Experimental Study On The Effects Of Decitabine Combined With Cisplatin On The DNA Methylation Status Of SOX2 Gene In Human Gastric Cancer

Purpose: In recent years, researches have demonstrated that SOX2 gene is closely related to the occurrence and development of gastric cancer and its malignant biological behavior. Based on our previous work, this study intends to investigate the effects of decitabine combined with cisplatin on the DNA methylation status of SOX2 gene in human gastric cancer cells and xenograft tumor models in nude mice, and explore the relationship between the DNA methylation status, m RNA and protein expression of SOX2 gene and the growth and proliferation of human gastric cancer cells and xenograft tumor models in nude mice.Methods: Human gastric cancer BGC-823 cells were routinely cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/m L penicillin, and 100 mg/m L streptomycin at 37°C in a humidified atmosphere containing5% CO2. Then decitabine, cisplatin and their combination treatment was carried out as indicated. Firstly, the effects of both the drugs alone and their combination on the growth and proliferation of BGC-823 cells in vitro were determined by WST-1 assay.Cell apoptosis was assessed by flow cytometry with Annexin V-FITC/PI double staining.Cell migration ability was analyzed by a standard 24-well Boyden chamber transwell assay. Meanwhile, Methylation-Specific PCR(MSP) detected the DNA methylation status of SOX2 gene in human gastric cancer cells. The expression levels of SOX2 m RNA and protein were determined by RT-PCR and Western blot, respectively.Furthermore, we established the xenograft tumor models in nude mice bearing human gastric cancer BGC-823 cells. Then decitabine, cisplatin and their combination treatment was carried out as the indicated administration schedule. The volume of xenograft tumor during the administration schedule was calculated, and the growth curve of xenograft tumor was drawn. After the administration schedule, Ki-67 expression of xenograft tumors was analyzed by immunohistochemistry(IHC) method.The DNA methylation status of SOX2 gene was detected by MSP. The expression levels of SOX2 m RNA and protein were determined by RT-PCR and Western blot,respectively.Results: Decitabine(5?M), cisplatin(1?M, 10?M, 10?M) and their combination treatment in vitro can inhibit the growth and proliferation and migration of BGC-823 cells in a time- and(or) concentration-dependent manner, and induce DNA demethylation of SOX2 gene promoter, and increase the expression levels of SOX2 m RNA and protein. Particularly, these effects can act synergistically by their combination treatment. In nude mouse xenograft tumor models bearing human gastric cancer BGC-823 cells, both decitabine(5 mg/kg) and cisplatin(6 mg/kg) can suppress the growth of xenograft tumors to some extent. Further, their combination treatment can apparently suppress the growth and significantly impair cell proliferation ability of the xenograft tumors. Likewise, both the drugs can induce DNA demethylation of SOX2 gene promoter, and increase the expression levels of SOX2 m RNA and protein in xenograft tumors. Notably, these effects can act synergistically by their combination treatment.Conclusion: At the cellular level in vitro and in nude mouse xenograft tumor models bearing human gastric cancer BGC-823 cells, decitabine combined with cisplatin can synergistically affect gene promoter methylation status resulting in demethylation and re-expression of SOX2 gene, then exert anti-tumor effect synergistically through mechanisms such as inhibiting cell growth and proliferation and migration, inducing cell apoptosis, and suppressing the growth and impairing cell proliferation ability of xenograft tumors.