Inhibition of CXCR4 activity with AMD3100 decreases invasion of human colorectal cancer cells in vitro

cancer cell line SW480 through down-regulation of VEGF and MMP-9 expression. Abstract AIM: To investigate the effect and mechanism of blockade of the CXC chemokine receptor-4 (CXCR4) signaling pathway by AMD3100, a small non-peptide CXCR4 inhibitor, on invasion and metastasis


INTRODUCTION
Stromal cell-derived factor-1 (SDF-1 or CXCL12) and its unique receptor CXC chemokine receptor-4 (CXCR4) have prominent roles in invasion and metastasis of a diverse number of cancers. The interaction between SDF-1 and CXCR4 has been shown to direct tumor cells to organ sites with high levels of SDF-1 expression, which suggests this molecular pair plays a key role in chemotaxis and homing of metastatic cells. Convincing evidence indicates elevated CXCR4 expression in primary tumors is associated with lymph node metastasis in breasts [1][2][3][4] , head and neck [5] , and colon [6] . Furthermore, CXCR4 expression is associated with intraperitoneal carcinomatosis of ovarian [7] and gastric [8] cancer. High or persistent expression of CXCR4 has been associated with poor prognosis in osteosarcoma [9] , epithelial ovarian cancer [10] , colon cancer [6,11,12] , esophageal carcinoma [13] , and melanoma [14] . Recently, intensive research has shown that binding of CXCL12 to CXCR4 plays a role in tumor angiogenesis by influencing the secretion of vascular endothelial growth factor (VEGF) [15] and increasing invasion associated with matrix metalloproteinase (MMP)-9 activation [16] . Therefore, interruption of the interaction between CXCR4 and SDF-1 has received considerable attention since it may provide a means of inhibiting the metastatic process.
AMD3100, a bicyclam molecule, has been identified as a specific inhibitor of CXCR4. It had originally been developed as an inhibitor of T-tropic human

Inhibition of CXCR4 activity with AMD3100 decreases invasion of human colorectal cancer cells in vitro
Ji-Kun Li, Liang Yu, Yun Shen, Li-Sheng Zhou, Yi-Cheng Wang, Jian-Hai Zhang immunodeficiency virus (HIV) infectivity and has been used to block HIV infection of T-tropic, X4-using, virus and induce X4 transformation to R5, which validate CXCR4 as a target for HIV therapy in clinical trials. Furthermore, AMD3100 has also been demonstrated to be an effective mobilizer of hematopoietic stem cells in both healthy volunteers and multiple myeloma and non-Hodgkin's lymphoma patients [17,18] . In recent studies, anti-CXCR4 treatment has suppressed primary tumor growth by inhibiting tumor angiogenesis, and has prevented lung metastasis of squamous cell carcinoma of the head and neck (SCCHN) in animal models [19] . Treatment of animals with AMD3100 resulted in decreased activation of the mitogen-activated protein kinase and AKT pathways in xenograft brain tumors, which are pathways downstream of CXCR4 that promote sur vival, proliferation and migration [20] .
However, the influence of various concentrations of AMD3100 on invasion and proliferation in human colorectal cancer cell lines has not been reported. The present study showed that inhibition of CXCR4, through using nonpeptide small molecule AMD3100, had significant antitumor activity, which represents a novel strategy for targeting highly metastatic colorectal cancer cells.

Cells
Human colorectal cancer (CRC) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Tumor cells were maintained in RPMI 1640, supplemented with 10% fetal calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator with an atmosphere of 5% CO2, 95% air at 37℃. The specific chemokine receptor CXCR4 antagonist, AMD3100 (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in sterile PBS (GIBCO, Carlsbad, CA, USA) as a × 1000 stock solution, and then diluted with the culture medium for experiments.

3-(4,5-dimethylthiazol-2-yl)-2.5-dipheny-tetrazolium bromide (MTT) assay
Cell viability was determined by MTT assay. Briefly, exponentially growing CRC cells were seeded in 96-well culture plates in serum-free medium at an optimal density. After 24 h incubation, either PBS or the indicated dose of AMD3100 was added for 2 h incubation in eight parallel holes. Then, CXCL12 (Peprotech, Rocky Hill, NJ, USA) was added daily at 20 ng/mL. MTT assays (Beyotime, Haimen, China) were performed after 24, 48 and 72 h of AMD3100 treatment. Absorbance was measured at 590 nm. The results were calculated as mean values of eight wells per treatment group.

Chemotaxis and invasion assays
Cell lines were assessed for migration utilizing a Boyden chamber chemotaxis assay. Chambers with 8 mm pore filters (HTS Transwell-24 System; Corning, Acton, MA, USA) were used. CXCL12 was added to the lower wells with 0.5% fetal bovine serum medium. Selected cells were www.wjgnet.com treated with PBS or AMD3100 15 min prior to assay performance. After 3 h incubation at 37℃, cells within the inserts were removed from the upper surface of the membrane using a moist cotton-tipped swab. Migratory cells on the lower surface of the membrane, which had migrated through the polycarbonate membrane, were fixed in 100% ethanol, washed with phosphate buffer solution, air dried and stained with crystal violet for 30 min, then rinsed several times with distilled water. Migration was quantitated by dissolving stained cells in 10% acetic acid, and an equal amount of the dye/solution mixture was transferred onto a 96-well plate for colorimetric reading of A590. Invasion assay was performed using Cell Invasion Kit (Chemicon, Temecula, CA, USA). The inserts had an 8-μm pore size polycarbonate membrane with a precoated thin layer of basement membrane matrix (ECMatrix, Temecula, CA, USA). After 24 h incubation, invasive cells on the lower surface of the membrane were stained and calculated as previous described. Assays were performed in triplicate.

Western blotting
Whole-cell protein and nuclear protein extracts from SW480 CRC cells were prepared with Cell lysis buffer for Western and IP (Beyotime, Haimen, China) and Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA), respectively, according to the manufacturers' instructions. Protein concentrations were determined using an assay kit (Bio-Rad, Hercules, CA, USA). Lysates containing 100 μg protein were mixed with loading buffer with 5% β-mercaptoethanol, and heated for 5 min at 100℃. Samples were separated by SDS-PAGE and transferred onto nitrocellulose membranes by semidry blotting. Membranes were incubated in TBS buffer for 1 h at room temperature, followed by hybridization with anti-CXCR4 antibody (Chemicon, Temecula, CA, USA); 1:1000 dilution), anti-MMP-9, anti-MMP-2 antibody, anti-VEGF antibody (Boster, Wuhan, China; 1:500 dilution) at 4℃ overnight. After three washes in TBS/0.1% Tween 20, the membranes underwent hybridization with a horseradish peroxidase-conjugated secondary antibody, rabbit IgG (Santa Cr uz Biotechnolog y, Santa Cr uz, CA, USA; 1:5000 dilution) for 1 h at room temperature. After three washes in TBS/0.1% Tween 20, signals were detected by chemiluminescence using the Western blotting luminol reagent (Santa Cruz Biotechnology).

Statistical analysis
Numeric data are presented as mean ± SD of three experiments. The paired student's t test or one-way ANOVA was used for comparing the differences between groups. Statistical significance was assigned if P < 0.05. Analyses were performed using SPSS 13.0 (SPSS, Chicago, IL, USA).

Expression of CXCR4 in intestinal epithelial cells
In our previous study, we analyzed CXCR4 expression in several cell lines (SW480, SW620, Lovo and IEC-6). In particular, our data showed lymph-node-metastasisderived cell line SW480 expressed CXCR4 at a high level, by using RT-PCR and Western blotting (Figure 1). We also examined mRNA expression of CXCL12, the ligand of CXCR4, by RT-PCR. We found no expression of CXCL12 mRNA in any of the colorectal cancer cell lines (data not shown).

Effect of AMD3100 on viability of CRC cells SW480
In our previous studies, we found no expression of CXCL12 mRNA in any of the CRC cancer cell lines. After 3 d incubation, CXCL12 greatly enhanced SW480 cells viability in the absence of serum ( Figure 2). The enhancing effect of CXCL12 on cell proliferation was strongly inhibited by treatment with different doses of AMD3100. In a dose-dependent fashion, the proliferation rate was reduced to 6.10 ± 0.13, 4.49 ± 0.22, 3.58 ± 0.13 respectively (P < 0.05). The effect of 100 and 1000 ng/mL AMD3100 was statistically significant (P < 0.01, n = 8) compared to that of the CXCL12 group (7.97 ± 0.811). Although a decrease in proliferation was also observed in the AMD3100 alone group compared to the serumfree cells (vehicle-treated cells), the inhibition rate was not significantly different, probably due to a specific effect of blocking CXCL12-CXCR4 interaction. The assay also revealed that, in 24 h, there was no significant difference in viability in any of the groups. Therefore, the cell invasion assay was performed at 24 h to remove its influence on cell viability.

Effect of AMD3100 on invasion of CRC cells
To evaluate the effects of inhibition of CXCL12-CXCR4 interaction on CRC invasion, we performed an in vitro invasion assay using AMD3100. After 24 h incubation, AMD3100 markedly reduced invasion of SW480 cells at concentrations of 100 and 1000 ng/mL (Table 1), by 28.43% (P < 0.05) and 77.23% (P < 0.01), respectively.

Effect of AMD3100 on chemotactic migration of CRC cells
The effect of AMD3100 on inhibiting CXCL12-induced migration of CRC cells was estimated by a classical chemotaxis assay. The selected CXCR4-positive cell line, SW480, did migrate in response to CXCL12 in a classical   Figure 2 Effect of AMD3100 on viability of CRC SW480 cells. After 24 h incubation, cells growing in 96-well plates were treated with AMD3100 for 2 h. CXCL12 was added at 20 ng/mL per day, and the MTT assay revealed that in serum-free medium or the absence of CXCL12, AMD3100-induced inhibition was relatively weak. CXCL12-induced cell proliferation was significantly suppressed by 100 and 1000 ng/mL AMD3100 in SW480 cells. Cell viability was not significantly affected by 10 ng/mL AMD3100 (compared to the unstimulated group). Data are mean ± SD of eight wells after 3 d incubation. Bars indicate mean ± SD of triplicate experiments.
www.wjgnet.com chemotaxis assay, with an optimal response at 100 ng/mL. After AMD3100 treatment, chemotactic activity of SW480 cells was reduced in a dose-dependent manner ( Figure  3B). The inhibition rate with AMD3100 at 10, 100 and 1000 ng/mL was 5.24%, 47.27% and 62.37%, respectively. The latter two achieved a significant difference compare to the control group (a, b and c in Figure 3A).

Effect of AMD3100 on expression of MMP-2, MMP-9 and VEGF in SW480 cells
The CXCL12-CXCR4 axis contributes to invasion and specific organ metastasis through regulation of its target genes, which have recently been shown to be VEGF and MMPs [7,16,25,26] . Therefore, we detected the effect of AMD3100 on expression of VEGF, MMP-2 and MMP-9. As shown in Figure 4, 24 h incubation with AMD3100 reduced MMP-9 and VEGF protein expression in a dosedependent manner in SW480 cells. RT-PCR demonstrated that the expression of MMP-9 but not MMP-2 and VEGF mRNAs in SW480 cells was significantly downregulated by 100 and 1000 ng/mL AMD3100. Densitometric analysis revealed the relative expression decreased to 17.58% ± 3.79% for MMP-9, and 39.44% ± 3.07% for VEGF, as compared to the controls (P < 0.05).

DISCUSSION
A growing body of literature has indicated CXCR4 is important in a variety of cancers, and more specifically, that this receptor can be a propitious target in treating cancer. In experimental systems, convincing evidence has shown that selective inhibition of CXCR4 suppresses CXCL12-induced migration of cancer cells, invasion, neoangiogenesis and metastases. Neutralizing the interactions of CXCL12 and CXCR4 by monoclonal antibody significantly impairs metastasis of breast cancer cells to regional lymph nodes and lungs [27] . Human breast tumor growth can be delayed by inhibiting CXCR4 with siRNA [28] . Similarly, CXCR4 antagonists, T140 analogs, inhibit SDF-1-induced migration of human breast cancer, leukemia and endothelial cells in vitro, in a manner relevant to tumor spread and angiogenesis [29] . The CXCR4 inhibitor RCP168 partially abrogates the protective effect of stromal co-culture system and greatly enhances chemotherapyinduced apoptosis in chronic lymphocytic leukemia cells [30] . Redjal  www.wjgnet.com kinases 1 and 2 and Akt, all of which are pathways downstream of CXCR4 that promote cell sur vival, proliferation and migration. Moreover, in vivo, combining sub-therapeutic doses of AMD3100 with a conventional cytotoxic agent in an order-dependent manner produces synergistic effects, which result from both a decrease in proliferation and an increase in apoptosis of tumor cells [31] .
Our previous studies have shown a significant association between CXCR4 expression and lymph nodal status and increased risk for metastasis in colorectal carcinoma. These initial observations prompted us to further question whether modulation of CXCR4 affects the invasive ability of CRC cells. In the present study, we demonstrated that AMD3100, a CXCR4-specific inhibitor, strongly suppressed CXCL12induced activity and invasion of the SW480 cell line, which expressed CXCR4 at a high level. Recently, researchers have found that CXCL12-CXCR4 signaling directly regulates MMP-9 expression in CRC cell lines [16] . In our present study, the use of AMD3100 markedly reduced the mRNA and protein expression of MMP-9 but not MMP-2 in SW480 cells, through blocking the CXCL12-CXCR4 signaling pathway. This suggests that inhibiting SDF-1/CXCR4 interaction may have a variety of therapeutic benefits in CRC patients. Meanwhile, we also observed a directional migration of CRC cells depending on the presence of extracellular matrix (ECM) proteins. AMD3100 significantly inhibited CXCL12-induced migratory activity and ECMdependent direct invasive ability.
Recent studies have also shown VEGF expression is associated with poor survival and prognosis in CRC, and that increased VEGF expression is correlated with increased microvessel density, local invasion, liver metastasis, and early recurrence after curative resection [20,32] . In the present study, we also found that AMD3100 significantly decreased mRNA and protein expression of VEGF in SW480 cells, which suggests that drug treatment may not only block CXCL12-induced angiogenesis, but also impair formation of microvasculature in metastasis stimulated by VEGF.
I n s e r u m -f r e e m e d i u m , a d d i n g C XC L 1 2 m ay significantly increase proliferation activity of tumor cells. This kind of protective microenvironment generally exists in various solid tumors, such as CXCR4-positive renal cancer and intracranial glioblastoma, which functionally improves cancer-cell viability, anti-apoptotic survival and chemoresistance [29] . Production of CXCL12 in tumor stroma may keep cancer cells tolerant to hypoxic or poor nutritional conditions. On the other hand, hypoxia-induced CXCR4 expression may activate downstream signal transduction, and subsequently regulate cancer cell biological behavior to adapt to hypoxia. In our study, we tested in the presence of CXCL12, the effect of various concentrations of AMD3100 on cell viability. Interestingly, although the treatment failed to decrease the number of cancer cells in serum-free medium, AMD3100 abrogated CXCL12 stimulation during cell proliferation in a dose-depended manner. Additionally, AMD3100 alone did not significantly affect cell proliferation compared with the unstimulated group, which suggests no autocrine growth stimulatory loops exist in this cell line.
In conclusion, CXCL12-CXCR4 signals are responsible for the invasion and metastasis of human CRC via regulation of cell viability and the expression of genes related to proteolysis and angiogenesis. Blocking the CXCL12-CXCR4 pathway may provide a novel means of preventing the invasion and metastasis of CRC.

ACKNOWLEDGMENTS
We thank Guo-Qiang Qiu, Chong-Zhi Zhou, and Jun-Wei Fan for providing excellent technical assistance.

Background
CXC chemokine receptor-4 (CXCR4) is by far the most common chemokine receptor overexpressed in human cancer cells. High-level expression of CXCR4 in various primary cancers is significantly associated with poor prognosis and extent of metastasis. Targeting CXCR4, therefore, is now regarded as a novel and efficient strategy for treating human cancer metastases. There are two categories of therapeutic strategy for targeting CXCR4, anti-CXCR4 monoclonal antibody and specific small molecular CXCR4 antagonists such as AMD3100, T140, ALX40-4C and CTCE-9908. Each of these inhibits CXCR4 via different mechanisms.

Research frontiers
Specific small-molecule CXCR4 antagonists such as AMD3100 may play an important role in the treatment of HIV infections, and many other pathological processes that are dependent on CXCL12-CXCR4 interactions (e.g. rheumatoid arthritis, asthma and cancer metastasis). Treatment of animals with AMD3100 results in decreased activation of the mitogen-activated protein kinase and AKT pathways in xenograft brain tumors. Administration of AMD3100 and 1, 3-bis (2-chloroethyl)-1-nitrosourea (BCNU) in an order-dependent manner produces synergistic cytotoxicity, which results from both a decrease in proliferation and an increase in apoptosis of glioblastoma cells.

Innovations and breakthroughs
We have demonstrated that CXCL12-induced activity and invasion of CRC cells were markedly suppressed by blocking the CXCL12-CXCR4 signaling pathway with AMD3100, a small non-peptide inhibitors of CXCR4. These findings strongly suggest CXCR4 plays an important role in CRC progression and may provide a novel and effective molecular target for treatment of CRC.

Applications
Our study showed that blockade of the CXCL12-CXCR4 axis affects the viability, invasion and migration of CRC cells by using various doses of AMD3100. The data also demonstrated the inhibitory effect was dose-dependent. It may be a good start to move from elucidation of the mechanism of action of chemokine receptors in cancer metastasis to development of novel therapeutic targets based on the CXCL12-CXCR4 axis.

Terminology
Chemokines are small secreted peptides that control adhesion and transendothelial migration of leukocytes, especially during immune and inflammatory reactions. They are divided into four subfamilies: CC, CXC, C and CX3C based on the position of their NH2-terminal cysteine residues, and bind to seven transmembrane domain G protein-coupled receptors. CXCL12, also known as SDF-1, belongs to the CXC chemokine family and CXCR4 is the only known physiological receptor for SDF-1.

Peer review
This is a very interesting study. The authors showed inhibition of CRC cell viability and metastasis by blocking CXCL12-CXCR4 interaction. Although this does not necessarily predict a positive result in human trials, the accumulation of these data may provide additional clues regarding some unanswered questions.