<title>1. INTRODUCTION</title> <p>Peroxisome proliferator-activated receptor gamma (PPAR<italic>γ</italic>) is
a member of
the ligand-inducible nuclear receptor superfamily. After activation, PPAR<italic>γ</italic> associates
with the 9-cis retinoic acid receptor (RXR) to form functional heterodimers, which
binds to the PPAR response element of the target genes and regulates the
expression of these genes. Previous documents have shown that the PPAR<italic>γ</italic>/RXR
signal pathway plays critical role in a variety of biological processes, including
adipogenesis, glucose metabolism, inflammation as well as inhibition of normal
and tumor cells growth [##REF##10549292##1##].</p> <p>Thiazolidinediones (TZDs) are synthetic agonists for PPAR<italic>γ</italic>. These PPAR<italic>γ</italic> ligands
were clinically used as antidiabetic drugs which could attenuate the insulin resistance
associated with obesity, hypertension, and impaired glucose tolerance in humans
[##REF##8922349##2##]. Recent studies have suggested that PPAR<italic>γ</italic> is a potential molecular target for
anticancer drug development, due to the increased expression of PPAR in several
cancer cells. It has been reported that TZDs could inhibit growth and induce
apoptosis in a variety of cancer cell lines. More importantly, TZDs exhibited
antitumor activities in vivo in the prevention of prostate, liver, and
pituitary cancers. Although increasing evidence
showed that TZDs are potential anticancer agents [##REF##15231248##3##], the mechanisms underlying
the antitumor effects are not well understood. TZDs were initially thought
to inhibit the cancer cells proliferation through regulation of expression of PPAR<italic>γ</italic>-mediated
target genes. However, recent evidence
revealed that the antitumor effects of TZDs exist via PPAR<italic>γ</italic>-independent
mechanisms in various types of cancers [##REF##16390557##4##–##REF##14563954##6##].</p> <p>We previously found the expression
of PPAR<italic>γ</italic> decreased in primary and metastatic
gastric carcinoma, compared with normal gastric tissues [##REF##17908458##7##]. Recent studies in
gastric cancer cells demonstrated that TZDs treatment resulted in significant growth
arrest both in cultured cell and in nude mice models [##REF##11044367##8##–##REF##10558888##12##]; however, the
effects of PPAR<italic>γ</italic> ligands on invasiveness and angiogenesis of gastric cancer are
still unclear. Therefore, this work was undertaken to investigate the effects
of PPAR<italic>γ</italic> agonists, such as rosiglitazone, on cell growth and the invasiveness in
human cell line SGC-7901, as well as on angiogenesis in vitro. </p>
<title>2. METHODS</title> <title>2.1. Cell culture</title> <p>Human gastric cancer cell line, SGC-7901, was obtained from the Type Culture Collection
of Chinese Academy of Sciences (Shanghai, China). Human
umbilical vein endothelial cells (HUVECs) were purchased from the Keygen Technology
Company (Najing, China). SGC7901 cells and HUVECs were
cultured in RPMI-1640 medium (GIBCO, Carlsbad, Calif, USA)
containing 10% fetal bovine serum (FBS) and 1% antibiotics (100 U/mL penicillin G, 100 <italic>μ</italic>g/mL streptomycin
sulfate, Sigma-Aldrich, Mo, USA).</p> <title>2.2. RT-PCR</title> <p>Total RNA was isolated using TRIzol
Reagent (Invitrogen, Carlsbad, Calif, USA)
according to the manufacturer's instructions. Reverse transcription reaction
was performed with random hexamer primers and a SuperScript Reverse
transcriptase kit (Invitrogen, Carlsbad, Calif, USA).
The sequences of specific primers were as follows: PPAR<italic>γ</italic> mRNA, forward, 5′-TCT CTC CGT AAT GGA AGA CC-3′, and reverse, 5′-GCA TTA TGA GAC ATC CCC AC-3′. MMP-2 mRNA, forward, 5′-GGC CCT GTC ACT CCT GAG AT-3′, and reverse, 5′-GGC ATC CAG
GTT ATC GGG GA-3′.
VEGF mRNA, forward,
5′-GAC AAg AAA ATC CCT GTG GGC-3′,
and reverse 5′-AAC GCG AGT CTG TGT TTT TGC-3′. <italic>β</italic>-actin mRNA, forward, 5′-CTT CTA CAA TGA GCT
GCG TA-3′, and reverse, 5′-TCA
TGA GGT AGT CAG TCA GG-3′.
PCR conditions were 94°C, 30 seconds, 55–57°C (depending on the primer set), 30 seconds,
and 72°C, 1 minute with 35 cycles using
Taq PCR MasterMix (Tianwei, Beijing, China). The resultant PCR products were
474 bp (PPAR<italic>γ</italic>), 243 bp (<italic>β</italic>-actin), 474 bp (MMP-2), and 169 bp
(VEGF). PCR products were electrophoresed on a 1.2% agarose gel and visualized
by ethidium bromide staining.</p> <title>2.3. Quantitative
real-time RT-PCR analysis</title> <p>The PCR
reactions were performed in a Brilliant
SYBR Green QPCR master mix (Stratagene, Calif, USA) according
to the manufacturer's instructions. The sequences of specific primers
were the same as for RT-PCR. After 10 minutes at 95°C to denature the cDNA, the cycling conditions were 95°C, 1 minute, 55–57°C (depending on the primer
set), 30 seconds, and 72°C, 1 minute with 40
cycles. The LightCycler software constructed the calibration curve by plotting
the crossing point (Cp), and the numbers of copies in unknown samples were
calculated by comparison of their Cps with the calibration curve. To correct
differences in both RNA quality and quantity between samples, the data were formalized
to those for <italic>β</italic>-actin.</p> <title>2.4. Western blotting</title> <p>The cells proteins were extracted
according to NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Pierce, Rockford, Ill, USA). Protein
concentration of each sample was assayed using BCA Protein Assay Reagent
according to manufacturer's instructions (Pierce Biotechnology, Rockford, Ill,
USA). Twenty
micrograms of proteins of different groups were separated in 10% SDS-PAGE, and
transferred onto PVDF membrane (Invitrogen, Carlsbad, Calif, USA). Five percent of milk
(blocking solution) was loaded over the membrane and incubated for 1 hour at
room temperature with agitation. The membranes were then incubated with the mouse
antihuman PPAR<italic>γ</italic> antibody at a dilution of 1:200 (Santa
Cruz, Calif, USA), the mouse antihuman
MMP-2 antibody (1:400, Neomarker, Calif, USA), the rabbit antihuman VEGF
antibody (1:200, Zymed, Calif, USA), and
the mouse antihuman <italic>β</italic>-actin
(1:200, Xiaxin, China) overnight at 4°C with agitation.
After being washed with 0.1% Tween 20 in Tris-saline, three times, the membranes were incubated with biotin-labeled
antirabbit or mouse IgG for 1 hour at room temperature with agitation. Reactive
protein was detected using ECL chemiluminescence system (Pierce, Rockford, Ill, USA).</p> <title>2.5. ELISA of secreted VEGF</title> <p>The effect of RGZ on VEGF release
in tumor cells was measured by ELISA. Cells grown in 90 mm plates were exposed to various concentrations
of RGZ (1–20 <italic>μ</italic>M) or
vehicle with or without GW9662 (2.5 <italic>μ</italic>M, pretreated 1 hour) for 24 hours. VEGF concentration in the supernatant was
measured using a VEGF ELISA kit (R &amp; D systems, Minneapolis, Minn, USA).</p> <title>2.6. Cell viability</title> <p>The viability of the cells was
assessed by MTT assay. Briefly, cells grown in 96-wells
were exposed to various concentrations of RGZ with or without GW9662 (2.5 <italic>μ</italic>M, pretreated 1 hour), for 24, 48, or 72 hours. Then, 20 <italic>μ</italic>L of MTT (5 mg/mL) was
added to each well, and cells were incubated continuously at 37°C for 4 hours. After
removal of medium, the crystals were dissolved in DMSO, and absorbance was
assessed at 570 nm with a microplate reader.</p> <title>2.7. Cell cycle and apoptosis analysis</title> <p>Cells treated with RGZ (1–20 <italic>μ</italic>M) or vehicle with or
without GW9662 (2.5 <italic>μ</italic>M,
pretreated 1 hour) for 48 hours were collected and fixed in cold 70% ethanol.
Then, the samples were treated with RNase, stained with 50 mg/mL propidium
iodide (PI), and analysed by EPICS Elite flow cytometer (Coulter Electronics, Fla, USA).</p> <title>2.8. Invasion assay</title> <p>The ability of cells to invade through a
Matrigel-coated filter was measured in transwell chambers (Corning, NY, USA). Polyvinylpyrrolidone-free
polycarbonate filters (pore size 8 <italic>μ</italic>m)
were coated with basement membrane Matrigel (50 <italic>μ</italic>L/filter) (BD, Bedford, Ohio, USA).
The membrane was washed in PBS to remove excess ligand, and the lower chamber
was filled with 0.6 mL of RPMI-1640 medium containing 10% fetal bovine serum
(FBS). Cells were serum-starved overnight (0.5% FBS), harvested with
trypsin/EDTA, and washed twice with serum-free RPMI-1640 medium. Cells were
resuspended in migration medium (RPMI-1640 medium with 0.5% FBS), and 0.1 mL migration
medium containing 1 × 10<sup>5</sup> cells was added to the upper chamber. After incubation with RGC (1–20 <italic>μ</italic>M) with or without GW9662
(2.5 <italic>μ</italic>M, pretreated
1 hour) at 37°C for 24 hours, the cells on the upper surface of the membrane were removed using
a cotton swab. The migrant cells attached to the lower surface were fixed in
10% formalin at room temperature for 30 minutes and stained with hematoxylin.
The numbers of migrated cells were counted under a microscope.</p> <title>2.9. Scratch wound-healing motility
assays</title> <p>Gastric cancer cells were seeded on 60 mm plates and allowed to grow to
confluence. Confluent monolayers were scratched with a pipette tip and
maintained under RGZ (1–20 <italic>μ</italic>M) with or without GW9662
(2.5 <italic>μ</italic>M, pretreated
1 hour) for 24 hours. Plates were washed once with fresh medium to remove nonadherent
cells and then photographed. The cell migration was evaluated by counting cells
that migrated from the wound edge.</p> <title>2.10. In vitro Angiogenesis assay</title> <p>The angiogenesis assays were performed as per the manufacturer's
instructions, that is, transfer 50 <italic>μ</italic>L
of ECMatrixTM solution to each well of a precooled 96-well tissue culture plate
on ice. Incubate at 37°C for 1 hour to
allow the matrix solution to solidify. Harvest human umbilical vein endothelial
cells (HUVECs) resuspend and Seed 5 × 10<sup>3</sup> cells per well onto the
surface of the polymerized ECMatrixTM. Incubate with RGC (1–20 <italic>μ</italic>M) with or without GW9662
(2.5 <italic>μ</italic>M, pretreated
1 hour) at 37°C for 12 hours. Inspect tube formation under an inverted light microscope at 100 X
magnification.</p> <title>2.11. Zymography</title> <p>Cells were cultured for 24 hours in serum-free
medium, washed twice, and finally treated with RGZ (1–20 <italic>μ</italic>M) with or without GW9662
(2.5 <italic>μ</italic>M, pretreated
1 hour) for a further 48 hours. The supernatants were collected and concentrated,
using centrifugal filter devices (Millipore Corp., Bedford, Mass, USA)
and the protein content was determined using BCA Protein Assay Reagent. Equal
amounts of protein (20 <italic>μ</italic>g) were mixed with SDS sample buffer without reducing
agents and incubated for 40 minutes at 37°C. For gelatinolytic activity, the assay
samples were separated on polyacrylamide gels containing 1mg/mL gelatin. After
electrophoresis, the gels were stained for 1 hour in a 45% methanol/10%
acetic acid mixture containing coomassie brilliant blue G250 and destained.
Zymograms were photographed after 10 hours of incubation at 37°C.</p> <title>2.12. Statistical analysis</title> <p>Data are expressed as mean ± standard
deviation (SD) of three independent experiments, each done in triplicate. Differences
between control and experiment groups were analyzed using the <italic>t</italic>-test. <italic>P</italic> &lt; .05 was considered
statistically significant.</p>
<title>3. RESULTS</title> <title>3.1. RGZ inhibited proliferation and
induced apoptosis in SGC-7901 cells through PPAR<italic>γ</italic>-dependent mechanism</title> <p>In SGC-7901 cells, the expression of PPAR<italic>γ</italic> was observed by RT-PCR and western blot (not
shown).</p> <p>RGZ (0.1–100 <italic>μ</italic>M) treatment for 24, 48, and
72 hours inhibited cells growth in a dose- and time-dependent manners in
SGC-7901 gastric cancer cell line as determined by MTT assay. Pretreatment with
the highly selective PPAR<italic>γ</italic> antagonist GW9662
(2.5 <italic>μ</italic>M) reversed the
effect of RGZ on cell viability (see ##FIG##0##Figure 1(a)##).</p> <p>To explore whether the growth inhibition of RGZ in SGC-7901 cells was
caused by apoptosis, we analyzed the sub-G1 population of the cells after
treatment with RGZ (1–20 <italic>μ</italic>M) for 48 hours. RGZ induced
apoptosis in a dose-dependent manner, which was also reversed completely by 2.5 <italic>μ</italic>M GW9662 treatment (see ##FIG##0##Figure 1(b)##).</p> <p>Furthermore, to determine whether the inhibitory effect of RGZ on cell
viability is associated with the arrest of the cell cycle, we analyzed the cell
cycle progression after treatment with RGZ (1–20 <italic>μ</italic>M) for 48 hours. RGZ
treatment increased the number of cells in the G1-G0 and decreased
the number of cells in the S phases in dose-dependent manner. The effects of
RGZ on cell cycle of SGC-7901 cells were also reversed by 2.5 <italic>μ</italic>M GW9662 (see ##FIG##0##Figure 1(c)##).</p> <title>3.2. RGZ inhibited SGC-7901 cells
migration and invasiveness through PPAR<italic>γ</italic>-independent mechanism</title> <p>After treatment with RGZ (1–20 <italic>μ</italic>M) for 48 hours, the number
of cells migrated to the scratched area was 60 ± 3.1 cells/mm<sup>2</sup>, 58 ± 2.7 cells/mm<sup>2</sup>, 49 ± 2.8 cells/mm<sup>2</sup>, 27 ± 2.9 cells/mm<sup>2</sup>, and 20 ± 1.9 cells/mm<sup>2</sup>, respectively, which were
significantly lower than those in control group (84 ± 3.4 cells/mm<sup>2</sup>). GW9662 treatment had no
effects on the cells migration with inhibition induced by RGZ. The number of
the cells migrated to the scratched area treated with GW9662 and RGZ (1–20 <italic>μ</italic>M) for 48 hours was 61 ± 1.8 cells/mm<sup>2</sup>, 53 ± 3 cells/mm<sup>2</sup>, 47 ± 2.5 cells/mm<sup>2</sup>,
29 ± 2.8 cells/mm<sup>2</sup>, 18 ± 3.2 cells/mm<sup>2</sup>, respectively, which
were not different from those in the groups treated with RGZ alone (see ##FIG##1##Figure 2(a)##).</p> <p>The effect of RGZ on the cells invasion through reconstituted basement
membranes was analyzed using Matrigel-coated invasion chambers. After treatment with RGZ (1–20 <italic>μ</italic>M) for 48 hours,
the cells attached to the lower surface of the filters were 256 ± 9 cells/mm<sup>2</sup>, 248 ± 7 cells/mm<sup>2</sup>, 219 ± 12 cells/mm<sup>2</sup>, 174 ± 11 cells/mm<sup>2</sup>, and 154 ± 10 cells/mm<sup>2</sup>, respectively,
which were significantly lower than those in control group (279 ± 9 cells/mm<sup>2</sup>). After cotreatment of the cells with
GW9662 and RGZ, the cells attached to the lower surface were 251 ± 29 cells/mm<sup>2</sup>, 238 ± 12 cells/mm<sup>2</sup>, 220 ± 7 cells/mm<sup>2</sup>, 166 ± 16 cells/mm<sup>2</sup>, and 148 ± 12 cells/mm<sup>2</sup>, respectively,
which were not different from those in the groups treated with RGZ alone
(see ##FIG##1##Figure 2(b)##).</p> <p>Metalloproteases (MMPs) have been
demonstrated to play a significant role in tumor cell invasion [##REF##11322833##13##]. In this
study, our results showed that RGZ inhibited the mRNA and protein expression
levels of MMP-2 in a
dose-dependent manner (see Figures ##FIG##2##3(a)##, ##FIG##2##3(c)##, and Tables ##TAB##0##1##, ##TAB##1##2##). Moreover, the gel zymography
results demonstrated that the activity of MMP-2 decreased after RGZ (1–20 <italic>μ</italic>M) treatment
for 48 hours in dose-dependent manner (see ##FIG##3##Figure 4(a)##). The inhibitory effects of
RGZ on MMP-2 were not affected by GW9662 treatment (see Figures ##FIG##2##3(b)##, ##FIG##2##3(c)##, and ##FIG##3##4(b)##).</p> <title>3.3. Effects of RGZ on angiogenesis in vitro</title> <p>Matrigel-plated HUVECs elongated and migrated in the presence
of VEGF and formed tubular networks. RGZ markedly suppressed the formation of the
tube-like structures of HUVEC cells in a dose-dependent manner (see ##FIG##4##Figure 5(a)##),
which was completely antagonized by GW9662 (see ##FIG##4##Figure 5(b)##). These results suggested
that rosiglitazone exhibits antiangiogenic activity via PPAR<italic>γ</italic>-dependent mechanism.</p> <p>To further determine whether the effect of RGZ on
angiogenesis is due to the down regulation of the tumor-secreted growth
factors, we measured the expression levels of VEGF in SGC-7901 cell cultured
medium, after treatment with various concentrations of RGZ. Our results
demonstrated that RGZ (1–20 <italic>μ</italic>M) did not
change the expression of mRNA and protein of VEGF in SGC-7901 cells (see Figures ##FIG##2##3(a)##, ##FIG##2##3(c)##, and ##TAB##0##Table 1##), but also the
results were confirmed by ELISA (see ##FIG##5##Figure 6##).</p>
<title>4. DISCUSSION</title> <p>As a
potential molecular target for anticancer drug development, PPAR<italic>γ</italic> and its
ligands have been extensively studied in the past several years. Previous
studies have shown that PPAR<italic>γ</italic> is expressed in several human gastric-cancer cell
lines, including MKN-7, MKN-28, MKN-45, and AGS. TZDs could inhibit these cancer cell lines
growths in vitro and in vivo [##REF##14960510##9##, ##REF##10558888##12##]. Also,
the growth inhibitory effects of TZDs on MKN45 cells depend on the PPAR<italic>γ</italic>
expression levels. The growth inhibition
of TDZs was more significant in the higher PPAR<italic>γ</italic> expressing cells. Moreover, Lu et al.
[##REF##15930296##10##] found that PPAR<italic>γ</italic> (+/ − ) mice were more susceptible to MNU-induced gastric
cancer than wild-type (+/+) mice, and troglitazone significantly reduced the
incidence of gastric cancer in PPAR<italic>γ</italic> (+/+) mice but not in PPAR<italic>γ</italic> (+/ − ) mice. All
these results indicated that TZDs inhibit the cancer cells growth via PPAR<italic>γ</italic>-dependent
mechanism. Our results demonstrated that RGZ, the most potent and selective
synthetic ligand of PPAR<italic>γ</italic>, inhibited SGC-7901
gastric cancer cells growth, caused G1 cell cycle arrest, and induced apoptosis in
a dose-dependent manner. The effects of RGZ on SGC-7901 cancer cells were
completely reversed by treatment with PPAR<italic>γ</italic> antagonist GW9662. These results
indicated that RGZ suppressed the SGC-7901 cancer cells growth in a PPAR<italic>γ</italic>-dependent
mechanism.</p> <p>In this study, we found that the RGZ inhibited invasion, migration, and
the secretion of MMP-2 of SGC-7901 cells. The inhibitory effects of RGZ on metastases and
MMP-2 activity were not directly mediated by PPAR<italic>γ</italic> activation,
since these effects were not reversed by GW9662 treatment. Our results were
consistent with the previous works on human adrenocortical cancer cell line
H295R [##REF##15585569##14##], pancreatic cancer cells [##REF##15479693##15##], and human myeloid leukemia cells [##REF##15838654##16##], which
showed that PPAR<italic>γ</italic> ligands act independently of PPAR<italic>γ</italic> activation in the invasion
suppression and down-regulation of MMP-2 activity. Recent papers
showed that PPAR<italic>γ</italic> regulated E-cadherin expression and inhibited
growth and invasion of prostate cancer [##REF##17015477##17##], and PPAR<italic>γ</italic> ligand
troglitazone inhibited transforming growth factor-beta-mediated glioma cell
migration and brain invasion [##REF##17541035##18##]. But
some studies have contrasting results that the PPAR<italic>γ</italic>, ciglitazone, induced cell invasion, through
activation of Pro-MMP-2, activation via the generation of ROS, and the activation
of ERK [##REF##17597617##19##], and that PPAR<italic>γ</italic> antagonists induced vimentin cleavage and inhibited
invasion in high-grade hepatocellular carcinoma [##REF##17786342##20##]. Further studies are
needed on the mechanism of PPAR<italic>γ</italic> in cancer and invasion.</p> <p>Recent
investigations suggested that PPAR<italic>γ</italic> ligands had inhibitory effects on tumor
cell lines, but the effects appear not to be entirely elicited by the direct
action on tumor cells. Inhibition of the neovascularization may be another
target of TZDs to suppress the growth of cancers. PPAR<italic>γ</italic> is expressed in
endothelial cells, and the PPAR<italic>γ</italic> ligands can inhibit the proliferation of these
cells induced by growth factors, or cause their apoptosis in vitro [##REF##10358055##21##–##REF##10854212##23##]. It has been
reported that PPAR<italic>γ</italic> ligands could inhibit choroidal, retinal, and corneal
neovascularization when administered intraocularly [##REF##10892878##24##–##REF##15721625##26##]. In addition,
systemic administration of rosiglitazone and troglitazone inhibits FGF2-induced
angiogenesis; thereby inhibiting primary tumor growth and metastasis [##REF##12370270##27##]. We observed
that RGZ inhibited
the angiogenesis of HUVECs in dose-dependent manner via PPAR<italic>γ</italic>
pathway. The effects RGZ on the endothelium suggest that RGZ may
regulate tumor growth by targeting non-cell-autonomous mechanisms.</p> <p>Previous studies [##REF##12475986##5##]
showed that suppression of angiogenesis could result from a decrease in the
local levels of stimulators (e.g., VEGF and FGF2) and/or an increase of
endogenous inhibitors of angiogenesis (e.g., thrombospondin) produced by tumor
cells. PPAR<italic>γ</italic> ligands suppressed
VEGF production in colon carcinoma [##REF##15289320##28##], human
breast cancer [##REF##17384282##29##], and human renal cell carcinoma cells [##REF##15780399##30##]. However, contradictory
results have also been reported in bladder and prostate cancer cells in which PPAR<italic>γ</italic> ligands
increased VEGF production [##REF##11980898##31##, ##REF##12239635##32##]. Inconsistent with the above documents, our
results showed that RGZ did not change the secretion of VEGF from SGC-7901.</p> <p>Taken together, our results demonstrated that RGZ inhibited growth and invasiveness
of SGC-7901 gastric cancer cells and angiogenesis in vitro via PPAR<italic>γ</italic>-dependent or -independent pathway. Further
study is needed to elucidate the
mechanisms by which RGZ exhibits different manner.</p>
<p>Recommended by Dipak Panigrahy</p> <p>Although thiazolidinediones (TZDs) were found to be ligands for peroxisome proliferators-activated receptor<italic>γ</italic> (PPAR<italic>γ</italic>), the mechanism by which TZDs exert their anticancer effect remains unclear. Furthermore, the effect of TZDs on metastatic and angiogenesis potential of cancer cells is unknown. Our results in this paper show that rosiglitazone inhibited SGC-7901 gastric cancer cells growth, caused G1 cell cycle arrest and induced apoptosis in a dose-dependent manner. The effects of rosiglitazone on SGC-7901 cancer cells were completely reversed by treatment with PPAR<italic>γ</italic> antagonist GW9662. Rosiglitazone inhibited SGC-7901 cell migration, invasiveness, and the expression of MMP-2 in dose-dependent manner via PPAR<italic>γ</italic>-independent manner. Rosiglitazone reduced the VEGF induced angiogenesis of HUVEC in dose-dependent manner through PPAR<italic>γ</italic>-dependent pathway. Moreover, rosiglitazone did not affect the expression of VEGF by SGC-7901 cells. Our results demonstrated that by PPAR<italic>γ</italic> ligand, rosiglitazone inhibited growth and invasiveness of SGC-7901 gastric cancer cells and angiogenesis in vitro via PPAR<italic>γ</italic>-dependent or -independent pathway.</p>
<title>ACKNOWLEDGMENTS</title> <p>This work was supported by the National Natural
Science Foundation of China (Grant no. 30671904, 30670949), China Postdoctoral
Science Foundation (no. 2004035181), and The Doctor Station of Ministry of
Education of China (no. 20060558010).</p>
<fig id="fig1" position="float"><label>Figure 1</label><caption><p>(a) RGZ (0.1–100 <italic>μ</italic>M) treatment for 24, 48, and
72 hours inhibited cell growth in a dose- and time-dependent manners in
SGC-7901 gastric cancer cell line, as determined by MTT assay, which was reversed
completely by 2.5 <italic>μ</italic>M GW9662 pretreatment for 1 hour. Cell
viability was expressed as the percentage of cells under control conditions (0 <italic>μ</italic>M
of RGZ or GW9662). (b) RGZ induced apoptosis in a dose-dependent manner,
which was also reversed completely by 2.5 <italic>μ</italic>M GW9662 pretreatment for 1 hour. (c) RGZ treatment increased the number of
cells in the G1-G0 and decreased
the number of cells in the S phases in dose-dependent manner, which was reversed
completely by 2.5 <italic>μ</italic>M GW9662 pretreatment for 1 hour. Values
are the means ± SD of three representative experiments.*Statistical significance (<italic>P</italic> &lt; .05 or
higher degree of significance) versus vehicle-treated controls.</p></caption></fig> <fig id="fig2" position="float"><label>Figure 2</label><caption><p>(a) Effect of RGZ on the migration and (b) invasion
of SGC-7901 gastric cancer cells, which was reversed completely by 2.5 <italic>μ</italic>M GW9662
pretreatment for 1 hour. Values are the means ± SD of three representative experiments.*Statistical significance (<italic>P</italic> &lt; .05 or
higher degree of significance) versus vehicle-treated controls.</p></caption></fig> <fig id="fig3" position="float"><label>Figure 3</label><caption><p>(a) RGZ (1–20 <italic>μ</italic>M) inhibited
the mRNA and (c) protein expression levels of MMP-2 in a dose-dependent manner, which were not affected by 2.5 <italic>μ</italic>M GW9662 pretreatment
for 1 hour (b), (c). RGZ (1–20 <italic>μ</italic>M) did not
change the expression of VEGF in SGC-7901 cells (a), (c).</p></caption></fig> <fig id="fig4" position="float"><label>Figure 4</label><caption><p>(a)The activity of MMP-2 was
decreased after RGZ (1–20 <italic>μ</italic>M) treatment
for 48 hours in dose-dependent manner. (b) The inhibitory effects of RGZ on MMP-2
were not affected by 2.5 <italic>μ</italic>M GW9662 pretreatment for 1 hour.</p></caption></fig> <fig id="fig5" position="float"><label>Figure 5</label><caption><p>(a) RGZ markedly suppressed the formation of the
tube-like structures of HUVEC cells in a dose-dependent manner, (b) which was
completely antagonized by 2.5 <italic>μ</italic>M GW9662 pretreatment
for 1 hour.</p></caption></fig> <fig id="fig6" position="float"><label>Figure 6</label><caption><p>RGZ had no effect on the secretion of VEGF of SGC-7901 cell.</p></caption></fig>
<table-wrap id="tab1" position="float"><label>Table 1</label><caption><p>Expression of MMP-2 and VEGF after RZD treatment
in SGC-7901 gastric cancers by real-time PCR.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">Rosiglitazone (<italic>μ</italic>mol/L)</th><th align="center" rowspan="1" colspan="1">PPAR<italic>γ</italic>
</th><th align="center" rowspan="1" colspan="1">MMP-2</th><th align="center" rowspan="1" colspan="1">VEGF</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0.132127 ± 0.045513</td><td align="center" rowspan="1" colspan="1">0.008912 ± 0.000133</td><td align="center" rowspan="1" colspan="1">0.61132 ± 0.078921</td></tr><tr><td align="left" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.121878 ± 0.034219</td><td align="center" rowspan="1" colspan="1">0.006003 ± 0.000331*</td><td align="center" rowspan="1" colspan="1">0.620255 ± 0.054671</td></tr><tr><td align="left" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">0.130134 ± 0.0521137</td><td align="center" rowspan="1" colspan="1">0.005486 ± 0.000541*</td><td align="center" rowspan="1" colspan="1">0.60728 ± 0.036799</td></tr><tr><td align="left" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">0.137778 ± 0.046222</td><td align="center" rowspan="1" colspan="1">0.005048 ± 0.000346*</td><td align="center" rowspan="1" colspan="1">0.599438 ± 0.076541</td></tr><tr><td align="left" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">0.141171 ± 0.038741</td><td align="center" rowspan="1" colspan="1">0.001924 ± 0.000189*</td><td align="center" rowspan="1" colspan="1">0.624165 ± 0.038966</td></tr><tr><td align="left" rowspan="1" colspan="1">20</td><td align="center" rowspan="1" colspan="1">0.143889 ± 0.061237</td><td align="center" rowspan="1" colspan="1">0.001298 ± 0.000267*</td><td align="center" rowspan="1" colspan="1">0.604246 ± 0.065679</td></tr></tbody></table></table-wrap> <table-wrap id="tab2" position="float"><label>Table 2</label><caption><p>Expression of MMP-2 and VEGF after RZD and GW9662
cotreatment in SGC-7901 by real-time PCR.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">Rosiglitazone (<italic>μ</italic>mol/L)</th><th align="center" rowspan="1" colspan="1">PPAR<italic>γ</italic>
</th><th align="center" rowspan="1" colspan="1">MMP-2</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0.14161 ± 0.055389</td><td align="center" rowspan="1" colspan="1">0.00975 ± 0.000533</td></tr><tr><td align="left" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.137738 ± 0.030102</td><td align="center" rowspan="1" colspan="1">0.008974 ± 0.000113*</td></tr><tr><td align="left" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">0.134614 ± 0.029881</td><td align="center" rowspan="1" colspan="1">0.006003 ± 0.000401*</td></tr><tr><td align="left" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">0.141156 ± 0.564569</td><td align="center" rowspan="1" colspan="1">0.00564 ± 0.000246*</td></tr><tr><td align="left" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">0.135666 ± 0.034887</td><td align="center" rowspan="1" colspan="1">0.002182 ± 0.000364*</td></tr><tr><td align="left" rowspan="1" colspan="1">20</td><td align="center" rowspan="1" colspan="1">0.129278 ± 0.019262</td><td align="center" rowspan="1" colspan="1">0.001712 ± 0.000178*</td></tr></tbody></table></table-wrap>
<table-wrap-foot><fn id="TF1"><p>*Statistical significance (<italic>P</italic> &lt; .05
or higher degree of significance) versus
vehicle-treated controls.</p></fn></table-wrap-foot> <table-wrap-foot><fn id="TF2"><p>*Statistical significance (<italic>P</italic> &lt; .05
or higher degree of significance) versus vehicle-treated controls.</p></fn></table-wrap-foot>
<graphic xlink:href="PPAR2008-649808.001"/> <graphic xlink:href="PPAR2008-649808.002"/> <graphic xlink:href="PPAR2008-649808.003"/> <graphic xlink:href="PPAR2008-649808.004"/> <graphic xlink:href="PPAR2008-649808.005"/> <graphic xlink:href="PPAR2008-649808.006"/>
[{"label": ["11"], "surname": ["Takeuchi", "Okumura", "Motomura", "Nagamine", "Takahashi", "Kohgo"], "given-names": ["S", "T", "W", "M", "N", "Y"], "article-title": ["Troglitazone induces G1 arrest by p27"], "sup": ["Kip1"], "italic": ["Cancer Science"], "year": ["2002"], "volume": ["93"], "issue": ["7"], "fpage": ["774"], "lpage": ["782"]}]
PPAR Res. 2008 Sep 15; 2008:649808