Effect Of XTSJF On DNA Methylation Of P16 Gene In Gastric Cancer Cells

Background:Gastric carcinoma (GC) is the most common cancer in eastern Asia and the third most frequent cancer across the world. Gastric Cancer has long been known to be a genetic disease.However, in recent years it has become increasingly obvious that genetic abnormalities are by no means the only mechanism by which gene expression becomes altered during tumourigenesis. Growing evidence now suggests that epigenetic factors, in particular DNA methylation, play a major role in carcinogenesis and sometimes even act as the only reason for inactivation of tumor suppressor genes in human cancers. CDKN2A (P16),a well-known tumor suppressor gene,was found to be highly methylated on it's promoter region in gastric cancer cells,leading to the transcriptional silence of gene.Demethylating agents 5-azacytidine and 5-Aza-CdR could inhibit cell proliferation of cancer by reversing DNA hypermethylation,but their usage were limited to myelodysplastic syndrome (MDS). Until now,there is no evidence that 5-azacytidine and 5-Aza-CdR could inhibit the growth of gastric cancer by reversing abnormal DNA hypermethylation without affecting DNA hypomethylation that often occurred to proto-oncogenes.Traditional Chinese herbal medicine(TCM) has been long used to treated tumours,and compared to the chemotherapeutic agents TCM could improve the patient's life quality with less side-effect. XTSJF is a very popular herbal medicine in china. XTSJF has been used in the treatment of gastrointestinal cancer,such as gastric carcinoma and esophageal cacinoma. It improved subjective symptoms and inhibit the proliferation of cancer cells. Many reports assayed the preventive and therapeutic effects of XTSJF on the experimental gastric carcimoma induced in rats by orthotopic transplantation.Many studies reported the effect of TCM on tumours,however, untill now there is few articles reported the effect of TCM on DNA methylation in carcinomas.The aim of this study was to assay the therapeutic effects of XTSJF decocton on gastric cancer cells. In order to understand the mechanism of XTSJF to inhibit proliferation of cancer cells, we also tested the drug's effect on the mRNA expression of P16 gene, and The DNA methylation level of P16 gene in gastric cancer cells.Objective:To explore the effect to tumorous growth,we study tumor development characteristic and proliferation of gastric cancer cell lines;we study the effect from mRNA level to DNA methylation level about CDKN2A(P16)by RT-PCR method,Methylight method (RT-PCR) from qualitation to quantitation with XiaoTanSanJieRecipe,we explored anti-tumor effect and mechanism from the aspect of epigenetics.Methods:1.10 male SD mouse models were divided into two groups:control group and TCM group,then treated with corresponding medicine respectively;8ml blood was got through arteria cruralis from each mouse after 5-day stomach lavage and then made into serum containing medicine;2.. Briefly,100μl cells(1×104cells/ml) were seeded per well in a 96-well flat-bottom plate. They were then treated for 24,48 and 72 h with appropriate concentrations of XTSJF pharmacological serum(20μl), normal rat serum (20μl),5-Aza-CdR (5μmol/L) and vehicle(20μl) in the presence of 10% FBS. We then added 10μl CCK-8 to each well to assess cell growth; Cell growth was assessed by CCK8 (cell counting kit 8) according to the manufacturer's instructions;3. Total RNA was isolated from MKN-45 and BGC823 cells according to the manufacturer's instruction after 72 hours treatment. then RT-PCR was performed to detect mRNA expression of PI 6;4. Total DNA was isolated from MKN-45 and BGC823 cells with DNA Mini kit according to the manufacturer's instruction after 72 hours treatment. then Methylight was performed to detect DNA methylation level of P16;5. 20 nude mouse models of human gastric carcinoma cell were made by using orthotopic transplantation. then they were diviede into two groups:control group and TCM group,then treated with corresponding medicine respectively 72 hours after orthotopic transplantation. Each dose was dissolved in 0.4 ml distilled water and administered twice a day intragastrally (i.g.) for 6 weeks, Each animal received one dose of XTSJF or vehicle daily for 6 weeks and then executed. Dietetic state and weight change of the tumor-bearing mice were observed and tumorous size was measured during administration;6. The experiment was terminated on the 6th week, The mice were sacrificed.we measured weight and volume of tumor taken from the mice and calculate the inhibitory rate;7. The tumours were taken on the 6th week,then we extract totle RNA from each tumour sample and detect the expression of PI 6 mRNA by RT-PCR.8. we detect the methylation level of P16 gene by Methylight. Results:1.Experimental animals had a good animation during the experimental period; and 20 ml pharmacological serum were got from each group of animals;2. Compared to cell proliferation of control group, those of 5-Aza-CdR group,XTSJF pharmacological serum group was degraded,the difference has statistical significance (P<0.05),and inhibiterary rate of cell proliferation was higher in XTSJF pharmacological serum group than that in 5-Aza-CdR group. Whereas there was no statistical significance between normal rat serum group and control group (P>0.05);3. We observed expression of P16 mRNA by RT-PCR method, control group is lower than XTSJF pharmacological serum group and 5-Aza-CdR group,the difference has statistical significance (P<0.05); 5-Aza-CdR group is higher than XTSJF pharmacological serum group, the difference has statistically significant (P<0.05);4.The methylation level of P16 gene is detected by methylight method:hypermethylation were observed in the promoter region of P16 gene in gastric cancer cell line MKN45 and BGC823(PMR>4); the methylation level were degraded after treated for 72hours with XTSJF pharmacological serum or 5-aza-dC; the methylation level of P16 gene is lower in 5-Aza-CdR group than in pharmacological serum group (P<0.05);P16 gene methylated was not observed in the gastric cancer line MKN45 after treated with 5-Aza-CdR for 72 hours;5. one mouse in the control group died of bowel obstruction on the 4th day of experiment. Emaciation, decreacing of movement and diet were observed in the control group on the 3th week, and in the TCM group the 4th week. The mice of the control group got a tumour lump on their right epigastric zone in earlier time than those of the TCM group, on the 6th week of experiment the mice got emaciation, limitation of movement and camptocormia, then they were sacreficed;6.The shape of tumours taken from the mice was round or oval, the cut surface looked like fish and had some ecptomas,and zone of necrosis was found in the center of some big tumours. The result of tumour weighing indicate that the TCM group have a higher inhibitory rate (54.82%) than the control group, the difference have a statistical significance(P<0.01)7. The result of RT-PCR indicated that expession mRNA of P16 was lower in the control group than in the TCM group which was treated with XTSJF for six weeks;the difference has remarkable statistical significance(p<0.01))8. The methylation level of P16 gene is detected by methylight method:hypermethylation were observed in the promoter region of P16 gene in control group (PMR>4); the methylation level were degraded after treated for 6 weeks with XTSJF decoction; the difference has remarkable statistical significance(p<0.01).Conclusion:1. Medical serum of XTSJF could depess the diffrentiation of gastric cancer cell lines;2. Decoction of XTSJF could depress the tumorous growth in mouse models of human gastric carcinoma cell;3. Both invivo and invitro study indicated that XTSJF could depress the tumorous growth by reversing DNA methylation and increasing the mRNA expession of P16 gene.