High CpG island methylator phenotype is associated with lymph node metastasis and prognosis in gastric cancer.

Several studies have found that the promoter CpG island is frequently methylated in gastric cancer. The CpG island methylator phenotype (CIMP) defines concordant methylation of multiple promoter CpG island loci in a subset of gastric cancer. However, the relationship between CIMP and lymph node metastasis in gastric cancer is unknown. Our study aimed to characterize the role of CIMP in lymph node metastasis. Clinical specimens from 120 patients were analyzed and PCR was used to detect the methylation status of five genes (ALX4, TMEFF2, CHCHD10, IGFBP3, and NPR1). We measured the level of mRNA for the five genes by real-time RT-PCR. Microsatellite instability and Helicobacter pylori infection status were assayed by capillary electrophoresis and real-time PCR, respectively. DNA methylation in the five genes was correlated with low expression of the respective mRNA. With CIMP as the dependent variable, CIMP-high gastric cancer tended to show more distant lymph node metastasis, higher pathologic tumor classification, more pathologic metastasis, and higher pathologic TNM status. Microsatellite instability and H. pylori status were not significant predictors of prognosis. CIMP-high gastric cancer showed significantly worse survival compared with that of CIMP-low/CIMP-negative gastric cancer (P < 0.001). Our results show that there is an association between CIMP status and lymph node metastasis in gastric cancer and CIMP-high was an independent prognostic factor.

[Curcumine inhibits migration and invasion of hepatic stellate cells by reducing MMP-2 expression and activity].

OBJECTIVE: To investigate the molecular mechanism of the inhibitory effect of curcumine on the migration and invasion of hepatic stellate cells (HSC). METHODS: Rat hepatic stellate cells were cultured and activated with ConA. Matrix metalloproteinase-2 (MMP-2) expression and activity was determined by Western blot and gelatin zymography. Migration and invasion of HSC was assessed by wound healing assay and modified Boyden chamber assay. RESULTS: Curcumine reduced the level and activity of MMP-2 expression in activated HSC in a dose-dependent manner. When treated with 25, 50 or 100 micromol/L curcumine, the expression of MMP-2 was reduced by 21.8%+/-5.1%, 65.5%+/-9.2% or 87.9%+/-11.5% (P < 0.05), and the activity of MMP-2 was also significantly reduced by curcumine. Migration and invasion of activated HSC was also inhibited by curcumine in a dose-dependent way. When treated with 25, 50 or 100 micromol/L curcumine, the migration of activated HSC was reduced by 27.5%+/-5.8%, 54.4%+/-7.6% or 67.1%+/-9.3% (P < 0.05), and the invasion of activated HSC was also significantly reduced by curcumine. CONCLUSION: Curcumine inhibits migration and invasion of activated HSC by reducing MMP-2 expression and activity.

Expression and prognostic impact of PRL-3 in lymph node metastasis of gastric cancer: its molecular mechanism was investigated using artificial microRNA interference.

High PRL-3 expression had been reported to have close association with lymph node metastasis (LNM) of gastric cancer. However, the prognostic significance of highly expressing PRL-3 in LNM of human gastric cancer and the role in the metastasis remain unclear. Our study examined PRL-3 expression both in the LNM (n = 107) and in the primary lesion (n = 137) of gastric cancer, and compared the overall survival rates. RNA interference, induced by recombinant plasmid pcDNA.rPRL3-miR expressing artificial PRL-3 miRNA, was employed to knockdown PRL-3 expression in human SGC7901 gastric cancer cells. Invasion assay and migration assay in vitro were conducted to determine the role of PRL-3 in the metastasis. The role of PRL-3 in the proliferation of SGC7901 cells and tumor growth were also determined. We observed that high PRL-3 expression was more frequently detected in the LNM than in the matched primary lesion (72.9 vs. 47.7%, p < 0.001). Furthermore, the overall survival rate of the patients with high expression of PRL-3 in the LNM was significantly less than those with moderate/low expression (p = 0.003). Importantly, knockdown of PRL-3 can significantly reduce both invasion and migration potencies of SGC7901 cells (p < 0.001), and significantly suppressed the proliferation of SGC7901 cells and slowed down the tumor growth (p < 0.001). It was concluded that high expression of PRL-3 in the LNM had a negative impact on the prognosis of the patients, and plays important roles in LNM of gastric cancer and the tumor growth, which can be a potential therapeutic target and a prognostic factor.

(-)-Epigallocatechin-3-gallate inhibits growth of gastric cancer by reducing VEGF production and angiogenesis.

AIM: To investigate the effect of (-)-epigallocatechin-3-gallate (EGCG) on growth of gastric cancer and its possible mechanism. METHODS: Heterotopic tumors were induced by subcutaneously injection of SGC-7901 cells in nude mice. Tumor growth was measured by calipers in two dimensions. Tumor angiogenesis was determined with tumor microvessel density (MVD) by immunohistology. Vascular endothelial growth factor (VEGF) protein level and activation of signal transducer and activator of transcription 3 (Stat3) were examined by Western blotting. VEGF mRNA expression was determined by RT-PCR and VEGF release in tumor culture medium by ELISA. VEGF-induced cell proliferation was studied by MTT assay, cell migration by gelatin modified Boyden chamber (Transwell) and in vitro angiogenesis by endothelial tube formation in Matrigel. RESULTS: Intraperitoneal injection of EGCG inhibited the growth of gastric cancer by 60.4%. MVD in tumor tissues treated with EGCG was markedly reduced. EGCG treatment reduced VEGF protein level in vitro and in vivo. Secretion and mRNA expression of VEGF in tumor cells were also suppressed by EGCG in a dose-dependent manner. This inhibitory effect was associated with reduced activation of Stat3, but EGCG treatment did not change the total Stat3 expression. EGCG also inhibited VEGF-induced endothelial cell proliferation, migration and tube formation. CONCLUSION: EGCG inhibits the growth of gastric cancer by reducing VEGF production and angiogenesis, and is a promising candidate for anti-angiogenic treatment of gastric cancer.

[(-)-Epigallocatechin-3-gallate reduces vascular endothelial growth factor expression in gastric cancer cells via suppressing activity].

OBJECTIVE: To investigate the molecular mechanism involved in the downregulation of vascular endothelial growth factor(VEGF) expression through the suppression of signal transducer and activator of transcription 3(Stat3) by(-)-Epigallocatechin-3-gallate (EGCG). METHODS: After human gastric cancer cells (AGS) were treated with IL-6 (50 µg/L) and EGCG(0, 5, 10, 25 or 50 µmol/L), the expression levels of VEGF, total Stat3(tStat3), and activated Stat3(pStat3) in tumor cells were examined by Western blotting. The influence of the inhibitor of Stat3 pathway on the IL-6-induced VEGF expression was investigated. VEGF protein level in tumor cell culture medium was determined by ELISA and VEGF mRNA expression in tumor cells by RT-PCR. Tumor cell nuclear extract was prepared and nuclear expression of pStat3 was detected. Stat3-DNA binding activity was examined with chromatin immunoprecipitation (ChIP) assay. RESULTS: IL-6 significantly increased VEGF expression in AGS gastric cancer cells. Compared with the group without IL-6, the expression and secretion of VEGF protein, and mRNA expression increased by 2.4 fold,2.8 fold, and 3.1 fold(all P<0.01), respectively. EGCG treatment markedly reduced VEGF protein, release and mRNA expression in a dose-dependent manner. When compared with the control group induced by IL-6, EGCG and AG490(a Stat3 pathway inhibitor) significantly inhibited VEGF expression induced by IL-6 (P<0.01). EGCG dose-dependently inhibited pStat3 induced by IL-6(P<0.05), but not tStat3 (P>0.05). Stat3 nuclear translocation and Stat3-DNA binding activity in AGS cells or that induced by IL-6 were directly inhibited by EGCG(P<0.05). CONCLUSION: EGCG reduces expression of VEGF in gastric cancer cells through the inhibition of Stat3 activity.

(-)-Epigallocatechin-3-gallate inhibits VEGF expression induced by IL-6 via Stat3 in gastric cancer.

AIM: To demonstrate that (-)-Epigallocatechin-3-gallate (EGCG) inhibits vascular endothelial growth factor (VEGF) expression and angiogenesis induced by interleukin-6 (IL-6) via suppressing signal transducer and activator of transcription 3 (Stat3) activity in gastric cancer. METHODS: Human gastric cancer (AGS) cells were treated with IL-6 (50 ng/mL) and EGCG at different concentrations. VEGF, total Stat3 and activated Stat3 protein levels in the cell lyses were examined by Western blotting, VEGF protein level in the conditioned medium was measured by enzyme-linked immunosorbent assay, and the level of VEGF mRNA was evaluated by reverse transcription polymerase chain reaction (RT-PCR). Stat3 nuclear translocation was determined by Western blotting with nuclear extract, and Stat3-DNA binding activity was examined with Chromatin immunoprecipitation (ChIP) assay. IL-6 induced endothelial cell proliferation was measured with 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazoliumbromide assay, in vitro angiogenesis was determined with endothelial cell tube formation assay in Matrigel, and IL-6-induced angiogenesis in vitro was measured with Matrigel plug assay. RESULTS: There was a basal expression and secretion of VEGF in AGS cells. After stimulation with IL-6, VEGF expression was apparently up-regulated and a 2.4-fold increase was observed. VEGF secretion in the conditioned medium was also increased by 2.8 folds. When treated with EGCG, VEGF expression and secretion were dose-dependently decreased. IL-6 also increased VEGF mRNA expression by 3.1 folds. EGCG treatment suppressed VEGF mRNA expression in a dose-dependent manner. EGCG dose-dependently inhibited Stat3 activation induced by IL-6, but did not change the total Stat3 expression. When treated with EGCG or AG490, VEGF expressions were reduced to the level or an even lower level in the tumor cells not stimulated with IL-6. However, PD98059 and LY294002 did not change VEGF expression induced by IL-6. EGCG inhibited Stat3 nucleus translocation, and Stat3-DNA binding activity was also markedly decreased by EGCG. Furthermore, EGCG inhibited IL-6 induced vascular endothelial cell proliferation and tube formation in vitro and angiogenesis in vitro. CONCLUSION: EGCG inhibits IL-6-induced VEGF expression and angiogenesis via suppressing Stat3 activity in gastric cancer, which has provided a novel mechanistic insight into the anti-angiogenic activity of EGCG.

[Epigallocatechin-3-gallate inhibits growth and angiogenesis of gastric cancer and its molecular mechanism].

OBJECTIVE: To investigate the inhibitory effect of epigallocatechin-3-gallate (EGCG) on growth and angiogenesis of gastric cancer and to explore its molecular mechanism. METHODS: Heterotopic tumor was established by subcutaneously injection with SGC-7901 cells in nude mice. Once the tumor was established, the mice were allocated randomly into two groups and received intraperitoneal injection of EGCG or phosphate buffered saline respectively. Tumor growth was measured by caliper in two dimensions, and angiogenesis was determined with tumor microvessel density (MVD) by immunohistochemistry. Protein levels of vascular endothelial growth factor (VEGF) and activation of signal transducer and activator of transcription 3(Stat3) in tumor cells and tumor tissues were examined by Western blot. VEGF release in tumor culture medium was determined by ELISA and VEGF mRNA expression in tumor cells by RT-PCR. RESULTS: Intraperitoneal injection of EGCG significantly inhibited the growth of gastric cancer[(0.32+/-0.08) g vs(0.81+/-0.12) g, t=7.24, P<0.01], and an average of 60.4% suppression of primary tumor growth was observed. Microvessel density in tumor tissues receiving EGCG treatment was also markedly reduced(15.2+/-4.3 vs 24.6+/-6.6,t=3.41,P<0.01),and an average of 38.2% suppression was observed. EGCG treatment markedly reduced VEGF protein level in vitro and in vivo. Secretion and mRNA expression of VEGF in tumor cells were also suppressed by EGCG in a dose-dependent manner. This inhibitory effect was associated with reduced activation of Stat3. Stat3 activation was dose-dependently suppressed by EGCG in tumor cells, and an average of 53.5% reduction was observed in tumor tissues, but EGCG treatment did not change total Stat3 expression. CONCLUSION: EGCG reduces expression of VEGF in gastric cancer by inhibiting activation of Stat3, thereby inhibits tumor growth and angiogenesis of gastric cancer.

Association of tyrosine PRL-3 phosphatase protein expression with peritoneal metastasis of gastric carcinoma and prognosis.

PURPOSE: In gastric carcinoma, high expression of PRL-3, a protein tyrosine phosphatase, is associated with lymph node metastasis. We studied the relationship between PRL-3 expression and peritoneal metastasis in gastric carcinoma. METHODS: Immunohistochemical analysis using the anti-PRL-3 antibody was done in 639 patients with gastric carcinoma including 89 with peritoneal metastases. We then compared the clinicopathologic characteristics of the PRL-3-positive and PRL-3-negative carcinomas. RESULTS: PRL-3 was expressed in 70.4% of the primary gastric carcinomas overall; in 80.9% of the cancers with peritoneal metastasis and in 68.7% of those without peritoneal metastasis (P = 0.020). PRL-3 expression was higher in peritoneal metastasis than in the corresponding primary gastric cancers (P = 0.028). PRL-3 expression was correlated with tumor stage (coefficient = 0.343, P = 0.01) and cancer progression, including lymphatic invasion (coefficient = 0.325, P = 0.02), extent of lymph node metastasis (coefficient = 0.322, P = 0.01), and peritoneal metastasis (coefficient = 0.316, P = 0.03). Patients who were PRL-3-negative had a better survival rate than those who were PRL-3-positive at all stages (stage I: log-rank P = 0.046, Wilcoxon P = 0.048; stage II: log-rank P = 0.035, Wilcoxon P = 0.041; stage III: log-rank P = 0.027, Wilcoxon P = 0.033; stage IV: log-rank P = 0.032, Wilcoxon P = 0.030). CONCLUSIONS: Peritoneal metastasis appears to be correlated with PRL-3 expression, tumor stage, lymphatic invasion, and extent of lymph node metastasis. PRL-3 expression was negatively correlated with prognosis in patients with gastric cancer.