Effects Of S-adenosylmethionine On Proliferation Of Gastric Cancer And Methylation Of C-myc And UPA Genes

Gastric cancer is one of the most prevalent malignant tumors, and the number of death from it is more than 150 thousand per year in China. Gastric cancer continue to be the most common fatal cancer recently in China. The carcinogenesis is known as a complicated process involved in polygene, poly-molecular change and multistage. And the most important events are the activation of oncogene and inactivation of tumor suppressor gene in the process. Recently, there are more and more attention to epigenetics, such as DNA methylation. DNA methylation, the covalent addition of a methyl group to the C-5 position of cytosine in the context of the CpG dinucleotide, is catalysised by the DNA methyltransferase enzyme, and has the significant role in the process of carcinogenesis and growth. DNA methylation is a process modified by genes, which is reversible and hereditary. The changes of methylation status, including the decrease of global genome methylation level and the abnomaly raise of CpG island partly methylation level, are important factors in carcinogenesis. These changes can lead to the activation of oncogene and inactivation of tumor suppressor gene, as well as the instability of genome.S-adenosylmethionine (SAM) is synthetized through S-adenosylmethionine synthetase, and is the primary methyl group donor for most biological methylation reactions. SAM is known as a demethylation suppressor and can reverse gene hypomethylation, induce gene hypermethylation. In recent studies, SAM has been found having the effect on chemopreventing and treating on cancers of liver, colon and breast, because it could promote apoptosis and suppress the growth of cancer cells. So far there were no reports on gastric cancer. For the reason, we investigated the potential benefits of SAM in inhibiting the growth of gastric cancer in vivo and in vitro. Abnomal expression of c-myc is closely correlated with carcinogenesis. And the methylation level of c-myc gene is decreased in the process of carcinogenesis such as liver cancer, gastric cancer, lung cancer. Urokinase-type plasminogen activator (uPA) is related with tumor metastasis. uPA is regard as a tumor trigger gene, for promoting the growth, invasion, transfer, and neovascularity of tumor. We studied the methylation status of gene promoter region and the expression of c-myc and uPA genes, trying to identify the anti-tumor mechanisms of SAM in gastric cancer.Methods1 Effects of SAM on proliferation of gastric cancer cell linesFor dose-response experiments, MKN-28, SGC-7901, BGC-823 and MKN-45 cells were incubated with increasing concentrations (0.5-32mmol/L) of SAM for 24h,48h and 72h. Cell growth was detected by MTT colorimetric method. The cell growth inhibition ratio and IC50 were calculated, and the restrain curves were ploted.2 Effects of SAM on cell cycle and apoptosis of gastric cancer cell linesThe four cell lines were incubated with different concentrations(0,2 and 4 mmol/L) of SAM for 72h. Cell cycle distribution and cell apoptosis were calculated by using of flow cytometry system.3 Effects of SAM on invasiveness and migration of gastric cancer cell linesThe four cell lines were incubated with different concentrations(0,2 and 4mmol/L) of SAM for 72h. Cells were incubated in the transwell caves at a density of 1×105/well for 24h, to investigate the invasiveness, after the polycarbonate membrane of transwell cave was covered by Matrigel. Or, cells were incubated at a density of 1×106/well for 20h, to investigate the migration. The number of cell which going through the membrane was counted.4 Effects of SAM on expression and methylation status of c-myc and uPA of gastric cancer cell linesThe four cell lines were incubated with different concentrations(0,2 and 4mmol/L) of SAM for 72h. RT-PCR analysis of c-myc and uPA mRNA expression in cells. Western blot analysis of c-myc and uPA protein expression in cells. MSP analysis of c-myc and uPA gene promoter region methylation status in cells.5 Establishment of nude mice model of tumor xenografts and treatment by SAMTumor xenografts were established by inoculation of SGC-7901 cells subcutaneously in BALB/c nude mice. The mice were randomized separated into three groups:low concentration group(192μmol/(kg-d) SAM), high concentration group(768μmol/(kg-d) SAM) and control group(NS), and treated by peritoneal injection for 15 days.6 Effects of SAM on growth and c-myc,uPA genes of xenograftsTumor volume, the weight of nude mice were recorded every three days. After gotten the samples,the changes of histopathology and apoptotic index were observed by HE. The metastasis of liver and lung of nude mice were observed. Immunohistochemistry, Western Blot and RT-PCR were used to analysis c-myc and uPA protein and mRNA expression. MSP analysis of methylation status of gene promoter regions in xenografts.Statistical AnalysisData were expressed as mean±SD. The results of the invasiveness and migration of cells, the immunohistochemistry of xenografts were evaluated by rank-sum test, The results of MSP of xenografts were evaluated by contingency table x2 test. The results of the rest were evaluated by ANOVA with SPSS 11.1. The statistical level of significance was set at P<0.05.Results1 The growths of MKN-28,SGC-7901,BGC-823 and MKN-45 cell were inhibited and the apoptosis cells were increasing with increase of SAM concentration and action time. The MTT assay showed that the proliferation of the four cell lines were restrained gradually (P<0.05) and the growth inhibition ratio were increasing after treated with SAM. The IC50 of cells of 72h were: MKN-28 IC50 6.37 mmol/L, SGC-7901 IC50 5.40 mmol/L, BGC-823 IC50 4.01mmol/L and MKN-45 IC50 4.77 mmol/L.2 The apoptosis cells were all significantly increased (P<0,01) after treated with SAM. In SGC-7901 and BGC-823, the cell percentages of G0/G1 phase were significantly increased (P<0.05 and P<0.01), whereas the cell proliferation indexes (PI) were significantly decreased (P<0.05 and P<0.01). But in MKN-28 and MKN-45, the cell cycle and the PI did not show any difference (P>0.05).3 The number of invasion and migration cell of the four cell lines were decreasing(P<0.01) and the invasion and migration inhibition ratio were all increasing, with increase of SAM concentration.4 The expressions of c-myc mRNA and protein in SGC-7901 and BGC-823, and the expressions of uPA mRNA and protein in the four cell lines were all significantly decreasing(P<0.05 and P<0.01) with increase of SAM concentration, and the hypomethylation of gene promoter regions were reversed. But the the expressions of c-myc mRNA and protein in MKN-28 and MKN-45, and the hypomethylation of gene promoter regions were no marked change(P>0.05).5 When nude mice were puting to death, the body weights of three groups were no difference (P>0.05). Compared with control group[(1018.22±223.07)mm3], the tumor volume of low concentration group[(618.51±149.27)mm3] and high concentration group[(444.32±118.51)mm3] were obviousily diminished(P<0.01), and the inhibitory rate of tumor growth of low and high concentration group were 39.26% and 56.36%, respectively.6 The xenografts pathology conformed with the characteristics of moderately differentiated human gastric carcinoma. The xenografts had more coagulative necrosis and apoptosis after treated with SAM. Apoptotic index had positive correlation with SAM concentration (P<0.01). The metastases of liver and lung of three groups were no found.7 In the xenografts, the expressions of c-myc and uPA mRNA and protein were all significantly weakening(P<0.05 and P<0.01), and the hypomethylation of gene promoter regions were improving (c-mycχ2=10.179, uPAχ2=11.667, all P<0.01), with increase of SAM concentration.Conclusions1 SAM can suppress the proliferation, invasion and migration, induce apoptosis, blocke at G1 phase of the gastric cancer cell lines, in a dose-and time-dependent manner in vitro. And SAM can reverse the hypomethylation of c-myc, uPA gene promoter regions, reduce their expression. The changes of proliferation, cell cycle, apoptosis, invasion and migration of gastric cancer cells may correlate with the effects of SAM on hypomethylation of c-myc and uPA gene promoter region and their expression.2 SAM can inhibit the growth, promote apoptosis, reverse the hypomethylation of c-myc and uPA gene promoter region, and reduce genes'expression of human gastric carcinoma xenografts. May be these effects result the inhibition of human gastric carcinoma in vivo.3 Our data demonstrate the inhibition of SAM on human gastric carcinoma in vivo and in vitro. The mechanisms of the effects may be SAM can reverse the hypomethylation of oncogenes and re-inactivate them by providing methyl groups.