also excluded from the study. Finally a total of 139 mothers met the criteria to enter the study.
Of the 139 women in the study, 68 were preeclamptic and 71 were normotensive. Among preeclamptic pregnancies were 35 male fetuses and 33 female fetuses. Of the 71 normal pregnancies, 35 were male bearing and 36 were female bearing. All pregnancies were at 30–38 weeks of gestation. The study was approved by the Ethical Institutional Committee of Lorestan University. Written informed consent was obtained from all participants. The including criteria was the patients who were primigravid, had third trimester pregnancy, BP = 140/90 mmHg, and proteinuria = 300 mg in a 24 h urine sample. Patients with renal diseases, chronic hypertension, renal and urinary infection, fetal disorders, multiple pregnancy, and immunologic diseases were excluded from the study. Pregnant women without the above criteria were considered as control group. A questionnaire was completed for each patient including patient's age, gestational age, parity, the history of hypertension, diabetes mellitus, hypertension in family, diabetes mellitus in family, and tobacco consumption. After taking informed consents from all pregnant women who entered the study, they were examined physically. Blood pressure and urine specimens were collected, and detailed ultrasonographic structural examinations of the fetuses were performed. Simultaneously, blood samples were drawn from an antecubital maternal vein prior to delivery at the time of admission (30–38 wk of gestation) for examination of hCG and total testosterone. A total of 10 CC peripheral venous blood was taken from each woman and sent to the central laboratory of Asali Hospital. Venous blood was collected by venepuncture in 10 mL silicone-coated Vacutainer blood-collecting tubes containing no additives between 7:00 AM and 9:00 AM in a fasting state. Blood was allowed to clot at room temperature and was then centrifuged for 20 minutes at 2000 g. Aliquots of the serum were then stored at −20°C until they were required for the assay.
Consistent with the definition of the ACOG, preeclampsia was defined as new-onset hypertension after 20 weeks of gestation, with a diastolic blood pressure of 90 mmHg or higher with concurrent proteinuria of 300 mg/24 hours or greater. Semiquantitative dipstick tests were used for measurement of proteinuria (1+ and 2+ corresponded to 300 mg/24 hours and 500 mg/24 hours, resp.). 10 human chorionic gonadotropin were measured by immunoradiometric assay (hCG IRMA KIT, IMMUNOTEC as Radova Prague). The Third International Standard for hCG was used as a reference standard. The assay has a detection limit of 3 IU/L. The intra-assay coefficient of variation was 3.3 and 7.0 for mean hCG levels at 21 and 1500 IU/L, respectively. The interassay coefficient of variation was 7.2 and 9.1 for hCG levels at 21 and 1500 IU/L, respectively.
Testosterone level was determined by a DRG Testosterone Eliza Kit (DRG Instruments GmbH, Germany). The test is based on a radioimmunoassay technique. The expected serum testosterone levels ranged between 0.3 and 2.8 nmol/L. The sensitivity was 0.1 nmol/L. The intra-assay coefficient of variation was 7.5 and 5.5 for testosterone levels at 1.6 and 26.5 nmol/L, respectively. The interassay coefficient of variation was 7.0 and 4.8 for testosterone levels at 1.2 and 23.3 nmol/L, respectively.
2.1. Statistical analysis
Laboratory tests results and other data (such as maternal age, gestational age, parity, type of pregnancy, systolic blood pressure, and diastolic blood pressure) were gathered in a questionnaire. Statistical analyses were performed using t-test and unparametric tests as appropriate, with statistical significance considered at P < 0.05. Data are presented as mean with standard error and 95% confidence interval (CI). SPSS version 15 was used to analyze the data.
3. Results
A total of 139 women with singleton pregnancies at 30–38 weeks of gestation entered the study. Maternal age was 14–38 years old, and parity was 0–3. In preeclamptic group, diastolic blood pressure was 90–110 mmHg and systolic blood pressure was 140–155 mmHg, and no difference was found between male and female fetuses (Table 1). Proteinuria was determined between +1 and +2, and it was not different between male and female fetuses. Mean serum hCG level was 16337 ± 2462 U/L in male-bearing normal pregnancies and 28774 ± 3197 U/L in female-bearing normal pregnancies. A statistically significant difference was found between the two mean values using t-test (P < 0.005). Mean serum hCG level was 31610 ± 3825 U/L in male-bearing preeclamptic pregnancies and 34480 ± 4559 U/L in female- bearing preeclamptic pregnancies. No significant difference was found between the two mean values using t-test (NS) (Table 2). Mean serum testosterone level in normal pregnancies was 2.85 ± 0.33 nmol/L for male fetuses and 2.50 ± 0.25 nmol/L for female fetuses. t-test showed no significant difference between the two mean values (NS). In pregnancies complicated with preeclampsia, mean serum testosterone level was 5.86 ± 0.78 nmol/L in male-carrying pregnancies and 3.92 ± 0.40 nmol/L in female-carrying pregnancies. The difference between mean values was statistically significant using t-test (P < 0.001) (Table 3). Mean serum hCG level in male-bearing pregnancies was significantly higher in preeclamptic (16337 ± 2462 U/L) than in normotensive pregnancies (31610 ± 3825) (P < 0.001) (Table 2). Mean serum hCG level in female-bearing pregnancies was not significantly different between normotensive (28774 ± 3197 U/L) and preeclamptic (34480 ± 4559 U/L) groups. Mean serum testosterone level in male-bearing pregnancies was significantly higher in preeclamptic group (2.85 ± 0.33 nmol/L) than in normotensive group (5.86 ± 0.78 nmol/L) (P < 0.001). Mean serum testosterone level in normotensive pregnancies with male fetus (2.50 ± 0.25 nmol/L) was found to be significantly lower than in female-bearing pregnancies complicated with preeclampsia (3.92 ± 0.40 nmol/L) (P < 0.001) (Table 3). Mean hCG serum level without considering fetus gender was shown to be 22643 ± 776 U/L in normal pregnancies and 33003 ± 339 U/L in preeclamptic pregnancies. The difference was shown significant using t-test (P < 0.001) (Table 4). Mean serum testosterone level without considering fetus sex was reported 2.67 ± 0.03 nmol/L in normal pregnancies and 4.92 ± 0.12 nmol/L for preeclamptic pregnancies (Table 5).
4. Discussion
These results are consistent with previous studies which also indicated that there is no gender difference in maternal serum hCG level during the first and second trimesters of pregnancy, but there is a shift from second to third trimester so that hCG levels significantly increase in female-bearing pregnancies and decrease in male-bearing pregnancies [9]. In a study by the American Association of Obstetricians and Gynecologists, it was reported that, in normal pregnancies, maternal serum hCG level is significantly higher in female-bearing pregnancies than in male-bearing pregnancies [12]. Several studies have found elevated hCG concentrations in maternal blood in preeclamptic pregnancies but without regard to fetal gender. Gurbuz et al. in a study in 2004 found that the maternal serum hCG level is a useful laboratory tool when managing and treating hypertensive disorders that complicate pregnancy. The serum hCG level is especially significant in severe preeclampsia and superimposed preeclampsia. Therefore, a high serum hCG level can be a helpful marker in the diagnosis and clinical management by preventing possible complications resulting from severe and superimposed preeclampsia [5]. In a study in Iran from 2001 to 2005, Basirat et al. found that maternal serum hCG level was significantly higher in preeclamptic pregnancies in comparison with normal pregnancies (P = 0.031) [6]. Bartha et al. in 2003 also indicated increased levels of maternal serum hCG in preeclamptic compared to normal pregnancies [14]. In the present study, the hCG levels in maternal blood in male-bearing preeclamptic pregnancies were significantly higher compared with that in male-bearing uncomplicated pregnancies, whereas in preeclampsia with female fetuses no significant increase in the maternal hCG level was observed. In a study in Turkey (2005), it was found that maternal serum hCG level at 24–28 weeks and 32–36 weeks of gestation is significantly higher in female-carrying pregnancies than male-bearing pregnancies (P < 0.004 and P < 0.001, resp.) [12]. Steier et al. in a study in 2002 found that in male-bearing pregnancies maternal serum hCG and testosterone levels are significantly higher in preeclamptic mothers than in normotensive mothers (P < 0.001). In female-bearing pregnancies, testosterone level was significantly higher in preeclamptic than normotensive mothers whereas hCG levels were not significantly different. Maternal serum levels of testosterone were significantly higher in preeclamptic pregnancies with male fetuses than with female fetuses (P < 0.001), whereas hCG levels were not significantly different. In normal pregnancies, female-bearing pregnancies had significantly higher serum hCG levels than male-bearing pregnancies (P < 0.005), whereas testosterone levels were not significantly different [9]. Elsmén et al. concluded that mean serum hCG level in male-carrying pregnancies was significantly higher in preeclamptic pregnancies than in normal pregnancies [13]. This finding was consistent with the study in America in which serum hCG level was found to be significantly higher in male-bearing pregnancies complicated with preeclampsia than normal ones (P < 0.001).
In the present study, mean serum hCG level was not significantly different between normal and preeclamptic pregnancies with female fetuses which was inconsistent with the results of the American Association of Obstetricians and Gynecologists. In fact, high maternal serum concentration of hCG in female-bearing pregnancies is not indicative of preeclampsia since this increment is also seen in normal pregnancies, whereas, in male-carrying pregnancies, high maternal serum levels of hCG can be a marker to predict preeclampsia. Mean serum hCG level was not significantly different in preeclamptic pregnancies with male and female fetuses which was in consistence with Steier et al.'s findings. Since there is an increment in maternal serum hCG levels in preeclamptic pregnancies with both male and female fetuses, so the difference between them is not clinically valuable. This finding suggested that the placenta seems to play a fundamental role in preeclampsia, as the condition improves rapidly after its removal. Examination of the placenta in pregnancies complicated by preeclampsia has revealed focal cellular necrosis with increased mitotic activity in the syncytiotrophoblast and cellular proliferation in the cytotrophoblast. A transformation of the cytotrophoblast into the syncytiotrophoblast also has been reported [15, 16].
These changes might explain the elevated maternal serum hCG levels in male-bearing preeclamptic pregnancies, but there is still no explanation for no increase in maternal serum hCG levels in female bearing with preeclampsia. In fact, high maternal serum concentration of hCG in female-bearing pregnancies is not indicative of preeclampsia since this increment is also seen in normal pregnancies, whereas, in male-carrying pregnancies, high maternal serum levels of hCG can be a marker to predict preeclampsia.
The serum levels of total testosterone increase throughout normal pregnancy and are primarily a result of progressive estrogen-induced increase in the concentration in sex-hormone-binding globulin concentrations [17, 18]. In preeclamptic pregnancies, however, studies have found lower maternal serum levels of estrogen than in normal pregnancies, so it is likely that other mechanisms mediate the maternal serum levels [19, 20]. In the present study, the maternal serum levels of total testosterone were significantly higher in preeclamptic than in normotensive pregnancies with male as well as with female fetuses (P < 0.001). Male-bearing preeclamptic pregnancies had significantly higher maternal serum testosterone levels than female-bearing pregnancies complicated by preeclampsia (P < 0.001).
The sources for the increased testosterone levels in maternal serum are unknown but could be the ovarian theca-interstitial cells and the maternal cortex, which might be stimulated by hCG throughout pregnancy. The fetal serum levels of testosterone are much lower than the maternal levels. Because of the stimulating effect of hCG on the fetal testis, the testosterone levels in male fetuses are significantly higher than in female fetuses. The fetal ovaries are regarded as hormone-ally inactive in the first part of pregnancy, but later they might have steroidogenic capacity [21–23].
In the present study, no correlations were found between maternal serum levels of hCG and total testosterone. In uncomplicated pregnancies, no significant gender differences were found. The significant increase in maternal serum testosterone levels in preeclamptic pregnancies with male fetuses as well as with female fetuses, and the significantly higher total testosterone maternal serum levels in male- than in female-bearing preeclamptic pregnancies were not related to maternal hCG levels only. In a study in 2006 in Athens, it was found that serum concentrations of total and free testosterone in preeclamptic pregnancies are significantly higher than in normal pregnancies (P < 0.01) [17]. Some other studies by Carlsen et al., Jirecek et al., and Gerulewicz et al. also showed increased levels of androgens in preeclamptic pregnancies in comparison with normal pregnancies [18–20]. It has been postulated that preeclampsia could result from a mutation in a paternally imprinted, maternally active gene. It is also known that only the paternal allele is expressed in human placenta [24, 25]. A paternal immunogenetic factor has been suggested, because significantly more preeclampsia is found in pregnancies with changed paternity [2, 26].
5. Conclusion
Our findings suggest the presence of an androgenic-mediated influence in the mechanism of preeclampsia. Considering high prevalence of preeclampsia and its complications on mother and fetus, most attempts are focused on prediction of the disease using detective indices. According to our results, there is a correlation between maternal serum hCG and testosterone levels and preeclampsia. Therefore, these tests can be used as routine during 30–38 weeks of gestation. High maternal serum concentrations of these markers can predict preeclampsia.
Table 1 Comparison of the clinical characteristics in normal and preeclamptic pregnancies with male and female fetuses.
Type of pregnancy
Normal Preeclamptic
Fetal gender
Male Female Male Female
Maternal age (year) Range 14–38 17–28 14–39 15–36
Mean ± SEM* 22.9 ± 0.9 21.2 ± 0.5 22.7 ± 1.2 22.5 ± 0.9
Parity (n) Range 0–3 0–2 0–3 0–3
Mean ± SEM 0.9 ± 0.2 0.6 ± 0.1 0.5 ± 0.1 0.5 ± 0.1
Gestational age (wk) Range 30–37 31–37 30–38 30–37
Mean ± SEM 33.5 ± 0.4 33.9 ± 0.3 34.1 ± 0.4 33.7 ± 0.4
Systolic blood pressure (mmHg) Range 100–130 95–130 140–155 140–155
Mean ± SEM 115.9 ± 1.6 113.5 ± 1.4 144.9 ± 0.8 145.5 ± 0.8
Diastolic blood pressure (mmHg) Range 65–85 60–85 90–100 90–100
Mean ± SEM 72.6 ± 1.2 70.6 ± 1.1 92.7 ± 0.5 92.9 ± 0.5
*SEM: standard error of the mean.
Table 2 Comparison of mean serum hCG level in normal and preeclamptic pregnancies with male and female fetuses.
Type of pregnancy Normal Preeclamptic
P value
Fetal gender Number hCG (mean ± SEM) Number hCG (mean ± SEM)
Male 35 16337 ± 313 35 31610 ± 355 <0.001
Female 36 28774 ± 326 33 34480 ± 470 NS
P value
<0.005
NS
Table 3 Comparison of mean serum testosterone level in normal and preeclamptic pregnancies with male and female fetuses.
Type of pregnancy Normal Preeclamptic
P value
Fetal gender Number Tes. (mean ± SEM) Number Tes. (mean ± SEM)
Male 35 2.85 ± 0.03 35 5.86 ± 0.05 <0.001
Female 36 2.50 ± 0.02 33 3.92 ± 0.03 <0.001
P value
NS
<0.001
Table 4 Comparison of mean serum hCG level between normal and preeclamptic pregnancies.
Type of pregnancy Number hCG (mean ± SEM)
P value
Normal 71 22643 ± 776 <0.001
Preeclamptic 68 33003 ± 339
Total 139 27711 ± 614
Table 5 Comparison of mean serum testosterone level between normal and preeclamptic pregnancies.
Type of pregnancy Number Testosterone (mean ± SEM)
P value
Normal 71 2.67 ± 0.03
Preeclamptic 68 4.92 ± 0.12 <0.001
Total 139 3.77 ± 0.11
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Arthritis Res TherArthritis Research & Therapy1478-63541478-6362BioMed Central ar34662193339110.1186/ar3466Research ArticleMast cells are the main interleukin 17-positive cells in anticitrullinated protein antibody-positive and -negative rheumatoid arthritis and osteoarthritis synovium Suurmond Jolien [email protected]ée Annemarie L [email protected] Mariëtte R [email protected] Edward F [email protected] Tom WJ [email protected] René EM [email protected] Annemie JM [email protected] Department of Rheumatology, Leiden University Medical Center, PO Box 9600, Albinusdreef 2, C1-R, NL-2300 RC Leiden, The Netherlands2 Department of Dermatology/Allergology, University Medical Center Utrecht, Heidelberglaan 100, NL-3584 CX Utrecht, The Netherlands2011 20 9 2011 13 5 R150 R150 7 3 2011 3 5 2011 20 9 2011 Copyright ©2011 Suurmond et al.; licensee BioMed Central Ltd.2011Suurmond et al.; licensee BioMed Central Ltd.This is an open access article distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/2.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Introduction
Mast cells have been implicated to play a functional role in arthritis, especially in autoantibody-positive disease. Among the cytokines involved in rheumatoid arthritis (RA), IL-17 is an important inflammatory mediator. Recent data suggest that the synovial mast cell is a main producer of IL-17, although T cells have also been implicated as prominent IL-17 producers as well. We aimed to identify IL-17 expression by mast cells and T cells in synovium of arthritis patients.
Methods
Synovial samples of anticitrullinated protein antibody-positive (ACPA+) and ACPA-negative (ACPA-) RA and osteoarthritis (OA) patients were stained for IL-17 in combination with CD117 (mast cells), CD3 (T cells) and CD68 (macrophages). Concentrations of IL-17 in synovial fluid were determined by ELISA.
Results
The number of IL-17+ cells in synovium was comparable in all groups. Although the vast majority of IL-17+ cells are mast cells, no difference in the percentage of IL-17+ mast cells was observed. Nonetheless, levels of IL-17 in synovial fluid were increased in ACPA+ RA patients compared to ACPA- RA and OA patients.
Conclusions
The synovial mast cell is the main IL-17+ cell in all three arthritis groups analyzed. These data are relevant for studies aimed at blocking IL-17 in the treatment of arthritis.
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Introduction
Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the synovial lining of the joint. In the majority of patients with established RA, anticitrullinated protein antibodies (ACPAs) can be found [1]. It is currently believed that ACPA+ and ACPA-RA are two different disease entities, each with its own pathogenesis [2].
Several cell types of the immune system play a role in the pathogenesis of RA. The presence of autoantibodies and the linkage of RA to human leukocyte antigen shared epitope alleles in ACPA+ RA indicate that the adaptive immune system plays a prominent role. However, cells of the innate immune system, such as mast cells, have also been implicated in pathogenesis of RA [3]. Indeed, the number of mast cells in synovial tissue is associated with inflammatory mediators such as histamine in synovial fluid [4].
Among the cytokines that are thought to be involved in RA, IL-17 has recently attracted considerable attention. IL-17 can induce production of other proinflammatory factors such as IL-6, IL-1, TNF and matrix metalloproteinases, leading to inflammation, breakdown of cartilage and bone erosion [5]. IL-17 deficient mice are less prone to develop experimental arthritis and blocking IL-17 can reduce both the onset and progression in these models [6]. In RA, high levels of IL-17 were found in synovial fluid, especially compared to OA patients [7]. The first proof-of-concept trial indicates that neutralization of IL-17 is a potential new target for the treatment of RA [8].
On the basis of the data described above, it is postulated that Th17 cells, through the production of IL-17 and other Th17-associated cytokines, play a prominent role in the inflamed synovium by perpetuating the inflammatory milieu observed in arthritis [6]. Interestingly, a recent study by Hueber et al. [9] indicated that the mast cell is the most abundant cell type expressing IL-17 in the synovial tissue of 10 RA patients. However, other studies have shown the presence of IL-17-producing T cells in RA patients [10]. Because previous investigators have reported that ACPA+ and ACPA- RA are distinct disease entities [2], our aim in the present study was to analyze which cell subsets express IL-17 in the synovial tissue of ACPA+ RA, ACPA-RA and OA patients.
Materials and methods
Patient samples
Synovial tissues were obtained from established ACPA+ (n = 34) and ACPA- (n = 25) RA patients who had undergone therapeutic arthroscopic lavage of an inflamed knee and knee or hip replacement surgery. Synovial tissues were obtained from patients with established OA (n = 29) who had undergone knee or hip replacement surgery. These tissues were fixed with 4% formaldehyde in PBS, stored in 70% ethanol and embedded in paraffin. Written informed consent was obtained from the patients, and the study was approved by the Leiden University Medical Center human ethics committee.
Synovial fluid was collected from established ACPA+ RA patients (n = 30) and ACPA- RA patients (n = 29) and from patients with established OA (n = 14) and stored at -20°C until analysis. Patient diagnoses of RA or OA were made according to the American College of Rheumatology criteria [11-13].
Immunohistochemistry
Synovial tissues were treated according to the method described by Schuerwegh et al. [14]. Slides were preincubated with 10% blocking buffer (10% normal horse serum/10% normal human serum in PBS) for 20 minutes and stained with polyclonal goat anti-human IL-17A (0.50 μg/mL; R&D Systems, Minneapolis, MN, USA) in 1% blocking buffer (1% normal horse serum/1% normal human serum in PBS/1% BSA) for one hour. For control sections, a matching isotype control (normal goat immunoglobulin G (IgG); Merck, Darmstadt, Germany) was used. Detection was performed using horse α-goat biotin (Vector Laboratories, Burlingame, CA, USA), avidin-biotin-peroxidase complex (VECTASTAIN Elite ABC Kit; Vector Laboratories) and 3, 3'-diaminobenzidine tetrahydrochloride-nickel chloride (Vector Laboratories).
For combined staining of IL-17 with CD117, CD3, CD4 or CD68, slides were stained for one hour with polyclonal rabbit anti-human CD117 (23 μg/mL; Dako, Glostrup, Denmark), monoclonal mouse anti-human CD3 (2.8 μg/mL; Dako), monoclonal mouse anti-human CD4 (7 μg/mL; Dako), monoclonal mouse anti-human CD68 (0.51 μg/mL; Dako) or matching isotype control (rabbit polyclonal Ig and mouse IgG1; Dako) in 1% blocking buffer. Detection of anti-CD117, anti-CD3, anti-CD4 or anti-CD68 was performed using alkaline phosphatase-conjugated anti-rabbit/anti-mouse Ig and Liquid Permanent Red (EnVision™ G|2 System/AP, Rabbit/Mouse (Permanent Red) Kit; Dako). The tissue sections were counterstained with hematoxylin.
Stained sections were coded and randomly analyzed. The mean number of single- and double-positive cells in 10 high-power fields (original magnification, ×400) was scored blindly by two observers.
Immunoassay for IL-17
Concentrations of IL-17A in synovial fluid were measured with an ELISA (PeproTech, Inc., Rocky Hill, NJ, USA) according to the manufacturer's instructions.
Statistical analysis
Differences between patient and control groups were analyzed using the Kruskal-Wallis and Mann-Whitney U tests. In all tests, P < 0.05 was considered significant.
Results
To determine the expression of IL-17 by mast cells, T cells and macrophages in synovial tissue, immunohistochemical staining was performed in synovial tissue sections of ACPA+ RA, ACPA- RA and OA patients (Table 1). Representative examples of the staining are shown in Figure 1. Isotype controls were negative (data not shown).
Table 1 Expression of IL-17 by mast cells, T cells and macrophages in synovial tissue
Demographics ACPA+ RA (n = 34) ACPA- RA (n = 25) OA (n = 29) P value
Age (years) 55 (32 to 80) 63 (19 to 80) 67 (42 to 83)
Gender (females/males) 22/12 14/11 21/8
Disease duration (years) 7 (0 to 28) 8 (0 to 24) Unknown
Median IL-17+ cells 21 (0 to 118) 12 (1 to 61) 17 (0 to 50) 0.381
IL-17+ mast cells (%) 97 (40 to 100) 96 (0 to 100) 93 (0 to 100) 0.969
IL-17+ T cells (%) 0 (0 to 100) 0 (0 to 24) 2 (0 to 49) 0.558
IL-17+ MØ (%) 0 (0 to 78) 4 (0 to 100) 1 (0 to 58) 0.382
Median MCs 28 (0 to 123) 19 (0 to 92) 25 (0 to 76) 0.198
MCs (CD117+) expressing IL-17 (%) 91 (13 to 100) 83 (0 to 100) 96 (4 to 100) 0.599
T cells (CD3+), n 21 (0 to 592) 10 (0 to 115) 11 (0 to 265) 0.609
T cells (CD3+) expressing IL-17 (%) 0 (0 to 60) 0 (0 to 66) 1 (0 to 78) 0.149
MØ (CD68+), n 71 (1 to 390) 53 (1 to 302) 66 (2 to 231) 0.634
MØ (CD68+) expressing IL-17 (%) 0 (0 to 16) 0 (0 to 14) 0 (0 to 11) 0.689
ACPA: anticitrullinated protein antibody; MC: mast cell; MØ: macrophage; OA: osteoarthritis; RA: rheumatoid arthritis. Results are expressed as medians (minimum-maximum). Mast cells are defined as CD117+ cells, T cells are defined as CD3+ cells and macrophages are defined as CD68+ cells.
Figure 1 Expression of IL-17 by immunohistochemistry. (A) Double-staining of IL-17 (black) and CD117+ mast cells (pink). (B) Double-staining of IL-17 (black) and CD68+ macrophages (pink). (C) Double-staining of IL-17 (black) and CD3+ T cells (pink). (D) Double-staining of IL-17 (black) and CD4 (pink). Representative examples are shown. In insets in parts (A) through (D), arrows indicate double-positive cells and arrowheads indicate single IL-17+ cells. The magnification of inset images are made digitally, and are 2× the magnification of the original figure which is made through a 400× magnification.
The median number of IL-17+ cells was slightly higher in ACPA+ RA patients than in ACPA- RA and OA patients, but this difference was not statistically significant (Figure 2A). Likewise, the total number of CD117+ cells was slightly higher in ACPA+ RA patients, although the difference was not statistically significant. There was no difference in the number of T cells (CD3+) or macrophages (CD68+) between the groups.
Figure 2 IL-17 in synovial tissue and synovial fluid. (A) Number of IL-17+ cells in synovial tissue of ACPA- and ACPA+ RA and OA patients. The results are expressed as the number of cells assessed in 10 high-power fields at ×400 magnification. (B) Levels of IL-17 in synovial fluid determined by ELISA. *P < 0.05 and **P < 0.01, both indicating statistically significant differences. ACPA, anticitrullinated protein antibody; OA, osteoarthritis; RA, rheumatoid arthritis.
To identify the source of IL-17 in synovium, double-staining of IL-17 with CD117 (mast cells), CD3 (T cells) and CD68 (macrophages) was performed. Interestingly, almost all IL-17-expressing cells were CD117+ in the synovial tissue of ACPA+ and ACPA- RA patients as well as OA patients. Only a small fraction of IL-17+ cells were CD3+ or CD68+ (Table 1). Furthermore, there were no differences in these percentages between the three groups. Because CD3 can be downregulated in activated T cells, we performed additional staining of IL-17 in combination with CD4 in six synovium samples (Figure 1D). The median percentage (minimum-maximum range) percentage of IL-17+ cells that were CD4+ was 0.4% (0.0% to 11.0%). The median (minimum-maximum range) percentage of CD4+ cells that were IL-17+ was 0.1% (0.0% to 0.7%). Taken together, these data indicate that IL-17 in synovium is expressed predominantly by mast cells.
Since immunohistochemistry does not reveal secretion of IL-17, an ELISA was performed with the synovial fluid of RA and OA patients. ACPA+ RA patients had significantly higher levels of IL-17 in synovial fluid compared to ACPA- RA and OA patients (Figure 2B).
Discussion
In this study, we have shown in a relatively large group of 59 RA and 29 OA patients that the majority of IL-17+ cells were mast cells and not T cells or macrophages. Interestingly, levels of IL-17 in synovial fluid were increased in ACPA+ RA patients. Because the expression of IL-17 in synovial tissue correlates strongly with the number of mast cells, it is conceivable that the increased level of IL-17 in the synovial fluid of ACPA+ RA patients results from the increased activity of mast cells in ACPA+ RA patients. Our data also show that IL-17 is not increased in all ACPA+ RA patients. Preliminary analysis of the characteristics of the RA patients with a high number of IL-17-producing cells shows that these patients tend to have higher serum ACPA titers and erythrocyte sedimentation rates at the time of diagnosis.
In this study, mast cells were identified as CD117+ cells. As described in Schuerwegh et al. [14], flow cytometric staining of synovial tissue revealed that all CD117+ cells express the high-affinity IgE receptor (FcεRI) and/or IgE. Therefore, CD117 alone can be considered a good mast cell marker in synovial tissue.
Although our results suggest that mast cells are the most prominent producers of IL-17 in synovial tissue, a clear limitation of this study is that only the expression of IL-17, and not active secretion, was studied. We do not know whether IL-17 is secreted by activated mast cells, as we were unable to isolate viable mast cells from synovial tissue. Nonetheless, Hueber et al. [9] showed IL-17 secretion by in vitro cultured mast cells, indicating that mast cells can readily produce IL-17. Because the samples of synovial fluid, in which higher levels of IL-17 were found, were from different patients than the samples of synovial tissue, it is unclear whether the increased levels of IL-17 correlate directly to the presence of IL-17+ mast cells in the same synovial tissue.
Our group previously found that IgE-ACPA can bind to FcεRI on basophils and that citrullinated proteins can directly activate basophils of ACPA+ RA patients. In addition, an increased number of degranulated mast cells was shown in the synovium of ACPA+ RA patients, indicating a higher activity of mast cells in these patients [14]. Because mast cells also express FcεRI, it is tempting to speculate that mast cells are also activated by citrullinated proteins present in the joint, thereby releasing IL-17, which contributes to the inflammatory milieu present in the inflamed synovium. However, there was no difference in the expression of IL-17 between ACPA+ and ACPA- RA patients in our study. Therefore, it is unclear whether the more activated state of mast cells that was found before [14] is related to the release of IL-17, as in our present study we were able to evaluate only the expression of IL-17 rather than its secretion.
Several studies have provided evidence indicating that IL-17-producing T cells in synovial tissue or fluid also contribute to inflammation. However, these T cells are not abundantly present in the synovial compartment. Indeed, even after strong nonspecific T-cell triggering, only a small minority of CD4+ T cells (about 1% to 10%) obtained from synovial fluid or synovial tissue produce IL-17, as shown by flow cytometry [10,15-17]. Furthermore, the antigen specificity of these Th17 cells in synovium is unknown; therefore, these cells can also be innocent bystanders that do not contribute to inflammation in the joint in vivo. In two studies in which immunohistochemical staining was performed, IL-17+ cells were identified as CD3+ cells. However, it is unclear how these results relate to our study, as in those previous studies cells were identified using single staining of consecutive sections and the positive cells in the overlying sections were not quantified, making it difficult to compare these contradictory results with the results of our study [18,19]. Two other studies in which microscopic analysis was performed showed that almost no CD3+ T cells in the synovium expressed IL-17. In agreement with our study, in one of these studies the cell types that did express IL-17 were found to be mainly mast cells [9]. However, the other study in which no CD3+ T cells were shown to express IL-17 identified IL-17+ cells as being mainly neutrophils and neutrophil precursors in the synovium of the facet joints [20]. Because we found the mast cells to be the main cell subset expressing IL-17 in synovium from the knee, it is possible that the cells expressing IL-17 might be different, depending on the site of the joint.
Because the production of IL-17 is highly restricted by transcriptional control via RORγT (retinoid acid receptor-related orphan receptor γt), which is also known to regulate the production of other Th17-associated cytokines, mast cells might also produce other Th17-related cytokines, such as IL-22. Furthermore, because mast cells can produce many other cytokines as well, blocking the activation of mast cells, such as by preventing their activation via the FcεRI through anti-IgE treatment, might lead to even more profound effects than blocking IL-17 alone in arthritis patients. Indeed, blocking TNF is a very successful therapy in RA, and mast cells are known to be important producers of TNF [21].
Conclusions
Our results show that IL-17 is expressed mainly by mast cells in the synovial tissue of both ACPA+ and ACPA- RA patients, as well as in OA patients. Selective activation of mast cells in ACPA+ RA patients might be responsible for the increased levels of IL-17 in synovial fluid. These data are relevant for new targeted therapies in arthritis, such as IL-17 blockade or the inhibition of mast cell activation.
Abbreviations
ACPA: anticitrullinated protein antibodies; BSA: bovine serum albumin; ELISA: enzyme-linked immunosorbent assay; IL: interleukin; OA: osteoarthritis; PBS: phosphate-buffered saline; RA: rheumatoid arthritis; TNF: tumor necrosis factor.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
JS carried out the experiments, performed the statistical analysis and drafted the manuscript. AD and MB carried out the experiments and contributed to the design and analysis of the study. EK, TH, RT and AS participated in the design and analysis of the study and helped to draft the manuscript. All authors read and approved the final manuscript.
Acknowledgements
JS's work is supported by the Dutch Arthritis Foundation. AJMS's and REMT's work is supported by the Netherlands Organization for Scientific Research (clinical fellow and Vici grants). AJMS's work is also supported by the Research Foundation Sole Mio and the Leiden Research Foundation (STROL). This work was further supported by a grant from the Centre for Medical Systems Biology (CMSB) within the framework of the Netherlands Genomics Initiative (NGI), FP06 AutoCure and FP07 MASTERSWITCH.
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Int J DentInt J DentIJDInternational Journal of Dentistry1687-87281687-8736Hindawi Publishing Corporation 2250589510.1155/2012/236409Clinical StudyA Prospective Study of Early Loaded Single Implant-Retained Mandibular Overdentures: Preliminary One-Year Results El-Sheikh Ali M.
1
*Shihabuddin Omar F.
2
Ghoraba Sahar M. F.
3
1Department of Prosthetic Dentistry, Faculty of Dentistry, Tanta University, Tanta 31111, Egypt2Department of Oral Maxillofacial Surgery, Dammam Dental Centre, Dammam Medical Complex, Dammam 31433, Saudi Arabia3Department of Oral Medicine, Periodontology, Radiology and Diagnosis, Faculty of Dentistry, Tanta University, Tanta 31111, Egypt*Ali M. El-Sheikh: [email protected] Editor: Dimitris N. Tatakis
2012 15 3 2012 2012 23640924 10 2011 17 12 2011 3 1 2012 Copyright © 2012 Ali M. El-Sheikh et al.2012This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Purpose. To investigate the predictability of simplifying mandibular overdenture treatment using one-stage surgery and early prosthetic loading of a single implant. Materials and Methods. Twenty edentulous patients with problematic existing mandibular dentures were treated. A single implant with a chemically modified surface (SLActive, Straumann AG, Basel, Switzerland) was placed into the mandibular midline. The patients were recalled at 3, 6 and 12 months. Clinical assessments and marginal bone loss using standardized radiographs were recorded. All complications, failures and maintenance were noted. Visual analog-scale questionnaires were used to record patient satisfaction in five categories. ANOVA was used to determine differences between means of marginal bone loss and different categories of patient staisfaction (P = 0.05). Results. The 20 early loaded implants were all surviving at the 12-month recall. All implants showed less than 1 mm of marginal bone loss by the end of the 1-year with a significant increase during the follow-up period. Few prosthetic problems were reported. Patient satisfaction was high with a significant increase in all comfort and functional parameters. Conclusions. These preliminary 1-year results indicate that early loading of a single chemically modified surface implant used to retain a mucosa-borne mandibular overdenture is a safe, reliable, and cost-effective treatment.
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1. Introduction
Rehabilitation of the completely edentulous mandible using implants to retain a fixed prosthesis is a predictable long-term treatment modality [1, 2]. High implant success rates have been achieved by Engquist et al. [3] (99%), Johns et al. [4] (96.2%), and Bergendal and Engquist [5] (100%), using 2 or more implants to anchor an overdenture. Two implant-retained overdentures with separated implants have been reported with high implant success rates (97–100%) and functional improvement [5–8]. There is consensus that 2 implants splinted [9–11] or unsplinted [6, 12, 13] in the interforaminal region of the mandible are sufficient to support an overdenture [14, 15]. Indeed, the McGill consensus statement suggested that the 2-implant overdenture should be the first choice of treatment for the edentulous mandible [16].
The success of the previous treatment modalities, while excellent, is unfortunately outside the financial scope of many compromised edentulous patients. A cost comparison study between an unsplinted 2-implant retained mandibular overdenture and a conventional complete mandibular denture showed the direct cost of the overdenture to be 2.4 times the cost of the complete denture [17]. It is, therefore, desirable for clinicians to be able to offer a significant functional improvement of the problematic mandibular complete denture in a cost effective manner. Concomitantly, a reduction in the overall time frame of clinical, technical, and maintenance procedures needed to achieve this goal would be advantageous [18].
Further directions with case reports and prospective studies point towards a more conservative approach: the use of only a single implant to support a mandibular overdenture [18–24]. Implant outcome and patient satisfaction has shown to be comparable whether one or two implants are used for support of mandibular overdentures [23, 24].
A chemically modified titanium surface, SLActive (Straumann AG, Basel, Switzerland), has been developed, using the well-documented topography of the sandblasted, large grit, acid-etched (SLA, Straumann AG) surface. The surface is chemically active, with high surface free energy, reduced atmospheric hydrocarbon contamination, and strong hydrophilicity; the water contact angle is 0° compared with 139.9° for the standard SLA surface [25, 26].
This surface showed enhanced bone formation and significantly increased cellular activity and proliferation of vascular structures compared with the conventional SLA surface in the first 14 days following implantation, as demonstrated by histological and immunohistochemical evaluation [27]. In vivo animal studies have demonstrated 60% greater bone formation at the SLActive surface compared with SLA, and there is earlier formation of more mature bone [28]. Mean removal torque values were also found to be consistently higher in the first 8 weeks, corresponding to the early healing period [29]. This evidence suggests increased implant stability in the critical early osseointegrated period. Therefore, early loading protocol (3-4 weeks) using SLActive implants has become more accepted and more prevalent for situations ranging from single-tooth replacement to full-arch restorations [30, 31].
The purpose of this study was to investigate the predictability of simplifying mandibular overdenture treatment using one-stage surgery and early prosthetic loading of a single chemically modified titanium surface (SLActive) implant.
2. Materials and Methods
2.1. Patient Selection
Twenty completely edentulous subjects, 12 men and 8 women, ranging from 52 to 70 years of age (mean age 62 years) were included in the study. These patients were treated in Dammam Dental Centre, Dammam Medical Complex (Dammam, Saudi Arabia) in the period from January to April 2010. All patients signed an informed consent form. Ethical approval for the project was granted by the Human Research Ethics Committee of The Dammam Medical Complex, Dammam, Saudi Arabia. The primary complaints among the patients referred to the clinic for treatment were related to poor retention of the mandibular denture, instability, denture sores, and phonetic problems.
Inclusion criteria dictated that the patient is completely edentulous for at least 2 years, has a maladaptive mandibular denture, and has sufficient bone for an implant of at least 10 mm length and 4.1 mm diameter. Exclusion criteria included any medical condition contraindicating implant surgery, logistic or physical reasons that could affect follow-up, psychiatric problems, and disorders to the implant site related to a history of radiation therapy to the head and neck, or bone augmentation.
2.2. Surgical Procedures
Thorough clinical evaluation of the proposed implant placement site was carried out. Preoperative panoramic, conventional lateral cephalometric, and periapical radiographs were used for radiographic evaluation of the placement site to avoid potential complications with important anatomy in this region.
The components used were regular platform soft tissue level implants (SLActive, Straumann AG, Basel, Switzerland) with a diameter of 4.1 mm. The Straumann 3.4 mm height retentive anchor with a titanium matrix provided the prosthetic anchorage. All patients were provided with a single implant of at least 10 mm in length inserted in the mandibular midline. Twelve patients were provided with 10 mm long implants while the remaining eight patients were provided with 12 mm long implants.
Bilateral mental nerve blocks and local infiltration in the labial and lingual sulcus were administered with lignocaine 2%. One-stage surgical approach was followed throughout the whole study (Figure 1). A minimal crestal incision (envelope type) was made, and a mucoperiosteal flap was raised, on both the labial and the lingual aspects, to enable adequate visualization of the lingual aspect of the mandible and to evenly divide the available keratinized tissue. This enabled the abutment to be surrounded by attached gingiva. The osteotomy was prepared using a standard bone drilling protocol, according to the manufacturer's directions with extreme care to avoid penetration of the lingual or inferior cortex. Bone quality was identified, and bone tap was used in types 1 and 2. Initial implant stability was tested manually by hand and insertion torques ≥35 Ncm were acceptable. Healing abutments of appropriate length were connected, and the mucosa was adjusted and sutured (4-0 Vicryl, Ethicon, Johnson & Johnson, Brussels, Belgium).
Any patients with implants lacking primary stability at this stage were excluded from further participation in the study and replaced; this was not considered an implant or treatment failure. Any patients with inadequate bone at the time of surgery were also excluded from further participation in the study. Patients excluded for these reasons were offered implantation using the conventional delayed loading protocol or another form of treatment.
Antibiotic (Augmentin 625 mg) and nonsteroidal anti-inflammatory (Ibuprofen 400 mg) medications were given to the patients every 8 hours for 5 days postoperatively. Immediately after surgery, the mandibular denture of each participant was modified and relined with a soft tissue conditioner (Viscogel, Dentsply, Konstanz, Germany). All patients were limited to a soft diet for 10 days and instructed to leave the denture out at night. The patients were instructed in a plaque control protocol at the time of implant placement and this was reinforced at subsequent reviews.
2.3. Prosthetic Procedures
Three weeks after implant placement, the healing abutment was removed and the retentive anchor was screwed to the implant (Figure 2). A torque of 35 Ncm was used for tightening the retentive anchor. Preliminary impressions for upper and lower arches were taken with stock trays using irreversible hydrocolloid (Hydrogum, Zhermack, Italy). The impression for the lower arch was taken directly over the retentive anchor. Secondary impressions were taken with autopolymerized acrylic resin special trays using vinyl polysiloxane impression material (Express, 3 M ESPE Dental Products, USA). The transfer pin was positioned in the lower impression before pouring it (Figure 3). Record blocks were fabricated on the duplicates of the master models for jaw registration. Teeth try-in and manufacturing of the acrylic dentures were carried out using standard prosthetic procedures. The titanium matrix (Figure 4) was incorporated in the final prosthesis using the direct technique inside the patient's mouth. Fabrication of the prostheses was finished in 1 week. Therefore, the maxillary complete denture and implant-retained mandibular overdenture (Figure 5) were delivered to the participants approximately 4 weeks after implant placement.
2.4. Radiographic Analysis
Standardized intraoral radiographs using a long cone technique of the implant were obtained. To provide a geometrically reproducible alignment, an index was recorded for each patient on the inserted mandibular overdenture with the use of vinyl siloxane material. With the aid of Hawe's sensor holder system (Kerr, KerrHawe SA, Switzerland), the radiographs were taken using direct digital imaging system (Trophy RVG, William Green Pty Ltd., Australia). Images were displayed on a computer screen with such a dimension and brightness that the observer could read comfortable and accurately the image. On each image, the implant-retentive anchor interface and first bone-to-implant contact were identified and marked with a cursor on the mesial and distal sides of the implant. The analysis program calculated and reported the distance between the two points with a degree of accuracy of ±0.01 mm. The same procedure was performed with all follow-up radiographs. The initial postoperative radiographs immediately after insertion of the new/final overdentures (baseline radiography) were compared with the follow-up radiographs 3, 6, and 12 months of functional loading. The vertical bone loss was calculated by subtracting the bone heights in the baseline radiographs from those of follow-up radiographs. Data were collected blindly by one experienced observer throughout the entire study.
2.5. Patient Satisfaction
Self-administered questionnaires that followed the Visual Analogue Scale (VAS) method were completed by patients preoperatively and at each scheduled recall to assess oral comfort and function [32]. Each VAS questionnaire consisted of a 100 mm line anchored at the beginning and end by opposing responses/statements such as “not at all satisfied” to “totally satisfied”. The participants marked a vertical line on the horizontal VAS line to indicate their feeling. Scores were determined by measuring the distance (in mm) from the left starting point of the line to the intersection of the response line. There were 10 questions, in 5 categories: general satisfaction (not at all satisfied, totally satisfied), social life (not at all improved the social life, totally improved the social life), mastication of hard food (not at all improved mastication of hard food, totally improved mastication of hard food), comfort (not at all comfort, totally comfort), and fit (not at all fit, totally fit).
2.6. Data Collection
The data collection (clinical and radiographic outcomes) of all patients was performed as follows: at the completion of the prosthetic treatment (baseline) and after 3, 6, and 12 months of functional loading.
2.7. Statistical Analysis
The measurements for the marginal bone loss were carried out on the mesial and distal surface for each implant and the mean was taken. The difference between the values at the baseline and the follow-up recall visits was based on the average bone loss value for each implant. The data were statistically analyzed using one-way repeated measures ANOVA followed by Tukey's test at 95% confidence level (SPSS for Windows, version 10.0, SPSS Incorporated, Chicago, IL, USA).
3. Results
Three patients were excluded from the study during the surgical placement of the implants. Two patients were excluded due to lack of primary stability and one due to inadequate bone. The 20 early loaded implants were all surviving at the 12-month recall. Prosthetic problems were relatively few with attachment functioning well at the 1-year recall and reline unnecessary. Two patients required the spring of the titanium matrix to be replaced with a new one due to loss of the retention approximately 9 months after functional loading. Plaque control was considered acceptable for most patients and considered relatively simple by the patients themselves. Calculus formation that impeded seating of the titanium matrix was encountered on 2 occasions and was further prevented by more diligent hygiene. Soft tissue health was visually assessed and was acceptable in all patients with no evidence of mucosal enlargement at recall appointments, as shown in Figure 6 (12-month follow-up).
All implants showed less than 1 mm of marginal bone loss by the end of the 1-year follow-up period. The mean vertical bone loss from baseline to 3 months was 0.30 mm (SD = 0.06). The mean vertical bone loss from baseline to 6 months was 0.63 mm (SD = 0.07). The mean vertical bone loss from baseline to 12 months was 0.93 mm (SD = 0.06). The comparison of the mean values of bone loss from 3 to 6 months, from 3 to 12 months, and from 6 to 12 months was statistically significant (P < 0.001).
The mean values and standard deviations (in mm) of comfort and functional parameters in the VAS questionnaires filled by all patients at pretreatment and all recall appointments were summarized in Table 1. Statistical analysis of these data showed a significant improvement in all parameters of oral comfort and prosthesis function (P < 0.001). Direct questioning indicated that common pretreatment problems, such as recurrent denture ulceration, had been eliminated and nonmasticatory functions such as yawning and laughing could be accomplished without complications.
4. Discussion
The conventional loading protocols for dental implants allow for a period of undisturbed healing after implant placement, to minimize the risk of healing and osseointegration complications. In recent years, shorter restoration times have become more common, especially due to increasing patient demands.
The purpose of this prospective study was to ascertain whether simplifying mandibular overdenture treatment using one-stage surgery and early prosthetic loading of a single implant would achieve acceptable implant success rates and provide the functional improvement expected using conventional techniques. While the study may be of limited duration, it provides sound support for the hypothesis that the single-implant mandibular overdenture can provide improved retention, stability, masticatory performance, and confidence for the maladaptive complete denture wearers. The limitations of the small sample size of 20 participants and the short follow-up period of 1 year need to be acknowledged, and the findings presented, therefore, must be interpreted cautiously.
Presurgical evaluation of the patient was simplified by using the relatively inexpensive panoramic, lateral cephalometric, and periapical radiographs. These important diagnostic aids, together with adequate visualization of the lingual surface of the bony ridge after flap evaluation, cannot be overstated in light of reports of life-threatening hemorrhage from the floor of the mouth during routine implant placement in this region [33, 34]. The present study on the early functioning single-implant overdenture showed excellent survival rates (100%) and dramatically improved patient-reported satisfaction levels in patients with pretreatment denture problems. These results are in agreement with the study by Alsabeeha et al. [30] which reported 100% of early loading for the Southern wide and Neoss regular implants. With respects to the 100% survival reported, the possibility that the authors are skilled clinicians experienced with this technique should be considered, so the single-implant procedure cannot be generalized to the entire practicing community. However, the procedures involved are not complex, provided that the mentioned protocol is followed. It is difficult to postulate whether two implants are twice as effective as one or even whether there is any discernable difference from a patient perspective.
In general, implant overdentures have a less controlled loading when compared to fixed prostheses [35]. It may be postulated that forces, both axial and lateral, generated by an overdenture on a single implant have the potential to be greater than those produced by a multiple implant-retained overdenture. Maeda et al. [36] examined the biomechanical rationale of a single implant-retained mandibular overdenture using an in vitro model. The model revealed statistically significantly smaller lateral forces to the ball abutments for single-compared to two-implant overdentures with molar loading. A higher load was observed when the denture was loaded in the midline region. No significant difference in three-dimensional denture base movement was observed between single- and two-implant overdentures in the midline and molar regions. They concluded that, overall, the single-implant overdenture had similar biomechanical effects to a two-implant overdenture in terms of lateral forces to the abutment and denture base movements under molar functional loads. However, the authors did stress the in vitro nature of the model and the need for follow-up studies performed in a clinical setting.
Prosthetic problems were relatively few compared to other studies [14, 15], with attachment functioning well at the 1-year recall and relines unnecessary. This is in agreement with the study by Liddelow and Henry [18] in which plastic caps and rubber O-rings were used. The present study used the titanium matrix with the spring. The inherent resilience with these kinds of attachments (i.e., plastic caps and rubber O-rings, and titanium matrix and spring) may allow more movement and, therefore, less strain and potential for wear. The other studies [14, 15] used metal retentive caps which may explain the occurrence of more prosthetic problems in these studies. The titanium matrix is, however, substantially small, resulting in an enough amount of denture base around the attachment. Therefore, no fracture of the denture bases at the attachment site was recorded in all cases. A denture with this type of attachment is primarily tissue borne and implant retained. From a prosthodontic perspective, if the implant is not placed in the ideal position, an unfavorable overbulking of the denture base would result.
Plaque control was considered acceptable for most patients and considered relatively simple by the patients themselves. Soft tissue health was acceptable in all patients with no evidence of mucosal enlargement at all recall appointments. The findings in the present study are in agreement with the studies of Liddelow and Henry [18] and Cordioli et al. [19], which also reported no mucosal enlargement during the follow-up period. Mucosal enlargement has been reported in other studies such as that by Engquist et al. [3], which had an incidence of 25%, and that by Wright et al. [11], which reported 35%.
The overall mean marginal bone loss after 1 year of function in the present study was less than 1 mm which is in agreement with previous studies [18–21]. The comparison of the mean values of bone loss from 3 to 6 months, from 3 to 12 months, and from 6 to 12 months was statistically significant. The explanation for that could be the presence of only one implant in the mandible which might have been subjected to excessive forces.
The cost of treatment for edentulous patients is a significant determinant of treatment acceptance, compared to other groups of patients. Any reduction in cost to the patient becomes more critical. The study measuring the cost of implant overdenture therapy has been done with a microcosting technique, which examines the direct cost to the patient and, also, indirect costs, such as time and transportation [17]. Measured in this way, the difference in cost between 1 and 2 implants would be primarily half the component costs, as the time differential from both the surgical and prosthodontics viewpoint would be minimal. The few prosthetic problems reported during the 1-year follow-up period are interesting from a maintenance cost standpoint. If this type of overdenture design and attachment component has a lower maintenance requirement, then this has favorable implications with respect to cost-effectiveness.
The preliminary 1-year report on this procedure indicates that it is a positive treatment modality, which could make it possible for completely edentulous patients with limited resources to benefit from an implant-retained prosthesis. It may well be considered to be the entry level treatment option for rehabilitation of the edentulous mandible in selected patients, especially the underprivileged geriatric groups. A limitation of this study is the lack of a comparison group with the more conventional 2-implant overdenture. Given the clear improvements and reduced costs with this modality, serious consideration for longer term and more extensive clinical trials is warranted. In long term, with favorable results, the McGill consensus statement may be challenged.
5. Conclusions
Within the limitation of this study and the preliminary nature of this 1-year report, it may be concluded that the early loaded, single implant-retained mandibular overdenture, using a chemically modified surface implant (SLActive), is an alternative treatment proposition for selected patients. The relatively simple treatment protocol and reduced component and laboratory involvement mean that a greater number of edentulous patients could benefit from this protocol. These preliminary findings must be confirmed by long-term randomized controlled clinical trials with a larger sample size and comparison groups (i.e., one versus two implants).
Figure 1 Surgical procedure of single implant placement in the mandibular midline.
Figure 2 Retentive anchor screwed into the implant 3 weeks after implant placement.
Figure 3 Transfer pin positioned in the impression before pouring.
Figure 4 Titanium matrix over the retentive anchor to be incorporated in the finished denture.
Figure 5 Finished mandibular overdenture with titanium matrix immediately before insertion.
Figure 6 Acceptable soft tissue health with no mucosal enlargement around the retentive anchor at 12-month recall.
Table 1 Comfort and functional parameters (Means and SDs in mm) at pretreatment and all recall examinations.
Pretreatment 3 months 6 months 12 months
General satisfaction 19.15 (2.41) 77.05 (3.85) 85.85 (3.31) 91.50 (1.70)
Social life 30.70 (1.75) 76.30 (2.47) 81.3 (3.26) 89.95 (2.04)
Mastication of hard food 19.70 (2.00) 75.65 (2.01) 86.00 (2.03) 90.15 (1.46)
Comfort 20.90 (1.65) 67.80 (2.35) 79.10 (1.80) 82.85 (3.23)
Fit 21.60 (1.93) 87.40 (2.21) 92.60 (1.64) 95.50 (1.28)
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22479585PONE-D-11-2209610.1371/journal.pone.0034277Research ArticleBiologyModel OrganismsAnimal ModelsMolecular Cell BiologyCytometrySignal TransductionMechanisms of Signal TransductionSignaling CascadesSignaling in Cellular ProcessesSignaling in Selected DisciplinesBak Compensated for Bax in p53-null Cells to Release Cytochrome c for the Initiation of Mitochondrial Signaling during Withanolide D-Induced Apoptosis WithaD Induced Bax and/or Bak Dependent ApoptosisMondal Susmita
1
¤
Bhattacharya Kaushik
1
Mallick Asish
1
Sangwan Rajender
2
Mandal Chitra
1
*
1
Cancer and Cell Biology Division, Council of Scientific and Industrial Research - Indian Institute of Chemical Biology, Jadavpur, Kolkata, West Bengal, India
2
Metabolic and Structural Biology Division, Council of Scientific and Industrial Research - Central Institute of Medicinal and Aromatic Plants, Lucknow, India
Zhivotovsky Boris EditorKarolinska Institutet, Sweden* E-mail: [email protected] and designed the experiments: SM CM. Performed the experiments: SM KB. Analyzed the data: SM KB CM. Contributed reagents/materials/analysis tools: RS. Wrote the paper: SM CM. Cell culture and preliminary screening of WithaD: AM.
¤ Current address: Department of Microbiology, Sammilani Mahavidyalaya, Baghajatin, Kolkata, India
2012 29 3 2012 7 3 e342774 11 2011 25 2 2012 Mondal et al.2012This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.The goal of cancer chemotherapy to induce multi-directional apoptosis as targeting a single pathway is unable to decrease all the downstream effect arises from crosstalk. Present study reports that Withanolide D (WithaD), a steroidal lactone isolated from Withania somnifera, induced cellular apoptosis in which mitochondria and p53 were intricately involved. In MOLT-3 and HCT116p53+/+ cells, WithaD induced crosstalk between intrinsic and extrinsic signaling through Bid, whereas in K562 and HCT116p53−/− cells, only intrinsic pathway was activated where Bid remain unaltered. WithaD showed pronounced activation of p53 in cancer cells. Moreover, lowered apoptogenic effect of HCT116p53−/− over HCT116p53+/+ established a strong correlation between WithaD-mediated apoptosis and p53. WithaD induced Bax and Bak upregulation in HCT116p53+/+, whereas increase only Bak expression in HCT116p53−/− cells, which was coordinated with augmented p53 expression. p53 inhibition substantially reduced Bax level and failed to inhibit Bak upregulation in HCT116p53+/+ cells confirming p53-dependent Bax and p53-independent Bak activation. Additionally, in HCT116p53+/+ cells, combined loss of Bax and Bak (HCT116Bax−Bak−) reduced WithaD-induced apoptosis and completely blocked cytochrome c release whereas single loss of Bax or Bak (HCT116Bax−Bak+/HCT116Bax+Bak−) was only marginally effective after WithaD treatment. In HCT116p53−/− cells, though Bax translocation to mitochondria was abrogated, Bak oligomerization helped the cells to release cytochrome c even before the disruption of mitochondrial membrane potential. WithaD also showed in vitro growth-inhibitory activity against an array of p53 wild type and null cancer cells and K562 xenograft in vivo. Taken together, WithaD elicited apoptosis in malignant cells through Bax/Bak dependent pathway in p53-wild type cells, whereas Bak compensated against loss of Bax in p53-null cells.
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Introduction
The primary goal of cancer chemotherapy is to trigger tumor-selective cell death [1] and the response of tumors to therapy to undergo cell death mainly depends on how fast tumor cell gets the signal to accomplish their programmed suicide. In this scenario, the ideal target of an anti-cancer agent might be mitochondria because perforation of mitochondrial membrane results in release of several death-promoting factors which ultimately either caspase dependently or independently execute cell death [2]. Therefore, regardless of the pathways involved, it is undoubtedly accepted that mitochondrial permeabilization is a central event in apoptosis and is undisputedly regulated by members of Bcl-2 family. Therefore, in a way, Bcl-2 family members and mitochondria are important targets of p53 [3]. When p53 encounter cellular stress, it restricts tumor development by responding to diverse signals for the ultimate benefit of the organism [4]–[5]. To achieve this cellular fate, p53 differentially activate or suppress definite sets of target genes and for this selection, multiple molecular mechanisms were involved. For example, p53 sensibly repress important anti-apoptotic proteins like Bcl-2, Bcl-xl and survivin whose ultimate outcome was identical to that of the activation of pro-apoptotic genes [6]–[8]. Simultaneously, p53 transactivates and upregulates different pro-apoptotic genes like Bid, Bax, Bak and Noxa [9]–[11], which mainly helped in mitochondrial membrane permeabilization. Besides that, Bid is a pro-apoptotic BH3-only protein which is cleaved and activated by caspase-8 or truncated by Granzyme B [12]. This truncated Bid (tBid) then interacts with Bax or ANT for the permeabilization of mitochondrial membrane and release cytochrome c along with Smac/DIABLO. Additionally, Bcl-xL has also been shown to inhibit tBid-induced cytochrome c release [13]. Therefore, Bid plays crucial role by combining receptor-mediated and mitochondria-mediated pathways through cross talk. Hence, chemotherapeutic agents targeting mitochondrial death are of immense importance, because this type of agents can enforce death in cells in which upstream signals normally leading to apoptosis have been disabled.
Withanolide D (C4b-C5b,C6b-epoxy-1-oxo-,20b, dihydroxy-20S,22R-witha-2,24-dienolide; WithaD) is a steroidal lactone isolated from the leaves of Ashwagandha (Withania somnifera Dunal, Solanaceae), one of the most reputed medicinal plant of Ayurveda [14]. The herb forms essential constituent of more than 100 traditional medicine formulations [15]–[19]. Earlier we have demonstrated that WithaD effectively induced apoptosis in leukemia (MOLT-4 and K562) and in primary cells from patients irrespective of their lineages. Also, we had shown that WithaD-induced apoptosis was through the early accumulation of ceramide by the activation of neutral-sphingomyelinase [20]. Here we wanted to explore the mitochondrial pathway as targeting a single pathway is unable to decrease all the downstream effect arises from signal cross talk.
We identified differences in activation pattern of intrinsic and extrinsic pathways in MOLT-3 and K562 cells, which was correlated with p53 status as revealed by HCT116p53+/+ and HCT116p53−/− cells. WithaD robustly enhanced p53 expression and also induced p53-dependent Bax and independent Bak upregulation. Additionally, WithaD elicited apoptosis through a Bax/Bak dependent way in p53-proficient cells, whereas Bak compensated against loss of Bax in p53-null cells. Moreover, WithaD induced in vitro growth-inhibitory activity against an array of p53 wild type (wt) and null cancer cells and inhibits tumor growth in athymic nude mice. Hence, we suggest that WithaD is a potent anti-cancer agent that induced mitochondria-mediated apoptosis both in p53wt and null cells.
Results
WithaD-mediated apoptosis commence through the involvement of mitochondria
To specify the role of mitochondria in WithaD-induced apoptosis, we first investigated the expression of pro- and anti-apoptotic molecules in leukemic cells (MOLT-3 and K562). Results showed that in both the cells, expression of Bcl-xl and Bcl-2 were reduced dose dependently after WithaD treatment. However, in MOLT-3 cells, WithaD showed a prominent increase in Bax and Bak levels. In contrast, Bax level remains unchanged after increasing WithaD treatment in K562, whereas Bak was significantly upregulated (Fig. 1B). These results suggested that in leukemia, mitochondria related Bcl-2 family proteins were differentially involved in WithaD-mediated cell death.
10.1371/journal.pone.0034277.g001Figure 1 WithaD-induced apoptosis occurs mainly through intrinsic pathway.
(A) Chemical structure of WithaD isolated from leaves of Withania somnifera. MOLT-3 and K562 cells were treated with (0–4 µM) WithaD for 15 hr and subsequently evaluated for the (B) expression of pro-survival (Bcl-xl, Bcl-2) and pro-apoptotic (Bak, Bax) Bcl-2 family members; (C) proteolytic processing of pro-caspase-9, -3 and -7 and the cleavage of PARP and (D) activation of caspase-8 and reduction of total Bid. In each experiment, β-actin served as the loading control. (E) For blocking assay, MOLT-3 and K562 cells were pre-incubated with or without IETD-FMK (20 µM), LEHD-FMK (20 µM) and DEVD-CHO (100 µM) for 1 hr followed by incubation for an additional 48 hr in presence of WithaD (2 µM) and % of apoptotic cells (annexinV+/PI+) were measured by flow cytometry. ‘*’ indicates the difference was statistically significant (P<0.005) in WithaD+z-LEHD-FMK and WithaD treated cells.
To locate the specific death cascade through which WithaD exerts its action, we investigated the key molecules of intrinsic and extrinsic pathways. Results showed that WithaD proteolytically cleaved inactive pro-caspase-9 (47 kDa) after 15 hr of treatment at 0.5 µM dose to form the active 35–37 kDa fragment in MOLT-3. Moreover, WithaD induced the proteolytic processing of executioner caspases-7 and -3 and also stimulated a dose-dependent hydrolysis of the 116 kDa PARP to 85 kDa fragment. In contrast, in K562 cells, the activation of pro-caspase 9, -7, -3 and PARP cleavage were only observed at higher concentration of WithaD (2 µM), suggesting the involvement of intrinsic pathway in both the cells, only the amount of WithaD required to activate the pathway was different (Fig. 1C).
Next, we tested the possibility of involvement of extrinsic pathway in WithaD-mediated apoptosis. In MOLT-3, proteolytic cleavage of pro-caspase 8 to its active 43 kDa fragment was observed within 0.5–1 µM WithaD treatment. Moreover, results showed significant reduction in total Bid expression with increased dose of WithaD. In contrast, in K562 cells, we did not detect any active caspase 8 fragments, only the reduced level of pro-caspase 8 was observed. Additionally, Bid level was also remain unaltered (Fig. 1D). These results suggest that, possibly caspase 9-mediated intrinsic pathway playing the central role in both the cells in WithaD-mediated apoptosis.
To confirm the possibility of involvement of intrinsic pathway in WithaD-induced cell death, we specifically inhibit the caspase -9, -8 and -3 and measured the apoptosis in MOLT-3 and K562 cells. Caspase -9 inhibition by LEHD-FMK significantly reduced WithaD-induced apoptosis both in MOLT-3 and K562, while caspase-8 inhibition by IETD-FMK only marginally affects (Fig. 1E). Additionally, caspase-3 inhibition by DEVD-CHO markedly reduced WithaD-induced apoptosis suggested WithaD-mediated specific activation of caspase cascade, which ultimately executed through caspase-3 activation. In summary, these results confirmed that the contribution of mitochondria-mediated pathway executed the WithaD induced apoptosis both in MOLT-3 and K562, although the accomplishment was different.
p53 is a critical mediator of WithaD-induced apoptosis
A consistent difference in the activation of intrinsic and caspase-8-mediated death receptor pathway in MOLT-3 and K562 cells along with difference in Bax activation prompted us to find the reason(s) behind this discrepencie(s). In intrinsic pathway, p53 target crucial subset of Bcl-2 family genes including Bax, Bid, Bcl-xl etc. [21] or induce the oligomerization of Bak at mitochondrial level. Therefore, we envisioned that the discrepancies between MOLT-3 and K562 may be due to the p53 status, as K562 are p53-null, whereas MOLT-3 is p53 wild type (wt). Therefore, we next assessed the effect of WithaD on the expression of p53 in MOLT-3 and K562 along with two other p53 expressing cells (HCT116 and U87MG). WithaD dose-dependently enhanced p53 expression in MOLT-3, HCT116 and U87MG whereas in K562 there was no p53 expression as expected (Fig. 2A).
10.1371/journal.pone.0034277.g002Figure 2 p53 is crucial in WithaD-induced apoptosis.
(A) Cells were treated with WithaD (0–4 µM) for 15 hr and p53 expression was evaluated in the lysate of MOLT-3, HCT116, U87MG and K562 cells by Immunoblot. (B) Status of p53 in HCT116 p53+/+ and stably knockdown HCT116p53−/− cells. (C) WithaD (0–4 µM) treatment led to proteolytic processing of pro-caspase-9, -3 and -7 and the cleavage of PARP as determined by immunoblot analysis after 15 hr in HCT116p53+/+ and HCT116p53−/− cells. (D) Effect of WithaD (0–4 µM) on activation of caspase-8 and reduction of total Bid after 15 hr of treatment in HCT116 p53+/+ and HCT116p53−/− cells. In each immunoblot, β-actin was served as the loading control. (E) Difference in WithaD (0–4 µM) mediated apoptosis induction in HCT116 p53+/+ and HCT116p53−/− cells for 24 hr. (F) Morphological changes as evaluated by phase contrast microscopy in HCT116 p53+/+ and HCT116p53−/− cells after 24 hr WithaD (2 and 4 µM) treatment.
Next we used HCT116p53+/+ and a stably p53 knockdown HCT116p53−/− cells and checked their p53 status (Fig. 2B). We then investigated the intrinsic and caspase-8 mediated death receptor pathways in these cells to specifically demonstrate whether p53 status really made any differences. In HCT116p53+/+ cells, WithaD induced the activation of caspase-9, caspase-3, caspase-7 and also stimulated the processing of PARP in similar manner as was observed in MOLT-3. Interestingly, activation of caspase-9 was only occurred at higher dose and caspase-3, caspase-7 and PARP cleavage proceed subsequently in HCT116p53−/− cells as was in K562 (Fig. 2C). In case of caspase-8-mediated death receptor pathway, active caspase-8 fragment was formed at 2.0 µM in HCT116p53+/+ cells, while in HCT116p53−/− cells, only the level of pro-caspase-8 was reduced. Additionally, reduction of total Bid expression was observed merely in the highest dose in 116p53−/− cells (Fig. 2D). Therefore, these results suggested that disparities in the activation of intrinsic and extrinsic pathway might be due to the variation in p53 status.
Next, we evaluated whether presence of p53 really made a difference in WithaD-induced apoptosis. Results showed that only 26.1% HCT116p53+/+ cells were viable at 24 hr at 5 µM dose whereas at identical conditions 43.8% HCT116p53−/− cells were viable (Figure S1). Similar differences were observed after 48 hr of WithaD treatment. Moreover, this trend of differences i.e. lower cell death in HCT116p53−/− compared to HCT116p53+/+ were also observed in annexinV-PI staining (Fig. 2E) which was further reflected in the changes of cell morphology (Fig. 2F). With increasing dose of WithaD, HCT116p53+/+ cells lost their adherent property, detached from the substratum and also rounded up. In contrast, HCT116p53−/− cells showed more adherences to its niche with extended normal cellular morphology. These results altogether confirmed that p53 crucially regulate WithaD-mediated apoptosis.
WithaD induced p53-dependent Bax and p53-independent Bak activation
Having established that p53 is a crucial mediator and mitochondria playing important role in WithaD-induced apoptosis, we next attempt to find the missing link between these two events. Hence, efforts were made to identify the role of different p53 downstream effector molecules related to mitochondrial apoptosis along with Bcl-2. Results showed that in HCT116p53−/− and HCT116p53+/+ cells, expression of Bcl-xl and Bcl-2 were reduced dose dependently after WithaD treatment. Being a p53 target molecule, Bcl-xl's reduction irrespective of p53 status could be explained by the fact that there may be other factors regulating Bcl-xl. Also, an increase in p21 level was observed in both the cells. However, WithaD showed a prominent dose-dependent increase in Bax and Bak levels in constitutive p53 expressing cells. In contrast, in p53-null cells, Bax level remains unchanged even at higher doses as was observed in K562, whereas under identical conditions Bak was significantly upregulated (Fig. 3A). These results suggested that possibly Bax was upregulated p53 dependently, while Bak upregulation was p53 independent. To scrutinized the p53 dependency of Bax, we specifically inhibited p53 expression with pifithrin-α in HCT116p53+/+ cells. A significant reduction of Bax expression was observed, while Pifithrin-α failed to affect the enhancement of Bak expression confirming p53-dependent Bax and p53-independent Bak activation (Fig. 3B).
10.1371/journal.pone.0034277.g003Figure 3 WithaD induced p53-dependent Bax and p53-independent Bak activation.
(A) Expression of p53 downstream effector molecules including Bcl-xl, Bcl-2, p21, Bak and Bax were evaluated by immunoblot assay after WithaD (0–4 µM) for 15 hr in HCT116p53+/+ and HCT116p53−/− cells. (B) HCT116p53+/+ cells were pre-incubated with pifithrin α (30 µM) for 1 hr followed by 15 hr WithaD (0–2 µM) treatment and the protein level of p53, Bax and Bak were evaluated by Western blot. In each blot, β-actin served as the loading control.
Bak functionally harmonizes for Bax in p53-null cells to release cytochrome c
To this end, we have established comparable different time scan for transmitting the intrinsic apoptotic signal and differential upregulation of Bax and Bak in p53 wt and null cells. These prompted us to further define the role of Bax and Bak in WithaD-induced mitochondrial apoptosis. Therefore, we further investigated Bax and cytochrome c level in both mitochondria and cytosol and Bak oligomerization in mitochondria (Fig. 4A). Release of cytochrome c in cytosol was enhanced along with decrease in Bax level in cytosolic fraction of HCT116 p53+/+ cells. In mitochondrial fraction, we observed significant accumulation of Bax and reduced cytochrome c level. Interestingly, we observed WithaD-induced dose-dependent oligomerization of Bak in mitochondria (Fig 4A). Initially at lower dose (0.5 µM), the level of monomeric (1×) and oligomerized (2× and 3×) Bak were minimal. Consequently, generation of 1×, 2× and 3× oligomeric Bak were enhanced as we have increased the dose of WithaD to 1–2 µM. However, at higher doses (3–4 µM), significant production of 3× oligomeric Bak and concurrently reduction of 1× and 2× form was observed.
10.1371/journal.pone.0034277.g004Figure 4 Cytochrome c was released before mitochondrial depolarization.
(A) To detect cytochrome c release, cytosolic and mitochondrial fraction of HCT116p53+/+ and HCT116p53−/− cells were separated as mentioned in materials and methods and electrophoresed on 15% SDS-PAGE and immunoblotted using anti-cytochrome c antibody. Cytosolic Bax and cytochrome c were evaluated by Western blot analysis and β-actin served as the loading control. Mitochondrial Bak oligomerization, Bax and cytochrome c were detected by Western blot and Cox IV served as mitochondrial loading control. (B) Mean fluorescence intensity (MFI) of FL1 was evaluated in HCT116p53+/+ and HCT116p53−/− after JC1 staining. Dose dependent treatment of WithaD (0–4 µM) revealed no significant changes in MFI value at15 hr. # considered not significant difference (P = 0.125) between untreated and WithaD (3 µM) treated cells.
In HCT116 p53−/− cells, cytochrome c level was enhanced in cytosolic fraction although Bax level remains unaltered. However, in mitochondrial fraction only basal level of Bax and reduced cytochrome c level was observed. Interestyingly, in HCTp53−/− cells a robust upregulation and generation of oligomeric Bak (3×) was observed in mitochondrial fraction which was higher when compared to HCT p53+/+ cells. These results suggested that both Bax and Bak were involved in cytochrome c release to activate intrinsic pathway in p53 wt cells, while only Bak was responsible for the same.
Cytochrome c release and mitochondrial membrane depolarization are two important events in intrinsic pathway mediated apoptosis. These two events are differentially regulated and induction of cytochrome c release is either dependent or independent on the detectable loss of the negative electrical gradient across the mitochondrial membrane [22]. Hence, we tested the possibility whether WithaD-induced cytochrome c release was through Bax and/Bak or due to mitochondrial membrane depolarization (Fig. 4B). We observed no detectable change in the MFI value of green fluorescence upto 2 µM, whereas even at 0.5 µM considerable release of cytochrome c in cyotsol was detected in both the cells. However, mitochondrial membrane potential loss may also be resulted due to the generation of ROS into the mitochondrial lumen. A recent study reports that production of ROS in mitochondria requires Bak in a Bax-independent manner. Additionally, the activation of Bak responsible for ROS production is dependent on the cytosolic presence of tBid [23]. Interestingly, although WithaD activated Bak, it was unable to produce early ROS either dose or time dependant manner (Figure S2). Therefore, it may be suggested that Bax and/Bak are responsible for cytochrome c release in cytosol rather than mitochondrial membrane depolarization.
To further delineate the role of Bax and Bak in WithaD-induced cell death, we took HCT116p53+/+ cells either wild type (HCT116Bax+) or homozygously deleted for the bax (HCT116Bax−) generating four sublines HCT116Bax+/Bak+, HCT116 Bax−/Bak+, HCT116Bax+/Bak− and HCT116Bax−/Bak−. Results showed p53 level enhanced dose dependently in all the cells and also Bax and Bak expression satisfy their sub-cell line status (Fig. 5A). Most importantly, WithaD dose dependently augmented the level of cytochrome c in cytosol in HCT116 Bax+/Bak+ whereas totally abolished the cytochrome c release from mitochondria in HCT116Bax−/Bak− cells having complete loss of Bax and Bak. Interestingly, single loss of Bax or Bak could release the cytochrome c in HCT116 Bax−/Bak+ and HCT116 Bax+/Bak−, only the intensity was lower than that of HCT116 Bax+/Bak+ cells.
10.1371/journal.pone.0034277.g005Figure 5 Bax and Bak were critical mediator of WithaD-mediated apoptosis.
(A) Expression of p53, Bax, Bak and Cytochrome c were evaluated in the lysate of HCT116 cells proficient for Bax and Bak (Bax+/Bak+), deficient for either Bax (Bax−/Bak+) or Bak (Bax+/Bak−) or deficient for both Bax and Bak (Bax−/Bak−) after WithaD (0–2 µM) treatment for 15 hr by Western blot analysis. Each sub-lines were treated with WithaD (0–4 µM) for (B) 24 and (C) 48 hr and viabilities were determined by MTT assay. (D) Apoptosis induction was assessed by flow cytometric detection of each sub-lines by 7-AAD staining. ‘*’ indicates the difference was statistically significant (P<0.005) between HCT116Bax+/Bak+ and HCT116Bax−/Bak− cells.
Next, we wanted to explore the role of Bax and Bak with WithaD-mediated mitochondrial apoptosis. Hence using all four cell lines, we determined the cellular viability and results showed that after 24 hr of WithaD (2 µM) treatment, HCT116Bax−/Bak− showed 68.12% viability, whereas HCT116Bax+/Bak+ showed only 42.65%. Interestingly, in HCT116 Bax−/Bak+ or HCT116Bax+/Bak− viabilities were 55.02 and 59.11% respectively (Fig. 5B). Similar trend was observed in 48 hr viability (Fig. 5C) and 7-AAD staining assays (Fig. 5D). These results suggested that WithaD elicited apoptosis through Bax/Bak dependent way in p53-functional cells, whereas in Bak dependent way in p53-null cells as was observed in K562.
WithaD inhibited in vitro and in vivo tumor growth in nude mice model
To check the contribution of p53 in WithaD-induced cell death, we have used a few p53wt (SiHa, HCT116, U87MG) cell lines along with MOLT-3 and p53-null (K562, H1299) cells of different cancer origins. Continuous exposure of different doses of WithaD for 24 and 48 hr revealed dose-dependent growth inhibition of p53wt cells, IC50 values being 0.75 µM, 0.9 µM, 0.75 µM and 1.0 µM for MOLT-3, HCT116, U87MG and SiHa respectively after 48 hr, whereas in p53-null cells reduction of cell growth was lower for K562 [19] and H1299 than that of the p53wt cells (Figure S3). This discrimination in sensitivity of p53wt and null cells were also reflected by the total disintegration of cell morphology and reduced cell density. Flow cytometric study revealed significant increase in numbers of annexinV-positive and both annexinV-PI- positive (Figure S4) cells in all the cancer cells indicating higher apoptosis in p53wt cells.
We have also examined the in vivo efficacy of WithaD against K562 xenograft in athymic nude mice. Tumor growth inhibition was most evident in mice treated with WithaD at 10 mg/kg/day, where ∼80% reduction in tumor size was observed, in contrast with mice treated with vehicle (Fig. 6A). The average body weights of the control and WithaD-treated mice did not differ significantly throughout the study (data not shown). Moreover, the WithaD-treated mice seemed healthy and did not exhibit signs of distress such as impaired movement or posture and indigestion. The average tumor volume in WithaD-treated mice was significantly lower compared with control mice on every day of tumor measurement (Fig. 6B). For example, on 6th day, the average tumor volume in control mice (1,080 mm3) was ∼5 fold higher compared with WithaD-treated mice (200 mm3). Consistent with tumor volume data, the average weight of the wet tumor was significantly lower in WithaD-treated mice compared with control mice (data not shown). To test whether WithaD-mediated inhibition of K562 xenograft growth in vivo was associated with reduced cell proliferation and/or increased cell death, tumor tissues from control and WithaD-treated mice were processed for PI positivity. Data from a representative mouse of each group were shown in Fig. 6C. The tumor cells from the WithaD-treated mice exhibited a significantly higher PI positivity compared with control tumors. Collectively, these results indicated that WithaD administration caused suppression of cellular proliferation and increased cellular death in the tumor. The histological data indicated that the minimal toxic effects over non-specific tissues (section of lungs, liver and spleen) of WithaD-treated nude mice. Results showed almost no toxic patches in the histological sections of spleen and lungs (Fig. 6D) after WithaD treatment. Only the liver was underwent some stress condition, which was identified by less packed density of the liver cells. Thus, WithaD administration significantly inhibited K562 xenograft growth in female nude mice minimally affecting the normal tissue.
10.1371/journal.pone.0034277.g006Figure 6 WithaD inhibit in vitro and in vivo K562 xenograft growth.
(A) WithaD could prolong the survival time of nude mice injected with K562. The tumor load of treated mice (10 mg/kg body weight) was visibly lower than untreated control mice. (B) WithaD significantly reduced tumor volume. Each point represents mean of three tumors. (C) Status of PI-positive cells in untreated and WithaD-treated tumor cells. PI-positive cells (▪) were determined with respect to PI unstained cells (□). (D) Tissue section of spleen, lungs and liver of nude mice (control and WithaD treated) determined by H&E staining and observed both in 20× and 40×.
Discussion
The discovery of anti-leukemic activity and a novel ceramide signaling of WithaD encouraged us to explore its ability as a multi-signal inducing anti-cancer agent and decipher the molecular mechanism of this natural product. Accordingly, the major findings of the present investigation in WithaD-induced cell death include (a) involvement of mitochondrial pathway, (b) p53 as critical mediator, (c) role of Bak and Bax in p53-null and wt cells and (d) demonstration of in vitro and in vivo growth-inhibitiory activity of WithaD fulfilling the criteria of a potent multi-faceted anti-cancer agent.
Apoptosis can be triggered through multiple signaling pathways, but the ultimate event by which physiological or chemotherapy induced cell death occurred is permeabilization of mitochondrial membrane. This ‘point-of-no-return’ in the cell death machinery is a complicated process and regulated mainly by the anti- and pro-apoptotic members of Bcl-2 family proteins. Hence the exploration of the role of Bcl-2 family members after WithaD treatment revealed significant upregulation of both Bax and Bak in MOLT-3 but Bak was upregulated only in K562, while Bcl-2 and Bcl-xl was downregulated in both the cells. Additionally, activation of caspases, cleavage of PARP, enhanced pro-caspase-8 proteolysis and dose dependent decrease of total Bid clearly indicated the activation of both intrinsic and extrinsic signaling in WithaD-mediated apoptosis in MOLT-3. In contrast, in K562 cells absence of early proteolytic processing of pro-caspase-8 and almost unchanged total Bid expression indicated towards the inactivation of death receptor signaling. Therefore, intrinsic pathway plays the central role in WithaD-mediated apoptosis in both the cells. Moreover, significant reduction in apoptogenic effect of WithaD after inhibition of caspase 9 further confirmed that WithaD-induced cell death commence mainly through mitochondrial pathway. However, a consistent difference exists in the commencement of intrinsic and caspase-8-mediated death receptor pathways in MOLT-3 and K562 cells. K562 being p53-null cell hinted us about the role of p53 in WithaD-induced cell death. p53 is not just a tumor suppressor protein that singly decide cells' fate, instead it is a central component which intricate network of signals and molecular interactions [24]. It has the ability to activate both the extrinsic and intrinsic apoptotic pathways. Extrinsic pathway is activated through the induction of Fas, DR5 and PERP [25]–[26] whereas in case of intrinsic pathway, p53 target Bcl-2 family proteins at mitochondrial level thus ultimately releasing cytochrome c [27].
WithaD have been shown to induce robust upregulation of p53 in MOLT-3 and two other cancer cells including HCT116 and U87MG having functional wild type p53. To exclude the variations in results due to different cell lines (MOLT-3 and K562) and to evaluate the role of p53 in WithaD-induced apoptosis, we used HCT116p53+/+ and HCT116p53−/− cells. Here we identified similar activation of intrinsic and death receptor signaling in HCT116p53+/+ cells as was in MOLT-3. Bid is a unique protein, playing the crucial role of maintaining the flow of death signal from cell surface to mitochondria. Activation of Bid mainly depends on either truncation by activated caspase 8 or transcriptional regulation by p53 [28]. Activated Bid then translocates to mitochondria and activates Bax and Bak to initiate intrinsic signal leading to apoptosome formation. Hence, p53 appears to promote the convergence of intrinsic and extrinsic pathways through Bid regulation [29]. Dose dependent decrease of total Bid in HCT116p53+/+ cells thereby clearly suggests that functional p53 simultaneously activate both intrinsic as well as extrinsic pathways intimated through Bid after WithaD treatment. Strikingly, in HCT116p53−/− cells, early processing of pro-caspase-8 was totally absent, although activation of caspase 9, -3, -7 and cleavage of PARP was observed. Moreover, almost unchanged Bid expression indicated towards the inactivation of death receptor signaling. In extrinsic pathway, the cell-surface receptor Fas (CD95/Apo-1) is a key component and in turn promotes cell death through caspase-8 [30]. However, Fas appears to be dispensable for p53-dependent apoptosis [31]. Therefore, the plausible explanation of the abrogation of extrinsic pathway is due to the absence of p53 in HCT116p53−/− as well as in K562. Furthermore, reduced sensitivity of p53-null (HCT116p53−/−) than p53wt (HCT116p53+/+) cells towards the apoptogenic effect of WithaD suggested a crucial role of p53 in WithaD-mediated apoptosis.
During the induction of mitochondrial apoptosis by a death stimulus, the role of p53 is manifold and therefore considerably difficult to follow [5]. p53 can target itself to the mitochondrial compartment or transactivate or trans-repress specific genes rendering its effect of mitochondrial death [4], [6]. Among them, Bak and Bax play the pivotal role of gatekeepers of mitochondrial integrity and cytochrome c release [32]. After WithaD treatment, significant upregulation of both Bax and Bak in HCT116 p53+/+ cells but upregulated Bak only in HCT116p53−/− cells suggested p53-independent Bak activation. Inhibition of p53 substantially reduced Bax but not Bak in HCT116 p53+/+ cells further confirming p53-dependent Bax and independent Bak activation. Therefore, activation of mitochondrial pathway in p53-null cells could be explained through the differential upregulation of Bax and Bak which is well correlated with p53 status.
Bax and Bak both can be activated either p53 dependently or independently. p53 can bind directly to Bak and induce a conformational change in the N-terminus encouraging the oligomerization thus allowing the release of cytochrome c and other pro-apoptotic proteins [27]. Similar function of p53 with Bax was also evident [33]. Alternatively, Degenhardt et al reported p53 independent role of Bax and Bak in tumor suppression [34]. Bax and Bak are also reported to have necessary function in staurosporin, UV radiation, etoposide, thapsigargin, and tunicamycin-induced apoptosis [31]. Here, reduced apoptosis in HCT116Bax−Bak− cells over HCT116Bax+Bak+ cells and marginal effect of single loss of Bax or Bak on the cell death clearly suggests that Bak can functionally complement for the loss of Bax and vice versa. Therefore when both Bax and Bak were present, WithaD-induced mitochondrial apoptosis was most potent as was observed in MOLT-3. In agreement with differential role of Bax and Bak, Bax translocation along with Bak oligomerization revealed perfect coordination with cytochrome c release in HCT116 p53+/+ cells. In striking contrast, no Bax translocation was found in mitochondria of HCT116p53−/− cells, although release of cytochrome c did not differ from HCT116 p53+/+ cells. A robust Bak upregulation and oligomerization further indicates toward the fact that in HCT116p53−/− cells, WithaD triggered mitochondrial apoptotic pathway that predominantly depends on Bak but not Bax. Loss of ΔΨm only at higher doses clearly indicated that cytochrome c release was an earlier event than mitochondrial ΔΨm dissipation where Bax and/or Bak were solely responsible for that.
In conclusion, WithaD elicited mitochondria-mediated apoptosis in malignant cells through a Bax/Bak dependent way in p53wt cells, whereas Bak compensated against loss of Bax in p53-null cells (Fig. 7). Hence, although extrinsic pathway and Bax were crippled due to absence of crucial p53, WithaD is able to recruit Bak which p53-independently can induce apoptosis in p53-null cells. Therefore, this study highlights a new possibility of using WithaD as alternative anti-cancer agent along with the existing chemotherapeutic agent which potentially target mitochondria-mediated apoptosis both in p53wt as well as p53-null malignant cells.
10.1371/journal.pone.0034277.g007Figure 7 Probable mechanism of WithaD induced apoptosis.
WithaD treatment induces p53 activation and mitochondrial apoptosis in p53wt cells in Bax and Bak dependent manner. However, in p53-deficient cells, lack of Bax function is complemented with Bak in WithaD-induced mitochondrial apoptosis.
Materials and Methods
Reagents
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma (St Louis, MO). The antibodies against p53, cytochrome c, caspase-3, FITC-annexin V, propidium iodide (PI), annexin V binding buffer, 7-AAD, BD Mitoscreen kit (JC 1) were from BD Bioscience (San Diego, USA). Antibodies against PARP, Bax, Bak, Bcl-2, Bcl-xl, Bid, p21, caspase 7, caspase-8, caspase-9 and HRP-secondary antibodies were from Cell Signaling Technology (USA). Cocktail protease inhibitor, z-VAD-fmk, z-IETD-fmk z-LEHD-fmk, z-DEVD-CHO were from Calbiochem. RPMI-1640, IMDM and fetal bovine serum (FBS) were from Gibco/BRL, USA.
Withanolide D
WithaD (M.W 470.6) was purified in high yields from the leaves of a well known medicinal plant Withania somnifera as described previously [35]. The pure compound was crystallized and analyzed by IR, mass, 1HNMR and 13C-NMR spectral analysis. The chemical structure of WithaD has been characterized as C28 steroidal lactone, namely C4β-hydroxyC5β,C6β-epoxy-1-oxo-,C20β,dihydroxy-20S,22R-witha-2,24-dienolide (Fig. 1A). WithaD was dissolved in absolute ethanol as 0.5 mM solution and stored at −70°C.
Cell lines and culture conditions
Chronic myelogenous leukemia (K562), colorectal carcinoma (HCT116), cervical carcinoma (SiHa), brain carcinoma (U87MG) and lung carcinoma (H1299) cells were purchased from ATCC. K562 cells were cultured in RPMI-1640 medium and rest of the cells were cultured in IMDM supplemented with 10% FBS and incubated in 5% CO2-95% air humidified atmosphere at 37°C. HCT116p53−/− cells were kindly provided by Dr. S. Roychowdhury (CSIR-IICB). HCT116Bax−/Bak− and HCT116Bax+/Bak− cell lines were a kind gift from Prof. G. Chinnadurai, Institute for Molecular Virology, USA. HCT116Bax−/Bak+ cells were a generous gift from Dr B. Vogelstein, Johns Hopkins University, USA. All these HCT116 sub-cell lines were cultured according to the originator [9].
Viability assay by MTT
Cells (1×104/250 µl/well) in log phase were seeded on 96-well tissue culture plates incubated with WithaD (0–5 µM) for 24 and 48 hr at 37°C. After incubation, MTT (0.1 mg/well) was added, and further incubated for 3–4 hr. After plate centrifugation, the resultant pellet was dissolved in DMSO. Absorbance of the resultant formazon was measured at 550 nm using a plate reader (Multiskan Ex, Thermo Electron Corporation).
Tumor xenograft study
Female nude mice of 6–7 weeks, having 20–22 gm of body weight were acclimated for 1 week in pathogen free condition. For subcutaneous xenograft study, mice were randomized in two groups; control and experimental, each group containing 5 mice. Exponentially growing K562 cells were suspended in 1∶1 RPMI-matrigel (BD bioscience) and 0.2 ml suspension containing 1×107 cells were injected s.c. on right flank of each mouse above the hind limb of each mice [36]. Tumor was allowed to develop for 20–25 days and tumor volumes were recorded till it reached 100–120 mm3. The mice were then injected i.p. with either vehicle (10% DMSO, 0.15 M NaCl) or vehicle containing 10 mg WithaD/Kg body weight per day for subsequent 8 days. Tumor volume was measured in a regular basis by external caliper and calculated as follows: L×W2/2 (mm3); where L = length, W = width. On the 9th day, mice were sacrificed and tumor xenografts were excised from each mouse. By collagenase type II-DNase I treatment, the tumor cells were isolated from tumor tissue. Control and experimental cells were then stained with PI and analyzed by flow cytometry.
Western blot analysis
Cells (1×106) were treated with WithaD (0–4 µM) for 15 hr and lysate were prepared by sonication (2 watt, 3 pulse). Equal amount of protein were electrophoresed on SDS-PAGE (10–15%) and electro-transferred to nitrocellulose membranes. The membrane was blocked by TBS-BSA, probed with primary antibody overnight at 4°C, washed with TBS containing 0.1% Tween-20 and incubated with the appropriate HRP-conjugated secondary antibody. Immunoreactive proteins were detected on X-ray films using the enhanced chemiluminescence system (Pierce, USA). For the detection of Bak oligomer, equal amount of mitochondrial fraction was boiled in sample buffer (-β-Me) and run on 12% SDS-PAGE and processed [37].
Apoptosis assay
Cells (1×106) were treated with WithaD (0–6 µM). Phosphatidyl-serine externalization was analyzed by double staining the cells with FITC-annexin V and PI (5 µg/ml) [38]. Alternatively, treated cells were stained with 7-AAD for 30 minutes. Cells were acquired and analyzed by CellQuest Pro software (BD FACSCalibur). For blocking assay, cells were separately pre-treated with caspase 8, 9 and 3 inhibitors IETD-FMK (20 µM), LEHD-FMK (20 µM) and z-DEVD-CHO (100 µM) respectively for 30 minutes at 37°C followed by WithaD treatment.
Sub-cellular fractionation
WithaD-treated cells (2×107) were harvested, washed and mitochondrial and cytosolic fractions were separated according to manufacturer's instructions (Pierce protein research products, USA). Protein content was measured by Lowry's method.
Mitochondrial depolarization assay
Mitochondrial transmembrane potential (Δψm) was determined using JC1 by Mitoscreen kit. Briefly, cells (1×106) were washed with PBS; JC1 (25 µM) was added and incubated in dark for 15 min at 37°C. Subsequently, they were washed with assay buffer and acquired immediately by flow cytometer [39].
Statement of Ethics
Animal experiments reported in the manuscript were performed with the approval of ‘Institutional Animal Ethics Committee’ of the National Institute of Immunology, New Delhi, India following the guidelines of CPCSEA. No research on humans has been carried out.
Statistical analysis
All the results were expressed as the mean ± S.D. of data obtained from three separate experiments. All statistical analysis was evaluated using graph pad prism software (San Diego). Data were analyzed by the paired t test, and P values less than 0.05 was considered statistically significant.
Supporting Information
Figure S1
HCT116p53+/+ and HCT116p53−/− cells were treated with WithaD (0–5 µM) for 24 and 48 hrs and viabilities were assessed using MTT.
(TIF)
Click here for additional data file.
Figure S2
WithaD fails to induce ROS production. (A) Effect of 1 µM WithaD in different time points (0–2.5 hr) on the generation of intracellular ROS by H2DCFDA in HCT116 wt cells. (B) Effect of different doses of WithaD (0–4 µM) after 1 hr on the generation of intracellular ROS by H2DCFDA in HCT116 wt cells.
(TIF)
Click here for additional data file.
Figure S3
Anti-proliferative effect of WithaD (0–5 µM) and vehicle treated control for 24 and 48 hr against cancer cell lines determined by MTT assay.
(TIF)
Click here for additional data file.
Figure S4 (A) WithaD (2 µM) treatment for 24 hr induced morphological changes and detachment of cancer cells from its substratum. (B) Flow cytometric analysis of annexin-V/PI in cancer cells treated with WithaD (2 µM) for 24 hr. WithaD treatment increased the percentage of annexin V+/PI− (down right quadrant) and annexin V+/PI+ (upper right quadrant).
(TIF)
Click here for additional data file.
We earnestly thank Director, Dr. C. Shaha, National Institute of Immunology, New Delhi, India for providing us the nude mice facility. We sincerely thank Prof. G. Chinnadurai, Institute for Molecular Virology, USA, Dr. B. Vogelstein, Johns Hopkins University, USA and Dr. S. Roychowdhury (CSIR-IICB) for their generous gift of cell lines.
Competing Interests: The authors have declared that no competing interests exist.
Funding: Council of Scientific and Industrial Research (CSIR) under IAP-0001, Systems Biology (HCP004), New Millennium Indian Technology leadership Initiative (NMITLI, TLP-004), CSIR - Indian Institute of Chemical Biology (IICB), Department of Biotechnology (DBT) under cancer Biology (GAP 235), Government of India supported this work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22509275PONE-D-11-2267310.1371/journal.pone.0034162Research ArticleBiologyBiochemistryGeneticsModel OrganismsAnimal ModelsMolecular Cell BiologyMedicineEndocrinologyGastroenterology and HepatologyOncologyMulti-Level Interactions between the Nuclear Receptor TRα1 and the WNT Effectors β-Catenin/Tcf4 in the Intestinal Epithelium Cross-Talk between TRα1 and WNT in IntestineSirakov Maria
1
¤a
Skah Seham
1
¤b
Lone Imtiaz Nisar
2
Nadjar Julien
1
¤b
Angelov Dimitar
2
Plateroti Michelina
1
*
¤b
1
Institut de Génomique Fonctionnelle de Lyon, Lyon, France
2
Laboratoire de Biologie Moléculaire de la Cellule, Université de Lyon, Université Lyon 1, Ecole Normale Supérieure de Lyon, Lyon, France
Katoh Masaru EditorNational Cancer Center, Japan* E-mail: [email protected] and designed the experiments: MS MP. Performed the experiments: MS SS INL JN. Analyzed the data: MS SS INL DA MP. Wrote the paper: MS MP. Read and approved the final manuscript: MS SS INL JN DA MP.
¤a Current address: Institut de Biologie et de Médecine Moléculaires, Université libre de Bruxelles, Bruxelles, Belgium
¤b Current address: Centre de Génétique et de Physiologie Moléculaire et Cellulaire, Université Lyon 1, Lyon, France
2012 3 4 2012 7 4 e3416216 11 2011 23 2 2012 Sirakov et al.2012This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Intestinal homeostasis results from complex cross-regulation of signaling pathways; their alteration induces intestinal tumorigenesis. Previously, we found that the thyroid hormone nuclear receptor TRα1 activates and synergizes with the WNT pathway, inducing crypt cell proliferation and promoting tumorigenesis. Here, we investigated the mechanisms and implications of the cross-regulation between these two pathways in gut tumorigenesis in vivo and in vitro. We analyzed TRα1 and WNT target gene expression in healthy mucosae and tumors from mice overexpressing TRα1 in the intestinal epithelium in a WNT-activated genetic background (vil-TRα1/Apc mice). Interestingly, increased levels of β-catenin/Tcf4 complex in tumors from vil-TRα1/Apc mice blocked TRα1 transcriptional activity. This observation was confirmed in Caco2 cells, in which TRα1 functionality on a luciferase reporter-assay was reduced by the overexpression of β-catenin/Tcf4. Moreover, TRα1 physically interacted with β-catenin/Tcf4 in the nuclei of these cells. Using molecular approaches, we demonstrated that the binding of TRα1 to its DNA target sequences within the tumors was impaired, while it was newly recruited to WNT target genes. In conclusion, our observations strongly suggest that increased β-catenin/Tcf4 levels i) correlated with reduced TRα1 transcriptional activity on its target genes and, ii) were likely responsible for the shift of TRα1 binding on WNT targets. Together, these data suggest a novel mechanism for the tumor-promoting activity of the TRα1 nuclear receptor.
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Introduction
The intestinal epithelium is a dynamic tissue that is continuously renewed through stem cells and committed progenitors, located in the crypts of Liberkün [1], [2]. The balance between proliferation and differentiation in the crypts is maintained by the fine cross-regulation among several pathways, including WNT, Hedgehog, Notch, BMP, and thyroid hormones (THs) [3]–[6]. These pathways are key players of intestinal homeostasis, and their deregulation is correlated with the onset of colorectal cancer [7]–[9]. The molecular basis of their action is only partially understood, and the mechanism of cross-regulation that occurs between these signaling pathways is still a puzzle. We previously demonstrated that TH signaling in the mouse intestine is involved in development, homeostasis and cancer susceptibility [10]#. THs act through nuclear hormone receptors, the TRs, which are encoded by THRa and THRb loci [11]. These transcription factors activate or repress the transcription of target genes by binding to specific DNA sequences called thyroid hormone response elements (TREs) [12], and they are involved in several cellular responses, such as cell proliferation, cell differentiation and apoptosis [5], [13]. TH signaling in the mouse intestine is mediated by TRα1, given that TRα0/0
[14] mice as well as hypothyroid mice have reduced cell proliferation in the crypts during development and in adulthood [15]. Recently, we showed that the constitutive TRα1 overexpression in the intestinal epithelium (vil-TRα1 mice) allows increased cell proliferation and adenoma development; this overexpression also enhances the intestinal tumorigenic process in an Apc mutated genetic background (vil-TRα1/Apc+/1638N mice) [9]. Interestingly, one of the molecular features of the vil-TRα1/Apc mice is the increased activity of the WNT pathway compared with that of the Apc mutants [9]. This is in agreement with our previous studies showing that TRα1 activates the WNT pathway via the transcriptional regulation of the genes Ctnnb1 and Sfrp2
[15], [16].
β-Catenin, the protein encoded by Ctnnb1, was originally identified as a structural component of cell adhesion complexes [17]. Upon increased cellular levels and nuclear accumulation, β-catenin interacts with transcription factors of the T-cell factor (Tcf) and lymphocyte-enhancer factor (Lef) families [18]. β-catenin/Tcf-Lef complexes are the downstream molecular effectors of the WNT signaling pathway. They bind to WNT response elements (WREs) in the genomic region of target genes involved in cell proliferation, survival, and migration [7], [19]. In the context of the intestinal epithelium, the β-catenin/Tcf4 complex regulates the expression of genes involved in the development and proliferation of normal and malignant epithelia [20], [21]. β-catenin, however, forms complexes with other transcription factors, including nuclear hormone receptors [22], [23]. This interaction modulates the transcriptional activity of the partners, depending on the specific tissue or nuclear receptor [24]–[28]. Our previous studies on the relationship between WNT and TRα1, combined with these observations, compelled us to investigate the physical interaction between TRα1 and β-catenin, the eventual involvement of Tcf4 in the complex, and its physiopathological relevance.
We established that the increased levels of the β-catenin/Tcf4 complex affects the transcriptional activity of TRα1 in vivo. We show that TRα1 interacts with the β-catenin/Tcf4 complex in the nuclei of Caco2 cells and that this interaction strongly reduces TRα1 functionality, but has a positive effect on the WNT downstream response in vitro. Finally, we observed the recruitment of TRα1 on the WRE regions in pre-cancerous and cancerous intestinal lesions.
Results
The expression of TRα1 direct target genes is impaired in vil-TRα1/Apc mice
We analyzed the expression profile of TRα1 target genes in normal mucosae or tumors from mice of different genotypes to gain additional insights on the cross-regulation between TRα1 and WNT pathways in gut tumorigenesis. Six-month-old vil-TRα1 [9], Apc+/1638N
[29] and double-mutant vil-TRα1/Apc [9] mice were analyzed. The vil-TRα1 mice are characterized by hyperproliferation in the intestinal crypts and a low rate of adenoma development in the small intestine and colon [9]. The Apc+/1638N develop a small number of adenocarcinomas, starting at approximately six months of age; these tumors occur primarily in the small intestine [29], [30]#. The vil-TRα1/Apc mice develop adenocarcinomas in the small intestine and colon at a higher frequency than do the Apc mutants. Moreover, the tumor appearance and progression to invasiveness are also accelerated in these mice. Finally, WNT activity in the tumors of these animals is increased compared with Apc mutants [9].
We compared the expression profiles of Ctnnb1 and Sfrp2, TRα1-direct target genes [15], [16], and of Ccnb1 and Cdc2a, TRα1-indirect target genes [16]. For Ctnnb1 and Sfrp2, we observed an increased mRNA levels in the vil-TRα1 mice compared with the WT mice, while in the normal mucosa or tumors of the Apc mice, the mRNA levels slightly decreased (Ctnnb1, Figure 1A) or did not change (Sfrp2, Figure 1B). However, in the vil-TRα1/Apc mice, the up-regulation of both genes was blunted, and the mRNA level unchanged (Ctnnb1, Figure 1A) or decreased (Sfrp2,
Figure 1B) in the tumors. When we examined the TRα1-indirect target genes Ccnb1 and Cdc2a, we observed that TRα1 overexpression stimulated the mRNA levels of these genes independently of the Apc mutation (Figure 1C, D). We also analyzed the Ctnnb1 and Sfrp2 mRNA expression in one-month-old animals. In fact, the tumorigenic program triggered by the loss of Apc heterozygosis [30] at that age is not yet accomplished, and the vil-TRα1/Apc mice do not yet show any signs of mucosal alteration. Interestingly, the mRNA expression level of TRα1-direct target genes in these animals was comparable with that of the vil-TRα1 mice, and in both cases, significantly higher than in the WT intestine (Figure S1A, B). We excluded the fact that the TRα1-reduced transcriptional activity is due to a lower availability of hormones, as the circulating TH level is not altered in the animals of different genotypes [9]. However, the intracellular concentration of TH may depend on T3 and T4 metabolism, which is locally regulated by the iodothyronine deiodinase selenoenzymes (Dio1, Dio2 and Dio3) [31], [32]. Next, we considered the possibility that the transcriptional activity of TRα1 was reduced by local hypothyroidism within the tumors and checked the levels of the three enzymes. However, when we analyzed the expression of the Dio mRNAs, we observed that only Dio1 was expressed, and its levels did not change in the intestine of different genotypes or in the tumors (Figure S1C), confirming that the cellular context was not hypothyroid. It is worth noting that in another context, Dio1 has been shown to be TH-responsive [31]; however, this difference may be due to the well-described tissue-specific regulation of TH target genes [10].
10.1371/journal.pone.0034162.g001Figure 1 Analysis of TRα1 target genes in mice of different genotypes.
RT-qPCR experiments were performed in the intestine of 6-month-old mice of the indicated genotype to analyze the mRNA levels of Ctnnb1 (A), Sfrp2 (B), Ccnb1 (C) and Cdc2a (D). Values represent fold change ± sd after normalization to the wild-type [WT] animals. *: P<0.05, **: P<0.001, compared with the WT; #: P<0.05, ##: P<0.01 compared with the vil-TRα1 animals, by two-tailed Student's t-test (n = 4). N, normal mucosa; T, tumor.
The Wnt3a ligand is not sufficient to affect TRα1 transcriptional activity in an ex vivo assay
We used intestinal epithelial primary culture to evaluate whether activation of the WNT pathway by an agonist could impair TRα1 transcriptional activity. To selectively modulate TRα1 and WNT, we treated the cells with T3 and the canonical Wnt3a ligand [33] alone or in combination. We observed that T3 and/or Wnt3a increased the number of proliferating cells (Figure 2A) and expressing nuclear β-catenin [Figure 2B]. The increase of both activated β-catenin protein level (Figure 2C) and Ccnd1 mRNA expression (Figure 2D), indicated that the cells were responding to the simple treatments as previously shown [15], [34]. When we investigated the expression of Ctnnb1 and Sfrp2, TRα1 direct target genes, both mRNA levels increased upon T3 treatment, as expected. Wnt3a had no effect on Ctnnb1 mRNA (Figure 2E) but stimulated Sfpr2 mRNA (Figure 2F). Finally, when the cells were treated with Wnt3a and T3, the TRα1 targets behaved as they did when treated only with T3, because they were not affected by the co-treatment with Wnt3a (Figures 2E, F). The expression of TRα1 or Tcf4 was not altered by T3 alone or in combination with Wnt3a (not shown).
10.1371/journal.pone.0034162.g002Figure 2 The Wnt3a ligand is not sufficient to impair TRα1 transcriptional activity ex vivo.
The primary cultures of intestinal epithelial cells were treated with 10 ng/ml of Wnt3a and/or 10−7 M of T3 for 24 hours. (A) The number of proliferating cells in the different experimental conditions was analyzed by Ki67 immunolabeling; all of the nuclei were labeled by Hoechst. The percentage of Ki67-positive nuclei was determined by counting under a fluorescence microscope (Zeiss Axioplan). The histograms represent the summary (mean ± sd) of the scoring of specific immunolabeling in two independent experiments each conducted in triplicate (n = 50). (B, C) Analysis of β-catenin in intestinal epithelial primary cultures by immunolabelling (B) and WB (C). Cells were in control, T3, Wnt3a and T3+Wnt3a conditions as indicated. Pictures in B show the fluorescent staining of the nuclei (blue), β-catenin (red) and the merging of each simple labeling. Bar: 15 µm. For the WB (C), we used a specific antibody allowing the detection of activated non-phosphorylated β-catenin [54], [55]. Actin was used as loading control. The image is representative of two independent experiments. Each lane represents whole protein extracts (50 µg/lane). (D–F) RT-qPCR analysis to evaluate mRNA levels of Ccnd1, Ctnnb1 and Sfrp2. Results are from three independent experiments, each conducted in duplicate. Values represent fold change ± sd after normalization to the control condition (Ctrl). *: P<0.05, **: P<0.01, ***: P<0.001 by two-tailed Student's t-test (n = 6).
Tcf7l2 (Tcf4) is overexpressed in vil-TRα1/Apc mice
The data obtained using the in vitro and in vivo approaches strongly suggested that β-catenin stabilization induced by Wnt3a is not sufficient to impair TRα1 transcriptional activity. Thus, the hypothesis that an increased level of both β-catenin and Tcf4 could affect TRα1 functionality was tested. We examined the mRNA expression of Tcf4 in the normal intestine and tumors from animals of different genotypes. The Tcf4 mRNA expression in vil-TRα1 or the normal mucosa of Apc mice was similar to that of WT animals (Figure 3A). However, the mRNA level significantly increased in the normal mucosa of the vil-TRα1/Apc mice and was even further upregulated in the tumors. We also observed an increased level of Tcf4 mRNA in the lesions from Apc mice compared with the healthy mucosa (Figure 3A). This trend was different from that observed for Lef1 mRNA, another transcriptional partner of β-catenin in the intestine [35], [36]. In fact, Lef1 mRNA level was significantly increased only in the lesions independently of the animals' genotype (Figure 3B).
10.1371/journal.pone.0034162.g003Figure 3 Analysis of Tcf7l2 (Tcf4) and Wnt target genes in mice of different genotypes.
RT-qPCR experiments were performed in the intestine of 6-month-old mice of the indicated genotype. (A) Tcf4, (B) Lef1, (C) c-Myc and (D) Ccnd1 mRNA levels were analyzed. Values represent fold change ± sd after normalization to wild-type (WT) animals. *: P<0.05, **: P<0.01 compared with the WT; $: P<0.05, $$: P<0.01 compared with the healthy mucosa of the same genotype; @: P<0.05, @@: P<0.01 compared with vil-TRα1/Apc N and T; #: P<0.05 compared with vil-TRα1, by two-tailed Student's t-test (n = 4). N, normal mucosa; T, tumor.
Finally, we analyzed the expression of Ccnd1 and c-Myc, the WNT target genes, and we observed their significant upregulation in the vil-TRα1 and in the normal mucosa of vil-TRα1/Apc mice compared with that of the WT or the normal mucosa of Apc mice [Figure 3C, D], in accordance with our previous results [9]. The expression level of these WNT target genes significantly increased in the tumors of both genotypes compared with the respective normal mucosae (Figure 3C, D).
The β-catenin/Tcf4 complex interferes with TRα1 functionality in vitro
The data described above strongly suggested that a negative feedback loop could occur between TRα1 and the β-catenin/Tcf4 complex. To test this hypothesis, we used a luciferase reporter system containing a synthetic tandem of TR-binding sites organized as DR4 (DR4-luc) [15] and monitored its response upon β-catenin and/or Tcf4 transfection. These experiments were performed in Caco2 cells maintained in T3-depleted serum with and without the addition of a supra-physiological concentration of T3 (Figure 4A). In the control condition, we observed a strong and significant increase in luciferase activity upon T3 treatment due to the presence of endogenous TRα1. It is worth noting that we observed a response to T3 addition in all conditions analyzed (Figure 4A). The transfection of β-catenin alone did not affect the DR4-luc response to the endogenous TRα1 stimulated by T3, while Tcf4 alone slightly reduced the reporter activity. Interestingly, Tcf4 in combination with β-catenin significantly reduced the T3 response of the endogenous TRα1 compared with the control (Figure 4A). As expected, the TRα1 transfection in the cells treated with T3 significantly enhanced the luciferase activity in comparison with the control (Figure 4A). This T3-mediated response in TRα1-transfected cells was slightly reduced in the presence of β-catenin, significantly decreased in the presence of Tcf4 and a stronger negative effect was observed by combining Tcf4 and β-catenin co-transfection.
10.1371/journal.pone.0034162.g004Figure 4 β-catenin/Tcf4 complex interferes with TRα1 functionality in luciferase assay in vitro.
(A) The DR4-luc luciferase reporter was transfected into Caco2 cells maintained in T3-depleted serum with or without supplementation with T3 as indicated, together with TRα1, Tcf4 or β-catenin expression vectors in different combinations. (B, C) The DR4-luc luciferase reporter (B) or TopFlash luciferase reporter (C) was transfected into Caco2 cells maintained in culture medium containing physiological concentrations of T3, together with the β-catenin/Tcf4 complex in the presence or absence of the TRα1 expression vector. Histograms represent mean ± sd from three independent experiments, each conducted in triplicate (n = 9). *: P<0.05, **: P<0.01 compared with the control condition (Ctrl); #: P<0.05, ##: P<0.01, compared with the TRα1 condition; $: P<0.05 compared with the TRα1+β-catenin condition; ££: P<0.01 compared with the β-catenin or β-catenin+Tcf4 condition, by two-tailed Student's t-test.
We also measured the DR4-luc activity in cells maintained in culture medium containing physiological concentrations of T3 [37] (Figure 4B). As expected, TRα1 transfection significantly induced the luciferase activity compared with the control. Moreover, this response was impaired by co-transfection of TRα1 with the β-catenin and Tcf4 vectors. The luciferase activity induced by TRα1 overexpression was not affected by β-catenin alone but was slightly reduced by Tcf4 (Figure 4B). We decided to verify if these combinatory transfections could also affect the activity of the WNT-reporter system TopFlash, which responds to the β-catenin/Tcf complex [38]. We observed a significant increase of TopFlash activity when TRα1 was transfected together with β-catenin or Tcf4 compared with the simple β-catenin or Tcf4 conditions (Figure 4C). We observed a slight up-regulation of the TopFlash activity, marginally significant, when TRα1 was transfected together with β-catenin/Tcf4. We verified the specificity of these responses by using mutated DR4-luc or FopFlash reporters (data not shown).
TRα1 binding on chromatin changes during gut tumorigenesis
Our findings strongly indicated that the increased level of the β-catenin/Tcf4 complex significantly reduced the TRα1 transcriptional activity both in cell lines and in vivo. To define the mechanism involved, we evaluated whether TRα1 could physically interact with β-catenin and/or Tcf4, as has been shown for other nuclear receptors [22], [23]. For this purpose, a co-immunoprecipitation (Co-IP) approach was employed to selectively target the endogenous proteins in the nuclei of Caco2 cells. After validating that TRα1 and β-catenin interact specifically in cell nuclei (Figure S2), we also analyzed the involvement of Tcf4. Interestingly, we observed that in the nuclei of the Caco2 cells the endogenous TRα1 is associated with β-catenin (Figure 5, upper and middle panels) as well as with Tcf4 (Figure 5, upper and lower panels). Moreover, all these interactions do not depend on T3 levels.
10.1371/journal.pone.0034162.g005Figure 5 Physical interaction between TRα1 and the β-catenin/Tcf4 complex.
Nuclear extracts from Caco2 cells, maintained in T3-depleted (−) or T3-supplemented (+) serum, were immunoprecipitated with antibodies directed against endogenous Tcf4, β-catenin or TRα1 and analyzed by WB by using the antibodies as indicated. Rabbit IgG was used as negative control. Ponceau red was used as whole-protein (50 µg/lane) loading control. Histone H1 was used to check the enrichment and was the loading control for the nuclear proteins in the inputs. The pictures are representative of at least three independent experiments.
We then investigated the presence of the three proteins on the chromatin, by using an in vivo chromatin immunoprecipitation (ChIP) approach to analyze the TREs and WREs of the specific target genes of TRα1 and WNT, respectively. The ChIP assay was performed on WT intestine and the normal mucosa or tumors from vil-TRα1/Apc mice using anti-TRα1, anti-Tcf4, anti-β-catenin antibodies or rabbit IgG (as negative control). As shown in Figures 6A and S3A, in the WT intestine, TRα1 binds to the promoter region of the Sfrp2 gene, which contains the TRE [16]; however, there was no recruitment of the β-catenin/Tcf4 complex independently of the genotype or the pathological condition (Figures 6A and S3A). This result was similar to that obtained by an in vitro EMS assay in which TRα1 binds to a DR4 element without forming a complex with β-catenin and/or Tcf4 (Figure S4). As the Sfrp2 expression profile suggested, the TRα1 chromatin occupancy changed between the WT and mutant intestine. In fact, in vil-TRα1/Apc mice, the TRα1-specific-DNA binding on the promoter was lost compared with the WT animals (Figures 6A and S3A). We confirmed this same profile of TRα1 chromatin occupancy on the Ctnnb1 TRE [15] (Figures 6B and S3B).
10.1371/journal.pone.0034162.g006Figure 6 Chromatin occupancy of TRα1, β-catenin and Tcf4 on genomic regions of target genes.
ChIP analysis was performed with chromatin isolated from the intestine of WT or vil-TRα1/Apc mice (healthy mucosa and tumor) and immunoprecipitated with anti-TRα1, anti-β-catenin, anti-Tcf4 antibodies or rabbit IgG (negative control). qPCR was performed using specific primers covering the TRE of Sfrp2 (A) and Ctnnb1 (B), the WRE of Axin2 (C) and c-Myc (D) or the promoters of Villin (E) and 36B4 (F); the Ppia gene was used as internal control. Data are representative of one of two experiments. Histograms represent the specific-DNA enrichment in each sample immunoprecipitated with the indicated antibody. The black bar in A–D delineates the threshold of binding specificity determined by the IgG non-specific binding. N, normal mucosa; T, tumor.
Next, we analyzed the specific DNA binding of Tcf4 and β-catenin on their target genes and the eventual presence of TRα1 in the same regions. A WRE has been described within the Axin2 promoter [39]. We observed that the Tcf4 occupancy on this promoter was similar in the different experimental conditions (Figures 6C and S3C) and that the presence of β-catenin on the WRE increased during tumorigenesis. More interestingly, TRα1 was not present on the Axin2 WRE region in the WT intestine but was clearly enriched on it, both in normal mucosae and the tumors of vil-TRα1/Apc mice (Figures 6C and S3C). We confirmed a similar trend of TRα1 binding on the WRE region of c-Myc, another classical direct WNT target [40] (Figures 6D and S3D), while no specific binding was detected on the Villin or 36B4 gene promoters (Figures 6E,F and S3E,F). Given the intriguing result of TRα1 binding to WRE regions, we used the software NUBISCAN (http://www.nubiscan.unibas.ch) to search for the presence of putative nuclear receptor binding sites in silico. No TREs were found within the Axin2 or c-Myc genomic regions containing the WREs analyzed (data not shown).
Discussion
The process of intestinal maturation and its homeostatic control by TH-TRα1 in mice results from a complex modulation of genes and signaling pathways, including the WNT, Notch and BMP [16]. The WNT pathway is considered a major signaling modulator of the physiological and pathological cell proliferation in the intestinal epithelium [41]#. Moreover, due to its direct action on epithelial stem cells, this pathway is also a main regulator of epithelial homeostasis [41]. Interestingly, the superfamily of the Nuclear Hormone Receptors (NHR), to which TRα1 belongs, displays several levels of functional and physical interactions with the WNT pathway [22], [23]. Although this interaction is relatively conserved among NHRs, there are however several differences. Some NHRs, such as VDR, PPARγ, and AR, are found in a complex with both β-catenin and Tcf/Lef, while others, such as ER and AR, can be found in a complex with Tcf/Lef in the absence of β-catenin [42]–[46]. The functional outcomes of these protein-protein interactions can also differ depending on the NHR. Generally speaking, β-catenin synergistically activates NHR activity, and NHR reciprocally deactivates or even represses β-catenin activity [22], [23]. This effect can be attributed to a large number of mechanisms involving E-cadherin expression, the competition between Tcf/Lef and NHRs for binding to β-catenin and/or p300 or the recruitment of co-repressors such as TLE, NCoR, and SMRT [22], [23]. However, these interactions are highly dependent on the cellular context and can create divergent effects. For example, PPARγ, whose expression and activity is negatively regulated by WNT in mesenchymal stem cells, undergoes positive regulation of the WNT signal in colon cancer cells [45]. A similar phenomenon is reported for VDR, which super-activates β-catenin activity in keratinocytes [42]#, but attenuates it in colorectal cancer [25], [28], [47]. In this context, the physical interaction between TRα1 and the transcriptional effectors of WNT was an attractive field of investigation. In fact, it had been previously shown that TRβ1 and β-catenin can interact directly to form a complex in thyrocytes [24]. This study, however, did not define the involvement of Tcf4 or the effect on TRβ1 activity. In this study, we have provided the first evidence that TRα1 interacts with β-catenin. Moreover, this interaction also involves Tcf4. There are two major differences between the interaction of TRα1/β-catenin/Tcf4 in the intestinal epithelium and that of TRβ1/β-catenin in thyrocytes. The first is the destabilization of the TRβ1/β-catenin complex by the hormone T3 [24], while we found no evidence for any T3-mediated effect on TRα1/β-catenin/Tcf4. The second is the action of the complex on the WNT pathway. In fact, TRβ1 induces the degradation of β-catenin [24], but TRα1 stabilizes it and activates WNT [9], [15]. It is worth noting that TRα1 and TRβ1 often play opposite roles [5], [48], and differences in the cell context may also explain these divergences.
Previously, we described several levels of complex relationships between WNT and the TRα1 pathways [10]#. First, we showed that TRα1 induces WNT activation and enhances cell proliferation [15], [16]. Second, we demonstrated a synergy between these two pathways during the process of intestinal tumorigenesis [9]. Finally, the third level of regulation outlined herein validates the positive action of TRα1 on WNT through a protein-protein interaction. In light of these new results, we propose that an intriguing feedback loop can also occur and that WNT over-activation likely decreases TRα1 transcriptional activity (proposed model of action in Figure 7). However, from our results, we cannot definitively conclude that this protein-protein interaction involves only the three partners we analyzed, as other factors that have not yet been identified may also participate in the complex.
10.1371/journal.pone.0034162.g007Figure 7 Proposed molecular model for the action of TRα1 in controlling the WNT signaling pathway in the intestine.
In physiological conditions (WT), TRα1 binding on TREs positively regulates the expression and stabilization of β-catenin, and then contributes to maintaining tissue homeostasis. In vil-TRα1 mice, there is an increased level of β-catenin expression and stabilization that leads to WNT activation and hyper-proliferation. In vil-TRα1/Apc mice, the stronger β-catenin stabilization and Tcf4 overexpression might be responsible of the shift in TRα1 binding from TREs to WREs. The physical interaction between TRα1 and β-catenin/Tcf4 we showed can explain its presence on the WREs. We speculate that this is a novel mechanism of WNT induction promoting the activation and/or the acceleration of the tumorigenic process. TREs, Thyroid hormone response elements; WREs, WNT response elements. Solid lines indicate genomic actions; dotted lines represent the speculative model of a negative regulatory loop involving non-genomic actions and eventually other(s) factor(s). Double black arrows indicate crypt width in WT and vil-TRα1 intestinal sections.
It has been shown that the functional interactions between signaling pathways involved in gut homeostasis can have a primary importance in the process of cell transformation. This is the case, for instance, of the synergy between WNT and Notch [49] or WNT and TRα1 [9]. In both examples, Notch and TRα1 induce increased cell proliferation, which is likely to determine an early mutagenic event in the Apc gene, and finally an acceleration of the tumorigenic process. We have shown that TRα1 interacts with the β-catenin/Tcf4 complex and speculate that this interaction might play a key role during the process of intestinal tumorigenesis by stabilizing the WNT effectors on their target genes. We could not observe, however, a clear-cut difference in the expression level of the WNT targets in the adenocarcinoma of different genotypes. One possibility is that once the tumoral program is completed, the molecular differences may be less apparent at the mRNA level. In addition, colon cancer is genetically heterogeneous and is composed of distinct subpopulations [50], [51]. Hence, interpretation of results from a comparative gene expression analysis in whole tumors can be hampered by such heterogeneity. In support of both assumptions, an upregulation of the WNT targets in the normal mucosa of the vil-TRα1/Apc mice is evident and might play a critical role during the early steps of gut tumorigenesis. With regard to the direct targets of TRα1, the overexpression of β-catenin/Tcf4 clearly inhibits their TRα1-dependent upregulation, but it does not affect the expression of the TRα1 indirect targets Ccnb1 and Cdc2a. It is probable that other transcription factors and compensatory mechanism(s) occurring in vivo may also be responsible for their expression levels.
Our results suggest that the binding of TRα1 to its DNA targets is shifted to the WNT targets when the β-catenin/Tcf4 protein levels increase (Figure 7). This condition is typical of intestinal cancers characterized by mutations in components of the WNT pathway (including Apc, Ctnnb1 and Axin2) 50,52 that allow for β-catenin increase and stabilization. With regard to Tcf4 expression, we observed its upregulation in the tumors of both Apc and vil-TRα1/Apc mice. Interestingly, we also observed an increased expression of Tcf4 in the normal mucosa of the vil-TRα1/Apc mice but not in the normal mucosa of the Apc animals. Taken together, our previous and present data strongly suggest that even if we refer to “vil-TRα1/Apc mice normal mucosa”, this may only be indicative of the morphological appearance. Indeed, from a molecular standpoint, it differs in several ways from the WT mucosa [9] (this paper) and clearly indicates an altered pre-cancerous status.
In conclusion, we have shown that in the context of intestinal lesions, TRα1 reshapes its function and induces specific genomic and non-genomic events. TRα1 transcriptional activity was negatively modulated by increased levels of β-catenin/Tcf4 and the binding to its target genes was also impaired. Interestingly, TRα1 was recruited to WNT target genes specifically within the lesions. This dual mechanism can have important repercussions and opens a new perspective in studying the sequential events of tumor development. An obvious speculation is that β-catenin/Tcf4 activity on their target genes could be reduced in the absence of TRα1. Our preliminary results from TRα0/0/Apc mice sustain this assumption (our unpublished observations); however, it is necessary to further confirm this result to account for the delay in or absence of tumor formation in these animals. A future challenge will be to define how complex cross-regulation and integrated networks, such as those involving the NHR and WNT pathways, can affect the cellular phenotype when signals are altered. These data will surely be of importance for translational research and could help explain the contradictory results concerning NHRs and carcinogenesis.
Materials and Methods
Animals and sample collection
We used vil-TRα1, vil-TRα1/Apc, Apc+/1638N and wild-type animals [9]. Mice were housed and maintained with approval from the animal experimental committee of the Ecole Normale Supérieure de Lyon (Lyon, France) and in accordance with European legislation on animal care and experimentation. Lyon University's “Comité d'Evaluation Commun au Centre Léon Bérard, à l'Animalerie de transit de l'ENS, au PBES et au laboratoire P4” (CECCAPP) and the “the Comité Régional d'Ethique pour l'Expérimentation Animale» (CREEA) approved all animal studies (agreement no. 0230). Animals were sacrificed, and the intestine (normal mucosae and tumors) was quickly removed. We recovered tumors and normal portions of the mucosa under a binocular microscope (Olympus) and froze them in liquid nitrogen for RNA extraction or the ChIP assay.
Intestinal epithelium primary cultures, immunolabelling and western blot
Intestinal epithelial primary cultures were derived from 4–6 day old neonatal mice according to Evans and colleagues [53]. Briefly, after sacrifice, the entire small intestine was removed. The epithelium was isolated as intact organoids by enzymatic dissociation using collagenase type XI (Sigma) and dispase (Boehringer Mannheim), followed by physical disaggregation and filtration on gauze. Organoids were plated in Dulbecco's modified Eagle medium (DMEM, Invitrogen) supplemented with 2.5% heat-inactivated fetal calf serum (Gibco), 20 ng/ml epidermal growth factor (Sigma), and insulin-transferrin-selenium diluted 1/100 (Sigma). Culture surfaces were coated with Matrigel™ Basement Membrane Matrix (BD Biosciences). For immunolabeling experiments, cover-slips were inserted in the wells before coating. For proliferation studies, 10 µM BrdU was added to the culture medium for an overnight incubation. The purity of the epithelial colonies was analyzed by immunolabeling for specific markers: epithelial cells: anti-cytokeratins (ICN); fibroblasts: anti-vimentin (Sigma); smooth muscle cells: anti-smooth muscle actin (Sigma). For treatment experiments, after 2 days of culture, the vehicle alone or 10−7 M T3 and/or 10 ng/µl of recombinant Wnt3a (R&D system) was added to the culture medium for 24 hours.
Immunolabeling was performed on 2% paraformaldehyde-fixed cultures, as previously described by Rezza and colleagues [32]. We used anti-Ki67 (Labvision MBL) and anti-β-catenin (Santa Cruz) primary antibodies.
Western blot (WB) was performed on whole-protein extracts obtained by homogenizing the samples in RIPA buffer, as described in [32]. Whole protein extracts (50 µg/lane) from cells maintained in the different experimental conditions were analyzed. We used anti-activated β-catenin (Upstate) and anti-actin (Sigma).
Cell lines and transfection experiments
This study was performed on the human Caco2 colorectal cancer cell line (from the American Type Culture Collection). Caco2 cells (50,000 cells/well in 24 multiwell plates) were cultured in DMEM supplemented with 10% heat- inactivated fetal calf serum. We used the following vectors: pGl2-DR4-Luc and pGl2-mutDR4-Luc (200 ng/well) and pGS5-TRα1 [15] (100 ng/well); TopFlash and FopFlash (Upstate; 200 ng/well); pCIneo-b-cateninXL (100 ng/well), Evr2-Tcf4E (100 ng/well; kind gift of Professor Waterman, UC Irvine, USA); pRL-CMV (1 ng/well; Promega). The vectors were transfected using the Exgen transfection reagent (Euromedex). For the T3 treatments, the cells were maintained in thyroid hormone-depleted serum [35]. T3 (10−6 M) or vehicle alone was added to the culture medium 24 hours before the end of the culture. Luciferase activity was measured 48 hours after transfection using the luciferase dual system (Promega).
Coimmunoprecipitation assay and western blot analysis
Coimmunoprecipitation (CoIP) studies were conducted on Caco2 cells using a slightly modified procedure described by Guigon and colleagues [24]. Nuclear and cytoplasmic extracts were pre-cleared with Protein A magnetic beads (Invitrogen) for 4 hours at 4°C. Immunoprecipitations of endogenous complexes were conducted overnight at 4°C with anti-Tcf4 (Santa Cruz), anti-β-catenin (Santa Cruz), anti-TRα1 [15] antibodies. As negative controls, the extracts were incubated with rabbit IgG (Promega). The complexes were washed and then recovered with Protein A magnetic beads. Immunoprecipitated proteins were subjected to SDS-PAGE and western blot analysis by using the different antibodies mentioned above. Whole proteins (50 µg/lane) on the blots were stained with Ponceau red. The nuclear extracts and loading in inputs were examined by using an anti-Histone H1 antibody (Santa Cruz).
RNA extraction and RTqPCR analysis
RNA was extracted from tissue samples using the Qiagen RNeasy Kit (Qiagen). To avoid the presence of contaminating DNA, DNase digestion was performed on all preparations. Reverse transcription was performed using MuMLV reverse transcriptase (Promega) on 1 µg of total RNA using random hexanucleotide priming (Promega) according to the manufacturer's instructions. For the primary cultures, RNA was extracted using the Absolutely RNA Nanoprep Kit (Stratagene). Reverse transcription was performed using the SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen) on 300 ng of total RNA. All of the cDNA samples were purified using the Qiagen PCR Purification Kit (Qiagen) before use for the qPCR experiments. The qPCR was performed with SYBR green PCR master mix (Qiagen) in an MxP3000 apparatus (Stratagene). The data from the qPCR were normalized to 36B4 levels for each sample. The primers are listed in Table S1A.
Electrophoretic mobility shift assay (EMSA)
Tcf4, β-catenin, TRα1 and RXRα full-length cDNAs were transcribed/translated in vitro using the quick TNT kit (Promega) according to the manufacturer's protocol. We performed the EMSA assay on the DR4-radiolabeled probe, as previously described [18], in a buffer similar to that of the CoIP: 3% glycerol, 20 mM HEPES, 3 mM DDT, 0.3% NP40, 100 mM KCl, protease inhibitor cocktail (Roche), 4 mM spermidine, and poly-dI-dC (1.5 µg). Where indicated, anti-TRα1 [15] and anti-Tcf4 (Santa Cruz) antibodies were included in the reaction mix.
Chromatin immunoprecipitation (ChIP)
The ChIP study was performed on 10 mg of samples collected from the intestine of vil-TRα1/Apc (healthy mucosa and tumors) and WT mice. The samples were minced with a razor blade and crosslinked with 1% formaldehyde at room temperature for 15 minutes. The crosslinking reaction was stopped by the addition of glycine to a final concentration of 0.125 M, and the samples were incubated at room temperature for 5 minutes. The samples were then centrifuged at 200 g to pellet the pieces. The fragments were washed once in cold PBS containing protease inhibitor cocktail (Roche) and then disaggregated by 10 strokes in a Dounce homogenizer. The resulting homogenate was centrifuged at 2000 g, the pellet recovered was then incubated in SDS buffer (1% SDS, 50 mM TRIS pH 8.1, 10 mM EDTA) on ice for 10 minutes. After 15 minutes of sonication (30 sec on/30 sec off cycles, Biodisruptor), the samples were centrifuged at maximum speed at 4°C, and the supernatant was recovered and quantified by Nanodrop (ND-1000 UV-Vis Spectrophotometer, NanoDrop Technologies). The same amount of each sample was diluted 1∶10 in dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl). After pre-clearing with Protein A magnetic beads (Invitrogen), each sample was divided into four samples, and the precipitation was performed using 9 µg of each antibody (anti-Tcf4, Santa Cruz; anti β-catenin, Santa Cruz; anti-TRα1 [15] or rabbit IgG). At the end of the reaction and washing steps, the complexes were recovered using protein A magnetic beads. The DNA was extracted after Proteinase K (Fermentas) and phenol-chloroform treatments. Specific DNA fragments were analyzed by qPCR using a SYBR green PCR master mix (Qiagen) in an MxP3000 apparatus (Stratagene). The primers designed to amplify the genomic regions containing: 1) TREs we previously described [15], [16]; 2) WREs described in literature [39], [40]; and 3) the villin and Rplp0 (36B4) promoters, as negative controls [15]. Primer sequences are listed in Table S1B; the Ppia gene was used in all reactions as internal control.
Supporting Information
Figure S1
RT-qPCR analysis in mice. (A, B) Analysis of TRα1-direct target genes in 1 month-old WT, vil-TRα1 and vil-TRα1/Apc mice. RT-qPCR analysis was performed to evaluate the mRNA levels of Ctnnb1 and Sfrp2. Values represent fold change ± sd after normalization to the wild-type (WT) animals. *: P<0.05, **: P<0.01 compared with the WT by two-tailed Student's t-test (n = 4). (C) Analysis of iodothyronine deiodinase selenoenzyme type 1 (Dio1) mRNA expression in WT, vil-TRα1, Apc and vil-TRα1/Apc mice. RT-qPCR experiments were performed in the intestine of 6-month-old mice. Values represent specific mRNA expression ± sd after normalization to 36B4 (n = 4). N, normal mucosa; T, tumor.
(PDF)
Click here for additional data file.
Figure S2
Physical interaction between TRα1 and the β-catenin in nuclei. Cellular lysates from Caco2 cells, maintained in T3-depleted (−) or T3-supplemented (+) serum, were fractionated into nuclear (N) and cytoplasmic (C) extracts. Lysates were immunoprecipitated with antibodies directed against endogenous β-catenin (upper panel) or TRα1 (lower panel), and the WB analysis of the indicated proteins was performed. Rabbit IgG was used as negative control. Ponceau red was used as whole-protein (50 µg/lane) loading control. Histone H1 was used to check the enrichment and was the loading control for the nuclear proteins in the inputs. The pictures are representative of at least three independent experiments.
(PDF)
Click here for additional data file.
Figure S3
Chromatin occupancy of TRα1, β-catenin and Tcf4 on genomic regions of target genes. ChIP analysis was performed with chromatin isolated from the intestine of WT or vil-TRα1/Apc mice (healthy mucosa and tumor) and immunoprecipitated with anti-TRα1, anti-β-catenin, anti-Tcf4 antibodies or rabbit IgG (negative control). qPCR was performed using specific primers covering the TRE of Sfrp2 (A) and Ctnnb1 (B), the WRE of Axin2 (C) and c-Myc (D) or the promoters of Villin (E) and 36B4 (F); the Ppia gene was used as internal control. Histograms represent the specific-DNA enrichment in each sample immunoprecipitated with the indicated antibody. The black bar in A–D delineates the threshold of binding specificity determined by the IgG non-specific binding. N, normal mucosa; T, tumor.
(PDF)
Click here for additional data file.
Figure S4
Electromobility Shift Assay on DR4 response element. (A) EMSA analysis was performed to examine the TRα1/β-catenin/Tcf4 complex formation on a DR4-radiolabeled probe. In vitrotranscribed/translated proteins were added to the mixture as indicated, and the DNA-protein complexes were separated by native gel electrophoresis. Specific antibodies were added to assess the protein[s] involved in the complex. In all of the conditions, only TRα1 alone binds to the labeled probe. (B) EMSA performed in the presence of the TRα1 transcriptional partner RXRα. It is worth noting that even in the presence of RXRα, there is no β-catenin/Tcf4 binding to TRα1 on a TRE.
(PDF)
Click here for additional data file.
Table S1 (A) Oligonucleotides used for the RT-qPCR study. (B) Oligonucleotides used for qPCR study after ChIP assay.
(PDF)
Click here for additional data file.
We gratefully acknowledge Nadine Aguilera for animal handling. We thank Professor Jacques Samarut for helpful discussion and scientific support at the early steps of this work. We are indebted to Professor Marian Waterman for the expression vectors.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by the Institut National pour le Cancer (grant INCA-2009-175) and the Ligue contre le Cancer Department du Rhone. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22509318PONE-D-11-2548410.1371/journal.pone.0034537Research ArticleBiologyBiotechnologyBiomaterialsBionanotechnologyEngineeringMechanical EngineeringNanoengineeringMaterials ScienceMaterial by AttributeNanomaterialsNanotechnologyBionanotechnologyNanomaterialsMedicineAnatomy and PhysiologyImmune PhysiologyCell Surface MoleculesOncologyBiotechnologyPhysiologyOncologyEngineering and TechnologyEquipmentOptical EquipmentLasersEngineering and TechnologyNanotechnologyNanoparticlesBiology and Life SciencesBiotechnologyBionanotechnologyEngineering and TechnologyNanotechnologyBionanotechnologyBiology and Life SciencesCell BiologyCell ProcessesEndocytosisBiology and Life SciencesCell BiologyCell ProcessesSecretory PathwayEndocytosisResearch and Analysis MethodsImaging TechniquesFluorescence ImagingPhysical SciencesPhysicsStates of MatterFluidsVaporsPhysical SciencesPhysicsClassical MechanicsPressureVapor PressureMedicine and Health SciencesOncologyCancer TreatmentImproved Cellular Specificity of Plasmonic Nanobubbles versus Nanoparticles in Heterogeneous Cell Systems Cellular Specificity of Plasmonic NanobubblesLukianova-Hleb Ekaterina Y.
1
Ren Xiaoyang
2
Constantinou Pamela E.
1
Danysh Brian P.
1
Shenefelt Derek L.
1
Carson Daniel D.
1
Farach-Carson Mary C.
1
Kulchitsky Vladimir A.
3
Wu Xiangwei
2
Wagner Daniel S.
1
Lapotko Dmitri O.
1
4
*
1
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas, United States of America
2
Department of Clinical Cancer Prevention, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America
3
Institute of Physiology, National Academy of Science of Belarus, Minsk, Belarus
4
Department of Physics and Astronomy, Rice University, Houston, Texas, United States of America
Roeder Ryan Keith EditorUniversity of Notre Dame, United States of America* E-mail: [email protected] the paper: EYLH DSW DOL. Conceived the mechanism of cell targeting as the nanoparticle cluster – plasmonic nanobubble threshold effect and designed the experimental set up: DOL. Designed cell models and discussed the results: DDC MCFC VAK XW DSW EYLH DOL. Performed the experiments (prepared and characterized the cells): EYLH XR PEC BPD DLS. Performed the experiments (characterized the nanoparticles and plasmonic nanobubbles): EYLH DOL.
Competing Interests: The authors have declared that no competing interests exist.
2012 3 4 2012 2 11 2017 7 4 e3453720 12 2011 1 3 2012 © 2012 Lukianova-Hleb et al2012Lukianova-Hleb et alThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.The limited specificity of nanoparticle (NP) uptake by target cells associated with a disease is one of the principal challenges of nanomedicine. Using the threshold mechanism of plasmonic nanobubble (PNB) generation and enhanced accumulation and clustering of gold nanoparticles in target cells, we increased the specificity of PNB generation and detection in target versus non-target cells by more than one order of magnitude compared to the specificity of NP uptake by the same cells. This improved cellular specificity of PNBs was demonstrated in six different cell models representing diverse molecular targets such as epidermal growth factor receptor, CD3 receptor, prostate specific membrane antigen and mucin molecule MUC1. Thus PNBs may be a universal method and nano-agent that overcome the problem of non-specific uptake of NPs by non-target cells and improve the specificity of NP-based diagnostics, therapeutics and theranostics at the cell level.
This work was supported by National Institutes of Health Grant R01GM094816 (DOL), P01 CA098912 (MCFC), MD Anderson Gynecologic Specialized Programs of Research Excellence (SPORE) in Uterine Cancer, Pilot Project Award 2P50CA098258-06 (DDC). Confocal microscopy was performed on equipment obtained through a Shared Instrumentation Grant from the National Institutes of Health (S10RR026399 01). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
Nanomedicine promises unique abilities to support diagnostic, therapeutic and theranostic functions at nanoscale, providing molecule- and cell-level resolution, specificity and selectivity. These functions are usually mediated through nanoparticles (NPs) that have to be delivered to specific molecular and cellular targets associated with a certain pathology or diagnosis. However, this strategic advantage of nanomedicine is compromised by the principal limitation in NP targeting. This is that no current method can deliver NPs only to target cells and molecules because some amount of NPs always accumulates non-specifically in non-target cells, thus reducing the specificity and selectivity of nanomedicine. The limited specificity of NP targeting, in turn, requires higher loads of NPs in order to achieve the desired diagnostic or therapeutic effect [1]–[8]. The high loads of NPs required for therapy delivery further induces non-specific accumulation and cause toxicity issues because most NPs are inorganic and of non-biological origin. Increased NP loads and low targeting specificity result in macro- rather than nano-resolution for NP medicines. Among NPs, those based on gold have been exploited most often. Gold NPs have low toxicity [9], [10] and have been used in a relatively wide spectrum of biomedical applications: optical [6], [11]–[13] and photoacoustic [14], [15] diagnostics, drug delivery [12], [16]–[19], the direct destruction of target cells through photothermal effects [1]–[7], [20]–[29], or in combination with chemotherapy [30]. In addition, gold NPs were applied in combination with other NPs such as drug carriers and diagnostic labels [31]–[37].
The specificity of NP targeting to specific (target) cells was improved by chemically attaching target-specific vectors to the gold NPs thus coupling NPs to specific target receptors at cellular membrane [12], [38]–[47]. This active targeting is more effective compared to passive targeting with “bare” non-functionalized NPs. However, many cellular receptors are widely expressed, albeit at very different levels, on target and non-target cells (bio-heterogeneity). As a result, a considerable number of actively targeted NPs will still get to non-target cells through various non-specific mechanisms [1]–[7]. Therefore, the targeting of NPs to cells so far cannot provide sufficient specificity, which slows the translation of nanomedicine to clinic.
The high biomedical specificity of NP-based effects could be achieved by activating them with a threshold mechanism that would efficiently discriminate between NPs in target and non-target cells. These effects are optical scattering, fluorescent and photoacoustic diagnostics, drug delivery and release, and photothermal therapeutics. Most of the current methods activate these NP effects in cells in a linear way without a threshold effect and thus such methods often cannot discriminate between target and non-target cells.
Recently we demonstrated a novel cell-level transient nano-phenomenon, the plasmonic nanobubble (PNB). This transient nano-event is triggered by the short pulsed optical heating of gold NPs and has a threshold of generation that is sensitive to multiple variables including clustering of NPs [48]–[51]. A PNB is a vapor nanobubble transiently induced around a superheated gold NP upon its activation with a short laser pulse whose energy is converted by the gold NP into heat through the mechanism of plasmon resonance [3]–[5], [12], [13], [49], [52], [53]. PNB generation threshold energy was found to depend upon NP structure, size and aggregation state and was found to be lowest for NP clusters, nanostructures with tightly aggregated NPs [48], [54], [55]. This unique physical property of PNBs allows their selective generation under low laser pulse fluence only around large clusters of NPs, while the same level of laser fluence was below PNB generation threshold for single NPs or their small clusters. As biomedical agents, PNBs demonstrated their potential for optical diagnostics [40], [56]–[58], delivery and on demand release of therapeutic and genetic cargo [59]–[62], elimination of target cells [38], [49], [63], [64], microsurgery [65], [66] and theranostics [40], [55], [63].
We hypothesized that combining the threshold nature of gold NP-generated PNBs with their biomedical properties could significantly improve the precision and specificity of gold NP-based biomedical effects (Figure 1). Despite extensive previous studies of PNBs and especially of gold NPs in cells, validation of this hypothesis requires direct comparison of the specificity of NPs and PNBs in target vs non-target cells, a study that has not occurred. To validate this hypothesis we compared the abilities of PNBs and gold NPs to discriminate between target and non-target cells under identical treatment conditions in six different in vitro models and molecular targets. We demonstrate an efficient and universal solution for overcoming the influence of non-specific accumulation of NPs in non-target cells and to achieve high cellular specificity of biomedical effects of NPs.
10.1371/journal.pone.0034537.g001Figure 1 PNBs and NPs in target (left panels) vs non-target (right panels) cells.
A: gold NP conjugates are collected at cellular membranes and are clustered during endocytosis resulting in the largest NP clusters in target cells. B: Excitation laser pulse (green) of low fluence induces PNBs only around the largest NP clusters (i.e. only in target cells) because the PNB generation threshold fluence for single NPs and small clusters (non-target cells) is higher than the fluence of the laser pulse. C: Optical scattering of the probe laser radiation (red) by PNBs provides its real-time imaging and monitoring in the individual cell (ID: image detector, RD: response detector).
Methods
1. Nanoparticles and their clusters
We have used three different types of gold nanoparticles (NP): commercially available 60 nm spheres (NSP) and fabricated 50 nm hollow gold nanoshells (NS) and 110 nm gold NS with silica core inside. Gold NSP were provided and conjugated with cell-specific antibodies by Bio Assay Work LLC (Ijamsville, MD). 50 nm hollow gold NSs were synthesized by galvanic replacement of gold on a silver core according to Zasadzinski et. al. [67] The advantages of this type of NP include low toxicity, reliable conjugation properties, relatively high photothermal efficiency and maximal plasmonic nanobubble (PNB) generation efficacy in the biologically safe near-infrared spectral region with reducing the PNB generation threshold laser fluence. The 110 nm gold NS with silica core inside were designed and fabricated as described in previously [4]. NSs structure and size were verified with TEM (JEOL 2010, Jeol Ltd., Tokyo, Japan). Optical density spectra of NPs were obtained in water with a spectrophotometer (the USB 650 Red Tide spectrometer, Ocean Optics, Inc, Dunedin, FL).
For the study of PNB generation around NP clusters in water, NS clusters were prepared by adding sodium chloride to a suspension of single NSs. Clusters were re-suspended in water to provide low surface density that provided exposure of only one NP cluster by a single laser pulse. Next, we added 5 µl polysterene microspheres of 7.6 µm diameter (Spherotech Inc., Lake Forest, IL) were added into 50 µl of the NS clusters suspension. These microspheres have been used as spacers between the two glasses (a standard microscope slide and coverslip of 18 mm diameter) to provide a specific height (7.6 µm) of the sample volume. Clusters were re-suspended in water so that their low surface density would exclude the exposure of closely located nanoparticles under laser irradiation. To minimize the effect of cluster size heterogeneity, we used only the clusters with close levels of pixel image amplitudes of their optical scattering images. Each cluster was positioned in the center of the excitation laser beam and was exposed to a single laser pulse.
2. Molecular targets and cell models
We studied the six cell models representing four molecular targets (Table 1). Each pair of cells represented target cells with high level of expression of specific molecular target and non-target cells with low level of the expression of such molecular target. Details on culturing cells and monitoring of the expression level of molecular targets can be found in Text S1 and Figure S1 and Figure S2. NP clusters were selectively formed in target cell though the two-stage mechanism (Figure 1a):
at the first stage we used target-specific antibodies to provide higher accumulation of gold NPs at the membranes of target cells compared to the NP accumulation at membranes of non-target cells. This stage did not provide desired specificity of the targeting but at the same time delivered much more NPs to target cells compared to non-target cells;
at the second stage we engaged receptor-mediated endocytosis so that target cells self-assembled the large clusters of gold NPs in their endosomal systems [38]–[46].
10.1371/journal.pone.0034537.t001Table 1 Cell models and conditions of their treatment with NP and laser pulses.
Incubation conditions Laser pulseparameters
Mole-cular target NP Vector Cells:target/non-target NP con-centratio, NP/ml Incu-bation time Dura-tion, ps Wave-length, nm Fluence, mJ/cm2
EGFR 50 nmNS Panitu-mumab HN31/NOM9 2.4*1010
24 h 70 820 30
EGFR 60 nmNSP Panitu-mumab HN31/NOM9 2.4*1010
24 h 70 532 60
EGFR 60 nmNSP C225 HN31/NOM9 2.4*1010
24 h 70 532 60
- 60 nmNSP none HN31/NOM9 2.4*1010
24 h 70 532 60
EGFR 110 nmNS C225 C42B/HS5 1.2*1011
30 min 500 787 38
MUC1 60 nmNSP 214D4 HES/HS5 2.4*1010
1 h 500 532 40
PSMA 60 nmNSP Anti-PSMA C42B/HS5 1.2*1011
30 min 500 532 60
CD3 60 nmNSP OKT3 CD3+ T-cells/CD3− BMC 1.2*1011
30 min 70 532 37
CD3 60 nmNSP OKT3 J32/JRT3-T3.5 1.2*1011
45 min 500 532 63
EGFR 60 nmNSP C225 A549/Fibro-blasts 6*1010
30 min 500 532 30
The targeting parameters such as the concentration of NPs and the incubation time were optimized to achieve maximal difference in NP uptake between the target and non-target cells (Table 1). Applied targeting method does not eliminate the non-specific uptake of NPs, however, it provides the formation of the largest NP clusters only in target cells for target-specific generation of PNBs. Table 1 resumes experimental conditions applied for each molecular target and cell model. Sphere- and shell-type NP conjugates did not induce any considerable cytotoxicity in either target or non-target cells within 48–72 h (see Supplementary Information for details). Both cell cultures were identically treated with NPs conjugated to target-specific antibodies.
3. Generation of plasmonic nanobubbles
PNBs were generated due to transient heating of gold NPs with single laser pulses to the temperatures well above the evaporation threshold for the liquid environment of NPs (Figure 1b). We employed single short laser pulses of 70 and 400 ps, 532 nm (for excitation of solid gold spheres) and 787 nm (for excitation of gold nanoshells) (PL-2250, Ekspla and and STH-01, Standa Ltd, Vilnius, Lithuania). A short laser pulse maximized the efficacy of NP heating by preventing three negative processes: thermal losses through thermal diffusion [68], NP photodamage [69] and attenuation of incident optical pulse by developing vapor bubble [70]. Such vapor nanobubble uses thermal energy generated by gold NPs through the mechanism of plasmon resonance [3]–[5], [12], [13], [49], [52], [53] and this thermal energy (1) determines maximal diameter and lifetime of PNB and (2) is determined by fluence of laser pulse [54], [55]. Fast adiabatic expansion of the PNB provides efficient thermal insulation of its environment from the internal heat [54], [71]. The described mechanism also explains the origin of the term “plasmonic nanobubbles”: such vapor bubbles get their energy through plasmon resonance of gold NPs and act at nanoscale as mechanical, optical and acoustic nano-agents. Optical generation and detection of the PNBs was performed with a photothermal laser microscope that we developed previously [40], [48]. The laser pulse fluence (10–90 mJ/cm2) was experimentally determined for each pair of the target and non-target cells to exceed the PNB generation threshold in target cells and to be below PNB generation threshold for non-target cells (see also Text S1 and Figure S3).
4. Optical detection of NPs and PNBs
To image and quantify the uptake of gold NPs by cells we imaged and measured optical scattering by gold NPs in individual cells. Amplitude of optical scattering signal correlates to the size of scattering nanoobject [54], [56] even if the latter is below optical diffraction limit and cannot be seen in a microscope. As a rule we used laser confocal microscopy (LSM 710, Carl Zeiss Microimaging Inc., Germany) to obtain the stack of several images per each cell (in case of A549 cells the NP scattering was imaged and quantified with regular inverted microscope). In each population (sample) 30–50 cells were analyzed and the population-average image pixel amplitudes were calculated for each cell sample. Excellent optical scattering properties of a PNB [54] were used for its imaging in water and cells (Figure 1c) with two probe laser beams, pulsed probe beam (576 nm, 70 ps, 0.1 mJ/cm2) and continuous probe laser (633 nm). This provided two independent signals and two optical metrics of PNB (Figure S3): optical scattering time-resolved image pixel amplitude and duration of optical scattering time response (measured independently and simultaneously with optical scattering image). First, time-resolved optical scattering was used for imaging of PNBs and analyzing of their brightness. The maximum pixel amplitude of PNB was used as PNB metric (see Text S1 detailed definitions). The second PNB metric was independently obtained with another, continuous, probe laser (Figure S3). The PNB-induced scattering of a part of the probe beam decreased its axial amplitude, resulting in a dip-shaped output signal of the photodetector that monitored the probe beam. Thus we registered the time response of the probe laser radiation to the transient scattering effect of the PNB. This mode provided the monitoring of PNB growth and collapse, and delivered the PNB lifetime that characterizes its maximal diameter [23], [24], [48], [54], [55]. All three described above metrics were obtained for individual cells and were averaged for each population of target and non-target cells. This provided maximal precision of NP and PNB analysis.
Results
1. The PNB creates a threshold response to the optical excitation of gold NPs
The physical mechanism of PNB specificity was studied by generating and analyzing single PNBs around individual gold NP clusters of variable size in water. NP clusters were prepared by aggregating gold NPs (hollow gold 50 nm nanoshells) in a high salt solution. We used single and clustered gold spheres (60 nm) and hollow shells (50–60 nm) (see Text S1 for details). Optical scattering imaging of NP clusters was used to characterize their size through the scattering image amplitude since optical scattering brightness correlates to the size of the scattering object [20], [72]–[74]. PNBs were detected around specific individual NP clusters with two simultaneous techniques, time-resolved optical scattering images and time response (see Text S1). Three PNB parameters, these being the probability of PNB generation, scattering image amplitudes and time response durations, were measured for individual PNBs as functions of the optical fluence of the excitation laser pulse and the brightness of the NP cluster. The excitation fluence that corresponded to the probability of PNB generation of 0.5 was defined as the PNB generation threshold. First we studied the dependence of the PNB threshold in a single pulse mode upon NP cluster size, measured through its scattering image amplitude (Figure 2a). We observed a significant reduction of the threshold fluence with the NP cluster size. Thus a low fluence was sufficient to generate PNBs around large NP clusters but was not sufficient to induce PNBs around single NPs or small NP clusters. This was demonstrated by exposing multiple NP clusters of various sizes to a single laser pulse of low fluence. We observed the selective generation of PNBs only around the largest NP clusters (Figure 2b) whose threshold was lower than the applied fluence. The PNB threshold for smaller clusters was above this fluence and, therefore, such small NP clusters did not return PNBs in response to optical excitation.
10.1371/journal.pone.0034537.g002Figure 2 Parameters of PNBs generated around gold NP clusters in water for gold nanoshells.
A: PNB generation threshold fluence of the excitation laser pulse as function of NP cluster size (measured through optical scattering amplitude of NP cluster image for individual clusters); B: PNB lifetime and scattering brightness as function of the NP cluster size (measured through optical scattering amplitude of NP cluster image) at specific fluence of the excitation pulse (778 nm, 22 mJ/cm2).
The dependence of the PNB threshold fluence upon cluster size can be explained through the mechanism of PNB generation around superheated NPs. Merged thermal fields of several tightly aggregated NPs form a common thermal field and vapor layer around the cluster. The initial vapor pressure in such a vapor layer is determined by the fluence of the laser pulse that is converted into heat by each NP in a cluster. Next, the external pressure of surface tension (that needs to be overcome to allow the expansion of the vapor) is inversely proportional to the radius of the vapor-liquid boundary [75]–[77] and, therefore, decreases with cluster size. We previously analyzed the mechanism of PNB generation around NP clusters versus single NPs [54]. In addition to the above thermal and hydrodynamic factors, NP clustering may enhance their optical absorbance [78], [79], thus additionally increasing the released thermal energy and the initial vapor pressure. All these factors cause the decrease of the PNB generation threshold fluence with cluster size. With the fluence of the excitation pulse below the threshold, the PNB does not emerge, and, therefore, creates no impact, unlike NPs (Figure 2b). Contrary to a gradual increase in optical scattering amplitude of NP clusters with their size, the PNB scattering signals responded to a threshold NP cluster size (Figure 2b). This resulted in the selective generation of PNBs only around the largest NP clusters, while no PNBs emerged around single NPs and small clusters under identical excitation conditions. This cluster-threshold mechanism of PNBs created a unique opportunity to improve the specificity of NP-based effects.
2. Cellular specificity of NPs and PNBs
Since the largest NP clusters can be selectively formed in target cells through the receptor-medicated endocytosis of NPs [38]–[46], we further studied the NP cluster-PNB mechanism in living cells in order to compare the cellular specificity of NPs and PNBs under identical conditions of NP targeting and optical excitation. Several different molecular targets were investigated in vitro in cell systems that included cells with a high level of molecular target expression (target cells) and a low level of the expression of the same molecular target (non-target cells).
We studied cell models representing lung (A549), head and neck (HN31), prostate (C4-2B), epithelial (HES, a WISH/HeLa derivative) and blood (Jurkat J32) cancers and also human T-cells that are used for gene therapies of cancer (see the detailed description of experimental models in Text S1). Both solid gold spheres (NSP) and gold nanoshells (NS) were conjugated to target-specific antibodies (Table 1, see also Text S1) and were administered under concentrations and incubation times that were experimentally optimized for each cell model for maximal uptake of NPs (see Text S1). After removing unbound NPs, the accumulation of gold NPs in individual cells was imaged and measured through gold NP-specific optical scattering (Figure 3a). Similar to previous experiments, we measured cell-averaged levels of scattering image amplitudes in target and non-target cells (Figure 4 row a). In all six cases we observed a higher level of NP signals in target cells, but all non-target cells also showed a significant level of NP uptake and formation of NP clusters (Figure 3a, 4a) so that the ratio of the NP signal for target versus non-target cells was below 10. However, higher pixel image amplitudes in target cells indicated the formation of the largest NP clusters in target cells.
10.1371/journal.pone.0034537.g003Figure 3 Images and signals of gold NPs and PNBs in co-culture of target (HN31, labeled with Green Fluorescent Protein for identification) and non-target (NOM9) cells identically treated with 60 nm gold NSP-C225 conjugates (specific to EGFR that is overexpressed in target cells).
A: overlay of bright field, fluorescent and scattering images shows target cells (green) and gold NPs (red) that can be found in both types of cells (the arrows show NP clusters in non-target cells); B: time-resolved scattering image of the same field shows PNB images (bright white spots) only in target cells; C,D: optical scattering time-responses of individual target (C) and non-target (D) cells show the PNB-specific signal only for target cell and the definition of the PNB lifetime of PNBs; time is measured from the moment of the exposure to the excitation laser pulse.
10.1371/journal.pone.0034537.g004Figure 4 Cell population-averaged levels of optical scattering signals obtained for individual target (solid bar) and non-target (hollow bar) cells in six cell models represented by target/non-target cells/molecular targets:
Squamous cell carcinoma, HN31/NOM9/EGFR (treated with 50 nm NS-Panitumumab conjugates); Lung cancer, A549/Fibroblast/EGFR (treated with 60 nm NSP-C225 conjugates); Epithelial cancer, HES/HS5/MUC1 (treated with 60 nm NSP-214D4 conjugates); Prostate cancer, C2-4B/HS5/PSMA (treated with 60 nm NSP-anti-PSMA conjugates); Leukemia, J32/JRT3-T3.5/CD3 and human T-cells, T-cell/BM/CD3 (treated with 60 nm NSP-OKT3 conjugates) for: Row A (red): gold NP amplitude of scattering image of gold NPs (a metric for the uptake of NPs by cells; Row B (purple): time-resolved scattering image amplitudes of PNBs; Row C (blue): PNB lifetimes. The ratio of the signals for target/non-target cell is shown for each parameter and cell model and indicates the cellular specificity of NPs (row A) and PNBs (rows B,C).
Next, target and non-target cells were identically treated with single laser pulses within the range of pulse fluences for PNB generation around NP clusters. For each cell model we experimentally determined the level of excitation pulse fluence that provided the generation of PNBs mainly in target cells and did not induce PNBs in non-target cells (Figure 3 b,c,d). The optical scattering images and time responses of individual cells were processed to compare the corresponding metrics for NP accumulation (Figure 4, row a) and PNB generation (Figure 4, row b,c) in target and non-target cells. Compared to NP signals, the PNB signals showed a much higher discrimination between target and non-target cells in all six. Cellular specificity of NPs and PNB was quantitatively shown through the ratios of the target cell signals to the corresponding signals in non-target cells (shown as colored numbers in each frame of Figure 4). Compared to NPs, the PNBs improved cellular specificity in some models by more than one order of magnitude. While the non-target cells showed significant uptake of NPs and even their potential aggregation into small clusters, no PNBs, or very small ones, were observed in non-target cells under identical treatment conditions (Figures 3 and 4).
The difference in cellular specificity of NPs and PNBs can be clearly seen in experiments with a co-culture of target (labeled with green fluorescent protein for identification) and non-target cells (Figure 3). At a specific fluence of the excitation laser pulse (25 mJ/cm2 at 778 nm) only target cells yielded PNBs while even adjacent non-target cells with gold NPs did not. Such a difference between NP and PNB signals was observed for all six cell models: adherent (HN31, HES, A549) and suspension (C4-2B, T-cells, Jurkat) cells, and for all molecular targets: receptors (EGFR, CD3, PSMA) and glycoproteins (MUC1). These results indicate the universal nature of the high cellular specificity of PNBs compared to that of gold NPs. Therefore, PNB provided better discrimination between target and non-target cells even when such cells were heterogeneously mixed.
3. Effects of the NP targeting vectors on the PNBs
The results reported above were obtained by using one type of antibody that was specific for the target cell in each cell model. In order to determine the role of the targeting vector in cellular specificity of PNBs, we completed three additional experiments in which the cellular specificity of NPs and PNBs was obtained for target (HN31) and non-target (cells) as a function of the targeting vector.
The first experiment compared NP and PNB signals for identical NPs with and without target-specific antibody (active vs passive targeting). We incubated cells identically with bare gold 60 nm spheres and with the conjugates of the same spheres to Panitumumab antibody that is specific against EGFR. The conditions of NP treatment were identical to those in Figure 4 and the laser treatment (532 nm, single pulse, 60 mJ/cm2) was identical for all cells. We observed no PNBs at all in target and non-target cells treated with “bare” NPs (Figure 5a) despite using an optical fluence that was above the PNB generation threshold even for the smallest clusters, but was below the PNB threshold for single NPs (Figure 2a). NP scattering amplitudes of both types of cells were comparable to those of the background scattering of intact cells. At the same time the cells treated with NP-Panitumumab conjugates (Figure 5a) returned a result similar to that observed earlier (Figure 4). PNB lifetime demonstrated a much higher contrast between target and non-target cells compared to scattering amplitudes measured for NPs. This experiment demonstrated that the important contribution of the targeting vector to NP uptake. The use of bare, non-conjugated NPs under the same targeting conditions (NP concentration and incubation time) was insufficient for achieving detectable NP effects.
10.1371/journal.pone.0034537.g005Figure 5 Influence of targeting vectors on NP scattering amplitude (red) and PNB lifetime (blue) in individual target (solid bars) and non-target (hollow bars) cells.
A: Target (HN31) and non-target (NOM9) cells identically treated with bare 60 nm gold NSPs and NSP-Panitumumab conjugates (antibody specific to EGFR that is overexpressed in HN31 cells); B: Effects of EGFR-specific antibodies C225 and Panitumumab as targeting vectors in HN31 cell model show 5 different combinations of the two antibodies; C: Effects of single and dual targeting antibodies against PSMA and EGFR (C225) in C4-2B cell model applied in combination with dual simultaneous optical excitation (so called rainbow PNB method) show synergistic enhancement of PNB lifetime in the rainbow mode.
The second experiment analyzed the effect of two anti-EGFR antibodies on NP and PNB signals for the same cell model of target (HN31) cells (Figure 5b). We applied two different antibodies, C225 and Panitumumab, that were separately conjugated to identical NPs (60 nm gold spheres). NPs were targeted in the following combinations that used identical concentrations of NPs and incubation times (Figure 5b). These combinations were (1) NP-C225, (2) NP-Panitumumab, (3) sequential targeting with Panitumumab alone and then with NP-C225, (4) sequential targeting with C225 alone and then NP-Panitumumab conjugates and (5) joint targeting of NP-C225 and NP-Panitumumab conjugates. NPs in cells were measured with NP-specific optical scattering amplitudes and the PNBs were characterized in the same cells with their lifetime measured under identical laser exposure to a single pulse (532 nm, 60 mJ/cm2) (Figure 5b). As can be seen from the data in Figure 5b, the PNB response to the variation of targeting conditions is more sensitive than that of NPs, whose uptake did not vary significantly. In particular, NSP-Panitumumab conjugate alone provided maximal PNB generation efficacy compared to all other combinations. Pre-treatment of the cells with free antibodies reduced the efficacy of PNB generation (lower lifetime) to possible blocking of EGFR with the administered free antibody during pre-treatment and, as a result, reduced uptake of NPs. It is interesting to note that the joint targeting of both NP conjugates also reduced the efficacy of PNB generation compared to Panitumumab alone (Figure 5b). This experiment demonstrated the superior sensitivity of PNBs compared to NPs to the targeting vector.
Finally, we compared the PNBs and NPs under the simultaneous combinatorial action of the two antibodies, each conjugated to a different NP type. This experiment employed the “rainbow” method of PNB generation which we recently developed [63], [80]. The method involves two different types of NPs being mixed in one cluster and simultaneously excited with two different laser pulses. We used the cell model of C4-2B cells described above. Gold nanospheres (NSP) of 60 nm, PNB excitation peak at 532 nm, and silica-gold nanoshells (NS) of 110 nm, PNB excitation peak at 787 nm, were conjugated to target-specific antibodies against PSMA [81] and non-specific antibody against EGFR (C225) as NSP-PSMA (spheres) and NS-C225 (shells), respectively. The target cells were incubated in three different combinations using identical nanoparticle concentration and incubation time. These combinations were (1) NSP-PSMA, (2) NS-C225 and (3) simultaneously with both conjugates. The uptake of all NPs was measured in individual cells through optical scattering for the three cases and did not show a significant difference (Figure 5c). PNBs were generated with two different simultaneous laser pulses of the same fluence of 38 mJ/cm2, each pulse wavelength matching the peak wavelengths of NSP and NS, respectively. In case 1, a single pulse was applied at 532 nm, in case 2 a single pulse was applied at 787 nm and in case 3 the two pulses, 532 nm and 787 nm, were simultaneously applied (Figure 5c). The laser fluence levels were adjusted to detect small PNBs in any of the three cases. Under such settings we detected similar small PNBs for cases 1 and 2 that employed one type of NP and one wavelength for optical excitation. However, in case 3, the PNB generated with the rainbow method showed a significant (about 10-fold) increase in the PNB lifetime compared to cases 1 and 2, which was much higher than the corresponding difference in NP scattering. The latter did not differ much for all three cases (Figure 5c). The PNB signal in case 3 apparently showed a synergistic enhancement of the PNB compared to cases 1 and 2, and even compared to the results presented above (Figure 4). Compared to Figure 4 (C4-2B cell, PNB lifetime) we increased the PNB lifetime with the rainbow mechanism by almost one order of magnitude, while at the same time reducing the laser fluence from 60 mJ/cm2 (Figure 4 for PSMA target) to 38 mJ/cm2. This was achieved through the simultaneous excitation of the two different plasmon resonances in co-localized NPs of two different types, NSP and NS. These were mixed in one cluster and simultaneously received two laser excitation pulses in the rainbow mode of PNB generation (details of the rainbow PNB method can be found in [63], [80]).
The three experiments described above demonstrate the role of NP targeting vectors in PNB generation. Depending on the vector employed, the PNBs varied from zero (for non-specific uptake of bare NPs) to the synergistic enhancement of the PNB in the rainbow mode.
Discussion
1. Mechanism of cellular specificity of PNBs
As can be seen from Figure 4, the PNB method can better discriminate between target and non-target cells compared to NPs. Cellular specificity of PNBs was more than one order of magnitude higher than that of NPs (this can be clearly seen by comparing the ratios of the corresponding signals for target and non-target cells). Such an effect was achieved through the cluster-threshold mechanism of PNBs that prevents the generation of PNBs around non-specifically targeted single NPs (and their small clusters). This is due to the dependence of the PNB threshold upon cluster size and the low optical fluence applied. Since a similar effect of PNB specificity was observed in the 6 different cell models and for the different molecular targets we conclude that the mechanism of such high specificity should be universal and can be applied to many other cell targets that express specific molecules. The universal nature of PNB specificity can be explained by (1) the threshold mechanism of PNB generation, (2) the dependence of the PNB generation threshold fluence upon the size of the NP cluster (Figure 2) and (3) the universal mechanism of NP clustering through receptor-mediated endocytosis [38], [39], [49]. The latter is responsible for the maximal size of the cluster that determines the minimal PNB threshold fluence (Figure 2a) and other parameters (Figure 2b) of a PNB generated in a cell targeted with NPs. Since NP clustering is a result of endocytotic internalization and concentrating of NPs into a cluster, this is a universal mechanism for any living cell. The size of NP cluster in a cell and its ability to generate the PNB depends upon several factors:
1.1. Targeting activity of NPs
Comparing NP and PNB signals in target (HN31) and non-target (NOM9) cells after active targeting with NP-Panitumumab conjugates (Figure 4) and passive targeting with “bare” identical NPs (Figure 5a) obtained under identical NP concentrations, incubation time and laser parameters, we concluded that the interaction of the targeting vector with specific cellular receptor is very important. The fact that passive targeting of both target and non-target cells returned no PNBs and the level of NP scattering was close to that of background means that the accumulation of bare non-conjugated NPs was very low and even endocytosis could not form a significant cluster due to the presumably low number of NPs accumulated at the cellular membrane. This assumption is fully in line with our previous data [39], [49] where we studied in detail the difference in cellular uptake of conjugated and bare gold NPs. These previous studies were performed only for one type of cell (only target cells were incubated without the comparison with non-target cells) and they directly showed a 10–100 fold reduction in the uptake of bare NPs [64] and a 20–80 fold increase in PNB generation threshold fluence compared to the same cells treated with conjugated NPs [38], [39]. Therefore, the combination of a sufficient number of specific receptors (typical for target cells) with NP vectors (active targeting) provides a maximal initial level of gold NPs accumulated at the cellular membrane.
1.2. Activity of endocytosis
Endocytosis works as a universal process that takes NPs from the cellular membrane and concentrates them into clusters in the endo-lysosomal system. Due to the well-known safety of the formation of clusters of gold NPs, this is a relatively safe process providing that such NPs do not carry any toxic molecules [12], [38]–[46]. Our previous studies showed a significant reduction in NP cluster size and a decrease in PNB signals in target cells in response to the suppression of endocytosis in cells that accumulated a sufficient amount of gold NPs at their membranes [49].
Therefore, the size of the NP cluster built by a cell depends upon the activity of the clustering process (endocytosis) and the amount of NPs available at the cellular membrane. This mechanism does not prevent the formation of NP clusters in non-target cells, as we observed in our experiments (Figure 4, row a), and we found that this is a general rule. However, we also observed that non-target cells could not build as large NP clusters as target cells (Figure 4, row a) and, therefore, the mechanism of formation of the largest NP clusters (as PNB sources) is target cell-specific. By adjusting the laser fluence to the level matching the largest NP clusters (as demonstrated in Figure 2a and b) we provided the generation of PNBs only around the largest NP clusters associated with target cells. At the same time this fluence was not sufficient to generate PNB in non-target cells regardless the formation of small NP clusters. This principle is directly demonstrated by Figure 2b and it explains the much higher cellular specificity of PNB compared to the receptor-mediated active targeting of NPs. In many cases we observed only one PNB per target cell. This assumes the formation of at least one large NP cluster that was earlier found to be sufficient to support diagnostic and therapeutic functions of PNB (see the next section).
Based upon our previous findings we may estimate the size of NP clusters in the range of 5–100 NPs or 100–600 nm (for NPs with the diameter 50–60 nm) [39], [64]. Therefore, we conclude that the PNB is a universal mechanism for discriminating between target and non-target cells that demonstrates much higher specificity compared to that of NP targeting and can be considered as a universal solution for overcoming unwanted effects of non-specific cellular uptake of NPs.
2. Feasibility of PNBs for medicine
The high cellular specificity of any nano-agent makes sense only providing that such an agent can support the required biomedical functions. While the study of the biomedical effects of PNBs is outside the scope of this work, we may point out several cell-level effects observed earlier. We recently demonstrated in vitro and in vivo how optical, acoustical and mechanical localized tunable impacts of PNBs support five biomedical functions that are determined by the maximal size of the PNB which, in turn, is precisely controlled through the fluence of the laser pulse. These functions are diagnostics [40], [56], [57], [82], delivery of intracellular and extracellular molecular cargo [59]–[62], mechanical destruction of target cells [38], [40], [49], [57], [61], [63], [64], microsurgery [64], [65] and theranostics (the method that unites diagnosis, treatment and guidance of the treatment in one connected procedure) [40], [57], [63], [80]. All these functions were activated on demand and realized with cell selectivity with a single laser pulse of specific fluence, wavelength and duration. Such a short activation mechanism allows the reduction of the duration of the biomedical procedures to nano- and micro-seconds. In addition, we observed that the generation of even a single PNB in the target cell was sufficient to achieve the desired biomedical effect. This required a single NP cluster with a maximum of 100 particles. Therefore, the PNB mechanism provides a significant reduction of the NP load by several orders of magnitude, compared to other diagnostic and therapeutic methods based on gold NPs [1]–[7], [11], [20]–[29], [83]–[90]. The clustering mechanism of NPs considered above is not limited to endocytosis and may also employ the capability of NPs to self-assemble in various structures under specific conditions including NP clustering at the cellular membrane due to the co-localization of the receptors, fusion of endosomes and other chemical and biological mechanisms [91]. The combinatorial use of NP targeting vectors (defined in many labs) with the rainbow mechanism of PNBs [63], [80] provides the potential for a further improvement in specificity of the PNB effect in the complex biological environment of the human body. In addition, the PNB mechanism can be generalized to other types of non-gold NPs by co-localizing gold NPs (as PNB sources) with other NPs (such as drug carriers, for example). Recently we demonstrated how the intracellular delivery of the commercially available anti-cancer drug, doxorubicin liposomes (Doxil), can be improved through the mixed administration and clustering of gold NPs with Doxil and the intracellular release of the drug with co-localized PNBs [62].
In summary, we demonstrated that the specificity of the optical activation of nanoparticles in target cells can be improved by more than one order of magnitude through the threshold mechanism of plasmonic nanobubbles (PNBs). Due to its threshold nature the PNB method effectively discriminates between target and non-target cells under the identical treatment of both with nanoparticles and optical radiation. By combining the threshold nature of PNBs and the enhanced accumulation and clustering of nanoparticles in target cells, we showed that PNBs, unlike nanoparticles, can be minimized or totally avoided in non-target cells despite the uncontrollable non-specific uptake of nanoparticles by such cells. The PNB method will be compatible with many existing nanomedicine technologies in development, and will significantly improve their precision and selectivity.
Supporting Information
Text S1 The detailed descriptions of cell models and the methods of plasmonic nanobubbles generation and detection.
(DOCX)
Click here for additional data file.
Figure S1 The expression level of EGFR in HN31 (cancer) and NOM9 (normal) cells as measured with Western blot method.
(TIF)
Click here for additional data file.
Figure S2 The expression level of CD3 receptor in human T-cells in the two cell samples of the peripheral blood mononuclear cells, target (CD3+) and non-target (CD3−) cells (the samples were stained with CD3-PE and analyzed on a Gallios Flow Cytometer from Beckman Coulter, Brea, CA).
(TIF)
Click here for additional data file.
Figure S3 Experimental scheme for plasmonic nanobubble (PNB) generation and detection: single gold NP clusters or individual cells in the sample chamber were mounted on the stage of inverted optical microscope; PNB generation was provided by the pulsed pump laser; a pulsed probe laser provided time-resolved optical scattering imaging of PNBs and a continuous probe laser provided the monitoring of the PNB size through its time-response.
(TIF)
Click here for additional data file.
Authors thank Professor Malcolm Brenner and Dr. Leslie Huye of Gene and Cell Therapy Center (Houston, TX) for their help with design of CD3 models and valuable scientific discussions, Professor Joe Zasadzinski of the University of Minnesota and Mr. Andrey Belyanin of Rice University for their help with synthesis of hollow gold nanoshells, Hannie and John Ford of BioAssayWorks, LLC for their help with conjugation of gold nanoparticles, Professor Leonid Metelitsa of Texas Children Hospital (Houston, TX) for design of Jurkat cell models, Professor Rebekah Drezek and Dr. V. Nammalvar of Rice University (Houston, TX) for their help in preparation silica-gold nanoshell. Antibodies to PSMA were kindly provided under a Material Transfer Agreement from the Memorial Sloan-Kettering Cancer Center. Ms. S. Parminter kindly copy-edited the manuscript.
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Emerg Infect Dis
Emerging Infect. Dis
EID
Emerging Infectious Diseases
1080-6040
1080-6059
Centers for Disease Control and Prevention
09-1583
10.3201/eid1604.091583
Letters to the Editor
Letter
Triple Reassortant Swine Influenza A (H3N2) Virus in Waterfowl
Triple Reassortant Swine Influenza A (H3N2) Virus in Waterfowl
Ramakrishnan Muthannan A.
Wang Ping
Abin Martha
Yang My
Goyal Sagar M.
Gramer Marie R.
Redig Patrick
Fuhrman Monte W.
Sreevatsan Srinand
University of Minnesota, St. Paul, Minnesota, USA (M.A. Ramakrishnan, P. Wang, M. Abin, M. Yang, S.M. Goyal, M.R. Gramer, P. Redig, S. Sreevatsan)
Sioux Nation Agriculture Center of Sioux Falls, Sioux Falls, South Dakota, USA (M.W. Fuhrman)
Address for correspondence: Srinand Sreevatsan, Department of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, 1365 Gortner Ave, 225 VTH, St Paul, MN 55108, USA; email: [email protected]
4 2010
16 4 728730
Keywords:
Influenza A virus
triple reassortant
H3N2
waterfowl
interspecies transmission
viruses
letter
==== Body
To the Editor: In 1998, a new lineage of triple reassortant influenza A (H3N2) virus (TR-H3N2) with genes from humans (hemmaglutinin [HA], neuraminidase [NA], and polymerase basic 1 [PB1]), swine (matrix [M], nonstructural [NS], and nucleoprotein [NP]), and birds (polymerase acidic [PA] and PB2) emerged in the U.S. swine population. Subsequently, similar viruses were isolated from turkeys (1,2), minks, and humans in the United States and Canada (3,4). In 2007, our national influenza surveillance resulted in isolation of 4 swine-like TR-H3N2 viruses from migratory waterfowl (3 from mallards [Anas platyrrhynchos] and 1 from a northern pintail [Anas acuta] of 266 birds sampled) in north-central South Dakota. We report on the characterization of these TR-H3N2 viruses and hypothesize about their potential for interspecies transmission.
Two of these isolates, A/mallard/South Dakota/Sg-00125/2007 (H3N2) and A/northern pintail/South Dakota/Sg-00126/2007 (H3N2), were recovered from the birds sampled in north-central South Dakota, 45°44′30′′N, 98°16′30′′W; 2 isolates, A/mallard/South Dakota/Sg-00127/2007(H3N2) and A/mallard/South Dakota/Sg-00128/2007(H3N2), were sampled at 45°46′30′′N, 98°15′30′′W. Viral RNA was extracted, reverse transcribed, and amplified; all segments were sequenced in entirety and submitted to GenBank under the identified virus names. Phylogenetic analysis showed significant nucleotide identities (99%–100%), differing only in 4 nucleotide positions: 1 each from PB1, PA, NP, and NS genes. Among 4 substitutions, 3 were nonsynonymous (PA, NP, and NS), and 1 (PB1) was synonymous. A1725G substitution in PB1 was identified in 2 isolates. C419T change was identified in 3 isolates (Sg-00125, Sg-00126, and Sg-00128), resulting in substitution of threonine by phenylalanine. Three isolates (Sg-00125, Sg-00126, and Sg-00127) carried an A at residue 149 of the NP gene (leading to S50N change) and 1 isolate (Sg-00128) had a G at that position (encoding serine). G809A change was present in the NP gene of 3 isolates (Sg-00125, Sg-00127, and Sg-00128). Genomes of the 4 isolates had high nucleotide and amino acid identities (>98%) with North American swine TR-H3N2 virus (A/swine/Iowa/533/99 [H3N2]). Phylogenetic analysis indicated that TR-H3N2 waterfowl and North American TR-H3N2 swine isolates belonged to a single cluster. The H3N2 subtypes from avian and swine isolates of our sequencing projects belonged to different clusters (Figure). Deduced amino acid sequences of all segments showed that these virus isolates shared common themes in virulence determinants to those previously reported for swine-like TR-H3N2 viruses (5).
Figure Phylogenetic analysis of hemagglutinin (HA) sequences from waterfowl strains isolated in this study (boldface), based on the HA gene sequences. The evolutionary associations were inferred in MEGA4.0 (www.megasoftware.net) by using the neighbor-joining algorithm with the Kimura 2-parameter gamma model and 1,000 bootstrap replications (shown on branch bifurcations). A) Evolutionary distances of waterfowl isolates from swine and avian HA (H3) sequences from the Minnesota Center of Excellence for Influenza Research and Surveillance (MCEIRS) sequencing project or Minnesota Veterinary Diagnostic Laboratory (D-Lab) database. B) Phylogeny of 230 strains, including Eurasian and North American lineages of influenza A (H3N2) viruses. Data suggest swine influenza virus (H3N2) ancestry in the waterfowl strains. GenBank accession numbers are shown. Scale bars indicate nucleotide substitutions per site.
Inasmuch as we identified a swine lineage virus in waterfowl, we first investigated laboratory contamination by using trace back and history of swine virus isolations during the time the surveillance samples were processed. No H3N2 subtype were isolated from swine sources in the Minnesota Veterinary Diagnostic Laboratory during this period. Furthermore, phylogenetic analysis of all HA segment sequences from isolates obtained in that 4-month period confirmed no contamination. We then investigated whether an ecologic niche existed for potential exposure of waterfowl to pigs. We identified a swine herd near the wildlife refuge area where the waterfowl sampling occurred. Pigs were housed outdoors, and the owner of this swine herd reported that geese and ducks inhabit the water ponds/stock dams/slough area to which the pigs had access. Contact with the local veterinarian and the South Dakota Veterinary Diagnostic Laboratory indicated no recent reports of influenza A (H3N2) episodes in the swine herd. In addition, this herd was not vaccinated for swine influenza.
The mode of transmission of swine-origin virus to waterfowl is not clear. In previously published cases, where swine influenza viruses have been identified in turkeys, the flocks were in close proximity to swine herds (2). Similarly, we identified a swine herd in north-central South Dakota where all 4 waterfowl were sampled. Respiratory secretions from the pigs possibly could have spread to birds through aerosols or droplets. It is also likely that swine and waterfowl shared common water sources, which contained feces from influenza-infected waterfowl or respiratory secretions from influenza-infected swine. This mode of influenza virus transmission from birds to pigs has been documented (6–9). Indeed, a waterborne source for transmission is most likely because influenza A virus can persist in water for several months depending on environmental factors such as pH, temperature, and salinity (10). Finally, because the swine herd in this area was housed outdoors in open pens, direct interaction with waterfowl was possible.
In late 2008, serum samples were collected from this swine herd. Hemagglutination inhibition test (1) showed that 10 of 19 samples reacted with all 4 waterfowl isolates; titers ranged from 10 to >640. Although low titers may have occurred because pigs were exposed to heterologous cross-reactive viruses, the high titers in most animals with positive serum samples suggest exposure to an influenza (H3N2) virus similar to that recovered from the waterfowl. Our data emphasize the need to investigate the possible role of waterfowl in the maintenance and transmission of influenza A viruses to humans and to lower mammalian species.
This work was funded in whole or in part with funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under contract no. HHSN266200700007C.
Suggested citation for this article: Ramakrishnan MA, Wang P, Abin M, Yang M, Goyal SM, Gramer MR, et al. Triple reassortant swine influenza A (H3N2) virus in waterfowl [letter]. Emerg Infect Dis [serial on the Internet]. 2010 Apr [date cited]. http://dx.doi.org/10.3201/eid1604.091583
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References
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2. Tang Y, Lee CW, Zhang Y, Senne DA, Dearth R, Byrum B, Isolation and characterization of H3N2 influenza A virus from turkeys. Avian Dis. 2005;49 :207–13 10.1637/7288-101304R 16094824
3. Olsen CW The emergence of novel swine influenza viruses in North America. Virus Res. 2002;85 :199–210 10.1016/S0168-1702(02)00027-8 12034486
4. Olsen CW, Karasin AI, Carman S, Li Y, Bastien N, Ojkic D, Triple reassortant H3N2 influenza A viruses, Canada, 2005. Emerg Infect Dis. 2006;12 :1132–5 16836834
5. Yassine HM, Al-Natour MQ, Lee CW, Saif YM Interspecies and intraspecies transmission of triple reassortant H3N2 influenza A viruses. Virol J. 2007;4 :129 10.1186/1743-422X-4-129 18045494
6. Karasin AI, Brown IH, Carman S, Olsen CW Isolation and characterization of H4N6 avian influenza viruses from pigs with pneumonia in Canada. J Virol. 2000;74 :9322–7 10.1128/JVI.74.19.9322-9327.2000 10982381
7. Karasin AI, West K, Carman S, Olsen CW Characterization of avian H3N3 and H1N1 influenza A viruses isolated from pigs in Canada. J Clin Microbiol. 2004;42 :4349–54 10.1128/JCM.42.9.4349-4354.2004 15365042
8. Ma W, Vincent AL, Gramer MR, Brockwell CB, Lager KM, Janke BH, Identification of H2N3 influenza A viruses from swine in the United States. Proc Natl Acad Sci U S A. 2007;104 :20949–54 10.1073/pnas.0710286104 18093945
9. Olsen CW, Karasin A, Erickson G Characterization of a swine-like reassortant H1N2 influenza virus isolated from a wild duck in the United States. Virus Res. 2003;93 :115–21 10.1016/S0168-1702(03)00073-X 12727349
10. Brown JD, Goekjian G, Poulson R, Valeika S, Stallknecht DE Avian influenza virus in water: infectivity is dependent on pH, salinity and temperature. Vet Microbiol. 2009;136 :20–6 10.1016/j.vetmic.2008.10.027 19081209 | 20350405 | PMC3321961 | CC BY | 2022-02-01 23:15:25 | yes | Emerg Infect Dis. 2010 Apr; 16(4):728-730 |
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22506026PONE-D-11-2201310.1371/journal.pone.0034549Research ArticleBiologyBiochemistryBioenergeticsEnergy-Producing OrganellesMolecular Cell BiologySignal TransductionSignaling CascadesApoptotic Signaling CascadeSignaling in Cellular ProcessesApoptotic SignalingCell DeathMedicineDermatologySkin NeoplasmsMalignant Skin NeoplasmsMelanomasMutual Regulation of Bcl-2 Proteins Independent of the BH3 Domain as Shown by the BH3-Lacking Protein Bcl-xAK
Regulation of Bcl-2 Proteins Independent of BH3Plötz Michael
1
Hossini Amir M.
1
Gillissen Bernhard
2
Daniel Peter T.
2
Stockfleth Eggert
1
Eberle Jürgen
1
*
1
Department of Dermatology and Allergy, Skin Cancer Center, University Medical Center Charité, Berlin, Germany
2
Department of Hematology, Oncology and Tumor Immunology, University Medical Center Charité, Berlin, Germany
Chandra Dhyan EditorRoswell Park Cancer Institute, United States of America* E-mail: [email protected] and designed the experiments: JE MP ES PD. Performed the experiments: MP AH. Analyzed the data: JE MP BG. Contributed reagents/materials/analysis tools: BG PD. Wrote the paper: JE MPE.
2012 10 4 2012 7 4 e345493 11 2011 2 3 2012 Plötz et al.2012This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.The BH3 domain of Bcl-2 proteins was regarded as indispensable for apoptosis induction and for mutual regulation of family members. We recently described Bcl-xAK, a proapoptotic splice product of the bcl-x gene, which lacks BH3 but encloses BH2, BH4 and a transmembrane domain. It remained however unclear, how Bcl-xAK may trigger apoptosis.
For efficient overexpression, Bcl-xAK was subcloned in an adenoviral vector under Tet-OFF control. The construct resulted in significant apoptosis induction in melanoma and nonmelanoma cell lines with up to 50% apoptotic cells as well as decreased cell proliferation and survival. Disruption of mitochondrial membrane potential, and cytochrome c release clearly indicated activation of the mitochondrial apoptosis pathways. Both Bax and Bak were activated as shown by clustering and conformation analysis. Mitochondrial translocation of Bcl-xAK appeared as an essential and initial step. Bcl-xAK was critically dependent on either Bax or Bak, and apoptosis was abrogated in Bax/Bak double knockout conditions as well by overexpression of Bcl-2 or Bcl-xL. A direct interaction with Bcl-2, Bax, Bad, Noxa or Puma was however not seen by immunoprecipitation. Thus besides BH3-mediated interactions, there exists an additional way for mutual regulation of Bcl-2 proteins, which is independent of the BH3. This pathway appears to play a supplementary role also for other proapoptotic family members, and its unraveling may help to overcome therapy resistance in cancer.
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Introduction
Apoptosis is a defined genetic death program that leads to ordered destruction of cellular components while membrane integrity is preserved [1]. It also represents a safeguard mechanism against tumor formation, due to the elimination of altered and mutated cells. Thus, apoptosis resistance is characteristic for tumor cells, and therapeutic strategies aim to overcome this resistance [2].
Two major apoptosis pathways (extrinsic and intrinsic) have been described in detail. Extrinsic pathways are initiated by binding of death ligands (TNF-α, CD95L and TRAIL) to cell surface receptors, leading to the formation of death-inducing signaling complexes, where initiator caspases 8 and 10 are activated [3], [4]. On the other hand, intrinsic/mitochondrial apoptosis pathways are triggered by intracellular signals such as by cellular or DNA damage. Key events are depolarization of the mitochondrial membrane potential (Δψm) and mitochondrial outer membrane permeabilisation (MOMP) resulting in cytochrome c release and subsequent activation of initiator caspase 9 [5]. Initiator caspases cleave and activate downstream effector caspases, which target a large number of death substrates to set apoptosis into work [6], [7].
Mitochondrial activation is critically controlled by the family of pro- and antiapoptotic Bcl-2 proteins [8]. These proteins share homology in four conserved regions termed Bcl-2 homology domains (BH) and in a transmembrane domain (TM). Antiapoptotic proteins as Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and Bfl-1/A1 enclose all four BH domains whereas proapoptotic Bcl-2 homologues subdivide in the Bax/Bak group characterized by BH 1–3, and the BH3-only group enclosing several proteins i.e. Bad, Bid, Bik/Nbk, Bim, Noxa and Puma. In present models, Bax and Bak drive MOMP and are neutralized by antiapoptotic family members. The BH3-only proteins contribute to the regulation either as sensitizers through inhibition of antiapoptotic Bcl-2 proteins or as direct activators of Bax and Bak [8], [9].
Mutual regulation and neutralization has been described as based on the formation of heterodimers between Bcl-2 family members. Thus, the BH3 domain of proapoptotic Bcl-2 proteins encloses an amphipathic α helix, which binds to a hydrophobic groove formed by BH1, BH2 and BH3 of antiapoptotic members [10]. In a rheostat model, the balance of pro- and antiapoptotic Bcl-2 proteins determines the fate of a cell [11]. In melanoma, apoptosis deficiency has been attributed to high expression of antiapoptotic Bcl-2 proteins [12], [13].
Alternative splicing further increases the number of the Bcl-2 family members. Thus, the bcl-x gene is expressed as a long antiapoptotic form (Bcl-xL) and a short proapoptotic form (Bcl-xS) [14]. We have recently described Bcl-xAK (atypical killer), a new proapoptotic splice product which encloses BH2, BH4 and TM. It completely lacks the BH3 domain, which has been regarded so far as indispensable for the proapoptotic function [15].
For unraveling the mechanism of Bcl-xAK-mediated apoptosis and exploring its possible therapeutic potential, we constructed an adenoviral vector, which mediates its efficient and conditional expression. We show that Bcl-xAK clearly activated the mitochondrial pathway, and its activity was critically controlled by both pro- and anti-apoptotic Bcl-2 proteins, despite the lack of BH3. Thus, a new model is suggested, in which Bcl-xAK acts as an atypical killer to trigger Bax/Bak-dependent apoptosis.
Materials and Methods
Cell culture and cell lines
Three representative human melanoma cell lines, SK-Mel-13 [16], Mel-2a and A-375 [17] were investigated. For analyzing the function of Bax and Bak, the prostate carcinoma cell line DU145 (DSMZ, Braunschweig, Germany) and the colon carcinoma cell line HCT116 (ATCC, Maryland, MD, USA) were used.
Parental DU145 cells are deficient for Bax and reveal only moderate expression of Bak. The cells had been reconstituted by EGFP-tagged Bax or Bak, resulting in DU145-EGFP-Bax and DU145-EGFP-Bak, as described previously [18]. HCT116 parental cells express both Bax and Bak. Isogenic sublines with either Bax knockout or Bak knockdown as well as Bax−/Bak− double knockdown cells had been kindly provided by B. Vogelstein (John Hopkins Cancer Center, Baltimore) [18]. Subclones of A-375 melanoma cells resulted from stable tansfection of a pIRES-Bcl-2 plasmid (A375-Bcl-2) or the pIRES empty plasmid (A375-Mock), as previously described [13]. The pIRES plasmid originated from Clontech (Palo Alto, California, USA).
Cell lines were cultured at 37°C, 5% CO2 in DMEM (Gibco, Karlsruhe, Germany) supplemented with 10% FCS and antibiotics (Biochrom, Berlin, Germany). For caspase inhibition, cells were preincubated for 1 h with 10 µM of the pancaspase inhibitior zVAD-fmk (R&D Systems, Wiesbaden, Germany), which binds the active sites of caspase-like proteases.
Construction of Bcl-xAK adenovirus
Bcl-xAK full-length cDNA [15] was subcloned into the Ad5 adenoviral vector pAd5-tTA, according to a strategy described previously [19]. In brief, the cDNA was inserted into the TRE-containing pHVAd2 shuttle vector. The resulting TRE-Bcl-xAK expression cassette was then inserted into pAd5-tTA by homologous recombination, thereby replacing the E1 region and creating pAdV-AK DNA (Fig. 1A). This was transfected into HEK293 cells, and adenoviral plaques corresponding to AdV-AK were propagated. Expression of Bcl-xAK after AdV-AK transduction was suppressed by addition of 1 µg/ml doxycycline to the culture medium (OFF condition), whereas omitting doxycycline resulted in promoter induction (ON condition). An adenoviral vector for expression of myc-tagged Bik/Nbk (Ad5-myc-Nkb-tTA=AdV-Nbk), used here as control, had been described previously [19]. A luciferase-encoding adenovirus (Ad5-CMV-Luc) served as mock control for adenovirus transduction and was applied at the same MOI [20].
10.1371/journal.pone.0034549.g001Figure 1 Efficient induction of cell death by Bcl-xAK.
(A) The structure of the adenoviral construct AdV-AK is shown. The adenoviral E1 region was replaced by the Bcl-xAK cDNA driven by a tetracyclin-responsive promoter (PTRE), and the E3 region was replaced by the tetracyclin-controlled transactivator (tTA) driven by a CMV promoter (PCMV). The tTA mediates Tet-OFF regulation. Striped boxes indicate the poly(A)+ regions. (B) Bcl-xAK expression as determined by Western blot analysis is shown in melanoma cell lines SK-Mel-13, A-375 and Mel-2a at 48 h after transduction with AdV-AK (MOI=50). Cells had received doxycycline (OFF condition) or were left without (ON condition). Equal protein loading was confirmed by β-actin. (C) Left, examples of cell cycle analysis after PI staining indicating sub-G1 apoptotic cell populations in Mel-2a at 48 h of transduction. Middle panel, detached and rounded cells indicating apoptosis are shown of Mel-2a at 48 h after transduction with AdV-AK under OFF and ON conditions. Right panel, chromatin condensation and nuclear fragmentation were visualized by bisbenzimide (DAPI) staining in Mel-2a at 48 h after AdV-AK transduction (MOI=50). D–F) Time course analyses of apoptosis (D, flow cytometry after PI staining), cytotoxicity (E, LDH release) and cell proliferation (F, WST-1 assay) are shown for SK-Mel-13, A-375 and Mel-2a cells at 24, 48 and 72 h after transduction with AdV-AK (50 MOI, +Dox=Off, −Dox=On). As positive controls for induced cytotoxicity, cell lines were completely lysed by triton X-100 (T=100%) or were treated with doxorubicin (D, 500 nM, 72 h). WST-1 values are expressed as percent of non-treated controls (=100%). (G) For comparison, apoptosis induction (sub-G1 cells) by AdV-Nbk is shown for Mel-2a cells at 24 h, 48 h and 72 h (MOI=50). AdV-Nbk shares the same backbone with AdV-AK. For induction, doxycycline was omitted (On). (H) A time course analysis of Bcl-xAK expression (3–48 h) after AdV-AK transduction and promoter induction is shown for Mel-2a, as determined by Western blot analysis. (I) Cell survival was determined according to calcein staining in Mel-2a cells at 48 h of Bcl-xAK induction. A shift to the left indicates calcein-negative (=non viable) cells. (J) Quantification of the calcein experiment. (D, E, F, G, J) Means and standard deviations of triplicate values of representative experiments are shown. A luciferase-encoding adenovirus (Ad5-CMV-Luc) applied at the same MOI served as mock control (M), for controlling adenovirus transduction. All experiments were performed at least twice, resulting in highly comparable results.
Apoptosis, cytotoxicity, cell proliferation and viability
For quantification of apoptosis, cell cycle analyses were carried out, and apoptotic cells corresponded to cell populations with hypodiploid nuclei [21]. Therefore, cells were seeded in 24-well plates (50,000 cells per well). After incubation, cells were harvested by trypsinisation, washed with ice-cold phosphate-buffered saline (PBS) and incubated for 1 h with the staining buffer, containing 0.1% sodium citrate, 0.1% triton X-100 and propidiumiodide (PI; 40 µg/ml; Sigma-Aldrich, Taufkirchen, Germany). The DNA content of nuclei was determined by using flow cytometry (FACSCalibur and CellQuest software; Becton Dickinson, Heidelberg, Germany). As a second assay for quantification of apoptosis, a cell death detection ELISA (Roche Diagnostics, Mannheim, Germany) was applied, which detects mono and oligonucleosomes formed in apoptotic cells. Cytotoxicity was determined in parallel by a cytotoxicity detection assay (Roche Diagnostics), which measures LDH activity in culture fluids. As positive controls for induced cytotoxicity, cells were completely lysed by triton X-100 or were treated with doxorubicin (500 nM, 72 h). Protocols for apoptosis ELISA and LDH release were according to the manufacturer with minor modifications [22].
Cell proliferation (as a product of cell number and mitochondrial activity) was quantified according to the cleavage of the water-soluble tetrazolium salt WST by mitochondrial dehydrogenases in viable cells (WST-1 assay, Roche Diagnostics). Cells were seeded in a density of 10,000 per 100 µl in 96-well plates, and treatments started after 24 h. At the time of analysis, WST-1 reagent was added and absorbance (450 nm) was determined in an ELISA reader. Data were reported in percent of non-treated controls. Cell viability at the single cell level was monitored by the life-cell labeling dye calcein-AM. Briefly, 105 cells were incubated with calcein (4 µM; eBioscience, Frankfurt, Germany) in serum-free growth medium (60 min, 37°C). After PBS washing, cell viability was determined by flow cytometry, comparing calcein-stained (viable) and unstained (dead) cells.
For identification of chromatin condensation and nuclear fragmentation in course of apoptosis, cells were harvested by trypsinisation, centrifuged on cytospins and fixed for 30 min in 4% formaldehyde. Cytospins were stained with bisbenzimide (Hoechst-33258; Sigma, Taufkirchen, Germany; 1 µg/ml, 30 min) and examined by fluorescence microscopy. Apoptotic cells were identified by fragmented nuclei or by bright blue-stained nuclei with condensed chromatin. For quantitative evaluation, fields with 100–200 cells were assessed in triplicates.
Cell transfection
Melanoma cells were seeded in six-well plates with 2×105 cells/well. For transient transfection, cells at a confluence of 50% were washed with serum-free Opti-MEM medium (Life Technologies, Carlsbad, CA, USA), followed by incubation at 37°C in Opti-MEM for 4 h with plasmid DNA (2.5 or 5 µg/ml) and 0.1% DMRIE-C (Life Technologies). Detailed protocols for transient cell transfection had been described previously [22]. Plasmid constructs of pcDNA3 (Invitrogen, Eugene, OR, USA) were used for transient transfection to express full length Bcl-xL and Bcl-xAK.
Mitochondrial membrane potential and ROS
For determination of the mitochondrial membrane potential (Δψm), the fluorescent dye JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolyl carbocyanine iodide) or the dye TMRM+ (Tetramethyl rhodamine methyl ester perchlorate) were used (both from Sigma-Aldrich). Cells were harvested by trypsinisation and stained for 15 min at 37°C with JC-1 (2.5 µM) or TMRM+ (1 µM), and changes of Δψm were determined by flow cytometry.
For measurement of intracellular ROS levels, the fluorescent dye H2DCFDA (2′, 7′- dichloro-dihydro-fluorescein-diacetate) was used. Cells were stained for 30 min with 15 µM H2DCFDA (Molecular Probes, Invitrogen), harvested by trypsinisation, resuspended in HBSS buffer (Biochrom, Berlin, Germany) and analyzed by flow cytometry. For ROS scavenging, N-acetyl cysteine (NAC, Sigma-Aldrich) was used in a concentration of 200 µM.
Assays for Bax/Bak activation
For determination of Bax and Bak clusters indicative for Bax/Bak activation, DU145 cells were used, which had been stably transfected for expression of EGFP-Bax or EGFP-Bak, respectively [18]. Cells were seeded, transduced with AdV-AK (MOI=50) and were cultured for 48 h with or without doxycycline. Bax and Bak clustering was demonstrated by a fluorescence microscope (Olympus BX50, Hamburg, Germany). For semi-quantitative evaluation, at least 500 cells of each condition were assessed.
For analysis of Bax/Bak conformational changes related to activation, primary antibodies specific for Bax/Bak N-terminal domains were applied in flow cytometry (Bax-NT, Upstate, Lake Placid, USA, #06-499; Bak-NT, Merck, Darmstadt, Germany, #AM04). Melanoma cells (105) were harvested by trypsinisation and fixed for 30 min with 4% paraformaldehyde in PBS. Cells were suspended in saponin buffer (1% FCS, 0.1% saponin in PBS) and incubated for 1 h at 4°C in the dark with antibodies Bax-NT (1∶100) or Bak-NT (1∶10). As secondary antibodies, goat anti-rabbit IgG (H+L)-FITC (Jackson Immuno Research, West Grove, USA) and goat anti-mouse IgG (H+L)-FITC (SouthernBiotech, Birmingham, AL, USA) were used. After washing and resuspension, cells were immediately measured by flow cytometry.
Western blot analysis
Detailed protocols for protein extraction and Western blot analysis had been described previously [22]. As a standard, 106 cells were harvested and dissolved in lysis buffer (150 mM NaCl, 1 mM EDTA, 0.5% SDS, 0.5% Nonidet P-40, 2 mM PMSF, 1 µM leupeptin, 1 µM pepstatin, 10 mM Tris-HCl, pH 7.5). For analysis of cytochrome c and mitochondrial localization of Bcl-2 proteins, cytosolic and mitochondrial cell fractions were separated by a mitochondria/cytosol fractionation kit (Alexis, Grünberg, Germany).
The following primary antibodies were used: procaspase-3 (Cell Signaling, Danvers, MA, USA; rabbit; 1∶1000), cleaved caspase-3 (Cell Signaling; rabbit; 1∶1000), caspase-8 (Cell Signaling; mouse; 1∶1000), caspase-9 (Cell Signaling; rabbit; 1∶1000), Bcl-xL (Santa Cruz, Heidelberg, Germany; mouse; 1∶200), mouse Bcl-2 (Santa Cruz; mouse; 1∶200), human Bcl-2 (Santa Cruz; mouse; 1∶200), Mcl-1 (Santa Cruz; rabbit; 1∶200), Bax (Santa Cruz; rabbit; 1∶200), Bak (Assay Biotechnology, Sunnyvale, CA, USA; rabbit; 1∶500), Bad (Cell Signaling; rabbit; 1∶1000), Puma (Epitomics, Burlingame, CA USA; rabbit; 1∶1000), Noxa (ProSci Incorporated, Poway, CA, USA; rabbit; 1∶500), cytochrome c (BD Biosciences, Heidelberg, Germany; mouse; 1∶1000), c-Myc (Calbiochem, Nottingham, UK; mouse; 1∶500), anti-porin 31 HL (VDAC; Calbiochem; mouse; 1∶5000), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Santa Cruz; mouse; 1∶1000), β-actin (Sigma-Aldrich; mouse; 1∶5000). As secondary antibodies, peroxidase-labeled goat anti-rabbit and goat anti-mouse antibodies were used (Dako, Hamburg, Germany; 1∶5000).
Immunoprecipitation with anti-Myc microbeads
Melanoma cells (106, SK-Mel-13) were transiently transfected with plasmids encoding myc-tagged Bcl-2 proteins (0.1% DMRIE-C, 5 µg/ml plasmid). After 24 h (for Bcl-xL and Bax) or 48 h (for Bcl-xAK), cells were harvested, washed with ice-cold PBS and resuspended in 1 ml of pre-cooled lysis buffer (150 mM NaCl, 1% triton X-100, 50 mM Tris-HCl, pH 8). Microbeads covered with monoclonal anti-myc antibodies were given to the lysate for magnetic labelling of the tagged proteins. Beads and bound proteins were captured on flow-through magnetic columns, washed 4× with buffer 1 (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8) and washed for another time with 20 mM Tris-HCl (pH 7.5). Proteins were eluted with hot (95°C) elution buffer (50 mM DTT, 1% SDS, 1 mM EDTA, 0.005% bromphenol blue, 10% glycerol, 50 mM Tris-HCl, pH 6.8). No secondary antibodies were needed. The mock control were melanoma cells transiently transfected with an empty pcDNA3 plasmid. The mock control proved that the anti-Myc beads do not result in any non-specific precipitates. Immunoprecipitation of myc-tagged proteins was carried out with the μMACS c-myc-tagged protein isolation kit (Miltenyi Biotec, Bergisch-Gladbach, Germany). Lysates and immunoprecipitates were investigated by Western blot analysis.
Results
Delayed but efficient apoptosis induction
For investigating the efficacy and mechanism of Bcl-xAK-mediated apoptosis, an adenoviral vector was constructed with the Bcl-xAK full length cDNA under control of a Tet-OFF promoter inserted into the adenoviral E1 region. The tetracycline/doxycycline repressible transactivator tTA was located in the adenoviral E3 region (Fig. 1A). The construct mediated high expression of Bcl-xAK in melanoma cell lines as shown for SK-Mel-13, A-375 and Mel-2a, when doxycycline was omitted (ON condition), whereas addition of doxycycline almost completely abolished Bcl-xAK expression (OFF condition, Fig. 1B).
Significant induction of apoptosis, as determined by counting hypodiploide sub-G1 cells, was seen in melanoma cell lines after transduction and promoter activation, whereas doxycyline strongly diminished apoptosis (Fig. 1D, examples shown in 1C left panel). Kinetic analyses revealed a delayed induction of apoptosis in the three cell lines, which increased to 12%–23% at 48 h and to 17%–37% at 72 h after transduction (Fig. 1D). In contrast, other proapoptotic Bcl-2 proteins induced apoptosis already at 24 h, as shown here for the BH3-only protein Bik/Nbk subcloned in the same adenoviral background (Fig. 1G). The delay in apoptosis induction by Bcl-xAK occurred despite its adenovirus-mediated high expression already at 6 h after transduction (Fig. 1H). Comparable results concerning increased DNA fragmentation were obtained by an apoptosis ELISA (data not shown).
In parallel with DNA fragmentation, clearly visible effects indicating apoptosis were evident, as reduced cell numbers, rounded and detached cells (Fig. 1C, middle panel). Chromatin condensation and nuclear fragmentation, typical hallmarks in apoptosis, were seen after bisbenzimide staining (Fig. 1C, right panel). At 48 h after transduction of Bcl-xAK, the cell numbers with atypical nuclei increased from 4% (Off) to 33% (On).
LDH release monitoring loss of plasma membrane integrity was determined to exclude early necrotic cell death. Indeed, LDH release was not significant at 48 h, when apoptosis was already induced, and it was less affected at 72 h, as compared to cytotoxicity controls (Fig. 1E). As determined by WST-1 assay, cell proliferation of Mel-2a cells was strongly decreased, reaching a loss of 60% at 72 h (Fig. 1F). Also cell viability, determined by calcein staining, was decreased (38% in Mel-2a at 72 h), as compared to 6% under Off conditions (Fig. 2I, J). Thus, Bcl-xAK triggered delayed but efficient induction of apoptosis in melanoma cells.
10.1371/journal.pone.0034549.g002Figure 2 Activation of caspases and mitochondria.
(A) Processing of caspase-3, -8 and -9 is shown in Mel-2a cells at 24 h and at 48 h after transduction with AdV-AK (MOI=50). Expression of Bcl-xAK was switched on in the absence of doxycycline (ON) or shut off with doxycycline (OFF). Equal protein loading (20 µg/lane) was confirmed by GAPDH. The whole experiment was performed twice. (B) Inhibition of apoptosis by preincubation with the pancaspase inhibitor zVAD-fmk (1 h, 10 µM) is shown. SK-Mel-13 cells had been transduced with AdV-AK (MOI=100, 48 h). Means and SDs of triplicate values of a representative experiment (one of two) are shown. (C) Decrease of the mitochondrial membrane potential (Δψm) is shown for Mel-2a cells at 48 h after transduction of AdV-AK, as determined by flow cytometry after JC-1 or TMRM+ staining. Cultures with doxycycline (OFF, grey) are compared to cultures grown in the absence of doxycycline (ON, open graphs). The experiment was performed three times, resulting in highly comparable results. (D) ROS levels were determined in Mel-2a cells at 24 h and 48 h after transduction with AdV-AK under ON and OFF conditions (flow cytometry after H2DCFDA staining). Below, parallel cultures were pre-treated for 1 h with 200 µM NAC before transduction. (E) Relative DNA-fragmentation rates (apoptosis) at 48 h with or without NAC were determined in parallel. Non-transduced cells (−/+NAC) are shown as additional controls (open bars). Values had been normalized with regard to non-treated controls, set to 1. Means and SDs of triplicate values of a representative experiment are shown (two independent experiments). (F) Expression levels of Bcl-2 proteins, of p53 and Survivin were determined by Western blot analysis in Mel-2a cells at 24 h and 48 h after transduction with AdV-AK (ON and OFF conditions). Equal protein loading (20 µg/lane) was confirmed by GAPDH.
Activation of caspases and mitochondria through adenovirus-encoded Bcl-xAK
Targeting of the caspase cascade was investigated in Mel-2a cells by Western blot analyses for the initiator caspases 8 and 9 as well as for the main effector caspase 3. Under conditions of high adenovirus-mediated expression of Bcl-xAK and strong apoptosis induction, also significant processing of these caspases was evident at 48 h of transduction (Fig. 2A). Underlining the role of caspases, Bcl-xAK-induced apoptosis was almost completely blocked by the pancaspase inhibitor zVAD-fmk (10 µM; Fig. 2B).
The effects on mitochondrial proapoptotic pathways were monitored by two distinct mitochondrial membrane potential (Δψm)-dependent dyes. Both JC-1 and TMRM+ revealed the same result, namely decrease of Δψm upon Bcl-xAK expression. Interestingly, loss of Δψm appeared already at 24 h after AdV-AK transduction, thus proving this as an early step in Bcl-xAK signal transduction, before apoptosis became evident (Fig. 2C). Reactive oxidative species (ROS) are regarded as an additional step in apoptosis regulation. Increased ROS levels were determined by flow cytometry after H2DCFDA staining and found in Mel-2a cells at 48 h but not at 24 h after transduction, thus characterizing this step likely as a consequence of apoptosis (Fig. 2D). Thus, increased ROS may further enhance the apoptotic effect, which was proven by pretreatment for 1 h with the antioxidant N-acetyl cysteine (NAC). Neutralization of ROS by NAC (Fig. 3D) resulted in a two-fold decrease of Bcl-xAK-induced apoptosis (Fig. 2E).
10.1371/journal.pone.0034549.g003Figure 3 Bcl-xAK-mediated apoptosis depends on Bax or Bak.
(A, C) Expression of Bax and Bak is shown by Western blot analysis in subclones of HCT116 and DU145, respectively. Equal loading was confirmed by incubation with β-actin. Two independent series of protein extracts revealed largely comparable expression. (B) HCT116 parental cells (Bax+/Bak+) as well as subclones (Bax−/Bak+), (Bax+/Bak−) and (Bax−/Bak−) were transduced with AdV-AK (MOI=50) and cultured under OFF or ON conditions. Relative DNA fragmentation values (apoptosis ELISA) were normalized according to the values of parental cells under OFF conditions (set to 1). (D) DU145 parental cells (Bax−/EGFP-Bak−) as well as subclones (Bax−/EGFP-Bak+) and (EGFP-Bax+/EGFP-Bak−) were transduced with AdV-AK (MOI=50, 100) and cultured under OFF or ON conditions. The percentages of apoptotic cells (sub-G1 populations) are shown, as determined by flow cytometry at 48 h after transduction. (B, D) Means and SDs of triplicate values of a representative experiment are shown (each two independent experiments). Statistical significance as determined by Student's t-test is indicated by asterisks (*, p<0.05; **, p<0.005), when comparing parental cells and subclones under ON conditions.
Despite the clear involvement of the mitochondrial pathway, levels of other Bcl-2 proteins remained rather stable after transduction with AdV-AK, as shown by Western blot analysis at 24 h and at 48 h for Bcl-2, Mcl-1, Bax, Puma and Noxa. Similarly, there were no significant changes of the levels of p53 or Survivin (Fig. 2F).
Dependency on Bax and Bak
To address the relation of Bcl-xAK-induced cell death to Bax and Bak, we used a HCT116-derived colon carcinoma cell model. This consisted of parental Bax+/Bak+ cells and sublines with either Bax knockout or Bak knockdown as well as Bax−/Bak− double knockdown cells (Fig. 3A). AdV-AK (50 MOI, 48 h) revealed strong apoptosis induction in parental cells, whereas both Bax and Bak single knockdown significantly diminished apoptosis, indicating that both proteins may be engaged by Bcl-xAK. In accordance, Bcl-xAK-induced apoptosis was completely abrogated in the double knockdown cells (Fig. 3B).
In a complementary approach, a DU145 prostate carcinoma cell model was applied. Parental cells are deficient for Bax and reveal only moderate activity of Bak. They had been reconstituted for either Bax or Bak expression by using EGFP-tagged copies (Fig. 3C). Parental DU145 cells were clearly non-responsive to AdV-AK, possibly indicating an endogeneous non-functional Bak. However, the reconstitution of either Bax or Bak strongly enhanced Bcl-xAK-mediated apoptosis, resulting in each case in more than 50% apoptotic cells. This again showed that Bcl-xAK can induce apoptosis via both Bax and Bak (Fig. 3D).
Formation of Bax/Bak clusters has been reported as related to proapoptotic function [23]. For monitoring this step, DU145 cells were used that had been stably transfected with EGFP-Bax and EGFP-Bak, respectively. In agreement with the function of both Bax and Bak, Bcl-xAK expression resulted in visible clustering of both EGFP-Bax and EGFP-Bak at 48 h after transduction. Clustering induced by Bcl-xAK was comparable to the effects of doxorubicin (2 µM, 24 h), used as positive control (Fig. 4A). Evaluations revealed Bax/Bak clusters in 20%–30% of cells, similar to apoptosis inductions at these conditions (Fig. 4B). In course of Bax/Bak activation, conformational changes may lead to exposure of their N-termini. Flow cytometry with N-terminus-specific antibodies (Bax-NT, Bak-NT) showed activation of Bax and Bak in 30% of Mel-2a cells in response to Bcl-xAK expression (Fig. 4C, 4D).
10.1371/journal.pone.0034549.g004Figure 4 Bax and Bak activation after Bcl-xAK overexpression.
(A) For investigation of Bax and Bak clustering, DU145 cells stably transfected for expression of EGFP-Bax or EGFP-Bak were transduced with AdV-AK and cultured for 48 h under OFF or ON conditions. Doxorubicin-treated cells (2 µM, 24 h) were used as positive controls. Examples of fluorescence microscope images taken at 48 h after transduction and promoter induction are shown. (B) A quantitative evaluation of Bax and Bak clustering was performed (means and SDs of triplicate values of a representative experiment). A second experiment revealed comparable results. (C) Bax and Bak activation upon Bcl-xAK expression was determined in Mel-2a at 48 after AdV-AK transduction (50 MOI), by flow cytometry after staining with conformation-specific antibodies against Bax and Bak N termini (Bax/Bak NT). The bars indicate the populations counted as positive for activated Bax and Bak, respectively. (D) A quantification of triplicate values (one experiment of two independent) is shown. Transduction with AdV-Nbk (50 MOI) is shown for comparison.
Abrogation of Bcl-xAK-mediated apoptosis by antiapoptotic Bcl-2 proteins
To address the role of antiapoptotic Bcl-2 proteins, A-375 melanoma cells stably transfected for Bcl-2 overexpression (A375-Bcl-2) were applied. These cells were completely protected against the proapoptotic effects of Bcl-xAK, whereas mock-transfected cells (A375-Mock) revealed about 30% apoptotic cells at 48 h of transduction with AdV-AK (Fig. 5A). A similar result was obtained after Bcl-xL overexpression. Transient transfection of a Bcl-xAK expression plasmid significantly enhanced apoptosis in SK-Mel-13 melanoma cells at 48 h, whereas the co-transfection of a Bcl-xL expression plasmid almost completely prevented Bcl-xAK-induced apoptosis (Fig. 5B). Thus, either one or these antiapoptotic proteins was sufficient to block Bcl-xAK-mediated apoptosis. Loss of Δψm was also seen in A375-Mock, which was completely prevented by Bcl-2 overexpression in A375-Bcl-2 (Fig. 5C).
10.1371/journal.pone.0034549.g005Figure 5 Bcl-2 and Bcl-xL block the proapoptotic effects of Bcl-xAK.
(A) Subclones of A-375 cells stably transfected with pIRES-Bcl-2 (A375-Bcl-2) or mock-transfected (A375-Mock) were transduced with AdV-AK under OFF or ON conditions. Non-transduced cells (−) were used as additional controls. Numbers of apoptotic cells (sub-G1 cell populations) were determined by flow cytometry after PI staining. (B) SK-Mel-13 melanoma cells were transiently transfected with either Bcl-xL or Bcl-xAK alone or with a combination of both (each 2.5 µg plasmid-DNA). Relative DNA fragmentation values, as determined at 24 h and 48 h after transfection, were calculated with respect to cells that had received only the transfection lipid (white bars). (A, B) Means and SDs of triplicate values of a representative experiment are shown (each two independent experiments). Overexpression of Bcl-2, Bcl-xL and Bcl-xAK, as determined by Western blot analyses, is shown in the insets. (C) The mitochondrial membrane potential (Δψm) was determined by flow cytometry after TMRM staining in A375-Mock and in A375-Bcl-2 at 24 h and 48 h. After transduction with AdV-AK, inducible and non inducible conditions were compared (On/Off). The experiment was performed three times, giving comparable results.
Mitochondrial translocation of Bcl-xAK is not prevented by Bcl-2
Hallmarks in mitochondrial apoptosis pathways are translocation of Bax and release of mitochondrial factors. Significant cytochrome c release was seen in Mel-2a and in A375-Mock at 48 h after AdV-AK transduction (Fig. 6A). Also higher levels of Bax were seen in mitochondrial extracts. In this assay however, Bax translocation and activation is underestimated as some cytosolic contaminations (up to 5%) were still left in mitochondrial fractions seen by the cytosolic marker GAPDH. This may explain the weaker bands of Bax already before induction of Bcl-xAK expression (Fig. 6B).
10.1371/journal.pone.0034549.g006Figure 6 Bcl-2 blocks Bcl-xAK-mediated cytochrome c release and Bax translocation.
Mel-2a, A375-Mock and A375-Bcl-2 cells were transduced with AdV-AK (MOI=50) and were kept under OFF and ON conditions. At 24 h and 48 h, cytosolic fractions (Cyto) and mitochondrial fractions (Mito) were isolated and analysed by Western blotting. Non-transfected controls (−) are shown as controls. The whole experiment was performed two times, resulting in highly comparable results. (A) Cytosolic extracts were analyzed for showing expression of Bcl-xAK and release of cytochrome c. Mitochondrial extracts serve as positive controls, the mitochondrial protein VDAC ruled out any contaminations of cytosolic extracts with mitochondria, and β-actin served as loading control. (B) Mitochondrial extracts were analyzed for showing mitochondrial translocation of Bcl-2 proteins. Here, cytosolic extracts served as controls, equal protein loading was confirmed by VDAC and the relative purity of mitochondrial extracts was examined by GAPDH. 5% of the total mitochondrial fractions and 2% of the total cytosolic fractions had been loaded on the gels.
The localization of Bcl-xAK itself appeared as an important step. When comparing 24 h with 48 h, the amount of Bcl-xAK in the cytosol significantly decreased at 48 h by 2–3-fold in all three cell lines. Equal loading of cytosolic extracts was proven by β-actin (Fig. 6A). The direct comparison of the mitochondrial extracts at 24 h and 48 h clearly showed almost no Bcl-xAK in Mel-2a and only weak bands in the two A-375 clones at 24 h. The mitochondrial localization of Bcl-xAK however strongly increased at 48 h (Fig. 6B). Simultaneous decrease of Bcl-xAK in the cytosol and its strong increase in mitochondria at 48 h clearly proved mitochondrial translocation of Bcl-xAK, which is suggestive as a critical step for induction of apoptosis. Importantly, the mitochondrial translocation of Bcl-xAK was not prevented by Bcl-2, whereas cytochrome c release and Bax translocation were completely blocked (Fig. 6A; B).
No interaction of Bcl-xAK with other Bcl-2 family members
For investigating whether Bcl-xAK might directly interact with other Bcl-2 proteins, SK-Mel-13 melanoma cells were transiently transfected with myc-tagged copies of Bcl-xAK, Bcl-xL or Bax. Following immunoprecipitation with anti-Myc microbeads, binding of Bcl-2, Bax, Bad, Noxa and Puma was investigated by Western blotting. Mock transfected cells were used as controls and ruled out non-specific precipitations by the microbeads. On the other hand, Myc-tagged proteins were efficiently immunoprecipitated, as seen in the pellet (P) fractions after incubation with the Myc antibody (Fig. 7A, panels 1–3).
10.1371/journal.pone.0034549.g007Figure 7 Co-immunoprecipitation analyses of Bcl-xAK with Bcl-2 family members.
(A) SK-Mel-13 melanoma cells were transiently transfected with each 5 µg of pcDNA3 plasmids encoding Bcl-xL, Bcl-xAK, Bax or empty vector (Mock). Cells lysates were immunoprecipitated with microbeads covered with anti-Myc antibody, and immunoprecipitates were analysed by Western blotting. Non-bound supernatants (S) were compared with the immunoprecipitated pellet fractions (P). Antibodies for immunodetection: anti- Myc, Bcl-2, Bax and Bad. The complete experiment was performed two times, which both gave the same result. (B) A model for apoptosis induction by Bcl-xAK is suggested. It is based on mitochondrial translocation of Bcl-xAK and activation of Bax/Bak. Bcl-2/Bcl-xL prevent Bax/Bak activation but not Bcl-xAK translocation. BH3-only proteins may mediate a BH3 domain-dependent pathway via inactivation of antiapoptotic Bcl-2 proteins and may also drive a BH3-independent pathway analogous to Bcl-xAK (see discussion part).
The binding analyses revealed characteristic interactions, thus proving the reliability of the assay. Thus binding of Bcl-2 to myc-Bax, binding of Bax to myc-Bcl-xL and myc-Bax as well as binding of Bad to myc-Bcl-xL were seen (Fig. 7A). Apoptosis, monitored in parallel, was induced by myc-Bax and myc-Bcl-xAK, whereas myc-Bcl-xL diminished basal apoptotic rates, thus providing a proof on the function of the transfected proteins (data not shown). However, no direct interactions of the five representatives of the Bcl-2 family were seen with Bcl-xAK (Fig. 7A), thus suggesting that Bcl-xAK displays its activation of Bax and Bak in an indirect way via a not yet defined step. In this pathway Bcl-xAK and antiapoptotic family members act independent of each other on Bax and Bak (Fig. 7B).
Discussion
Pro- and antiapoptotic Bcl-2 proteins are critically involved in apoptosis regulation by controlling mitochondrial cell death pathways [5]. Their already high number is further increased by differential splicing, leading to an enhanced complexity. Thus, up to 10 splice products have been reported for the bim gene, of which BimS, BimL and BimEL have been characterized. Also eight splice products with different domain structures have been reported for the bax gene, of which Bax-α is best characterized [24], [25]. Another example is given by the bcl-x gene, which is expressed in four reported isoforms with different activities. Besides Bcl-xL (long), antiapoptotic functions have also been reported for Bcl-xES (extra short) [26], [27]. In contrast, Bcl-xS (short) and Bcl-xAK (atypical killer) exert proapoptotic functions [14], [15]. Alternative splicing is a target of specific regulations. Thus, the switch from Bcl-xL to Bcl-xS in response to genotoxic stress was related to an ATM/CHK2/p53-dependent pathway [28]. The pathway, which triggers Bcl-xAK expression, is not yet defined.
Bcl-2 proteins are categorized in three subfamilies according to different domain structures, enclosing antiapoptotic proteins (BH 1–4), the Bax/Bak group (BH 1–3) and BH3-only proteins [9]. The bcl-x splice products, however, reveal unique structures. Thus, Bcl-xS encloses BH3 and BH4 [24], whereas Bcl-xAK encloses BH2 and BH4 [15]. Despite the BH3 domain has been regarded as indispensible for proapoptotic functions [12], we had previously categorized Bcl-xAK as proapoptotic based on a moderate induction of apoptosis in melanoma cells (two-fold), after plasmid transfection [15]. For unraveling Bcl-xAK-mediated pathways, we have constructed an adenoviral vector, which drives its high and conditional expression under Tet-OFF control. With this efficient expression system, Bcl-xAK induced apoptosis in up to 40% of melanoma and in 50% of non-melanoma cells. In its efficacy, Bcl-xAK was comparable to the BH3-only protein Bik/Nbk, which was available in the same adenoviral backbone [19].
Under AdV-AK-mediated high expression of Bcl-xAK, significant caspase activation became evident, in contrast to previous findings under moderate expression of Bcl-xAK
[15]. Thus, caspase activation by Bcl-xAK in melanoma cells appeared as dependent on its expression level. Initiator caspases of both extrinsic and intrinsic pathways (caspase-8, and −9) were cleaved. However, caspase-8 may also be activated downstream of caspase-3 in a described amplification loop [29], which is suggestive for Bcl-xAK.
Bcl-2 family proteins are particularly involved in the control of mitochondrial apoptosis pathways, which can be induced by overexpression of BH3-only proteins as well as by overexpression of Bax or Bak [18], [30], [31]. Also, Bcl-xAK resulted in significant decrease of mitochondrial membrane potential and in cytochrome c release, thus clearly indicating parallels to other proapoptotic Bcl-2 proteins. Although Bax/Bak-independent mechanisms were also discussed [32], mitochondrial activation is mainly related to Bax or Bak function [9]. Here again, Bcl-xAK revealed typical characteristics of proapoptotic Bcl-2 proteins, namely a strong dependency on either Bax or Bak. Both proteins share a similar structure and related functions [33]. Some proapoptotic Bcl-2 proteins show preference for activating either Bax or Bak, as Bik/Nbk and tBid go via Bax [9], [18] and Bcl-xS goes via Bak [34]. For Bcl-xAK, however, Bak expression could compensate for loss of Bax and vice versa, and apoptosis induction was abolished only in Bax/Bak double deficient cells. This suggests that Bcl-xAK may may drive more general changes at the mitochondrial membrane rather than selectively targeting a specific protein.
Importantly, after transduction all melanoma cells were responsive to Bcl-xAK, as the whole cell population showed reduced Δψm, increased ROS as well as activated Bax and Bak. However, certain thresholds may prevent full apoptosis induction in the majority of cells. This may be related to the activity of antiapoptotic Bcl-2 family members, which may block Bax and Bak. Thus, overexpression of Bcl-2 abrogated apoptosis induced in melanoma cells by Bik/Nbk [35], [36], and Bcl-xL inhibited Bax-induced apoptosis in mouse embryonic fibroblasts [37]. These antiapoptotic activities had been described as dependent on BH3-mediated heterodimerization. However, also the proapoptotic effects of Bcl-xAK were completely inhibited by Bcl-2 or Bcl-xL. This may depend on the inhibition of Bax and Bak by the antiapoptotic proteins, rather than on direct inhibition of Bcl-xAK. In agreement, Bcl-2 could not prevent Bcl-xAK mitochondrial translocation.
Highly characteristic for Bcl-xAK-induced apoptosis was a time delay of 48 h, whereas other Bcl-2 proteins as Bik/Nbk and Bcl-xS induced apoptosis in melanoma cells already at 24 h [35], [36]. In general, proapoptotic signaling as mutual regulation of Bcl-2 proteins, cytochrome c release and caspase activation are rather quick cellular events [38]. The time delay of Bcl-xAK in contrast to other proapoptotic Bcl-2 proteins is indicative for an indirect mechanism enclosing a time-consuming step. No relation was seen to the expression of other Bcl-2 proteins. Rather, Bcl-xAK mitochondrial localization appeared as a critical step, and membrane transport may play a regulatory role therein. Whereas Bcl-xAK was cytosolic at 24 h, it translocated to mitochondria at 48 h, when apoptosis was induced. Also other proapoptotic Bcl-2 proteins have to translocate to mitochondria to exert their proapoptotic activities, as shown for tBid and Bax [5], [39]. Thus, apoptosis by Bcl-xAK appeared as tightly linked to its presence in mitochondria, where it resulted in Bax and Bak activation.
An interesting finding was that loss of Δψm preceded translocation of Bcl-xAK and MOMP. The relation between Δψm and MOMP is still a matter of discussion; one effect may precede the other or they may even occur independently of each other [40], [41]. Loss of Δψm may result from uncoupling of the mitochondrial electron transport chain which may lead to Bax and Bak oligomerization [42]. Mitochondrial dynamics appears as another important level, which may be influenced by Bcl-xAK overexpression. Mitochondrial dynamics may contribute to the control of MOMP, which is further dependent on Bax [43]. Formation of large Bax/Bak clusters has been suggested, which may translocate to mitochondrial constriction sites, to drive MOMP [23]. Clustering of Bax and Bak was clearly induced in response to Bcl-xAK, thus further relations to mitochondrial fission and fusion may be expected.
For BH3-only proteins, different mechanisms have been suggested to explain their proapoptotic activities. In the neutralization/displacement model, BH3-only proteins bind antiapoptotic family members, to release Bax or Bak [5]. This activity is based on BH3, which binds to the hydrophobic groove of antiapoptotic Bcl-2 proteins [44]. According to a second model, BH3-only proteins may also directly bind and activate Bax or Bak, which has been shown for tBid, Bim and Puma [38], [45], [46]. This activity is also regarded as BH3-dependent. Thus, direct, although week binding of Bim to Bax has been shown, which was abrogated by the replacement of the Bim BH3 [30]. Also peptides of the BH3 domains of Bid, Bim and Puma were able to drive direct activation of Bax [45]. Both ways of apoptosis induction can not apply to Bcl-xAK, due to its lack of BH3.
A third way of apoptosis induction has been recently suggested. It is explained by a general remodelling of the mitochondrial outer membrane, and it was also seen after intercalation of BH3-only proteins, which resulted in Bax activation [47]. Of note, this proapoptotic activity appeared as independent of the BH3 domain. Thus for the BH3-only protein Bnip3, the transmembrane domain (TM) has been proven as essential for its proapoptotic activity, whereas BH3 could be mutated without major effect on apoptosis induction [48]. Also for BimS, deletion or point mutation of its BH3 on one hand prevented the interaction with Bcl-2 and Bax but remained largely without effect on apoptosis induction. BimS mutants still localized to mitochondria, suggesting that this was the critical step, and indeed, when the TM was deleted, the proapoptotic activity was lost [49]. Also for Bcl-xAK, mitochondrial translocation appeared as the critical step. A deletion analysis for Bcl-xAK may become particularly helpful for identification of proapoptotic domain(s) independent of BH3, as overlapping functions with BH3 are here excluded.
Thus, the characterization of Bcl-xAK strongly supports speculations on proapoptotic pathways that are mediated by Bcl-2 proteins but act independent of the BH3 domain. These pathways are nevertheless critically dependent on Bax and Bak as well as on antiapoptotic Bcl-2 family members. As shown here for melanoma, colon and prostate carcinoma cells, activation of these pathways can be effective in cancer cells. Bcl-2 proteins are of critical importance for therapy resistance in cancer, as particularly seen in melanoma [2]. Thus, new pathways for regulating Bcl-2 protein activity are of particular interest and may become useful for targeting so far therapy-refractory tumors, such as melanoma.
Competing Interests: The authors have declared that no competing interests exist.
Funding: The study was supported by the Sonnenfeld-Stiftung, Berlin. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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BMC Cell BiolBMC Cell BiolBMC Cell Biology1471-2121BioMed Central 1471-2121-13-82244346810.1186/1471-2121-13-8Research ArticleAurora kinase-C-T191D is constitutively active mutant Khan Jabbar [email protected] Sanaullah [email protected] Sobia [email protected] Ijaz [email protected] Shahid Niaz [email protected] Department of Biological Sciences, Gomal University Dera Ismail Khan, Dera Ismail Khan, Pakistan2 Institute of Genetics and Development, University of Rennes1, Rennes1, France3 Department of Zoology, Kohat University of Science and Technology, Kohat, Pakistan4 Department of Zoology, Islamia College Peshawar (A Public Sector University), University Campus, Jamrod Road, Peshawar 25120 Khyber Pakhtunkhwa, Pakistan5 Institute of Biotechnology and Genetic Engineering, Khyber Pakhtunkhwa University of agriculture Peshawar, Khyber Pakhtunkhwa, Pakistan2012 26 3 2012 13 8 8 29 12 2011 26 3 2012 Copyright ©2012 Khan et al; licensee BioMed Central Ltd.2012Khan et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background
Aurora kinases (Aurora-A, B and C) belong to a family of conserved serine/threonine kinases which are key regulators of cell cycle progression. Aurora-A and Aurora-B are expressed in somatic cells and involved in cell cycle regulation while aurora-C is meiotic chromosome passenger protein. As Aurora kinase C is rarely expressed in normal somatic cells and has been found over expressed in many cancer lines. It is suggested that Aurora-C-T191D is not hyperactive mutant.
Result
Aurora-C-T191D variant form was investigated and compared with wild type. The overexpression of Aurora-C-T191D was observed that it behaves like Aurora-C wild type (aurC-WT). Both Aurora-C-T191D and aurC-WT induce abnormal cell division resulting in centrosome amplification and multinucleation in transiently transfected cells as well as in stable cell lines. Similarly, Aurora-C-T191D and aurC-WT formed foci of colonies when grown on soft agar, indicating that a gain of Aurora-C activity is sufficient to transform cells. Furthermore, we reported that NIH-3 T3 stable cell lines overexpressing Aurora-C-T191D and its wild type partner induced tumour formation when injected into nude mice, demonstrating the oncogenic activity of enzymatically active Aurora kinase C. Interestingly enough tumour aggressiveness was positively correlated with the rate of kinase activity, making Aurora-C a potential anti-cancer therapeutic target.
Conclusion
These findings proved that Aurora C-T191D is not hyperactive but is constitutively active mutant.
Aurora-COncogeneCentrosomeMultinucleationTumour
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Background
Aurora kinases are a conserved family of serine/threonine kinases that are pivotal to the successful execution of cell division. Three Aurora kinases (Aurora-A, -B, and -C), which share sequence homology in their central catalytic kinase domains, have been identified in mammals [1]. All the three mammalian Aurora kinases are implicated as mitotic regulators and due to their elevated expression profiles detected in many human cancers, have generated significant interest in the cancer research field.
Aurora-C is predominantly expressed in the testis [2,3] and is mainly restricted to meiotically dividing spermatocytes [4] and mouse oocytes [5]. Aurora-C is also associated with inner centromere protein (INCENP) in male spermatocytes. Moreover, it is reported that overexpressed Aurora-C kinase behaves like a dominant negative kinase for Aurora-B leading to a cytokinesis defect [6]. Aurora-C disrupts the chromosome passenger protein complexes necessary for cytokinesis. Aurora-C can fulfil the role of Aurora-B in centromere assembly, kinetochore- microtubule attachment, the spindle assembly checkpoint and cytokinesis and, thus, possibly, Aurora-C regulates mitosis by the same mechanisms as Aurora-B in those somatic tissues in which it is overexpressed. Additional potential roles for Aurora-C in somatic tissues could include cooperative or modulating functions in mitosis, or non-mitotic functions such as gene regulation via phosphorylation of histone H3 [7]. Overexpression of Aurora-C in cancerous tissues and cell lines also raises questions about its potential role in carcinogenesis and its effect on the proliferative capacity of tumour cells [8,9]. The expression levels of Aurora-C, Aurora-B and Aurora-B splice variants are commonly altered in tumour cell lines and tissues [10-13]. These alterations in expression have been associated with tumourigenesis, tumour metastasis and tumour aggression. Aurora kinase inhibition by small molecules has been intensively studied recently as a possible cancer therapy [10,14-18].
It is reported that Aurora-C-T191D is hyperactive mutant and its relative activity is sevenfold higher than the activity of Aurora-C-WT [19]. But we report that Aurora-C-T191D is not hyperactive but is constitutively active and behaves exactly like its partner Aurora C-WT.
Methods
Construction of vectors
Human aurora-C cDNA was obtained from pET21b-aurora-C [6] by BglII/EcoRI digestion and inserted into pEGFP-C3 plasmid (Clonetech USA). Green fluorescence protein (GFP) -aurC-WT DNA was used as a template to obtain K72R, expressing kinase dead GFP-tagged aurC and GFP-aurC-T191D, expressing the constitutively active GFP-tagged aurC by double PCR site directed mutagenesis (Quick change site-directed mutagenesis kit, Stratagene USA), following manufacturer's instructions. The GFP-alone empty vector pEGFP-C3 was used as a control.
Cell line and transfection
Mouse NIH-3 T3 cells were used in all experiments. Cells were grown in Dulbecco's Modified-Eagle Medium (DMEM) (Gibco USA) containing 10% Fetal Bovine Serum- (PAA France) and 1% Penstrep (GIBCO- 10000 units/ml penicillin + 10000 units/ml streptomycin). Cells were transfected in Lipofectamine™ 2000 transfection reagents (Invitrogen USA) with GFP-aurC-WT, GFP-aurC-CA, GFP-aurC-KD and GFP-alone plasmid DNA, following manufacturer's instructions. For establishment of stable cell line, 800 μg/ml Geneticin G-418 (PAA France) was added in culture media, changing the media twice a week. Clonal selection was performed after 14 days, keeping the cells under continuous pressure of Geneticin G-418.
Kinase assay
Equal number of stable Cells of GFP-aurC-WT, GFP-aurC-CA, GFP-aurC-KD and GFP-alone were lysed in L-buffer (1% NP40, 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 20 mM Tris-HCl pH 7.5, 1 mM AEBSF, 0.2% Okadaic Acid, Protease inhibitor cocktail (Roche Germany) and 1 mM Na3VO4). Cell lysates were sonicated and incubated on ice for twenty minutes. The lysates were centrifuged at 13000 rpm for 15 minutes and supernatants were pre-cleared with protein G-sepharose beads (GE Healthcare USA) for twenty minutes at 4°C. The pre-cleared lysates were incubated with 5 μg of Anti-GFP antibody (Roche) and protein G-sepharose beads for two hours at 4°C. The lysates were again centrifuged at 13000 rpm for five minutes at 4°C and the pellets were washed three times with L-buffer containing 500 mM NaCl. The pellets were resuspended in L-buffer and divided into three aliquots, one for kinase assay, on aliquot for western blotting and the third aliquot was saved as a backup at -20°C. The aliquots to be used for kinase assay were washed three times with kinase buffer (50 mM Tris-HCl, pH 7.5, 25 mM NaCl, 10 mM MgCl2, 0.1% Triton).
The pellets were resuspended in 20 μl of kinase buffer containing additional 1 mM DTT, 10 μM ATP, 5 μCi γ32P ATP 3000 Ci/mmol (Amersham Pharmacia Biotech USA) and 4 μg of histone H3 (Millipore 14-494 USA). The reaction mix was incubated at 30°C for 30 minutes. Proteins were then separated on 12.5% SDS-polyacrylamide gel electrophoresis. The gel was stained with coomassie blue, dried and analysed by a phosphorimager (Molecular Dynamics USA).
Soft agar assay
Nine clones each of GFP-aurC-WT and GFP-aurC-CA, and four clones each of GFP-aurC-KD and GFP-alone were tested with this in vitro transformation assay. 10,000 cells/well in a 6-well plate in triplicate were grown in 2 ml top agar containing 2X DMEM media, 20% fetal bovine serum and 1% agarose. Geneticin-G-418 was added 24 hours after seeding. Media were changed twice a week. Thirty days after seeding, well plates were stained with 0.005% crystal violet dye and the numbers of colonies were counted.
Immunofluorescence
105 cells were grown on 12 mm glass cover slips in a 12-well plate. Cells were washed with PBS and fixed with cold methanol for 10 minutes at -20°C. Fixed cells were washed three times with TBS and then saturated with 1%BSA + 0.1%Tween20 prepared in PBS for 1 hour at room temperature. Primary antibodies in 1%BSA + 0.1%Tween20 in PBS were added on the cells (mouse anti-gamma tubulin, GTU-88-T6557, 1:2500 (Sigma USA); rabbit anti-phospho histone H3 ser-10- 06570, 1:1000 (Millipore USA), rabbit anti-GFP- 632375, 1: 2000 (Clonetech USA) for 2 hours at 4°C, on slow agitation and then washed three times for 10 minutes with TBS. The cells were then incubated with secondary antibodies (anti-mouse Alexa-555, 1: 1000; anti-rabbit Alexa-555, 1:1000; anti-rabbit Alexa-488, 1:1000 (Invitrogen USA) for 1 hour at room temperature on slow agitation, protected from light, washed again with TBS, three times for 10 minutes and then mounted with mounting media- Prolong-Gold (Invitrogen USA), containing DNA staining dye, DAPI. Images were collected using Leica DMRXA2 fluorescent microscope with 63× oil immersion Plan-Apochromat numerical aperture 1.32 objective. Photographs were taken using a black and white cool snap ES camera (Roper Scientific Canada) and images were processed using Metamorph Software (Universal Imaging USA). A minimum of 600 cells was counted for each condition.
Western blotting
Cells were lysed in RIPA buffer (1% NP40, 50 mMTris HCl, pH 7.5, 150 mM NaCl, 0.25% Sodium deoxycholate, 2 mM EGTA, Protease Inhibitor cocktail (Roche USA). Cell lysates were sonicated and cleared by centrifugation at 13000 rpm for 20 minutes. Proteins were quantified by Bradford method (BioRad USA). Cell lysates were boiled for 5 minutes at 95°C in Laemmli sample buffer. Equal amounts of protein samples were loaded onto 10% SDS-PAGE gel for electrophoresis and then transferred onto nitrocellulose membrane. Membranes were blocked with 5% milk-TBST for 1 hour at room temperature and incubated overnight at 4°C with primary antibodies {Mouse anti-GFP, 1:1000, (Sigma USA); Rabbit polyclonal anti-aurC, 1:250, (Zymed USA)}. Membranes were washed three times for 10 minutes each with TBST and then incubated for 1 hour at room temperature with secondary antibodies {(Anti-mouse coupled with HRP, 1:5000; Anti-rabbit coupled with HRP, 1:30000, (Jackson USA)}. Membranes were washed again with TBST as stated above and then revelation was done with chemiluminiscent, Pico or Dura (Pierce USA).
Tumour growth
Female nude mice of 3 weeks age, housed in microisolator units under controlled humidity and temperature were fed with sterile diet and water. Stable cell clones to be injected were stained overnight with DilC18(3) (FluoProbes USA) prior to injection. Seven million cells of each were injected subcutaneously in the abdominal region of each mouse. Each mouse was injected with two different clones, one on each side of the abdomen. Tumour sizes were monitored every 10 days by direct observation and the day of sacrifice, using Kodak image station 2000 (Kodak USA) by an excitation of 535 nm that detected cells stained with DilC18(3). Images were then analysed, using Kodak Molecular Imaging Software. Tumour volumes were then determined according to the formula, L × W × H × π/6, shown in mm. Mice were sacrificed when the tumour size reached 1-2 mm3 or two months after injection. Tumours were removed, put immediately in liquid nitrogen and then stored at -80°C for further analysis.
Immunohistochemistry
Ten-micrometer thick frozen sections of tumours or remaining injected cells were cut on a cryostat (Leica, Milton Keynes, UK) and mounted onto uncoated glass slides. Classical Feulgen staining or Hemalin counterstaining were performed. Immunohistochemistry was performed with rabbit monoclonal KI-67 (1.200, Epitomics, clone SP6) and anti-phospho histone- H3 ser-10 (Millipore USA) and anti-HRP (Jackson USA) secondary antibodies.
Statistical analysis
Non-parametric Mann-Whitney test was performed and the results were considered statistically significant for a p-value under 0.05.
Results
Establishment of GFP-aurC stable cell lines
NIH-3 T3 cells were transiently transfected with GFP-aurC-WT (Wild type), GFP-aurC-CA (constitutively active) and GFP-alone. The expression of GFP-aurC protein was controlled by western blotting 24 hours after transfection with two different antibodies, anti-GFP and anti-aurC (Figure 1A, B). GFP-aurC was identified in GFP-aurC-WT, GFP-aurC-CA and GFP-aurC-KD at 65 KDa with anti-GFP and anti-aurC antibodies. This band is not present in GFP-alone samples. However, we identified GFP-alone at 29 KDa only with anti-GFP-alone antibody.
Figure 1 Western blots, showing GFP-aurC and GFP-alone proteins after 24 hours of transient transfection with GFP-alone, GFP-aurC-KD GFP-aurC-CA and GFP-aurC-WT plasmid DNA with mouse Anti-GFP antibody (A) and with rabbit Anti-aurC antibody (B). Western blots showing the level of expression of GFP-aurC protein in three stable clones of GFP-aurC-KD (KD1 to KD3), four stable clones of GFP-aurC-CA (CA1 to CA4), three stable clones each of GFP-aurC-WT (WT1 to WT3) and GFP-alone (GFP1 to GFP3) illustrating the different level of expression of GFP-aurC and GFP proteins by different clones. The antibody used was mouse anti-GFP (C& D) and anti-β tubulin antibody as a loading control (E & F); (G) Kinase assay GFP-aurC-WT, GFP-aurC-CA and GFP-alone clones, using histone-H3 as a substrate. (H 1,2,3,4) The left column shows DAPI stained cells and the right column shows phosphorylated cells with Histone-H3 ser-10. (H-1) GFP-aurC-WT and (H-2) GFP-aurC-CA (H-3) GFP-aurC-KD. (H-4) GFP-alone (I) Histogram shows the percentage of cells with phosphorylation on histone H3 of GFP-aurC-WT, GFP-aurC-CA, GFP-aurC-KD and GFP-alone.
Stable cell lines were generated for GFP-aurC-WT, GFP-aurC-CA and GFP-alone. The level of expression of GFP-aurC and GFP-alone proteins was checked in all stable cell clones with anti-GFP antibody (Figure 1C-F). The level of expression was varied from clone to clone.
Overexpressed GFP-aurC-WT and GFP-aurC-CA are active kinases
Kinase activity of GFP-aurC was controlled in vitro, GFP-aurC-WT, GFP-aurC-CA, GFP-aurC-KD and GFP-alone proteins were immunoprecipitated with anti-GFP antibody and histone-H3-ser10 was used as a substrate. Both the GFP-aurC-WT and GFP-aurC-CA showed kinase activity but the GFP-alone did not show any kinase activity (Figure 1G). We also checked the kinase activity of GFP-aurC-WT, GFP-aurC-CA and GFP-alone in vivo in stable cell lines and the phosphorylation of Histone H3 was assayed. The number of positive cells for Histone H3-serine-10 phosphorylated was found almost two fold higher in GFP-aurC-WT and GFP-aurC-CA compared to GFP-alone (Figure 1H, I). Four clones were assayed for each condition.
Overexpression of active GFP-aurC results in abnormal centrosome number and polyploidy
We used γ-tubulin staining, a centrosomal marker to assess abnormal centrosome amplification (more than two centrosomes per cell) and DNA staining (DAPI) to assess multinucleation (more than one nucleus per cell). It was found that the percentage of cells with abnormal centrosome amplification in GFP-aurC-WT and GFP-aurC-CA was almost 5 times higher than GFP-alone in transiently transfected NIH-3 T3 cells (Figure 2A, B, E, F). Same ratio between GFP-aurC-WT and GFP-aurC-CA was found and compared to GFP-alone in stable cell lines. For multinucleation, we found that the percentage of multinucleated cells in GFP-aurC-WT and GFP-aurC-CA was 5 times higher than multinucleated cells in GFP-alone in transiently transfected NIH-3 T3 cells. Same difference in GFP-aurC-WT and GFP-aurC-CA was found and compared to GFP-alone stable cell lines, showing a clear difference between the two populations i.e. GFP-aurC-WT + GFP-aurC-CA and GFP-aurC-KD + GFP-alone (Figure 2C, D, E, G). It was showed that overexpression of active GFP-aurC results in both abnormal centrosome amplification and multinucleation.
Figure 2 Abnormal centrosome amplification and multinucleation. The immunoflorescent microscopy images (A-G) show abnormal centrosome amplification and multinucleation observed in GFP-aurC-WT, GFP-aurC-CA with negative control GFP-alone. (A&B) more than two centrosomes/cell appeared as white dots with anti-γ tubulin staining in GFP-aurC-CA and GFP-aurC-WT respectively. (C) and (D) show multinucleation (more than one nucleus/cell in GFP-aurC-CA and GFP-aurC-WT respectively. (E) Two centrosomes per cell and only one nucleus/cell in G2 phase of GFP-aur-KD. (F) histogram showing the percentage of cells with more than 2 centrosomes/cell of 96 hours after transient transfection in GFP-aurC-CA, GFP-aurC-WT and GFP-alone cells. (G) histogram shows the percentages of multinucleated cells of 96 hours after transient transfection in GFP-aurC-CA, GFP-aurC-WT and GFP-alone.
Aurora kinase C and in vitro transformation
The ability of GFP-aurC was assessed to transform cells in soft agar assay with GFP-aurC-WT and GFP-aurC-CA, and GFP-alone NIH-3 T3 stable cell clones. Nine clones each of GFP-aurC-WT and GFP-aurC-CA and four clones of GFP-alone were tested for growth on soft agar (Figure 3A, B). All the clones of GFP-aurC-WT & GFP-aurC-CA formed a large number of foci of colonies. In contrast, stable cell clones of GFP-alone formed negligible number of small colonies. The data showed that only active overexpressed GFP-aurC has the potential to transform NIH-3 T3 cells.
Figure 3 Soft agar assay, tumour formation and immunohistochemistry. (A) Foci of colonies of GFP-aurA, GFP-aurC-CA, GFP-aurC-WT stable cell lines and very negligible number of very small colonies of GFP-alone in soft agar assay. (B) Histogram of the average number of colonies. (C) Visualization of the tumours formed by injecting GFP-aurC-CA and GFP-aurC-WT stable cell lines, and the remaining injected cells of GFP-alone on the day of sacrifice using Kodak image station 2000. (D) Rabbit monoclonal KI-67, a proliferation marker from late G1 to M-phase staining (E) anti-phospho histone-H3 ser-10 (Millipore) and anti-HRP (Jackson) secondary antibodies and (F & G) Feulgen staining showing prometaphase defects, metaphase defects, lagging chromosomes at anaphase, and cytokinesis defect.
Aurora kinase C and in vivo transformation
To test whether NIH-3 T3 cells overexpressing GFP-aurC were able to induce neoplastic transformation in vivo, eight clones each of GFP-aurC-WT and GFP-aurC-CA and four clones each of GFP-alone were injected (seven million cells of each clone) subcutaneously in Swiss nu/nu mice. Tumours sizes were monitored every 10 days after injection by both direct and indirect measurements. The direct method used was measurements by vernier calliper and the indirectly through fluorometry in live mice. Cells stained with DilC18 (3) dye were excited through skin and the emission signals were used to calculate tumour sizes. We observed a correlation between tumour volumes (Figure 3C-G).
The proliferation status of cells within tumours was analyzed after sacrifice by using different markers. For Ki-67 (a proliferation marker from G2/M phase), more than 60% of cells overexpressing GFP-aurC-WT and GFP-aurC-CA were positive for Ki-67 but less than 2% of the injected cells of GFP-alone were positive for Ki-67 (Figure 3D). Feulgen staining of tumours induced by GFP-aurC-WT and GFP-aurC-CA showed abnormal figures of mitosis such as abnormal prometaphase (92%), abnormal metaphase (90%) (Figure 3F) lagging chromosomes (85%) and cytoplasmic bridges (80%) (Figure 3G). No such types of abnormalities were observed in cells overexpressing GFP-alone.
Immunostaining of phosphor-histone H3-serine-10 was used to evaluate the percentage of cells in M-phase. More than 16% of cells overexpressing GFP-aurC-WT or GFP-aurC-CA were histone H3 positive whereas less than 2% of cells overexpressing GFP-alone were positive for histone H3 (Figure 3E). Thus the histological analysis of these tumours confirmed high proliferation rate of both GFP-aurC-WT and GFP-aurC-CA and chromosomal abnormalities.
Discussion
All the three members of Aurora kinase family have been detected in human cancers when they are overexpressed [10-12]. In this study, whether or not aurora-C-T191D mutant is constitutively active, was in question. We compared the potential to induce cell growth in soft agar and tumour of stable cell lines overexpressing GFP-aurC-WT, GFP-aurC-T191D (GFP-aurC-CA expressing the constitutively active GFP-tagged aurC) and GFP as a control.
We showed in vitro kinase assays that the relative activity of histone H3 phosphorylation by GFP-aurC-CA was the same as that by GFP-aurC-WT (Figure 1G-I). These results are in contrast to those previously described [20]. This might be due to the reason that we used mouse NIH3T3 cell line. The GFP-aurC-KD did not phosphorylate Histone H3.
Abnormal expression of Aurora kinases causes abnormal centrosomes amplification and multinucleation [6,17,21]. Both Aurora-A and Aurora-B overexpression phenotypes are aggravated in the absence of active p53 [6,22]. An elimination of the p53-dependent checkpoint may be evoked [23] to explain centrosome amplification and multinucleation induced by Aurora-C. Moreover, overexpressed Aurora-C kinase behaves like a dominant negative kinase for Aurora-B leading to cytokinesis defect that could explain the multinucleation phenotype observed in Aurora-C overexpressing cells [6]. We demonstrated that the overexpression of only active GFP-Aurora-C-CA or Aurora-C-WT induces centrosome amplification and multinucleation (Figure 2).
Although all Aurora kinases are found overexpressed in cancer cells, their direct implication in oncogenesis varies. During interphase Aurora-C localizes to the centrosomes just like Aurora-A, both of them demonstrating oncogenic potentials. Moreover, centrosome amplification, a common feature of Aurora-A and Aurora-C overexpression, is a frequent event in almost all types of solid cancer [24-26]. Interestingly, the kinase activity of Aurora-A is not essential for induction of centrosome amplification, however, the oncogenic transformation requires kinase activity. Aurora-B by itself cannot induce transformation of cells but augments Ras-mediated transformation [27,28]. Aurora-B and -C have overlapping functions and compete each other for their substrates and other chromosome passenger proteins [11]. INCENP and Survivin have stronger affinity for Aurora-B than for Aurora-C [11] but interestingly Aurora-C can complement the functions of Aurora-B in mitotic cells. Although it is likely that the oncogenic activity of Aurora-C is related to its interphase function (Aurora-A like) rather to its mitotic function related to its chromosome passenger behaviour (Aurora-B like) this remains to be deciphered. Similarly we found that the overexpression of Aurora-C induces tumour formation when injected into nude mice, but this needs kinase activity (Figure 3).
It is demonstrated that through both in vitro and in vivo transformations, overexpression of Aurora-C-CA and Aurora-C-WT in somatic cells has an oncogenic potential and have almost equal relative activity. Thus GFP-aurC-CA is constitutively active kinase mutant, at least in mouse NIH-3 T3 cells, and not hyperactive mutant as has been described earlier in Hela cells and in U2OS cells. Here we used human Aurora-C gene in mouse NIH3T3 cells that needs further to be explored, at least mouse Aurora-C gene in mouse cells.
Conclusion
On the basis of above stated results and analysis, we thus concluded that at least in NIH-3 T3 cells, the human Aurora C-T191D is constitutively active mutant, and not hyperactive mutant.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
JK designed and performed the experiments, SK participated in discussion of the data and draft of the manuscript. SA, SNK and IA review the manuscript. All authors read and approved the final manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22509406PONE-D-12-0041310.1371/journal.pone.0035324Research ArticleBiologyNeuroscienceCellular NeuroscienceNeuronal MorphologyMolecular NeuroscienceSignaling PathwaysMedicineAnesthesiologyAnesthetic MechanismsGeneral AnesthesiaNeurosurgical CarePerioperative Critical CareSurgeryAnesthesiaNeurosurgeryTrauma SurgeryPropofol Prevents Autophagic Cell Death following Oxygen and Glucose Deprivation in PC12 Cells and Cerebral Ischemia-Reperfusion Injury in Rats Propofol Prevents Autophagic Cell DeathCui Derong
1
Wang Li
1
Qi Aihua
2
Zhou Quanhong
1
Zhang Xiaoli
1
Jiang Wei
1
*
1
Department of Anesthesiology, Shanghai Sixth People's Hospital Affiliated with Shanghai Jiaotong University, Shanghai, China
2
Department of Postgraduate School, Soochow University, Suzhou, China
Xie Zhongcong EditorMassachusetts General Hospital, United States of America* E-mail: [email protected] and designed the experiments: DC LW WJ. Performed the experiments: DC AQ QZ XZ. Analyzed the data: DC LW. Contributed reagents/materials/analysis tools: DC AQ XZ. Wrote the paper: DC LW WJ.
2012 11 4 2012 7 4 e353244 1 2012 12 3 2012 Cui et al.2012This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Background
Propofol exerts protective effects on neuronal cells, in part through the inhibition of programmed cell death. Autophagic cell death is a type of programmed cell death that plays elusive roles in controlling neuronal damage and metabolic homeostasis. We therefore studied whether propofol could attenuate the formation of autophagosomes, and if so, whether the inhibition of autophagic cell death mediates the neuroprotective effects observed with propofol.
Methodology/Principal Findings
The cell model was established by depriving the cells of oxygen and glucose (OGD) for 6 hours, and the rat model of ischemia was introduced by a transient two-vessel occlusion for 10 minutes. Transmission electron microscopy (TEM) revealed that the formation of autophagosomes and autolysosomes in both neuronal PC12 cells and pyramidal rat hippocampal neurons after respective OGD and ischemia/reperfusion (I/R) insults. A western blot analysis revealed that the autophagy-related proteins, such as microtubule-associated protein 1 light chain 3 (LC3-II), Beclin-1 and class III PI3K, were also increased accordingly, but cytoprotective Bcl-2 protein was decreased. The negative effects of OGD and I/R, including the formation of autophagosomes and autolysosomes, the increase in LC3-II, Beclin-1 and class III PI3K expression and the decline in Bcl-2 production were all inhibited by propofol and specific inhibitors of autophagy, such as 3-methyladenine (3-MA), LY294002 and Bafilomycin A1 (Baf),. Furthermore, in vitro OGD cultures and in vivo I/R rats showed an increase in cell survival following the administration of propofol, as assessed by an MTT assay or histochemical analyses.
Conclusions/Significance
Our data suggest that propofol can markedly attenuate autophagic processes via the decreased expression of autophagy-related proteins in vitro and in vivo. This inhibition improves cell survival, which provides a novel explanation for the pleiotropic effects of propofol that benefit the nervous system.
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Introduction
Propofol is a widely used intravenous anesthetic. In addition to its sedation/hypnotic properties, propofol displays neuroprotective effects [1]–[3]. As an activator of GABAA receptors, an inhibitor of NMDA receptors and a modulator of calcium influx through slow calcium channels, propofol improves the neurological outcome. In a rat cerebral ischemia model, propofol treatment was shown to decrease the infarct size in the hippocampus [4]. In addition, propofol administration also decreased the apoptotic rate and improved cell viability in hypoxic neuronal cultures [2], [3]. Moreover, propofol has a phenolic hydroxyl (OH) group, which is similar to that of vitamin E (α-tocopherol) and demonstrates antioxidant activity by scavenging free radicals [5], [6]. On the organelle and tissue level, the treatment of rat brain oxidative stress injury with propofol confers neuroprotective effects through an inhibition of lipid peroxidation [7]. Although, such pleiotropic mechanisms have been suggested to contribute to propofol-mediated neuroprotection, its capabilities are still not completely understood.
Recent evidence suggests that autophagy is activated in the pyramidal neurons of the rat hippocampus upon ischemic insult [8]–[10]. Autophagy is an evolutionarily conserved and highly regulated homeostatic process by which cytoplasmic macromolecules and organelles are degraded for removal or turnover through the lysosomal system [8]. However, excessive autophagy results in neuronal cell damage [8], [9]. The involvement of autophagy in neurodegenerative disorders is demonstrated by increased autophagic vacuoles [8], [9], with associated high levels of Beclin-1-phosphatidylinositol-3 kinase class III (class III PI3K) lipid-kinase-Vps34 and low levels of anti-apoptotic cellular Bcl-2 in pathological settings [11].
Apoptosis has been implicated in the delayed neuronal death induced by ischemia and has been extensively studied [12], [13]. However, autophagy could also mediate the execution of ischemia/reperfusion (I/R) injury-induced neuronal cell death, particularly in the hippocampus [8]–[10]. Therapies developed to target autophagy might have a beneficial effect on brain I/R injury.
Given the pleiotropic effects of propofol on nervous system function, we investigated the role of autophagy in propofol-mediated neuroprotection in vitro and in vivo. Our results are the first to show propofol-attenuated autophagic cell death in hypoxic neuronal PC12 cells and the rat hippocampus after I/R insult.
Results
Activation of Autophagy in Neuronal PC12 Cells after OGD Injury
In vitro ischemia was induced in cultured neuronal PC12 cells by OGD, which is a condition used to mimic in vivo metabolic inhibition [14]. Transmission electron microscopy was used to identify ultrastructural changes in neuronal PC12 cells at 0.5, 1, 3, 6 and 12 h after OGD insult. The control cells contained organelles, nuclei and chromatin with normal morphologies (Fig. 1AA). At 0.5–6 h after OGD, the PC12 cells contained many vesicles with the typical morphological features of autophagosomes. A number of isolated double or multi-membrane structures, which engulfed cytoplasmic fractions and organelles, were observed in the cytoplasm (Fig.1AB–AF, as indicated by the broad arrows).
10.1371/journal.pone.0035324.g001Figure 1 Ultrastructural changes in PC12 cells after OGD injury.
(A) The PC12 cells were subjected to OGD for 0.5 (AB), 1 (AC), 3 (AD), 6 (AE) and 12 h (AF) and were fixed for TEM examination. TEM micrograph features: Typical cytoplasms and nuclei in the control cells (AA, normal mitochondria indicated by black arrowheads). Isolated membrane and double- or multi-membrane autophagosomes in the PC12 cells following OGD treatment (AB–AF, as indicated by broad arrows). The mitochondria displayed swelling, dilation and cristae disruption (AB, AD and AE, as indicated by black arrowheads). The lysosomes were darkly stained, indicating the activation of lysosomes (AF, as indicated by narrow arrows). Cell shrinkage, nuclear condensation, loss of cellular organelles and vacuolization (AE and AF). N, nucleus; Broad arrows represent autophagosomes; Narrow arrows represent lysosomes; Black arrowheads represent mitochondria; Scale bar = 1 µm. (B) Quantitative analysis of the number of intact mitochondria, autophagosomes and lysosomes in the OGD and control groups. Three samples in each group and ten fields for each sample were examined. The results are expressed as the mean ± SD. Statistical comparisons were conducted using an ANOVA followed by the Tukey test. *p <0.05 vs. control group.
A quantitative analysis of the cytoplasmic components showed a significant increase in the number of autophagosomes at 1–3 h after OGD (Fig. 1B). When the autophagosomes fused with the lysosomes, their inner membranes disappeared, and the autophagosomes became single-membrane autophagic vacuoles at 6–12 h after OGD (Fig.1AE and AF, as indicated by the broad arrows). The mitochondria displayed swelling, dilation and cristae disruption (Fig. 1AD and AE, as indicated by the black arrowheads), and the number of intact mitochondria was drastically decreased in a time-dependent manner (Fig. 1B). The lysosomal staining was darkened (Fig. 1AF, as indicated by the narrow arrows), and the number of lysosomes was obviously increased at 6 h after OGD (Fig. 1B), indicating the activation of lysosomes. Moreover, morphological features of apoptosis (Fig. 1AE) and necrosis (Fig. 1AF), such as cell shrinkage, chromatin condensation and damaged organelles with deteriorated membranes, were also observed at 12 h after OGD.
Increased Expression of Autophagy-related Proteins in PC12 Cells Following OGD
Class III PI3K has been previously shown to activate autophagy, although the precise contribution of this specific substrate kinase to the regulation of LC3-II elevation under different cellular contexts is unclear. Our findings revealed that the class III PI3K pathway is involved in LC3-II upregulation, which promotes autophagy. The results demonstrated that the protein levels of class III PI3K were significantly upregulated and peaked at 1 h after OGD (Fig. 2A and C).
10.1371/journal.pone.0035324.g002Figure 2 Increased class III PI3K (A and C), Beclin-1 (B and D), Bcl-2 (E and G) and LC3-II (F and H) expression in PC12 cells after OGD injury.
PC12 cells were cultured, and OGD was induced as described in the methods. The cells were harvested for western blot analysis at various times after OGD. The optical densities of the respective protein bands were analyzed using Sigma Scan Pro 5 and normalized to the loading control (GAPDH). The results are expressed as the mean ± SD from three independent experiments. Statistical comparisons were conducted using an ANOVA followed by the Tukey test. *p < 0.05, **p < 0.01 vs. control group.
Beclin-1, a homologue of yeast Atg6, forms a protein complex with PI3K within the autophagosome [15]. The results demonstrated that the protein levels of Beclin-1 were significantly upregulated and peaked at 3 h after OGD (Fig. 2B and D).
Bcl-2 is an important anti-apoptotic protein. Previous studies have shown that excitotoxic and ischemic insults result in an autophagy-dependent decrease of Bcl-2 protein levels [8], [16]. As shown in Figure 2E and G, a marked reduction in Bcl-2 protein levels was observed 3 h following OGD treatment.
LC3 is required for the formation of autophagosome membranes [17]. The cytoplasmic form of LC3 (LC3-I) is diffusely distributed in the cytoplasm [18]. but is modified and concentrated in the autophagosomes during autophagy activation [17], [19]. When associated with autophagosomes, LC3 typically exhibits a shift in electrophoretic mobility from 18 to 16 kDa and is commonly referred to as LC3-II [17]. LC3-II is a common marker of autophagosomes in mammalian cells [17]. As shown in Figure 2, a dramatic increase in the LC3-II/LC3-I ratio was observed in PC12 cells 3 h after OGD treatment (Fig. 2F and H).
Propofol Reduced the OGD-induced Cell Death
To determine the influence of propofol on OGD-induced cell injury, PC12 cells were treated with propofol or 3-MA during OGD. A concentration of 20–50 µmol/L of propofol or 20 mmol/L of 3-MA [8] effectively blocked the activation of autophagy, as evidenced by the inhibition of LC3-II production (Fig. 3A and B).
10.1371/journal.pone.0035324.g003Figure 3 PC12 cells were treated with propofol (10, 20, 50 µmol/L) and 3-MA 20 mmol/L) during OGD treatment, and the cells were harvested 6 h later.
(A) Immunoblotting LC3 in PC12 cells after OGD. Propofol at concentrations of 20 and 50 µmol/L and 3-MA at a concentration of 20 mmol/L effectively blocked the activation of autophagy, as evidenced by inhibiting the production of LC3-II. (B) The optical densities of the respective protein bands were analyzed using Sigma Scan Pro 5 and normalized to the loading control (GAPDH). The results are expressed as the mean ± SD from six independent experiments. Statistical comparisons were conducted using an ANOVA followed by the Tukey test. **p < 0.01 vs. control group; #
p < 0.05 vs. OGD-treated group.
Lactate dehydrogenase (LDH) leakage was measured as an indicator of OGD-induced injury in PC12 cells [20]. The results showed that LDH leakage was markedly increased at 6 h after OGD. Propofol treatment resulted in a small but significant decrease in LDH leakage in a dose-dependent manner (Fig. 4A). Bafilomycin A1 (Baf) is a selective inhibitor of vacuolar H+-ATPase and therefore inhibits the maturation of autophagosomes. The results of the present study showed that the PC12 cell viability was decreased sharply 6 h after OGD. Propofol treatment significantly increased the cell viability of PC12 cells in a dose-dependent manner (Fig. 4B).
10.1371/journal.pone.0035324.g004Figure 4 Inhibition of autophagy reduced OGD-induced PC12 cell injury.
(A) Inhibition of OGD-induced lactate dehydrogenase (LDH) leakage by propofol and 3-MA. PC12 cells were treated with propofol (10, 20, 50 µmol/L) or 3-MA (20 mmol/L) during OGD treatment, and the cells were harvested 6 h later. Supernatants and cell lysates were prepared as described in the methods. LDH leakage was detected with a LDH assay kit according to the manufacturer’s protocol. The results are presented as the mean ± SD from three independent experiments. Statistical comparisons were conducted using an ANOVA followed by the Tukey test. (B) Inhibition of the OGD-induced reduction in PC12 cell viability by Baf. The PC12 cells were treated with various doses of propofol (10, 20 or 50 µmol/L) and Baf (4 µmol/L) for the indicated time, and cell viability was analyzed with an MTT assay. The results are presented as the mean ± SD from three independent experiments. Statistical comparisons were conducted using an ANOVA followed by the Tukey test. **p < 0.01 vs. control group; ##
p < 0.01 vs. OGD-treated group.
The Effect of Propofol on the Expression of Autophagy-related Proteins in PC12 Cells Following OGD
Autophagy is primarily regulated by one central pathway: the class III PI3K-Beclin-1-Bcl-2-dependent mechanism. To explore how propofol regulates OGD-induced autophagy, we analyzed the expression of several autophagy-related proteins involved in this pathway in the OGD-injured PC12 cells. OGD injury resulted in a significant increase of Beclin-1 and LC3-II expression as compared with the control group (Fig. 5B, D, F and H). In addition, class III PI3K, which positively mediates autophagy, was greatly upregulated in the OGD-injured PC12 cells. However, treatment with propofol and/or LY294002 significantly decreased Beclin-1 and class III PI3K expression in PC12 cells (Fig. 5A–D), suggesting that OGD-induced autophagic cell death is dependent on the formation of the class III PI3K sub-complex containing Beclin-1.
10.1371/journal.pone.0035324.g005Figure 5 Blockade of the inhibition of autophagy activation by propofol.
PC12 cells were treated with propofol and the class III PI3K relatively selective inhibitor LY294002 (50 µmol/L) during the OGD insult and were harvested 6 h later for western blot analysis. (A, B, E, F) Immunoblot analyses of OGD-injured PC12 cells. The PC12 cell homogenates were analyzed by western blotting using a specific antibody against each autophagy-related protein. The expression of GAPDH was also examined for the protein loading control. (C, D, G, H) Quantification of class III PI3K, Beclin-1, Bcl-2, LC3-I and LC3-II expression. Each protein (class III PI3K,class III PI3K, Beclin-1, Bcl-2, LC3-Ι and LC3-II) shown in Fig. 5A, B, E, F was quantified after a densitometric scan and normalized to GAPDH. The optical densities of the respective protein bands were analyzed using Sigma Scan Pro 5 and normalized to the loading control (GAPDH). The results are expressed as the mean ± SD from three independent experiments. Statistical comparisons were conducted using an ANOVA followed by the Tukey test. **p < 0.01 vs. control group; #
p < 0.05, ##
p < 0.01 vs. OGD-treated group.
To further confirm the influence of propofol on the response of the class III PI3K-Beclin-1-Bcl-2 interaction to OGD-induced autophagy in the presence of propofol, the cells were transiently transfected with small interference RNA (siRNA) against Beclin1 for 0–72 h (Fig. 6A, B), which is a principal regulator in the formation of autophagosomes and the initiation of autophagy through the class III PI3K pathway or the inhibition of autophagy through the Bcl-2 pathway. We observed a significantly increased interaction between Beclin-1 and class III PI3K, leading to Beclin-1-dependent autophagic cell death, while the administration of propofol promoted Bcl-2 protein expression and significantly decreased class III PI3K protein expression in the OGD-injured PC12 cells (Fig. 5A, C, E, G and Fig. 7A-F). These observations suggest that the decreased expression of Beclin-1 and class III PI3K or the increased Bcl-2 expression by propofol in the OGD-injured PC12 eventually inhibits autophagy.
10.1371/journal.pone.0035324.g006Figure 6 PC12 cells were transfected with Beclin-1 siRNA at specific concentration (100 nM) for various times.
(A) The inhibition of Beclin-1 protein expression by siRNA transfection was measured by immunoblot assay with an anti-Beclin-1 antibody. GAPDH was used as the loading control. (B) Quantification of Beclin-1 expression. The Beclin-1 protein shown in Fig. 6A was quantified after a densitometric scan and normalized to GAPDH. The optical densities of the respective protein bands were analyzed using Sigma Scan Pro 5 and normalized to the loading control (GAPDH). The results are expressed as the mean ± SD from three independent experiments. Statistical comparisons were conducted using an ANOVA followed by the Tukey test. *p < 0.05, **p < 0.01 vs. 0 hour group.
10.1371/journal.pone.0035324.g007Figure 7 The expression of autophagy-related proteins during 6 h of OGD after transfection with Beclin-1 siRNA at specific concentration (100 nM) for 48 h.
(A, B, E) Immunoblot analyses of OGD-injured PC12 cells. The PC12 cell homogenates were analyzed by western blotting using a specific antibody against each autophagy-related protein. The expression of GAPDH was also examined as the protein loading control. (C, D, F) The quantification of class III PI3K, Bcl-2, LC3-I and LC3-II expression. Each protein (class III PI3K, Bcl-2, LC3-I and LC3-II) shown in Fig. 7A, B, E was quantified after a densitometric scan and normalized to GAPDH. The optical densities of the respective protein bands were analyzed using Sigma Scan Pro 5 and normalized to the loading control (GAPDH). The results are expressed as the mean ± SD from three independent experiments. Statistical comparisons were conducted using an ANOVA followed by the Tukey test. *p < 0.05, **p < 0.01 vs. the 0 hour group.
The Injury of Hippocampal Pyramidal Neurons Following I/R
Studies have reported an early decline in the number of hippocampus CA1 pyramidal neurons following severe ischemic insults [13], [21]. To specifically investigate the temporal effects of I/R on hippocampal pyramidal neuron function, we measured the number of pyramidal neurons in the CA1 region of the hippocampus following severe ischemic insults at various time points using histochemical techniques. The results revealed that, as compared with the control group (Fig. 8A), there was no robust change in the number of hippocampal pyramidal neurons at 1 h after ischemia (Fig. 8B). This result was consistent with the observation the lack of LC3 expression in the CA1 hippocampus at that time point (Fig. 9A). However, the number of hippocampal pyramidal neurons was reduced in the ischemic CA1 hippocampus at 3 h (Fig. 8C) and was further decreased at 6–24 h after ischemia (Fig. 8D, E and F). Moreover, the damaged pyramidal neurons in the ischemic CA1 hippocampus exhibited various stages of fragmentation (Fig. 8F), which is characteristic of dying cells, indicating that the hippocampal pyramidal neurons were damaged and died in a time-dependent manner following I/R.
10.1371/journal.pone.0035324.g008Figure 8 The neuronal damage and histological characteristics of necrotic neurons were assessed by a histological examination.
Thionine staining of brain sections at the level of the hippocampus from sham (n = 6) rats and rats sacrificed at 1, 3, 6, 12, or 24 h after I/R (n = 6 per time point). (A) Sham brain sections revealed normal neurons in the hippocampus. (B–C) Sections from 1 h and 3 h after a 10-min period of brain ischemia showed neuronal loss, and a small number of neurons in the CA1 area exhibited the morphological criteria of necrosis, displaying cell shrinkage, nuclear condensation, and fragmentation. (D–F) A dramatic loss of neurons was observed in the CA1 cell layer at 6, 12, and 24 h after ischemia. (H) Quantitative analysis of the number of pyramidal neuronal cells. The number of pyramidal neurons in the ischemic hippocampus was significantly decreased in the ischemic rats compared to the sham rats. The data are expressed as the percentage of sham-operated group animals and as the mean ±SD, n = 6. The statistical analysis was performed using a one-way ANOVA. *p < 0.05, **p < 0.01 vs. sham group. Scale bars: lower magnification (G), 50 µm; higher magnification (A-F), 500 µm. so, stratum oriens; sp, stratum pyramidal; sr, stratum radiatum.
We also measured the expression of LC3 ΙΙ by immunohistochemistry at various time points following the ischemic insult (Fig. 9A). LC3 ΙΙ is a marker for autophagic vacuoles (AVs). When the hippocampal pyramidal neurons were examined by fluorescence microscopy after I/R treatment, the immunohistochemistry-labeled AVs appeared as distinct puncta distributed throughout the cytoplasm, the perinuclear regions and the processes. As compared with the control group (Fig. 9A), there was a significant increase in the number of LC3 II-labeled vesicles at 1 h (Fig. 9A and B), which peaked at 3–6 h after I/R treatment (Fig. 9A and B), suggesting an induction of AV formation in hippocampal pyramidal neurons after I/R.
10.1371/journal.pone.0035324.g009Figure 9 LC3-II was detected with a monoclonal anti-LC3-II-FITC antibody (green).
The sections were taken from the infarct regions of the ipsilateral hippocampus 1, 3, 6, 12 and 24 h following I/R. I/R increased the LC3-II-positive cells and LC3-II protein levels in the ischemic hippocampus after I/R in rats. I/R was induced by two-vessel occlusion. Representative photomicrographs of LC3-II immunofluorescence. Immunofluorescence of LC3-II was performed at 0–24 h after I/R. Images (magnification 200x) were taken from the same part of the ischemic hippocampus. (B) The quantitative analysis of the number of LC3-II-positive cells. The number of LC3-II-positive cells in the ischemic hippocampus was significantly increased in the ischemic rats compared to the sham rats. The data are expressed as percentage of the sham-operated animals and as the mean±SD, n = 6. The statistical analysis was performed using a one-way ANOVA. *p < 0.05, **p < 0.01 vs. sham group.
Activation of Autophagy in Rat Hippocampal Pyramidal Neurons after I/R
The ultrastructural changes in rat hippocampal pyramidal neurons were observed by transmission electron microscopy at 1–24 h after I/R. The smooth cytoplasmic, normal appearance of the mitochondria (Fig. 10AA, as indicated by black arrowheads), nuclei and chromatin were observed in the control hippocampal pyramidal neurons. After the I/R insult, the pyramidal neurons exhibited typical signs of autophagic/lysosomal activation and apoptosis, as shown in Figure 10AB–AF. Autophagosomes were observed as C-shaped double membrane structures or double membrane vacuoles (Fig. 10AB–AF, as indicated by broad arrows). The most abundant autophagosomes were observed at 3 h after I/R (Fig. 10C and G). Occasionally, autophagosomes with engulfed organelles were observed. The fusion of autophagosomes with lysosomes was occasionally observed (Fig. 10AD, as indicated by asterisks). The mitochondria displayed swelling, dilation and cristae disruption (Fig. 10AC, AD and AE, as indicated by black arrowheads), and the number of intact mitochondria were dramatically decreased in a time-dependent manner (Fig. 9G). Lipid drops were phagocytized by lysosomes that were darkly stained (Fig. 10AD and as indicated by narrow arrows), and the quantitative analysis of the change in numbers of lysosomes also showed that the number of lysosomes was markedly increased 6 h after I/R (Fig. 10G), indicating the activation of lysosomes. The loss of organelles and cytoplasm vacuolization was apparent 6 h after the I/R insult (Fig. 10AD). In addition, both apoptotic and necrotic morphological features were observed in the same cell; e.g., cell shrinkage, large chromatin clumping, nuclear condensation/fragmentation, swollen cytoplasm, damaged organelles and deteriorated membranes were observed in the same pyramidal neurons at 24 h after I/R (Fig. 10AF). The quantitative analysis of the cytoplasmic components also revealed that the number of lysosomes was markedly increased at 6 h after I/R (Fig.10B), indicating the activation of lysosomes.
10.1371/journal.pone.0035324.g010Figure 10 Ultrastructural changes in rat hippocampal pyramidal neurons at 1 (AB), 3 (AC), 6 (AD), 12 (AE) and 24 h (AF) after I/R.
(A) Transmission electron microscopy (TEM) images showing: the normal appearance of the cytoplasms, organelles and nuclei in the control hippocampal pyramidal neurons (AA, normal mitochondria indicated by black arrowheads); C-shaped double-membrane structures and double-membrane autophagosomes in the hippocampal pyramidal neurons following I/R treatment (AB–AF, as indicated by broad arrows); swelling, dilation and cristae disruption in mitochondria (AC, AD and AE, as indicated by black arrowheads); and activated lysosomes (AD, as indicated by narrow arrows). An autophagosome fused with a lysosome (AD, as indicated by an asterisk). The coexistence of morphological features of necrosis and apoptosis in the same neurons (AF). N, nucleus; Broad arrows represent autophagosomes; Narrow arrows represent lysosomes; Black arrowheads represent mitochondria; Asterisks represent autolysosomes. Scale bar = 1 µm. (B) The quantitative analysis of the number of intact mitochondria, autophagosomes and lysosomes in the ischemic and control groups. Three rats in each group and 10 fields for each rat were examined. The results are expressed as the mean ± SD. The statistical comparisons were conducted using an ANOVA followed by the Tukey test. *p < 0.05 vs. control group.
Propofol Reduced the I/R-induced Death of Cells
To further address the function of propofol in the cerebral ischemia-induced damage of hippocampal pyramidal neurons in vivo, we examined the effects of propofol (i.p., 10, 50, and 100 mg/kg ) or 3-MA (i.c.v., 600 nmol) on the number of CA1 hippocampus pyramidal neurons using histochemical techniques and the number of LC3 II-positive cells using immunofluorescence at 12 h after I/R. The results showed that the number of pyramidal neurons in the ischemic pyramidal layers of the CA1 hippocampus was dramatically decreased, and the number of LC3 II-positive cells was dramatically increased, in the ischemic rats (Fig. 11B). In contrast, propofol (i.p., 50 and 100 mg/kg) or 3-MA significantly increased the number of pyramidal neurons and decreased the number of LC3 II-positive neurons (Fig. 11A). These results suggest persistent and excessive autophagy/lysosome activation induced by the in vivo ischemia of pyramidal neurons, and the inhibition of autophagy by propofol mildly increased cell survival.
10.1371/journal.pone.0035324.g011Figure 11 Propofol increased the number of the hippocampal pyramidal neurons and decreased the expression of the LC3-II protein and the number of lysosomes and autophagosomes in the ischemic hippocampus after I/R in the rats.
I/R was induced by 2-vessel occlusion. Propofol 10, 50, or 100 mg/kg was administrated intraperitoneally and 3-MA (600 nM) was administered intracerebroventricularly 10 min after the onset of ischemia. (1) Sham-operated group (control group, Cont); (2) I/R group; (3) I/R+ 3-MA 600 nmol group; (4) I/R + propofol 10 mg/kg group; (5) I/R + propofol 50 mg/kg group; (6) I/R + propofol 100 mg/kg group; (7) I/R + vehicle (intralipid, 100 mg/kg) group. Histochemical, immunohistochemical and TEM morphological analyses were performed at 12 h after I/R. (A) Quantitative analysis of the number of hippocampal pyramidal neurons. The number of hippocampal pyramidal neurons in the ischemic hippocampus was significantly increased in the propofol- and 3-MA treated rats, compared to the ischemic rats. (B) Quantitative analysis of the number of LC3-II-positive neurons. The number of LC3-II-positive neurons in the ischemic hippocampus was significantly decreased in the propofol- and 3-MA treated rats compared to the ischemic rats. The data are expressed as percentage of the sham-operated animals and as the mean±SD, n = 6. Statistical analyses were performed using a one-way ANOVA. **p < 0.01 vs. control group; ##
p < 0.01 vs. I/R-treated group. (C) Quantitative analysis of the number of intact mitochondria, autophagosomes and lysosomes in the ischemic group, the propofol- or 3-MA treated group and the control group. Six rats in each group and 10 fields for each rat were examined. The results were expressed as the mean ± SD. Statistical comparisons were conducted using an ANOVA followed by the Tukey test. *p < 0.05 vs. control group. #p < 0.05 vs. I/R-treated group.
Quantitative analysis of the number of intact mitochondria, autophagosomes and lysosomes in the ischemic model, propofol-treated, 3-MA-treated and control groups (Fig 11C) revealed that the number of autophagosomes and lysosomes in the ischemic hippocampus was dramatically increased, and the number of was intact mitochondria was dramatically decreased in the ischemic rats. In contrast, the administration of propofol (i.p., 50 and 100 mg/kg) or 3-MA significantly decreased the number of autophagosomes and lysosomes and increased the number of intact mitochondria.
There were no significant changes in the pH, the arterial carbon dioxide (PaCO2) or oxygen (PaO2) concentrations or blood glucose concentrations before and after the intracerebral ventricular injection of 3-MA or the intraperitoneal injection of propofol in any of the groups (Table 1).
10.1371/journal.pone.0035324.t001Table 1 Physiological parameters.
Groups Time MAP(mmHg) PH PaCo2(mmHg) Po2(mmHg) GI(mg/dl)
Cont Baseline 105±6 7.38±0.02 35.6±2.9 146.2±18.2 156±23
Ischemia 103±5 7.43±0.03 36.8±2.8 141.5±19.3 166±16
Recovery 107±5 7.37±0.03 38.5±2.2 140.5±13.2 165±21
I/R Baseline 105±6 7.40±0.02 38.3±2.6 144.3±11.8 158±22
Ischemia 39±3a
7.42±0.03 36.9±2.8 136.6±16.5 161±19
Recovery 110±5 7.39±0.03 39.3±3.2 139.8±19.2 163±18
I/R+3-MA(600nM) Baseline 105±6 7.37±0.02 38.3±3.1 138.6±15.5 165±18
Ischemia 41±2a
7.43±0.03 38.6±2.3 143.3±16.8 155±19
Recovery 116±4 7.39±0.02 38.9±3.1 140.3±18.3 158±21
I/R+Prop(10mg/kg) Baseline 105±6 7.39±0.03 37.8±2.6 142.3±16.9 163±20
Ischemia 38±3a
7.41±0.02 38.5±2.5 143.5±18.6 159±18
Recovery 113±6 7.43±0.03 39.5±2.1 141.6±16.2 156±17
I/R+Prop(50mg/kg) Baseline 108±3 7.39±0.03 38.6±3.3 143.3±18.3 159±16
Ischemia 39±2a
7.41±0.01 39.6±3.8 143.5±16.5 160±15
Recovery 115±6 7.42±0.03 40.5±2.3 143.3±11.5 161±18
I/R+Prop(100mg/kg) Baseline 105±6 7.39±0.02 39.6±2.9 141.6±18.5 162±16
Ischemia 41±3a
7.39±0.02 40.8±3.1 142.6±16.9 169±15
Recovery 110±4 7.42±0.03 39.8±2.6 144.3±12.8 168±19
I/R+Int(100mg/kg) Baseline 105±6 7.38±0.02 40.3±3.2 143.6±15.8 173±18
Ischemia 39±2a
7.41±0.03 39.9±2.8 143.8±18.5 169±26
Recovery 113±5 7.42±0.01 38.9±2.1 140.9±16.9 165±21
All values are mean±SD. Arterial blood gas tensions include PaO2, PaCO2, PH and GI.
MAP, mean arterial pressure; PaO2, arterial oxygen pressure; PaCO2, arterial carbon dioxide pressure; GI, glucose.
a Controlled parameter.
Effect of Propofol on the Expression of Autophagy-related Proteins During I/R
The brain I/R injury resulted in a significant increase in Beclin-1 and LC3-II expression as compared with the control group (Fig. 12B, D, F, H). In addition, class III PI3K, which positively mediates autophagy, was greatly upregulated in the I/R-injured brains. However, the administration of propofol significantly increased Bcl-2 expression and prevented I/R-dependent Beclin-1and class III PI3K protein expression after I/R injury (Fig. 12A-D, E, G). The western blot analysis showed a decreased conversion of LC3-I to LC3-II in the hippocampi of the propofol-treated group at 12 hours after I/R injury (Fig. 12F, H).
10.1371/journal.pone.0035324.g012Figure 12 Inhibition of autophagy activation by propofol.
I/R was induced by 2-vessel occlusion. Propofol 100 mg/kg was administrated intraperitoneally and 3-MA (600 nM) was administered intracerebroventricularly 10 min after the onset of ischemia. The rats were treated with propofol (100 mg/kg) and the autophagy inhibitor 3-MA (600 nM) during the I/R insult and were sacrificed 12 h later for western blot analysis. (A, B, E, F) Immunoblot analyses of the I/R-injured hippocampus. The hippocampus homogenates were analyzed by western blotting using an antibody specific against each autophagy-related protein. The expression of GAPDH was also examined as the protein loading control. (C, D, G, H) The quantification of class III PI3K, Beclin-1, Bcl-2, LC3-I and LC3-II expression. Each protein (class III PI3K, Beclin-1, Bcl-2, LC3-I and LC3-II) shown in Fig. 12A, B, E, F was quantified after a densitometric scan and normalized to GAPDH. The optical densities of the respective protein bands were analyzed using Sigma Scan Pro 5 and normalized to the loading control (GAPDH). The results were expressed as the mean ± SD from six independent experiments. Statistical comparisons were conducted using an ANOVA followed by the Tukey test. **p < 0.01 vs. control group; ##
p < 0.01 vs. I/R group.
Discussion
The major findings of the present study are that propofol therapy achieves a greater inhibition of autophagic cell death in both in vitro and in vivo models of neuronal ischemia.
We demonstrated the positive effect of propofol on the inhibition of OGD-induced autophagosomes in neuronal PC12 cells. The formation of such autophagosomes is essential for autophagic cell death [8]–[10], as demonstrated by the increased numbers of LC3-II-positive neurons and the increased expression of class III PI3K and Beclin-1, which are key proteins in autophagy induction [22], [23].
The prevention of neuron death by the inhibition of autophagy after hypoxic-ischemic injury has been documented to be dependent on an autophagy induction-related gene, Atg7. The present results indicate that a group of factors including class III PI3K, Beclin-1 and Bcl-2 are also engaged in the neuroprotection of propofol against OGD-induced damage in neuronal PC12 cells. The experimental evidence supporting such an argument includes the inhibition of class III PI3K-Beclin-1, the formation of autophagosomes, and the increase in the level of Bcl-2 by propofol in vitro. The role of autophagy in neurodegeneration and neuroprotection is elusive. Rapamycin, an autophagy-inducing drug, can provide protection in models of neurodegenerative diseases [24], which indicates that neurodegeneration is inhibited by autophagy [25]. However, excessive autophagic responses could become hazardous and harmful. Indeed, it has been demonstrated that mutations in lysosomal surface proteins and a variety of deficits in lysosomal enzymes are able to cause prominent neurodegeneration. The results of the present study revealed that the formation of AVs in both OGD-exposed PC12 cells and I/R-injured hippocampal neurons in rats was associated with a reduced number of cells, indicating that autophagy-related processes may promote cell death. This result agrees with those of Li et al [26], who showed that the inhibition of autophagy with lithium reduced brain injury after hypoxia-ischemia in neonatal rats. The present data also indicate that autophagic cell death was attenuated by propofol, adding a new neuroprotective mechanism for this agent that has not been reported previously.
A number of mechanisms have been associated with the neuroprotective effects of propofol, including [27] the reduction in the cerebral metabolic rate of oxygen, the antioxidant-based removal of lipophilic and hydrophilic radicals [1], [5], the activation of γ-aminobutyric acid type A receptors [28], the inhibition of glutamate receptors [29], and the reduction of the extracellular glutamate concentrations by inhibiting Na+ channel-dependent glutamate release [30] or the enhancement of glutamate uptake [2], [31]. In this study, our results demonstrated that propofol significantly reduced the degree of cell damage induced by OGD injury in neuronal PC12 cells. We found that OGD-induced cell death is associated with the activation of autophagy through the expression of class III PI3K, Beclin-1 and LC3-II, and the accumulation of autophagic vacuoles. This autophagic cell death was inhibited by the administration of propofol through the reversal of the activation mechanism during OGD.
To further validate our findings in vitro, we used a two-vessel occlusion model in rats to induce brain injury because forebrain ischemia is often expected in a clinical setting. This model could imitate cerebral ischemia resulting from acute bleeding, cardiac arrest and certain types of shock [13]. In this study, our results demonstrated that propofol significantly reduced the degree of hippocampus damage induced by I/R injury in rats. In our I/R model, the neuroprotection of propofol was less effective than that reported in models of transient focal ischemia [4], [13]. This difference could be attributed to the model itself; our model is more severe because we used a single injection of propofol rather than continuous infusion. Additionally, propofol reduced the expression of class III PI3K and Beclin-1 and increased the expression of Bcl-2. Previous studies [32] found that the brain protective effect of propofol during I/R was mediated by the inhibition of Bcl-2 dissociation from Beclin-1, resulting in a significant decrease in autophagic cell death. The interaction of Beclin-1 with Bcl-2 was diminished by I/R injury and was rescued by propofol to levels comparable with those observed in the control. These results also suggested that propofol might modulate autophagy via class III PI3K-Beclin-1-Bcl-2 dependent pathways.
There are a number of issues in this study that still must be clarified. (1) Because we used a recovery interval of 12 or 24 h for PC12 cells exposed to OGD and for rats after I/R, we cannot exclude a transient neuroprotective effect for propofol, as reported for other anesthetics [33]. However, in an incomplete cerebral ischemia and reperfusion model, propofol offered long-term neuroprotection [34]. In addition, the early evaluation of the neuroprotective effects of propofol seem to indicate the long-term improvement of brain function in rats exposed to mild brain ischemia [7]. (2) The concentrations of propofol that were used in OGD-injured PC12 cells have been reported in previous publications [35], [36], but are considered to be high compared with the commonly used clinical concentration. The total amount of propofol administered in I/R rats was in accordance with the amount used in the study by Arcadi et al [37]. A single intraperitoneal injection of 50 or 100 mg/kg propofol could significantly attenuate CA1 injury after global ischemia in rats. These doses are also considered to be high. (3) It is still unclear how propofol directly modulates the expression of autophagy-related genes and the activation of lysosomes when the brain is exposed to the I/R injury. Therefore, further in vivo and in vitro studies focusing on the regulation of autophagy-related genes and lysosomal activation will contribute to the development of specific drugs that can be used to treat and/or prevent autophagy-mediated neuronal death. Despite these limitations, our study shows that propofol is neuroprotective in PC12 cells exposed to OGD in vitro, potentially through the inhibition of autophagy activation and maturation. In a severe model of forebrain cerebral ischemia in vivo, propofol reduces the extent of the injury of hippocampal pyramidal neurons and prevents ultrastructural changes.
In summary, the present results indicated that the negative effects of OGD and I/R, including the formation of autophagosomes and autolysosomes, the increases in LC3-II, Beclin-1 and class III PI3K expression and the decrease in Bcl-2 production were all inhibited by propofol. Furthermore, in vitro OGD cultures and I/R rats exhibited an increase in cell survival following the administration of propofol. These results also suggest that autophagy might represent a novel mechanism by which I/R damage induces cell death, and the inhibition of autophagy activation and maturation by propofol might reduce I/R injury in brain. Our findings suggest a novel strategy for the development of a novel therapy for damage due to brain hypoxia.
Materials and Methods
Preparation and Incubation of Neuronal PC12 Cells
Neuronal PC12 cells were obtained from the Key Laboratory of Neurobiology, Institute of Medicine, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and cultured in RPMI 1640 medium (Gbico/Life Technologies Ltd, Paisley, Scotland) supplemented with 10% fetal bovine serum (Gibco/Life Technologies Ltd, Paisley, Scotland) and 7.5% horse serum (Gibco/Life Technologies Ltd, Paisley, Scotland) in a humidified incubator (Hira Sawa Work, Japan) at 37°C and 5% CO2. For the survival experiments, the PC12 cells were seeded (2–103 cells/well) on 96-well plates in culture medium supplemented with 10 nM mouse 7S nerve growth factor (NGF) (Sigma, Saint Louis). After 3 days, additional NGF was added (10 nM). After 6 days of culture with NGF, more than 95% of the cells appeared to be morphologically differentiated with neurites at least twice the length of the cell body diameter; the cells were exposed to combined oxygen and glucose deprivation (OGD) at 0, 0.5, 1, 3, 6 and 12 h on the seventh day.
Oxygen and Glucose Deprivation (OGD) Treatment and Assessment of PC12 Cells Injury
Combined oxygen and glucose deprivation (OGD) was performed as described previously [14], [38]. Briefly, ischemia was introduced by a buffer exchange to Hanks solution, which is an ischemia-mimetic solution (in mmol/L: 140 NaCl, 3.5 KCl, 0.43 KH2PO4, 1.25 MgSO4, 1.7 CaCl2, 5 NaHCO3, 20 HEPES, pH 7.2–7.4) and subsequently, the culture dishes were placed in a hypoxic incubator chamber (Billups-Rothenberg) equilibrated with 95% N2/5% CO2 at 37°C for 0.5, 1, 3, 6 and 12 h. The buffered Hanks solution was previously gassed with 95% N2/5% CO2 for 30 min. The control cells were incubated in RPMI 1640 medium solution and run in parallel for each condition for the indicated time periods corresponding to those of the experimental groups. Solutions of 10% Intralipid at a concentration of 50 µM (used as a vehicle control, Sigma, St Louis, MO) and propofol (10, 20 or 50 µM) (AstraZeneca UK Limited) were preincubated for 10 min before and during OGD stimulation. 3-MA (20 mM) (Sigma, 08592(fluka) [8], a specific inhibitor of autophagosome formation, was added as a positive control. For the western blot analysis of the effects of propofol on autophagy-related proteins, the PC12 cells were cultured in 60 mm dishes, harvested and probed for autophagy-related proteins after 0, 0.5, 1, 3, 6 and 12 h of OGD.
Transmission Electron Microscopic Analyses of Autophagosomes in PC12 Cells after OGD Injury
The PC12 cells were cultured in 60 mm dishes and treated with OGD for 0.5, 1, 3, 6 and 12 h. After treatment, the cells were fixed with 4.0% paraformaldehyde in phosphate-buffered saline (PBS) and then post-fixed with 2.0% glutaraldehyde in 0.1 mol/L PBS and preserved at 4°C for further processing. When the processing resumed, the cells were post-fixed in 1% osmium tetroxide in PBS, dehydrated in graded alcohols, embedded in Epon 812, sectioned with an ultramicrotome, and stained with uranyl acetate and lead citrate. The sections were examined using a transmission electron microscope (Technai 10; Philips).
Cytotoxicity Assay
Lactate dehydrogenase (LDH) leakage not only occurs during necrosis but also during apoptosis [39]. Because 3-MA interferes with the MTT assay, LDH leakage was assessed as an index of cell death after the PC12 cells were treated with OGD [20], [40]. To examine the contribution of propofol to the OGD-induced death of PC12 cells, the cells were treated with propofol (10, 20 or 50 µmol/L) and 3-MA (20 mmol/L) during OGD. LDH leakage was measured 6 h after OGD. Briefly, after OGD treatment, the supernatant of the cell culture was harvested. The PC12 cells were rinsed with PBS and lysed with 1% Triton X-100 at 37°C for 30 min. The supernatants and cell lysates were prepared following the manufacturer’s instructions for the LDH assay using a cell viability assay kit (Nanjing Jiancheng Bioengineering Institute, A020). The absorbance value (A) at 440 nm was determined with an automatic multiwell spectrophotometer (Bio-Rad Laboratories, Hercules, CA, USA). LDH leakage was calculated using the following formula: LDH leakage (%) = (A positive/A positive blank)/(A negative/A negative blank) × 100% [41].
Cell Viability Assay
PC12 cell viability was determined with a 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, M5655use) assay 6 h after OGD treatment in accordance with a previously described method [42]–[44]. To examine the contribution of propofol to OGD-induced PC12 cell death, PC12 cells were treated with propofol (10, 20 or 50 µmol/L) and the autophagy inhibitor Bafilomycin A1 (Baf, 4 µmol/L, Alexis Biochemicals, Alx-380–030) during OGD. Briefly, the MTT solution was added to the culture medium (final concentration = 500 µg/mL) at the end of the OGD treatment. The reaction was terminated by the addition of 10% acidified SDS (100 µL) to the cell culture 4 h after the MTT addition. The absorbance value (A) was measured at 570 nm using a multiwell spectrophotometer (Bio-Rad Laboratories). The percentage of cell death was calculated using the following formula: cell death (%) = (1-A of experiment well/A of control well) × 100%.
Assay of the Effects of Propfol on Autophagy-related Proteins
To confirm whether propofol blocks the autophagic process, the effects of propofol on autophagy-related proteins were assessed. The OGD time (6 h) and the final concentration of propofol (50 µM) were determined by pilot studies and the average blood propofol concentration during human brain surgery in our clinical project [6], [45]. For the inhibitory experiments, the cells were pre-incubated with a selective PI3K inhibitor (LY2940002, final concentration 50 µM, Cell Signaling Technology, #9901) for 10 min, and then treated with OGD and/or propofol or Intralipid. These drugs were diluted in serum-free medium prior to their addition to the cultures. The cells were randomized into seven groups: Group 1, control (untreated); Group 2, cells were subjected to 6 hours of OGD; Group 3, cells treated with OGD and propofol (50 µM); Group 4, cells treated with OGD and Intralipid (50 µM); Group 5, cells treated with OGD and LY294002 (50 µM, prepared 10 min prior to the OGD treatment); Group 6, cells treated with OGD and LY294002 (50 µM) and propofol (50µM); and Group 7, cells treated with OGD and LY294002 (50 µM) and Intralipid (50 µM). For the western blot analysis of the effects of propofol on autophagy-related proteins, the PC12 cells were cultured in 60-mm dishes and harvested after 6 h of OGD.
Transfection of Cells with Beclin1 siRNA
The cells were transiently transfected with small interference RNA (siRNA) against Beclin-1 (Refseq number: NM-001034117, NM-053739; siRNA ID: 195717; Ambio INC, Austin, TX, USA), a principal regulator in the formation of autophagosomes and the initiation of autophagy through the PI3K class III pathway, using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA). The cells transfected with siRNA #3 were used as a negative control. The transfected cells were randomized into four groups followed by immunoblot assay: Group 1, control (untreated); Group 2, subjected to 6 h of OGD; Group 3, treated with OGD and propofol (50 µM); and Group 4, cells treated with OGD and Intralipid (50 µM). For the western blot analysis of the effects of propofol on autophagy-related proteins, the PC12 cells were cultured in 60-mm dishes and harvested after 6 h of OGD.
Animal and Surgical Protocol
Male Sprague-Dawley rats weighing approximately 250–300 g were purchased from the Experimental Animals Center of Shanghai Jiaotong University (certificate No. 201000082, Grade II) and surgically prepared for I/R injury as described previously [13]. All the procedures were performed in accordance with the Guide for Care and Use of Laboratory Animals published by the National Institutes of Health (Guide for the Care and Use of Laboratory Animals, 1996). The Animal Research Committee of Shanghai Jiaotong University in China approved the protocol.
All the rats were fasted for 8–12 h, and water was provided ad libitum; other conditions were constantly controlled. Anesthesia was induced in a Plexiglas chamber with 4% halothane; the animals were then tracheally intubated and mechanically ventilated with 1.5% halothane in 30% O2/70% N2O. No muscle relaxants were given during the anesthesia. The left femoral artery was cannulated to monitor the blood pressure and to collect the blood, and the right external jugular vein was used for drug administration and for blood reinjection. Digital thermistor probes (Multi-thermistor Meter D321; Technol Seven, Yokohama, Japan) were placed in the rectum to monitor the core temperature, which was maintained at 37±0.5°C using an electrically heated blanket. The arterial blood samples were collected for blood gas analysis after the isolation of the bilateral common carotid arteries from the carotid sheaths using a ventral midline incision. If the blood gas parameters were PO2 90–140 mmHg, PCO2 35–45 mmHg, pH 7.35–7.45, GI 150–180 mg/dl, cerebral ischemia was induced by clamping the common carotid arteries with small vascular clips and inducing hypotension (MAP: 40±5 mm Hg) by withdrawing and injecting blood for 10 min. Forebrain ischemia was confirmed by an EEG indicating the complete suppression of electroencephalographic activity. Thereafter, the clips were removed, and the withdrawn blood was reinfused. At the end of the anesthesia process, the vascular catheters were removed, and the wounds were sutured. The endotracheal catheter was extubated until there was a recovery of spontaneous respiration and the righting reflex.
Sham-operated rats underwent the same procedures, except for the I/R. To observe the time course for the histochemical and immunohistochemical analysis following I/R, the animals were sacrificed at 0 (sham-operated rats, Sham), 1, 3, 6, 12 and 24 h post-I/R (n = 5/group) by transcardial perfusion of 0.9% normal saline, followed by 4% paraformaldehyde in 100 mM phosphate-buffered saline. To study the effects of propofol and the autophagy inhibitor 3-methyladenine (3-MA) by histochemical, immunohistochemical and transmission electron microscopic analyses, the rats received an intracerebral ventricular injection of 600 nmol 3-MA (purchased from Sigma, 08592 (Fluka) and dissolved in normal saline by heating the solution to 60–70°C immediately before injection), an intraperitoneal injection of propofol (10, 50, and 100 mg/kg) or an intraperitoneal injection of vehicle (Intralipid, 100 mg/kg) 10 min after I/R and were sacrificed 12 h after I/R. The left femoral artery was cannulated to measure the arterial pH, PaCO2, PaO2 and blood glucose concentration. These parameters (Table 1) were measured before and during I/R and 60 min after I/R. The body temperature was closely monitored with a rectal probe and maintained at 37.0 ± 0.5°C with a heating pad (Institute of Biomedical Engineering, CAMS, BME-412A Animal Regulator, 308005669) during and after surgery until recovery from anesthesia.
Transmission Electron Microscopy of Autophagosomes in the Hippocampus after I/R Injury
To observe the time course of the I/R-induced formation of autophagosomes and morphologic changes in the organelles by TEM, the rats were transcardially perfused with phosphate-buffered saline (PBS) (pH 7.4) followed by PBS containing 4% paraformaldehyde (pH 7.4) 1, 3, 6, 12 and 24 h after I/R. To study the effects of the propofol and 3-MA by TEM, the rats were sacrificed 12 h after I/R. The brain tissue samples of 1 cubic millimeter that were removed from the ischemic core of the hippocampus were first immersed in 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.2), post-fixed in 1% osmium tetroxide in 0.1 mol/L phosphate buffer (pH 7.4), dehydrated in graded ethanol series, and flat embedded in Araldite. Ultrathin sections (40–60 nm thick) were placed on grids (200 mesh), and double-stained with uranyl acetate and lead citrate. The sections were observed under a Philips CM-120 electron microscope (Philips).
Histochemical Analyses
The rats were deeply anesthetized with pentobarbital (25 mg/kg i.p.) and fixed by cardiac perfusion with 4% paraformaldehyde buffered with 0.1 mol/L phosphate buffer (pH 7.2) containing 4% sucrose for light microscopy. For light microscopy, the brain tissues were quickly removed from the rats and further immersed in the same fixative for 2 hours at 4°C. The samples processed for paraffin embedding were cut into 5-µm thick sections using a semi-motorized rotary microtome and placed on silane-coated glass slides. For routine histological studies, the paraffin sections were stained with thionine. For the light microscopy observations, semithin sections were cut 1-µm thick with an ultramicrotome (Ultracut N; Reichert-Nissei, Tokyo, Japan) and stained with thionine.
Immunohistochemical Analyses
The rats were deeply anesthetized with pentobarbital (150 mg/kg i.p.) and then perfused transcardially with 4% paraformaldehyde in 0.1 mol/L PBS (pH 7.4) 6 or 24 h after I/R. Immunohistochemistry was performed on 18-µm thick cryostat sections. For immunofluorescence labeling, the sections were preincubated for 45 minutes in 15% serum and 0.3% Triton X-100 in PBS and then incubated overnight at 4°C with the primary antibody (Anti-LC3B antibody, polyclonal, Abcam, ab64781) in 1.5% serum and 0.1% Triton in PBS, washed in PBS, and incubated for 2 h in fluorochrome-coupled secondary antibody (FITC, Yeasen, Lot: 94766) at room temperature. The sections were then rinsed in PBS and mounted with FluorSave with the nuclear stain 4′,6′-diamidino-2-phenyl indole dihydrochloride (DAPI) (Sigma, 32670; 5µg/ml). A LSM 510 Meta confocal microscope was used for the confocal laser microscopy. The confocal images were displayed as individual optical sections. For the double labeling experiments, the immunoreactive signals were sequentially visualized in the same section with two distinct filters, with acquisition performed in separated mode. The sections were viewed under high power (200×) with a fluorescence microscope (Eclipse TE 2000, Nikon) with a Nikon digital camera, and the images were visualized in a computer monitor. For the quantification of LC3-II immunostaining, 10 microscopic fields (200× magnification) in each section across ischemic hippocampus regions in the ipsilateral hemisphere were analyzed. Three sections were used for each animal. The number of cells with LC3-II immunoreactivity in each field was counted by an examiner who was blind to the experimental conditions.
Protein Preparation and Immunoblotting
We deeply anesthetized the rats with an overdose of pentobarbital (100 mg/kg i.p.) and subsequently cut and quickly removed the brain tissues from ischemic hippocampus area and the corresponding area of sham-operated rats. We immediately placed all of the tissue into dry ice-cold collecting tubes and stored them at -80°C until further analysis. The PC12 cells were cultured in 60-mm dishes and harvested and rinsed twice with ice-cold PBS after OGD. We later homogenized these tissue samples and cells in cold Radio Immunoprecipitation Assay lysis buffer (Beyotime Corporation, Nanjing, Jiangsu, China) with a 1% protease-inhibitor cocktail (Sigma-Aldrich, USA), followed by centrifugation at 14,000×g for 10 min at 4°C. We determined the protein concentration using a BCA protein assay kit (Beyotime Corporation, China). After heating the aliquots of protein (30 µg) in SDS-PAGE protein loading buffer (Beyotime Corporation, china) at 95°C for 10 min, we separated them on SDS-PAGE gels and transferred the proteins to PVDF membranes (Millipore Corporation, Billerica, MA, USA) for immunoblotting. We incubated the membranes in blocking buffer (5% milk in Tris-buffered saline [TBS] with 0.1% Tween 20) for 1 h at room temperature, followed by an overnight incubation 4°C with primary antibodies against class III PI3K (Monoclonal, Cell Signaling Technology, #3358), Beclin-1 (polyclonal, Cell Signaling Technology, #3738), LC3 (polyclonal, MBL International, PD014), and Bcl-2 (polyclonal, Cell Signaling Technology, #2870). We then washed off the primary antibody three times in TBS, incubated the membranes with horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:5000, Santa Cruz Corporation) for 2 h at room temperature, and washed them three times in TBS. We detected the immunoreactive blots with enhanced chemiluminescence (Amersham Bioscience, Piscataway, NJ, USA) and visualized them on X-ray film (Kodak, Shanghai, China). GAPDH (1:2000; monoclonal, Cell Signaling Technology, #2118) was used as the loading control. The signal intensity of primary antibody binding was quantitatively analyzed with Sigma Scan Pro 5 and was normalized to a GAPDH loading control. The statistical analyses were performed by a one-way analysis of variance (ANOVA) followed by the Tukey test. The differences were considered significant when p< 0.05.
Statistical Analysis
We analyzed the data using SAS software (Version 8.01, SAS Institute Inc., Cary, NC, USA) and reported the results as the mean±SD. We analyzed the variance in neuronal damage and the number of LC3-II-positive cells in rat hippocampal pyramidal neurons at a given testing time using a one-way ANOVA. For the between-group variance in the ultrastructural changes and the immunoblot analyses of the PC12 cells or rat hippocampal pyramidal neurons at a given testing time, we performed an ANOVA followed by the Tukey test. We considered a result statistically significant when P<0.05.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported through grants from the Youth Scientific Research Project, Bureau of Health, Shanghai, China (2009Y039), the B. Braun Anesthesia Scientific Research Project (2009 to Derong Cui) and Shanghai Jiaotong University Medical and Technology Fund (2009 to Li Wang). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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PLoS OnePLoS ONEplosplosonePLoS ONE1932-6203Public Library of Science San Francisco, USA 22530024PONE-D-11-2606910.1371/journal.pone.0035453Research ArticleBiologyBiochemistryProteinsProtein InteractionsTransmembrane Transport ProteinsMicrobiologyBacterial PathogensGram PositiveHost-Pathogen InteractionMedical MicrobiologyMicrobial PathogensMedicineInfectious DiseasesBacterial DiseasesMycobacteriumTuberculosisA β-Lactamase Based Reporter System for ESX Dependent Protein Translocation in Mycobacteria Reporter for ESX-Dependent Protein TranslocationRosenberger Tobias
1
Brülle Juliane K.
1
Sander Peter
1
2
*
1
Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland
2
Nationales Zentrum für Mykobakterien, Zurich, Switzerland
Nigou Jérôme EditorFrench National Centre for Scientific Research - Université de Toulouse, France* E-mail: [email protected] and designed the experiments: TR PS. Performed the experiments: TR JKB. Analyzed the data: TR JKB PS. Wrote the paper: TR PS.
2012 18 4 2012 7 4 e3545319 12 2011 16 3 2012 Rosenberger et al.2012This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.Protein secretion is essential for all bacteria in order to interact with their environment. Mycobacterium tuberculosis depends on protein secretion to subvert host immune response mechanisms. Both the general secretion system (Sec) and the twin-arginine translocation system (Tat) are functional in mycobacteria. Furthermore, a novel type of protein translocation system named ESX has been identified. In the genome of M. tuberculosis five paralogous ESX regions (ESX-1 to ESX-5) have been found. Several components of the ESX translocation apparatus have been identified over the last ten years. The ESX regions are composed of a basic set of genes for the translocation machinery and the main substrate - a heterodimer. The best studied of these heterodimers is EsxA (ESAT-6)/EsxB (CFP-10), which has been shown to be exported by ESX-1. EsxA/B is heavily involved in virulence of M. tuberculosis. EsxG/H is exported by ESX-3 and seems to be involved in an essential iron-uptake mechanism in M. tuberculosis. These findings make ESX-3 components high profile drug targets. Until now, reporter systems for determination of ESX protein translocation have not been developed. In order to create such a reporter system, a truncated β-lactamase (‘bla TEM-1) was fused to the N-terminus of EsxB, EsxG and EsxU, respectively. These constructs have then been tested in a β-lactamase (BlaS) deletion strain of Mycobacterium smegmatis. M. smegmatis ΔblaS is highly susceptible to ampicillin. An ampicillin resistant phenotype was conferred by translocation of Bla TEM-1-Esx fusion proteins into the periplasm. BlaTEM-1-Esx fusion proteins were not found in the culture filtrate suggesting that plasma membrane translocation and outer membrane translocation are two distinct steps in ESX secretion. Thus we have developed a powerful tool to dissect the molecular mechanisms of ESX dependent protein translocation and to screen for novel components of the ESX systems on a large scale.
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Introduction
Tuberculosis (TB) is a chronic, contagious disease caused by several members of the Mycobacterium tuberculosis complex [1]. Although marginalized in America and central Europe, TB remains an overwhelming burden on humanity. Approx. 2 billion people - about a third of the world's population - are carrier of the pathogen, making TB a pandemic. Among these people, 5–10% develop symptoms and become infectious themselves [2]. An estimated 1.7 million infected people die from TB every year.
Subversion of the normal progression of the phagosomal compartment into an active, bactericidal lysosomal compartment is one of the key features of M. tuberculosis pathogenicity [3], [4]. As an additional resistance factor, mycobacteria possess a nearly impenetrable cell envelope which protects the bacteria against physical and chemical stress. This cell envelope also plays a crucial role in intrinsic drug resistance in pathogenic mycobacteria, and is one of the key features of persistence in latency [5]. The unique mycobacterial cell envelope consists of a phospholipid bilayer plasma membrane (PM), followed by a periplasmatic space (PP) with two electron dense layers of unconfirmed identity [6]. The inner of these layers, located proximal to the PM, appears to be granular. The outer layer represents at least a part of the peptidoglycan-arabinogalactan polymer [7]. Furthermore, the bacteria posses an additional membrane called mycobacterial outer membrane (MOM). The MOM is mainly composed of long chain mycolic fatty acids (C60-C90) with free intercalating glycolipids. It is covalently linked to the arabinogalactan-peptidoglycan layer [8] and presents a veritable permeability barrier. The outermost layer of the cell envelope is a capsule composed of polysaccharides, proteins and small amounts of lipids [9].
Protein secretion is essential for all bacteria in order to interact with their environment. In monoderm bacteria export systems that translocate proteins across the PM are sufficient. In diderm bacteria such as the Gram-negatives or mycobacteria secreted proteins have to overcome not only the PM but also the second hydrophobic permeability barrier – the outer membrane. Both the general secretion system (Sec) and the twin-arginine translocation system (Tat) are functional in mycobacteria [10], [11], [12]. Interestingly, it has been shown that mycobacteria possess an accessory, non-essential SecA2 protein (compared to the housekeeping SecA1) which is involved in the export of a specific subset of proteins, e.g. the superoxide dismutase SodA [13]. Furthermore, a high amount of small, highly immunogenic proteins lacking a classical secretion signal peptide has been found in the culture filtrate of M. tuberculosis
[14]. These proteins have a size of approx. 100 amino acids (aa) and share a Trp-Xaa-Gly (WXG) motif [15]. The most prominent members of this protein family are the early secreted antigen target, 6 kDa (ESAT-6, EsxA) and the culture filtrate protein, 10 kDa (CFP-10, EsxB). Genes coding for WXG-100 family proteins are found in the genome of all mycobacteria and in a wide range of actinobacteria and low G+C content Gram-positives [15]. Most of these WXG-100 proteins are predicted to be exported by a novel type of protein translocation system in mycobacteria often referred to as Type VII secretion system (T7SS) [16] or more general as WXG-100 secretion systems (WSS) [17], [18]. In mycobacteria, where the T7SS have been discovered first, the protein translocation machineries as well as the corresponding genomic regions are commonly referred to as ESAT-6 secretion systems (ESX). This term will be used throughout this article. In M. tuberculosis five paralogous ESX regions are annotated (ESX-1 to ESX-5). Several of these ESX regions are also present in other members of the genus Mycobacterium
[19]. Outside the genus Mycobacterium, multiple ESX regions have not been found so far. ESX gene clusters typically also encode proteins of the mycobacterial specific enigmatic PE and PPE families, named after their N-terminal proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) motifs [20].
The first ESX protein translocation system found in M. tuberculosis (ESX-1) is required for full virulence [21]. Most genes of ESX-1 are located in the region of difference-1 (RD-1), which is prominently absent in the attenuated vaccine strain Mycobacterium bovis BCG [22] and in Mycobacterium microti
[23]. The exact function of ESX-1 and its translocation substrates are not fully understood yet. Among the reported functions are block of phagosome maturation [24], suppression of proinflammatory cytokine production [25] and disruption of membranes [26]. Furthermore, it has been shown that ESX-1 translocation plays an essential role in mycobacterial DNA transfer in M. smegmatis
[27]. Deletions in the ESX-1 gene cluster seem to increase the donor function, whereas the same deletions abolish reception of transferred DNA. Functional studies on the ESX-2 and ESX-4 systems have not been published so far. Whole genome studies suggest that neither of these two systems is required for in vitro growth or virulence [28], [29]. Mycobacterium leprae is missing both the ESX-2 and ESX-4 region from its minimal genome [19]. These findings indicate a negligible function of these two ESX clusters. ESX-3 is present in all mycobacterial genomes sequenced so far. In M. tuberculosis and in M. smegmatis several studies have shown that ESX-3 is involved in iron import [30], [31]. Siegrist et al. found evidence, that iron bound to secreted mycobactin can not be utilized in ESX-3 deletion strains [32]. Although M. smegmatis tolerates ESX-3 deletions quite well, the system seems to be essential in M. tuberculosis since corresponding deletion mutants could not be generated so far [28], [31]. ESX-5 is the phylogenetically youngest ESX region and restricted to slow growing, pathogenic mycobacteria [19]. It has been show to be involved in the secretion of a set of diverse PPE and PE_PGRS proteins [33]. ESX-5 has been linked to virulence in the fish pathogen M. marinum
[33], [34] and was suggested to play a role in cell death in M. tuberculosis related pathogenesis [35].
The M. tuberculosis ESX-1 system (and supposedly the ESX-5 system) is required for full virulence, therefore discovering and characterizing its components is a focal point of mycobacterial virulence research. However, there is a growing interest in the essential ESX-3 system of M. tuberculosis
[32], [36], [37], [38], [39] since its components represent promising new targets for antimycobacterial drugs. The absence of ESX-protein-translocation reporter limits characterization of ESX systems, especially the identification of components not encoded in the ESX region. In this study we present such a reporter system for studying ESX protein translocation.
Materials and Methods
Bacterial strains and growth conditions
Mycobacterium smegmatis SMR5, a derivative of M. smegmatis mc2155, carrying a non-restrictive rpsL mutation conferring streptomycin resistance [40] was grown on Middlebrook 7H10 agar supplemented with 10% oleic acid albumin dextrose catalase (OADC, Difco) and in liquid Middlebrook 7H9 supplemented with 10% OADC and with Tween 80 (0.05%) to avoid clumping. When appropriate, antibiotics were added at the following concentrations: ampicillin, 120 µg/ml; streptomycin, 100 µg/ml; hygromycin, 50 µg/ml; kanamycin, 50 µg/ml. The cells were grown at 37°C. Strain designations were as follows: M. smegmatis SMR5, M. smegmatis ΔblaS, a derivative of M. smegmatis SMR5 with deleted blaS (MSMEG_2658) locus and M. smegmatis ΔblaS ΔeccD
3, a derivative of M. smegmatis ΔblaS with an additional deletion in eccD
3 (MSMEG_0623).
Plasmids
A series of heterologous fusion protein constructs were cloned and expressed, using the epichromosomal, high copy number shuttle vector pOLYG [41]. All fusion constructs consist of a truncated form of the TEM-1 β-lactamase (‘Bla TEM-1), originally identified in a clinical isolate of E. coli
[42], and a secreted protein from M. tuberculosis or M. smegmatis. The truncated form of the β-lactamase (from here on referred to simply as BlaTEM) lacks the N-terminal 19 amino acids (aa) comprising the secretion signal peptide. It is no longer exported on its own. The construction and schematic organization of the reporter constructs is shown in
figure 1
. Unmarked targeted gene deletions were generated using the mycobacterial suicide vector pMCS5 (MobiTec) with an additional hygromycin resistance cassette [43] and a rpsL gene [40]. The deleted β-lactamase blaS from M. smegmatis was complemented in trans using the integrative vector pMV361-blaS, a derivative of pMV361 [44]. All plasmids used in this study are listed in table S1 (supporting material).
10.1371/journal.pone.0035453.g001Figure 1 Schematic representation of the reporter fusion constructs.
Shown are only the promoter regions, the truncated β-lactamase (blaTEM) and the fused ORF with restriction endonuclease sites for cloning (not drawn to scale). Additional epitope tags for anti HA antibodies are also annotated. The roman numbers on the left side correspond to the vector numbers. Along with blaTEM the vector pI-blaTEM contains the ORF encoding the 40 aa N-terminal sec-secretion signal peptide of FbpB (Rv1886c). The vector pII-blaTEM originated from pI-blaTEM and contains the additional 285 codons of the mature FbpB. In the plasmid pIII-blaTEM blaTEM is C-terminally fused to the ORF fbpB (325 codons). In pIV-blaTEM and pV-blaTEM the ORF esxG (Rv0287, 96 codons) is either C- or N-terminally fused to blaTEM. pVI-blaTEM originates from pIV-blaTEM with esxG substituted for esxB (Rv3874, 99 codons). In pVII-blaTEM and pVIII-blaTEM esxG is substituted for either esxUmt (Rv3445c, 104 codons) or esxUms (MSMEG_1538, 102 codons). 0 stands for pOLYG-blaTEM expressing only the truncated version of Bla TEM-1 (267 aa) lacking the 19 aa secretion signal peptide (BlaTEM). Note that the constructs 0 and I-III are expressed by the fbpB promoter (368 bp region upstream of the start codon) whereas the esx constructs are under the control of the esxGmt promoter (500 bp region upstream of the start codon).
Targeted gene replacements
Replacement of the targeted genes in this study - MSMEG_2658 (blaS) and MSMEG_0623 (eccD
3) - were done by application of the rpsL counter selection strategy [40]. Therefore, electrocompetent streptomycin-resistant M. smegmatis SMR5 derivatives were transformed with the suicide plasmids pMCS5-rpsL-hyg-ΔblaS and pMCS5-rpsL-hyg-ΔeccD
3. These vectors carry approx. 1 kbp genomic regions adjacent to the targeted gene and an unmarked deletion in the targeted gene. Transformants were grown on hygromycin containing media and afterwards counter selected using streptomycin. A point mutation in the rpsL gene coding for the ribosomal protein S12 renders M. smegmatis SMR5 streptomycin resistant. Thus, transformants integrating the knock out plasmid that encodes the rpsL
+ locus by single cross-over homologous recombination become sensitive to streptomycin again. A second recombination event results in deletion of the target gene (or revision to wild type) and restores the streptomycin resistant and hygromycin sensitive phenotype. The deletion mutant strains were confirmed by Southern blot analysis using specific DNA probes (
Fig. 2
).
10.1371/journal.pone.0035453.g002Figure 2 Strategy for targeted generation of mutants and Southern blot analysis.
(A) Schematic drawing of the blaS (MSMEG_2658) genomic region of the wild-type and the knock out strain (not drawn to scale). Southern blot analysis confirms the deletion of blaS from the genome of M. smegmatis. Genomic DNA of M. smegmatis was digested with restriction endonuclease MluI. A DIG labeled PCR fragment from one of the flanking regions was used as a probe. The wild-type band (wt) was calculated to be 3.39 kbp. The 3′ single cross over (sco) bands correspond to lengths of 9.451 kbp and 2.483 kbp, respectively. The knock out strain (Δ) corresponds to a length of 2.483 kbp. (B) Schematic drawing of the eccD
3 (MSMEG_0623) genomic region of the wild-type and the knock out strain (not drawn to scale). Southern blot analysis confirms the deletion of eccD
3 from the genome of M. smegmatis ΔblaS. Genomic DNA was digested with restriction endonuclease Tth111I. The wild-type band (wt) was calculated to be 4.309 kbp. The 5′ single cross over (sco) bands correspond to lengths of 10.316 kbp and 4.309 kbp, respectively. The knock out strain (Δ) corresponds to a length of 5.680 kbp.
Minimal inhibitory concentration (MIC) determination
Broth microdilution tests were performed in a microtiter plate in a total volume of 100 µl. Bacterial strains were pre-cultured in 7H9 broth supplemented with Tween and OADC as described above. Freshly grown cultures were diluted to an OD600 of 0.015, and incubated in 7H9 broth in the presence of 2-fold serial dilutions of ampicillin in the range between 400 µg/ml and 0.8 µg/ml. The minimal inhibitory concentration (MIC) is defined as the drug concentration at which no visible growth is observed by eye after an incubation time of 72 h, corresponding to 24 generations.
Preparation of cell extracts and Western blot analysis
M. smegmatis from 10 ml cultures were harvested and resuspended in phosphate-buffered saline (PBS). After a washing step, the cells were disrupted and homogenized by sonication in an ice bath (Elma, Transsonic T460H) for 1–2 hours. Culture filtrate was concentrated using the Amicon Ultra-15 system (Millipore). Proteins were separated by SDS-PAGE (12.5%) and analyzed by Western blot. Antibodies against the β-lactamase were purchased at Antikoerper-online.de.
Results
Targeted inactivation of blaS (MSMEG_2658)
M. smegmatis is naturally resistant to β-lactam antibiotics due to the presence of an exported β-lactamase as demonstrated by Flores et al.
[45] and confirmed by us (
Tab. 1
). In order to use a β-lactamase as a reporter in M. smegmatis, its native major β-lactamase BlaS (MSMEG_2658) had to be disrupted first. The genomic blaS was inactivated by targeted gene replacement using the suicide vector pMCS5-rpsL-hyg-ΔblaS. A Southern blot analysis confirming the deletion of blaS is shown in
figure 2
. The resulting strain M. smegmatis ΔblaS was about 16 times more susceptible to ampicillin than the wild-type strain (
Tab. 1
).
Reporter vectors
Deletion of its native β-lactamase renders M. smegmatis ΔblaS highly susceptible to ampicillin. This is a prerequisite to use β-lactamases as selectable reporters. We constructed and expressed fusion constructs using the TEM-1 β-lactamase (Bla TEM-1) [42]. Compared to other β-lactamases, Bla TEM-1 has the significant advantage of being compatible with both Sec and Tat signal sequences, because it does not have to be folded prior to its translocation as shown by McCann et al.
[46]. In order to investigate protein translocation in M. smegmatis, particularly ESX-secretion, eight reporter vectors (pI-blaTEM to pVIII-blaTEM) were constructed. These vectors contain promoters and parts of or entire open reading frames (ORF) of Sec- and ESX-dependent secreted proteins from M. tuberculosis (
Fig. 1
). In case of pVIII-blaTEM the Esx part (EsxU) of the fusion protein is derived from M. smegmatis. The ORF of each secreted protein was ligated in frame with the ORF of blaTEM (a truncated version of bla TEM-1 without the 19 codons coding the secretion signal peptide). The backbone plasmid for all reporter plasmids was the shuttle vector pOLYG [41]. The vectors were created in a modular design, facilitating the construction of similar constructs as explained in
figure 1
. pOLYG-blaTEM expressing only the truncated form of Bla TEM-1 was constructed as a negative control. BlaTEM without additional secretion signal is suspected to be localized in the cytoplasm and therefore not to confer ampicillin resistance.
Expression of the reporter fusion constructs and ampicillin susceptibility testing in M. smegmatis ΔblaS
Cell extract and culture supernatant was analyzed for the presence of the reporter protein. Expression of the reporter constructs at the protein level was shown by Western blot analysis with an antibody specific for Bla TEM-1. BlaTEM-1 was readily detected in the cell extracts (
Fig. 3
) but not in the concentrated culture supernatant (data not shown). Ampicillin susceptibility of the β-lactamase (BlaS) knock out strains with or without reporter plasmids was determined by minimal inhibitory concentration (MIC) assays. The results for each strain are shown in
table 1
. All strains were also tested for susceptibility towards hygromycin (resistance conferred by backbone of the pOLYG vectors) and kanamycin (an unrelated antibiotic). The MIC determination shows that all plasmid bearing strains were equally resistant to hygromycin (MIC>500 µg/ml) compared to the not transformed parental strains which were equally susceptible to hygromycin (MIC 20 µg/ml). This suggests a similar reporter plasmid copy number in all strains. All strains – parental and reporter – were found to be equally sensitive to kanamycin (MIC 12.5 µg/ml). Kanamycin binds to the 30S subunit of bacterial ribosomes - an intracellular target (data not shown). Due to the results of the kanamycin resistance determination it is unlikely that the introduced genetic alteration generally heightened the membrane permeability for antibiotics.
10.1371/journal.pone.0035453.g003Figure 3 Protein expression of BlaTEM reporter constructs shown by Western blot analysis.
Fusion-proteins expressed from the nine reporter vectors (pOLYG-blaTEM (0) and pI-blaTEM to pVIII-blaTEM) were separated using SDS-PAGE (12.5% Tris-HCl gels) and blotted onto PVDF membrane. The membrane was incubated with an antibody against the β-lactamase TEM-1. (A) Protein expression of pOLYG-blaTEM (calculated mass 31.8 kDa) and the three Sec dependent FbpB β-lactamase fusion-constructs (pI-blaTEM, pII-blaTEM and pIII-blaTEM). Their masses were calculated to be 31.5 kDa, 33.3 kDa, 61.5 kDa and 62.2 kDa, respectively. The apparent molecular weight corresponds to the mass of a mature protein without secretion signal sequence. (B) Expression of β-lactamase fusion products associated with one of the three ESX protein translocation systems in M. smegmatis ΔblaS: EsxG (ESX-3), EsxB (ESX-1) and EsxU (ESX-4) expressed from pIV-blaTEM (40.7 kDa), pVI-blaTEM (41.7 kDa), pVII-blaTEM (42.3 kDa) and pVIII-blaTEM (42.3 kDa). Although the C-terminally fused “EsxG-BlaTEM” (from pV-blaTEM; 41.5 kDa) and both ESX-4 associated N-terminally fused “BlaTEM-EsxU” are translated, no ampicillin resistant phenotype could be observed in the MIC assays. As a positve control (+) lysate from E. coli expressing Bla TEM-1 from pGEM-T easy (Promega) was used. The negative control (−) consists of lysate resulting from M. smegmatis ΔblaS with pOLYG.
10.1371/journal.pone.0035453.t001Table 1 Ampicillin MIC of M. smegmatis ΔblaS strains with or without reporter plasmid.
strain plasmid reporter MIC (µg/mL) amp
M. smegmatis SMR5 none none 200–400
M. smegmatis ΔblaS
none none 12.5–25
M. smegmatis ΔblaS-blaS
pMV361-blaS (integrated) BlaS 200–400
M. smegmatis ΔblaS
pOLYG-blaTEM
BlaTEM 12.5–25
M. smegmatis ΔblaS
pI-blaTEM
a
Sec-BlaTEM 100–200
M. smegmatis ΔblaS
pII-blaTEM
a
Sec-BlaTEM-‘FbpB 200–400
M. smegmatis ΔblaS
pIII-blaTEM
a
FbpB-BlaTEM 200–400
M. smegmatis ΔblaS
pIV-blaTEM
b
BlaTEM-EsxG >400
M. smegmatis ΔblaS
pV-blaTEM
b
*
EsxG-BlaTEM 12.5–25
M. smegmatis ΔblaS
pVI-blaTEM
c
BlaTEM-EsxB 100–200
M. smegmatis ΔblaS
pVII-blaTEM
d
BlaTEM-EsxUMt
12.5–25
M. smegmatis ΔblaS
pVIII-blaTEM
d
BlaTEM-EsxUMs
12.5–25
Predicted export systems:
a ) Sec,
b ) ESX-3,
c ) ESX-1,
d ) ESX-4.
* Export signal supposedly not recognized since internal.
The MIC data show that the ampicillin sensitive M. smegmatis ΔblaS strain can be complemented with pMV361-blaS, thereby restoring its natural resistance. M. smegmatis ΔblaS transformed with pOLYG-blaTEM (a vector expressing a truncated version of Bla TEM-1 without secretion signal sequence) remains susceptible to ampicillin. Insertion of the Sec signal sequence from the fibronectin binding protein B (Antigen 85B, Rv1886c) FbpB (pI-blaTEM) confers ampicillin resistance (MIC 100–200 µg/ml). The resistance is even higher when in addition to the signal peptide the entire FbpB is fused to BlaTEM. The expression cassette: Sec-BlaTEM-FbpB is expressed by pII-blaTEM (MIC 200–400 µg/ml). FbpB-BlaTEM is expressed by pIII-blaTEM and also confers high level ampicillin resistance (MIC 200–400 µg/ml). These results coincided with results from McCann et al.
[46].
In order to investigate the functionality of each of the three annotated ESX systems of M. smegmatis (ESX-1, ESX-3 and ESX-4) we created a new reporter system by fusing BlaTEM with a M. tuberculosis protein supposed to be specific for each system. EsxB (CFP-10, Rv3874) located in the ESX-1 cluster, EsxG (Rv0287) located in the ESX-3 cluster and EsxU (Rv3445c) located in the ESX-4 cluster were chosen. The vector pIV-blaTEM expressed fusion protein BlaTEM-EsxG and the vector pV-blaTEM the reverse constellation, i.e. EsxG-BlaTEM (
Fig. 1
). pVI-blaTEM expressed BlaTEM-EsxB and pVII-blaTEM expressed BlaTEM-EsxU. Both pIV-blaTEM [BlaTEM-EsxG] and pVI-blaTEM [BlaTEM-EsxB] conferred ampicillin resistance in M. smegmatis ΔblaS. Transformation of the vector pV-blaTEM could not confer ampicillin resistance despite expression of the fusion protein (
Fig. 3B
). Vector pVII-blaTEM was also unable to confer ampicillin resistance. We hypothesized that the secretion signal of EsxU from M. tuberculosis (EsxUMt) was not recognized by M. smegmatis ESX-4. Therefore we exchanged EsxUMt with EsxU from M. smegmatis (EsxUMs) (MSMEG_1538) resulting in the reporter vector pVIII-blaTEM. Nevertheless, pVIII-blaTEM also was not able to confer ampicillin resistance in M. smegmatis ΔblaS despite expression of the fusion protein (
Fig. 3B
).
Targeted inactivation of EccD3 (MSMEG_0623) and ampicillin susceptibility testing in M. smegmatis ΔblaS ΔeccD
3
EccD
3 encodes the supposed pore protein of the ESX-3 secretion system [19]. Using M. smegmatis ΔblaS as parental strain, a deletion in eccD
3 was generated resulting in strain M. smegmatis ΔblaS ΔeccD
3. Inactivation of EccD3 did not affect susceptibility to ampicillin as compared to the parental strain (
Tab. 2
). A Southern blot confirming the deletion of eccD
3 region is shown in
figure 2
.
The strain deficient in EccD3 (M. smegmatis ΔblaS ΔeccD
3) was transformed with the reporter vectors. Ampicillin susceptibility of M. smegmatis ΔblaS ΔeccD
3 double knock out strain was determined in the same fashion as in the single knock out strain M. smegmatis ΔblaS. The results are shown in
table 2
. As expected, the strain expressing the ESX-3 related construct BlaTEM-EsxG was no longer able to confer ampicillin resistance thus confirming the specificity of the reporter construct. Recombinants expressing reporter constructs of the Sec- or ESX-1 pathway were not affected by the deletion of EccD3 with respect to the ampicillin resistance phenotype. These results were confirmed by plating the strains on 7H10 Amp (120 µg/ml) and are shown in
figure 4
.
10.1371/journal.pone.0035453.g004Figure 4 Growth of M. smegmatis strains on an agar plate containing ampicillin.
Freshly grown cultures were diluted to an OD600 of 0.015. 1 µl of each strain was streaked in a separate sector on a Middlebrook 7H10 plate containing ampicillin [120 µg/ml]. The plate was incubated for 3 days at 37°C. The parental strain in the sectors 1–5 is M. smegmatis ΔblaS. The parental strain in sectors 6–8 is M. smegmatis ΔblaS ΔeccD3. Additional roman numbers correspond to the reporter vector in each strain (cf.
tables 1
+
2
). Reporter constructs: (−): none; 0: BlaTEM; II: Sec-BlaTEM-‘FbpB; IV: BlaTEM-EsxG; and VI: BlaTEM-EsxB. Note: The reporter construct BlaTEM-EsxG from pIV-blaTEM (sector 7) is no longer able to confer ampicillin resistance when a key component of ESX-3 (EccD3) is deleted. In contrast, constructs specific for Sec (sector 6) and ESX-1 (sector 8) dependent translocation still confer an ampicillin resistance phenotype.
10.1371/journal.pone.0035453.t002Table 2 Ampicillin MIC of M. smegmatis ΔblaS ΔeccD
3 strains with or without reporter plasmid.
strain plasmid reporter MIC (µg/mL) amp
M. smegmatis ΔblaS ΔeccD
3
pOLYG-blaTEM
BlaTEM 12.5–25
M. smegmatis ΔblaS ΔeccD
3
pI-blaTEM
a
Sec-BlaTEM 100–200
M. smegmatis ΔblaS ΔeccD
3
pII-blaTEM
a
Sec-BlaTEM-‘FbpB 200–400
M. smegmatis ΔblaS ΔeccD
3
pIII-blaTEM
a
FbpB-BlaTEM 200–400
M. smegmatis ΔblaS ΔeccD
3
pIV-blaTEM
b
BlaTEM-EsxG 12.5–25
M. smegmatis ΔblaS ΔeccD
3
pVI-blaTEM
c
BlaTEM-EsxB 100–200
Predicted export systems:
a ) Sec,
b ) ESX-3,
c ) ESX-1.
Discussion
In monoderm Gram-positives, protein translocation processes are export processes. In these bacteria, protein export equals protein secretion, since the bacteria do not possess an outer membrane [17]. The majority of protein secretion in monoderm Gram-positives is conducted by the general secretion system (Sec) and the twin arginine transport system (Tat) [38]. In some Gram-positives, protein translocation can also be performed by WXG100-family secretion systems (WSS). WSS are especially found in all diderm Gram-positives, where the Sec, Tat and supposedly WSS are export systems. In mycobacteria, where WSS were described first, the terms type VII secretion system (T7SS) and ESAT-6 secretion systems (ESX) are most commonly used. The substrate translocated by the ESX system is ultimately found in the culture supernatant and thus extracellular. Actual proof that ESX are responsible for one step secretion similar to the Gram-negative type III and type IV secretion systems has yet to be given. A new bioinformatics approach suggests EccB1 and/or EccE1 as MOM channels for ESX-1 substrates [47]. However it is very well possible that an independent, hypothetical translocation machinery is located in the mycobacterial outer membrane handling the second translocation step for all the exported proteins destined for secretion into the extracellular milieu. Much progress has been made in elucidating the mycobacterial protein translocation, however there are still many puzzle pieces missing.
In 2005, Flores et al. identified the major secreted β-lactamases of M. tuberculosis (BlaC, Rv2068c) and of M. smegmatis (BlaS, MSMEG_2658) [48]. The β-lactamase knock out strains were later exploited to investigate protein secretion in M. tuberculosis and M. smegmatis, respectively. The Tat translocated BlaC [10] and the Sec translocated β-lactamase TEM-1 (Bla TEM-1) [46] - originally identified in a clinical isolate of E. coli
[42] - were used as reporter in these strains. Bla TEM-1 fulfils all criteria for an export-reporter enzyme. It is small and can easily be fused to other proteins. Bla TEM-1 is inactive in the cytoplasm and can confer ampicillin resistance in a M. smegmatis ΔblaS strain when exported [46]. For these reasons we used a truncated version of Bla TEM-1 (BlaTEM) – lacking secretion signal peptide – as a reporter in our BlaS knock out strain (M. smegmatis ΔblaS). The minimal inhibitory concentration (MIC) of ampicillin of M. smegmatis ΔblaS was about 16-fold lower compared to the wild-type strain. This ampicillin susceptible M. smegmatis ΔblaS became the parental strain for all other strains. A first series of reporter constructs was generated by C-terminal fusion of the N-terminally truncated Bla TEM-1 (lacking its native secretion signal peptide) to several variants of the fibronectin binding protein B (FbpB; antigen 85b of M. tuberculosis). FbpB is secreted via the Sec pathway. Ampicillin resistance was conferred due to expression and export of the heterologous BlaTEM proteins. M. smegmatis ΔblaS expressing BlaTEM without a signal peptide remained sensitive to ampicillin. These results coincided with results from McCann et al.
[46].
We established an ESX-specific reporter system by fusing blaTEM N-terminally and in frame with genes encoding proteins associated with the ESX translocation systems such as esxB (cfp-10, Rv3874), esxG (Rv0287) and esxU (Rv3445c). The reporter constructs with their suspected translocation machineries are drawn in
figure 5
. The constructs BlaTEM-EsxB and BlaTEM-EsxG conferred ampicillin resistance in M. smegmatis ΔblaS. EsxB possesses a C-terminal secretion signal sequence [49]. Here we showed that EsxG bears an analogous sequence which is suspected to be specific for ESX-3 protein translocation. The N-terminal fusion-construct EsxG-BlaTEM did not confer ampicillin resistance. Together, these results suggest that the translocation signal sequence has to be located at the C-terminus. Since we could not detect the BlaTEM fusion products in the supernatant using the β-lactamase antibody in Western blots (data not shown), we suspect, that the reporters are exported and remain in the PP. These findings indicate that ESX-dependent secretion comprises two discrete steps, translocation across the PM and subsequently translocation across the MOM. Dissection of these steps is possible since translocation into the PP confers a selectable phenotype. The subsequent translocation step of the reporter constructs may be disturbed because of the heterologous expression, the size of the fusion protein, its folding, or absence of an interaction partner. Interestingly both EsxB and EsxG from M. tuberculosis seem to be translocated by M. smegmatis without co-expression of the proposed heterodimer partner proteins EsxAMt and EsxHMt. Eventually EsxBMt and EsxGMt can bind to EsxAMs and EsxHMs and translocate together. Alternatively, translocation does not require a dimerization step as long as a C-terminal secretion signal is attached to the translocation substrate.
10.1371/journal.pone.0035453.g005Figure 5 Schematic drawing of the cell envelope and the known protein export systems of M. smegmatis.
Displayed is a simplified model of a cell envelope (without outer capsular layer) as suggested by cryo-electron tomography images [6], [9]. PM stands for the phospholipid bilayer plasma membrane. The periplasmatic space (PP) contains two electron dense layers of unconfirmed identity L1 and L2. The additional membrane is the mycobacterial outer membrane (MOM). The general secretion system (Sec) is colored brown as is one of its substrate: FbpB. FbpB contains a cleavable Sec signal peptide represented as an attached oval. The twin arginine translocation pathway (Tat) is represented in green. One of its substrate is the mayor secreted β-lactamase BlaS containing a cleavable Tat signal peptide. BlaS is missing in the ΔblaS knock out strain. The ESX protein translocation systems 1, 3 and 4 of M. smegmatis are drawn together with their suggested heterodimeric substrates: EsxA/B, EsxH/G and EsxT/U. EsxB, EsxG and EsxU posses a C-terminal secretion signal represented as a small attached circle. All reporter constructs used in this study are schematically represented and lettered with the corresponding roman number (cf.
Fig. 1
). The β-lactamase BlaTEM fusion partner is represented as a red box (Bla). Solid arrows indicate a functional translocation event, dotted arrow indicate that translocation function has not been demonstrated so far. The grey oval with the question mark drawn in the MOM represents a yet unknown translocation system for crossing the second permeability barrier.
To test specificity of the ESX system for translocation reporters we created an ESX-3 deletion strain (eccD
3, MSMEG_0623) of M. smegmatis ΔblaS. BlaTEM-EsxG was no longer able to confer ampicillin resistance in M. smegmatis ΔblaS ΔeccD
3, indicating that EsxG is specifically translocated by ESX-3. In contrast Sec- and ESX-1 reporter constructs still conferred an ampicillin resistance phenotype. This indicates that other translocation systems are not affected by EccD3 deletion.
BlaTEM-EsxU was unable to confer ampicillin resistance even though it was expressed. To test if merely the homology between the secretion signal of EsxUMt (Rv3445c) and EsxUMs (MSMEG_1538) was too low, the two genes were exchanged in the BlaTEM constructs. Nonetheless BlaTEM-EsxUMs was not able to confer ampicillin resistance in M. smegmatis ΔblaS. EsxU was never found in culture filtrates of M. tuberculosis
[50] and also does not seem to be exported in our experiments. Therefore, we speculate that ESX-4 in both M. tuberculosis and in M. smegmatis is either not transcribed or not functional as a translocation system anymore. The absence of the genes coding for an AAA+ ATPase (EccA) and the transmembrane protein (EccE) in the ESX-4 operon supports the latter hypothesis. Also missing are genes coding for PE and PPE proteins.
In conclusion we have established a reporter system for functional investigation of ESX protein translocation. The reporter system works with both ESX-1 and ESX-3 substrates. Since ESX-3 is essential in M. tuberculosis but not in M. smegmatis, we have now an excellent format for studying components of ESX-3 dependent protein translocation. The reporter system facilitates identification and confirmation of novel components of ESX protein translocation systems for plasma membrane transport by a genetic approach. Furthermore, we have for the first time a tool for high throughput screening of drugs interfering with crucial components of the ESX system (ESX-1 or ESX-3), which could give us a new edge in fighting drug resistant M. tuberculosis strains as proposed by Feltcher et al.
[38].
Supporting Information
Table S1
Plasmids used in this study.
(DOC)
Click here for additional data file.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported in part by the University of Zurich, the Swiss National Foundation (31003A_135705) and the European Union (EU FP-7 NewTBVac, project No. 241745). No additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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ScientificWorldJournalScientificWorldJournalTSWJThe Scientific World Journal2356-61401537-744XThe Scientific World Journal 2256675810.1100/2012/125608Research ArticleChemical Speciation and Potential Mobility of Heavy Metals in the Soil of Former Tin Mining Catchment Ashraf M. A.
1
*Maah M. J.
1
Yusoff I.
2
1Department of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia2Department of Geology, University of Malaya, 50603 Kuala Lumpur, Malaysia*M. A. Ashraf: [email protected] Editor: María Carmen Yebra-Biurrun
2012 1 4 2012 2012 12560822 10 2011 14 12 2011 Copyright © 2012 M. A. Ashraf et al.2012This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.This study describes the chemical speciation of Pb, Zn, Cu, Cr, As, and Sn in soil of former tin mining catchment. Total five sites were selected for sampling and subsequent subsamples were collected from each site in order to create a composite sample for analysis. Samples were analysed by the sequential extraction procedure using optical emission spectrometry (ICP OES). Small amounts of Cu, Cr, and As retrieved from the exchangeable phase, the ready available for biogeochemical cycles in the ecosystem. Low quantities of Cu and As could be taken up by plants in these kind of acidic soils. Zn not detected in the bioavailable forms while Pb is only present in negligible amounts in very few samples. The absence of mobile forms of Pb eliminates the toxic risk both in the trophic chain and its migration downwards the soil profile. The results also indicate that most of the metals have high abundance in residual fraction indicating lithogenic origin and low bioavailability of the metals in the studied soil. The average potential mobility for the metals giving the following order: Sn > Cu > Zn > Pb > Cr > As.
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1. Introduction
Within the terrestrial ecosystem, soils play a major role in element cycling and accumulate heavy metals in concentration orders of magnitude higher than in water and air. Meanwhile, soils are the reservoir for many harmful constituents, elemental and biological, including heavy metals and trace metals, henceforth referred to as just metals [1]. Total metal content of soils is useful for many geochemical applications but often the speciation (bioavailability) of these metals is more of an interest agriculturally in terms of what is biologically extractable [2]. Speciation is defined as the identification and quantification of the different, defined species, forms, or phases in which an element occurs [3] and is essentially a function of the mineralogy and chemistry of the soil sample examined [4]. Quantification is typically done using chemical solutions of varying but specific strengths and reactivity to release metals from the different fractions of the examined soil [5]. In terms of bioavailability, various species of metals are more biologically available in the ecosystem [6]. Bioavailability and the mobility of metals are also related to each other, then higher the concentration of mobile toxic metals (Cu, Pb, Cd, and Al) in the soil column which increases the potential for plant uptake, and animal/human consumption [3, 7, 8].
Heavy metals take part in biogeochemical cycles and are not permanently fixed in soils; therefore, assessment of their distribution in soils is a key issue in many environmental studies [9]. Heavy metals are included in soil minerals as well as bound to different phases of soil particles by a variety of mechanisms, mainly absorption, ion exchange, coprecipitation, and complexation. Moreover, soil properties such as contents of organic matter, carbonates, oxides as well as soil structure and profile development influence the heavy metal mobility [10]. The knowledge of the binding of metals with the different soil phases and components is of major interest to assess the connections with other biotic and abiotic elements of the environment [11]. Nevertheless, as Cabral and Lefebvre indicate, the metal speciation is a more complex task that determination of total metal contents [12].
It is widely recognized that to assess the environmental impact of soil pollution, the determination of the metal speciation will give more information about the potential for release of contaminants and further derived processes of migration and toxicity [13, 14]. Therefore, in geo-environmental studies of risk assessment, chemical partitioning among the various geochemical phases is more useful than measurements of total heavy metals contents [15, 16]. Among the procedures to determine element speciation, those based on sequential extraction are the most widely used [14]. These works are of interest in environmental studies to inform on the interactions with other components of the biosphere as well as to outline areas of potential toxicity and to provide information on the soil micronutrient levels for agricultural use [17]. To assess the binding of heavy metals to the main fractions in soils, a five-step sequential extraction procedure based on the capacity of some extracting reagents to remove the heavy metals retained from the geochemical phases has been used [4].
It is generally recognized that information about the physio-chemical forms of the elements is required for understanding their mobility, pathways, and bioavailability. Therefore, identification and quantification of the different species or forms of phases in which the heavy metals occur is very important to determine their bioavailability in the environment.
2. Study Area
Bestari Jaya catchment is located at 3° 24′ 40.41′′ N and 101° 24′ 56.23′′ E. It is a part of Kuala Selangor district, located in Selangor, biggest state of the country. District Kuala Selangor has three main towns, namely, Mukim Batang Berjuntai, Mukim Ulu Tinggi, and Mukim Tanjung Karang. Bestari Jaya is located in Mukim Batang Berjuntai. Tin mining activities has ceased from last ten years, now sand mining. The catchment has total of 442 small and big mining lakes and ponds. Bestari Jaya has a tropical humid climate, with very little variations in temperature throughout the year. The average temperature of the area is 32°C during day and 23°C at night [18].
3. Material and Methods
3.1. Soil Sampling
Five sampling sites were selected in the catchment according to their edaphic characteristics to conduct this study (Figure 1). Edaphic is a nature-related to soil. Edaphic qualities may characterize the soil itself, including drainage, texture, or chemical properties such as pH [19]. To obtain a representative average sample for each site, a total of 5 samples composed from 25 subsamples were collected at each site. Samples were extracted till an average depth of 45 cm by using an automatic core driller [20].
3.2. Soil Analysis
The composite samples of soil were air-dried and milled so as to pass through a 2 mm sieve, homogenized and stored in plastic bags prior to laboratory analysis. The pH was measured in a 1 : 2.5 soil/H2O suspension using a waterproof pH/ORP meter [21]. Cation Exchange Capacity (CEC) was measured following the standard procedure [22] and the texture was analyzed by the hydrometer method as described by Gee and Bauder [23]. Organic matter was determined by the Walkley and Black procedure [6]. All parameters were determined in triplicate. For the analysis of metals homogenised soil samples were ashed in a muffle furnace at 400°C for 1 hour and were digested by microwave assisted acid digestion [24]. Solutions from digested soil samples were stored in 100 mL high-density polyethylene samples bottles at 4°C until analysis. Speciation analysis was done through sequential extraction procedure on 1 g soil by using inductively coupled plasma optical emission spectrometry ICP-OES (Varian) (Perkin Elmer AA Analyst) [4]. Concentrations, obtained after three measurements per element, are expressed in mg/kg. Working standards for chemical analyses were prepared from Perkin-Elmer stock solutions.
3.3. Sequential Extraction Procedure
The Tessier procedure was selected because it is well documented, widely used [4], and it has been adapted to the study of soils and dusts [25]. Therefore, this five-step procedure allows comparison of the results obtained. Problems such as variability in extraction efficiency, inaccuracy in differentiation among geochemical phases and overlapping of the chemical partitioning between the different extraction steps are well known and they have been widely reported in the literature [14, 26]. Nevertheless, usefulness of the sequential procedure to inform about the relative bonding of metals in different solid phases and therefore the forms likely to be released in the soil solution under different environmental conditions is widely recognized [27, 28].
Following the sequential extraction procedure of Tessier, the chemical partitioning of heavy metals allows to distinguish five fractions [4] representing the following chemical phases: exchangeable metals, bound to carbonates, bound to Fe–Mn oxides, bound to sulphides and organic matter and residual fraction. The procedure was carried out with an initial weight of 1 g of the sieved dry soil sample. Deionized water was used in preparing stock solutions and for each step of the leaching procedure that was obtained from a Millipore Milli-Q plus system.
To check the results of the sequential extraction, the sum of the different fractions for each element was compared with the results obtained from the total digestion. The recovery rates were very good for iron and chromium (around 95%), whereas lower recovery rates (65–80%) were obtained for zinc and manganese. As quality control, duplicate analyses as well as analyses at an external laboratory were performed on five selected samples. The sequential extraction procedure is next described.
Fraction 1—Exchangeable Fraction
Samples (1 g) of soil were extracted at room temperature for 1 hour with 16 mL of magnesium chloride solution (1 M MgCl2) at pH 7. Soil and extraction solution were thoroughly agitated throughout the extraction. This is mainly an adsorption-desorption process. Metals extracted in the exchangeable fraction include weakly adsorbed metals and can be released by ion-exchange process. Changes in the ionic composition of the water would strongly influence the ionic exchange process of metal ions with the major constituents of the samples like clays, hydrated oxides of iron, and manganese [29]. The extracted metals were then decanted from the residual soil.
Fraction 2—Bound to Carbonates
The metals bound to carbonate phase are affected by ion exchange and changes of pH. The residue of Fraction 1 was extracted with 16 mL of 1 M sodium acetate/acetic acid buffer at pH 5 for 5 hours at room temperature. Significant amount of trace metals can be coprecipitated with carbonates at the appropriate pH. The extracted metal solution was decanted from the residual soil. The residual soil was used for the next extraction.
Fraction 3—Bound to Oxides
The residue from fraction 2 was extracted under mild reducing conditions. 13.9 g of hydroxyl amine hydrochloride (NH2OH·HCl) was dissolved in 500 mL of distilled water to prepare 0.4 M NH2OH·HCl. The residue was extracted with 20 mL of 0.4 M NH2OH·HCl in 25% (v/v) acetic acid with agitation at 96°C in a water bath for 6 hours. Iron and manganese oxides which can be present between particles or coatings on particles are excellent substrates with large surface areas for absorbing trace metals. Under reducing conditions, Fe (III) and Mn (IV) could release adsorbed trace metals. The extracted metal solution was decanted from the residual soil which was used for the next extraction.
Fraction 4—Bound to Organics
The residue from fraction 3 was oxidized as follows: 3 mL of 0.02 M HNO3 and 5 mL of 30% (v/v) hydrogen peroxide, which has been adjusted to pH 2, was added to the residue from fraction 3. The mixture was heated to 85°C in a water bath for 2 hours with occasional agitation and allowed to cool down. Another 3 mL of 30% hydrogen peroxide, adjusted to pH 2 with HNO3, was then added. The mixture was heated again at 85°C for 3 h with occasional agitation and allowed to cool down. Then 5 mL of 3.2 M ammonium acetate in 20% (v/v) nitric acid was added, followed by dilution to a final volume of 20 mL with de-ionized water. Trace metals may be bound by various forms of organic matter, living organisms, and coating on mineral particles through complexation or bioaccumulation. These substances may be degraded by oxidation leading to a release of soluble metals. The extracted metal solution was decanted from the residual soil which was used for the next extraction.
Fraction 5—Residual or Inert Fraction
Residue from Fraction 4 was oven dried at 105°C. Digestion was carried out with a mixture of 5 mL conc. HNO3 (HNO3, 70% w/w), 10 mL of hydrofluoric acid (HF, 40% w/w) and 10 mL of perchloric acid (HClO4, 60% w/w) in Teflon beakers. Fraction 5 largely consists of mineral compounds, where metals are firmly bonded within crystal structure of the minerals comprising the soil. To validate the procedure, the instrument was programmed and it carried out metal detection by displaying three absorbance readings and what was reported was the average. Blanks were also used for correction of background and other sources of error. Apart from calibration before use, quality checks were also performed on the instrument by checking the absorbance after every ten sample runs.
3.4. Quality Assurance of Data
In order to verify the accuracy of the sequential extraction method, certified soil reference material CRM027-050 Certified Material (Resource Technology Corporation, USA) and was analyzed concurrently with the soil samples. Recovery of metal was 99% for tin, 97% for arsenic, 112% for copper, 108% for chromium, 99% for zinc, and 94% for lead and the coefficient of variation was between 3% and 7% when analyzed in triplicate (Table 5). An internal check on the results of the microwave extraction procedure, the sequential extraction procedure was performed by comparing the sum of the 4 steps (acid-soluble + reducible + oxidizable + residual) from the sequential extraction procedure with the total metal content from the microwave-assisted acid digestion procedure (Table 5). The recovery of the sequential extraction procedure was calculated as follows:
(1) Recovery=Fraction 1+Fraction 2+Fraction 3+Residual fraction Total digestion ×100.
4. Results
Morphological characteristics of the soil of Bestari Jaya are shown in (Table 1), whereas physical and chemical characteristics of the soil are shown in (Table 2). It shows mean values of the characteristics of top 45 cm of soil of the catchment. The results of the sequential extraction of soil samples conducted in August 2011 are summarized in (Tables 3 and 5). With respect to the soil reference material CRM 027-050, the results shown in (Table 5) indicate that the sums of the 4 fractions are in agreement with the total metal contents with satisfactory recoveries (94–112%). Correlations between pH, carbonate, organic matter and clay percentages and contents of Pb, Cu, Cr, Zn, As, and Sn in the five chemical phases of the sequential extraction have been established and analysed in (Table 4). Table 6 shows the correlations between pH, carbonate, organic matter and clay percentages and contents of Pb, Cu, Cr, Zn, As, and Sn in the five chemical phases of the sequential extraction for soil samples. The percentages of metals in the fractions are represented graphically in (Figure 2) while (Figure 3) represents charts of the average potential mobility for the metals.
5. Discussion
5.1. Characteristics of Soil
Bestari Jaya catchment has alluvium soil having alluvial tin deposits. Alluvium is loose, unconsolidated soil, which is then eroded, deposited, and reshaped by water in some form in a nonmarine setting [30]. Alluvium is typically made up of a variety of materials, including fine particles of silt and clay and larger particles of sand and gravel and often contains a good deal of organic matter. When this loose alluvial material is deposited or cemented into a lithological unit, or lithified, it would be called an alluvial deposit. Bestari Jaya has alluvial deposits containing tin ore (cassiterite) [31]. Cassiterite, also called tinstone, heavy, metallic, hard tin dioxide (SnO2) that is the major ore of tin. It is colourless when pure, but brown or black when iron impurities are present [32].
5.2. Morphology
Morphologically, there is no profile development in the tintailings as a whole; as such horizon differentiation is nonexistent. This is because the deposits are young and therefore not much affected by soil forming processes, as the mine ceased operation only about 10 years ago. These soils could thus be classified as Entisols [33]. Generally, the sandy deposits occur in the well-drained areas, while the slimes occur in the depressions, where the water table varies from 50–60 cm depth. During the rainy season the water table in the depressions could rise to the surface and cause flooding [34].
The clayey materials appear to be reddish, with colour notation of 5YR 5/6 or redder, this could be due to leaching of some iron-rich materials. In some areas, the textural composition changes with depth, especially at sites 3 and 5. At site 5 (Table 1), the top 30 cm of the deposits are clayey, but at 30–45 cm they are sandy with a sand content of more than 95%. At best, we can describe the texture of tintailings as variable both vertically and horizontally.
5.3. Physical and Chemical Properties
Table 2 shows mean values of the characteristics of top 45 cm soil samples from ex-tin mining area, Bestari Jaya. pH values ranged from acidic to neutral (4.8–7.2). The pH was acidic in the location (S4-S5), while it was slightly acidic at (S1–S3). The carbonate percentages were in a broad range and the organic matter values were less than 10%. According to SISS, criteria, the soil of the location (S4-S5) mine dumps can be considered poor in organic matter [35]. The CEC represents the ability of the soils to absorb or release cations and, consequently, is an important parameter in sites contaminated by heavy metals. Organic matter and clay minerals are responsible for the CEC. CEC ranged from low 17.81 to high 26.98 cmol/kg. According to Conesa, pH and ECs are the most important factors [36] because under acidic conditions the tailings matrix will dissolve more salts [37]. Due to the moderately acidic and saline conditions of the soil, pH and ECs could be the limiting factors for plant establishment in the studied zone. At all sampling locations, the soil showed a sandy texture. Sandy substrates generally present oxidizing conditions however, in this case, the water-saturation state of soils and the flooding of sediments explained the reducing environment [31].
Taking the case of the sandy deposits, it is noted that the sand content is very high with values exceeding 90%. This condition results in excessive draining and intensive leaching of bases in the soil and these will be reflected in the low CEC [32]. The presence of too much sand in particular soil will slow down the process of soil structure development and as such the soil will retain a single-grain structure unless remedial steps are undertaken quickly. The slimes however are subjected to a different set of conditions. The clay content is quite high with values exceeding 40% in some cases. Currently the structure is rather massive but in time to come the structure may develop, especially if organic matter is present [33]. The development of structure is possible in the presence of clay, silt and sand in a favourable ratio.
Generally, the pH of these soils is low with values of 6 or less. The low pH values are possibly due to the presence of high metal contents in the area. Similar results were reported for the pH of tintailings in Dengkil, Selangor, where the pH was reported to be 2.8–3.0 [38]. Low pH values may reduce the availability of the most micronutrients. This rather low pH can easily be overcome by using organic fertilizer. Continuous application of organic fertiliser can increase the pH to a more favourable level for crop growth as well as increase the organic contents of the soil [34].
The CEC is low in these soils even for with more than 50% clay content. The value at sites 1 and 3 17 cmol(+)/kg dry soil or less. This is related to the mineralogy, in which kaolinite, mica, and chlorite are found to be dominant in the clay fraction [32]. The CEC of these soils can probably be improved somewhat by incorporating organic matter into the soil. Organic carbon is far too low compared to normal soils under Malaysian conditions. This is somewhat related to the recent nature of the deposits which have just been exposed by the mining operation. Incorporation of organic manure (cow dung) and/or agricultural waste from the factory (POME) is considered essential to improve the organic matter content of the soils. In so doing, soil structure development is encouraged and more nutrients are added [34].
5.4. Metal Speciation of Soil
Metal chemical speciation carried out by sequential extraction of the metals is essential to the metal mobility [4]. The results (Table 3) obtained show that the amounts of heavy metals extracted from each fraction vary widely. The order of mobility of the metals considering their abundance in the fractions is: exchangeable > bound to carbonate > bound to oxides > bound to organics > residual [4]. Oxides exist as nodules and cement between particles. These oxides hold trace metals and can be mobilized under reducing and acidic conditions. The organic phase is relatively stable in nature but can be mobilized under strong oxidizing conditions due to degradation of organic matter [4, 39]. Cr is mostly abundant in exchangeable fraction in all the samples. The abundance of Cr in exchangeable phase is 07.98% while in the residual fraction the abundance was 39.24%. Abundance of Cr in other geochemical phases was very low. This means that Cr was more mobile in this environment than other metals that are mostly abundant in the remaining four geochemical phases. Zn is mostly abundant bound to oxides with abundance of 44.87% while in the residual fraction the abundance was 33.75% in the dry season and 34.37% in the rainy season. Abundance of Zn in other fractions was low. Zn in this environment was more mobile than the metals that were mostly abundant in the Residual fraction. This is in agreement with Zerbe et al. [40]. Copper can easily complex with organic matters because of high formation of organic-Cu compounds [39]. The result of sequential extraction shows that Cu is mostly abundant (51.33%) in the fraction bound to organics. The abundance of Cu in residual fraction was 28.34%. Heavy metals with high abundance in the phase bound to organics are more available than heavy metals in the residual fraction. The remaining heavy metals, as shown in (Figure 2) had the highest abundance in the residual fraction as follows: Cd—67.32%; Pb—95.63%; Sn—36.87%; Fe—67.73%. This agrees with Ramirez et al., 2005, who reported that Cd, Fe, Mn, Ni, and Pb were mostly associated with the residual phase [41]. The results of the sequential extraction show that most of Cr, Fe, and Zn are strongly retained in the residual phase in all soils. These heavy metals are contained in the crystal lattices of minerals with strong bindings and consequently they will not be released into the environment. In the case of Mn, its higher proportion (80%) is associated to oxides and carbonates and only in the case of a change in the redox conditions towards reductive ones, Mn would be released from oxides and if conditions became acidic, it would be released from carbonates. Therefore, such changes could only be expected from an anthropogenic impact. Metals present in the residual fraction are a measure of the degree of environmental pollution. The higher the metals present in this fraction, the lower the degree of pollution [42]. Sum of concentrations of metals in different geochemical phases can be used to express the potential mobility of metals. The potential mobility of a metal can be assessed by adding up the results of the exchangeable phase, carbonate phase, oxide, and organic phase of that metal [39]. As shown in (Figure 3), the potential mobility of Cr in soil was 63.25%. The exchangeable phase represents the mobile and bioavailable heavy metal fraction. In this phase, the heavy metals have the more labile bounds and can be easily released into the environment. The presence of heavy metals in this phase where they can be taken up by plants from the soils is the most hazardous to the ecosystem. In our soils, only little Fe, Mn, and insignificant amounts of Zn detected in four soils are present in this phase. From the result of the sequential extraction in all the soil samples studied, Cr was mostly abundant in the exchangeable fraction and the abundance was lower in the residual fraction. Low abundance of a metal in the residual phase compared with its abundance in other geochemical phases indicates higher mobility of the metal in the environment. This shows that Cr would easily be released to the environment and highly toxic. High abundance of Zn bound to oxides accounts for the high potential mobility of Zn (66.25%) (Figure 3). Cu has the highest potential mobility (71.66%) of all the heavy metals due to higher decomposition and decay of organic matter, therefore, high formation of organic-Cu compounds. The potential mobility of heavy metals with high abundance in residual fraction were: Cr 32.68%; Pb 24.37%; Cu 21.44%, Zn 27.03%; As 15.10%; Sn 63.13%. These values were low compared with the values obtained for heavy metals that were more abundant in other geochemical phases.
According to Jones and Jarvis, 1981, processes of metal mobilization-immobilization are affected by a variety of soil properties [43]. To examine this influence, correlations between pH, carbonate, organic matter and clay percentages and contents of Pb, Cu, Cr, Zn, As, and Sn in the five chemical phases of the sequential extraction have been established for all soils. As can be seen in (Table 4), apart from Cr associated with the organic phase, no significant correlations between the carbonate contents and the heavy metals were found. Between pH and the heavy metals, the only significant relationships are the positive correlation with Fe from the carbonate phase and the negative one with Fe from the organic phase. Zinc from the exchangeable phase and Mn and Cr both from the organic phase are positively related to organic matter. Iron and Cr from the organic phase are also positively related to clay contents. Although the size of data set does not allow for better results, in general the main trends reported in the literature, such as positive correlations with organic matter and clays have also been observed in our soils [44, 45].
5.5. Potential Mobility of Heavy Metals in Soil
Average potential mobility of the heavy metals in the soil is shown in (Table 6). It is also shown graphically in (Figure 3). The average potential mobility of Cr was 64.84%. High potential mobility indicates high reduction in concentration soil. Zn is mostly abundant bound to oxides and the average potential mobility of Zn was 65.94%. Under reducing and acidic conditions, Zn will easily be mobilized to the environment. Therefore compared with other metals that were mostly abundant in the residue fraction Zn was more available and toxic in the environment. The average potential mobility of Cu is 61.86%. Under strong oxidizing conditions, due to degradation of organic matter, Cu becomes very available and toxic in the environment. More metals were present in this fraction than in the other fractions which show that the degree of pollution in the environment is presently low. Pb had the least Average potential mobility (28.08%). Change in concentration was low because Pb in soil in this environment was relatively immobile.
From Table 6 and Figure 3, the order of average potential mobility for soil agrees with the percentage reduction in concentration. High-average potential mobility indicates high percentage reduction in concentration. However, values of the percentage reduction in concentration for metals sometimes might be controlled not only by its speciation, changes in pH and salinity, but also by unknown factors [39]. Metals with anthropogenic origin are mainly extracted in the first step of sequential extraction procedures while lithogenic metals are found in the last step of the process corresponding to the residual fraction [41]. Metals at Bestari Jaya catchment, from the results of sequential extraction, were mostly anthropogenic origin due to mining activities. Analyzed heavy metals such as Pb, Cu, Cr, Zn, As, and Sn were highly abundant in the residue, implying that they were not derived from anthropogenic sources. The Bestari Jaya catchment was also subjected to heavy metal analysis and was found to contain high concentrations of Sn, As, Pb, and Zn due to anthropogenic sources such as mining activities and forest cultivation [46].
6. Conclusion
The ex-mining land of Bestari Jaya can be classified into sandy, clayey (slime), and a mixture of sandy and clayey deposits. The sandy deposit occurs in the well-drained areas, while the slime occurs in the poorly drained areas. These soils contain low amounts of bases, phosphorus, nitrogen, and organic carbon. The pH and CEC are also low. This study served to evaluate the distribution, retention, and release of Pb, Cu, Cr, Zn, As, and Sn in the selected soils of the former tin mining catchment Bestari Jaya. The results obtained from speciation studies showed that most of the metals considered had the highest abundance in the residual fraction. This indicates that the metals were immobile. The largest proportions for Pb, Cu, and Sn were extracted in the residual phase, in which metals are strongly retained in the soil minerals. This shows that soil in the environment was not likely to be polluted by these metals. Cr was found to be highly abundant in the exchangeable fraction, indicating that it could be easily released to the environment from soil. It was also likely of high toxicity in the environment. The relatively high metal content in the catchment may represent some indirect environmental risk due to clay dispersion, and disaggregated soil particles bearing heavy metals may eventually reach and accumulate in water bodies. The average potential mobility of Zn, Cu, Pb, and Sn in the soil samples studied were quite high implying that under favourable conditions they can be released to pollute the environment. The average potential mobility of the metals arranged in decreasing order was as follows: soil: Sn > Cu > Zn > Pb > Cr > As. The results of the speciation have given the present status of metal pollution and the potential pollutants in the catchment. Further research can be carried out on the speciation of heavy metals in other environmental components in the vicinity of the Bestari Jaya catchment.
Acknowledgments
The work reported in this paper was carried out in Analytical Laboratory, Department of Chemistry, and some of the facilities were utilised from Hydro-Geological Laboratory, Department of Geology, University of Malaya, Kuala Lumpur, Malaysia, through UM Research Grant vide no. PV039/2011B. Thanks also to the Ministry of Higher Education Malaysia (MOHE) for the financial support.
Figure 1 Map of Bestari Jaya catchment.
Figure 2 Percentages of metals in different fractions.
Figure 3 Potential mobility of soil.
Table 1 Morphological properties of the soil of Bestari Jaya catchment.
Site no. Soil properties (August 2011)
S1
Sandy loam∗Entisol-10 YR 5/6∗poorly drainedSand∗deep
S2
clay loam∗Entisol-5 YR 5/6∗well drainedloam∗deep
S3
Silty loam∗Entisol-10 YR 3/6∗water table 50 cm Clay loam∗deep
S4
Sand∗Entisol-10 YR 5/6∗well drained%Sand∗deep
S5
Sand∗Entisol-10 YR 4/4∗water table 60 cm Flat silt clay∗deep
Table 2 Chemical and physical characteristics of soil.
Location Sand % Silt % Clay % Textural class pH ECs (dSm−1)b
CaCO3
OMc% CEC (cmol(+)/kg)d
S1 60 ± 14 29 ± 9 11 ± 3 Sand 6.1 ± 0.02 18 ± 8 5.7 ± 0.8 8.39 ± 0.7 17.81 ± 4
S2 65 ± 13 26 ± 8 9 ± 4 Sand 6.3 ± 0.01 17 ± 7 18.4 ± 0.6 9.91 ± 0.4 19.43 ± 3
S3 67 ± 11 21 ± 9 12 ± 3 Sand 6.7 ± 0.01 11 ± 5 6.9 ± 1.2 7.98 ± 0.5 13.76 ± 3
S4 59 ± 13 30 ± 7 11 ± 2 Sand 5.1 ± 0.01 12 ± 4 12.1 ± 2.4 5.13 ± 0.3 26.98 ± 3
S5 68 ± 13 20 ± 8 12 ± 3 Sand 4.9 ± 0.01 10 ± 5 2.5 ± 0.9 4.78 ± 0.4 24.42 ± 3
± Standard deviation, n = 5; belectrolytic conductivity; corganic matter; dcation exchange capacity.
Table 3 Heavy metals speciation in the soil.
S/N Sample ID Pb Cu Cr Zn As Sn
Fraction 1: Exchangeable mg/kg (ppm)
1 S1 03.45 02.99 03.46 02.56 01.23 12.34
2 S2 05.67 04.37 02.91 01.98 00.92 11.87
3 S3 04.34 03.76 04.23 03.67 02.11 14.17
4 S4 06.88 05.23 02.40 02.59 01.74 15.88
5 S5 03.24 04.65 03.21 03.99 00.86 12.23
Mean 04.71 04.20 03.22 02.95 01.37 13.29
%age 09.34% 06.44% 07.98% 09.55% 11.53% 14.64%
Fraction 2: Bound to carbonates mg/kg (ppm)
1 S1 08.29 11.29 08.97 06.87 02.87 25.98
2 S2 11.34 08.34 09.37 05.49 03.29 28.56
3 S3 09.88 07.35 10.10 09.30 04.21 30.25
4 S4 07.78 08.34 06.56 05.34 02.39 28.99
5 S5 10.84 09.23 09.23 06.53 02.65 25.40
Mean 09.62 08.91 08.64 06.70 03.06 27.83
%age 18.34% 15.44% 17.98% 19.51% 27.53% 33.64%
Fraction 3: Bound to oxides mg/kg (ppm)
1 S1 16.23 15.88 10.00 10.76 04.21 53.23
2 S2 18.29 19.24 14.21 12.34 05.53 59.65
3 S3 12.87 17.30 15.99 13.21 03.92 61.23
4 S4 19.02 11.02 16.37 10.98 05.67 58.34
5 S5 17.25 14.83 12.31 15.23 09.34 50.85
Mean 16.73 15.65 13.77 12.50 05.73 56.61
%age 23.76% 26.19% 24.15% 23.98% 28.21% 42.12%
Fraction 4: Bound to organics mg/kg (ppm)
1 S1 32.14 23.56 18.91 25.88 06.72 79.34
2 S2 29.39 27.91 16.28 28.76 09.36 80.05
3 S3 24.26 30.20 23.22 34.75 10.67 68.32
4 S4 30.22 26.87 29.90 30.04 07.49 76.51
5 S5 28.95 31.64 24.24 29.80 06.99 81.49
Mean 28.99 28.03 22.51 29.84 08.24 77.14
%age 26.16% 28.91% 30.75% 33.58% 40.53% 52.40%
Fraction 5: Residual or inert mg/kg (ppm)
1 S1 48.21 62.51 32.44 54.38 11.23 102.34
2 S2 52.78 70.46 21.96 78.23 14.24 098.34
3 S3 61.10 49.80 38.75 59.00 10.96 112.87
4 S4 57.91 53.77 50.02 63.22 08.34 118.34
5 S5 43.55 50.24 43.25 49.30 20.34 101.45
Mean 52.71 57.35 37.24 60.62 13.02 106.66
%age 57.56% 52.98% 61.45% 63.12% 59.52% 71.29%
Table 4 Correlation between pH, Carbonates, organic matter content, and clay percentages with contents of heavy metals (mg/kg).
Metals with fractions pH CO3
2−
OM Clay
%
Pb Exchangeables −0.280 0.324 0.487 0.123
Carbonates 0.653 0.086 −0.561 −0.572
Oxides −0.353 −0.361 −0.235 0.183
Organics −0.686 0.138 0.466 0.651
Residuals 0.141 0.282 0.077 0.096
Cu Exchangeables −0.223 0.558 0.221 0.246
Carbonates 0.484 0.138 −0.348 −0.049
Oxides −0.331 0.268 0.530 0.278
Organics −0.292 0.420 0.698 0.416
Residuals 0.408 0.085 −0.137 −0.077
Cr Exchangeables −0.198 0.174 0.613 0.150
Carbonates 0.404 −0.176 −0.232 0.051
Oxides −0.375 −0.474 −0.037 0.251
Organics −0.341 0.094 0.193 0.293
Residuals 0.243 −0.044 −0.166 −0.039
Zn Exchangeables −0.223 0.558 0.221 0.246
Carbonates 0.484 0.138 −0.348 −0.049
Oxides −0.331 0.268 0.530 0.278
Organics −0.292 0.420 0.698 0.416
Residuals 0.408 0.085 −0.137 −0.077
As Exchangeables −0.198 0.174 0.613 0.150
Carbonates 0.404 −0.176 −0.232 0.051
Oxides −0.375 −0.474 −0.037 0.251
Organics −0.341 0.094 0.193 0.293
Residuals −0.223 0.558 0.221 0.246
Sn Exchangables 0.484 0.138 −0.348 −0.049
Carbonates −0.331 0.268 0.530 0.278
Oxides −0.292 0.420 0.698 0.416
Organics 0.408 0.085 −0.137 −0.077
Residuals −0.198 0.174 0.613 0.150
Table 5 Results of analysis of standard reference materials (SRM) in comparison with certified values.
Standard reference material Analysed SRM value Certified SRM value Recovery%
Sequential extraction
SRM-027-050-Soil (n = 3)
Step 1
As 4.19 ± 0.40 4.98.0 ± 0.38 88
Cr 3.13 ± 0.10 2.87 ± 0.13 85
Zn 4.08 ± 0.79 3.94 ± 0.39 100
Cu 34.24 ± 1.6 46.73 ± 1.8 69
Pb 73.11 ± 2.00 64.20 ± 2.00 101
Sn 178 ± 5.00 195 ± 2.00 103
Step 2
As 3.08 ± 0.30 3.12 ± 0.34 90
Cr 2.89 ± 0.14 2.98 ± 0.19 85
Zn 100.18 ± 4.00 88.84 ± 3.00 92
Cu 102.38 ± 2.0 129.0 ± 2.5 101
Pb 62.30 ± 3.00 65.28 ± 3.00 99
Sn 112 ± 4.00 136 ± 5.00 100
Step 3
As 1.18 ± 0.23 1.02 ± 0.32 95
Cr 100.12 ± 4.00 105.00 ± 5.00 106
Zn 52.28 ± 2.00 65.74 ± 3.00 103
Cu 29.28 ± 2.0 36.32 ± 2.5 100
Pb 10.10 ± 2.00 8.80 ± 3.00 102
Sn 70 ± 2.00 72 ± 3.00 100
Step 4
As 0.18 ± 0.03 0.20 ± 0.52 120
Cr 73.33 ± 5.00 60.56 ± 4.00 128
Zn 98.00 ± 5.00 87.34 ± 4.00 120
Cu 20.98 ± 0.88 19.93 ± 2.00 115
Pb 14.23 ± 0.90 10.53 ± 1.00 104
Sn 40 ± 4.00 35 ± 2.00 111
Total digestion
CRM-027-050-Soil (n = 3)
As 9.86 ± 1.00 10.78 ± 1.00 97
Cr 157.95 ± 4.00 180.45 ± 4.00 108
Zn 251.16 ± 4.00 250.06 ± 5.00 99
Cu 198.18 ± 4.00 235.75 ± 3.00 112
Pb 143.91 ± 2.00 155.31 ± 2.00 94
Sn 390 ± 5.00 381 ± 4.00 99
Table 6 Analysis of heavy metals speciation.
Fraction Description Pb Cu Cr Zn As Sn
1 Exchangeable 09.34% 06.44% 07.98% 09.55% 11.53% 14.64%
2 Bound to carbonates 18.34% 15.44% 17.98% 19.55% 27.53% 33.64%
3 Bound to oxides 23.76% 26.19% 24.15% 23.98% 28.21% 42.12%
4 Bound to organics 26.16% 28.91% 30.75% 33.58% 40.53% 52.40%
5 Residual or inert 57.56% 52.98% 61.45% 63.12% 59.52% 71.29%
Sum of concentration of metals (mg/kg) 22.55 22.82 17.07 22.52 06.28 56.30
Potential mobility 52.73% 54.88% 42.34% 52.12% 16.72% 89.14%
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