二甲双胍增强Ishikawa细胞对顺铂化疗敏感性的实验研究

摘要

目的 观察二甲双胍体外增强顺铂对子宫内膜癌Ishikawa细胞化疗敏感性的作用,并探讨可能的作用机制.方法 实验分为空白对照组、二甲双胍组(培养液中二甲双胍终浓度为10 mmol/L)、顺铂组(培养液中顺铂终浓度为20 μmol/L)、联合组(培养液中二甲双胍和顺铂的终浓度分别为10 mmol/L和20 μmol/L),各组作用24 h,CCK-8法和细胞克隆形成法检测细胞增殖抑制率和存活率,金氏公式评价二药协同效果;Transwell法检测细胞侵袭力;流式细胞仪检测细胞周期分布及凋亡率;蛋白质印迹法检测葡萄糖调节蛋白(GRP)78蛋白表达水平.结果 二甲双胍、顺铂均可抑制Ishikawa细胞增殖、诱导细胞凋亡、降低细胞存活率和侵袭力;二甲双胍可阻滞细胞周期于G0/G1期,顺铂单独或联用二甲双胍可阻滞细胞周期于G2/M期,并能下调GRP78蛋白表达水平(P<0.05),二药联用上述作用效果更为显著(P<0.01).结论 二甲双胍能够增强顺铂对子宫内膜癌Ishikawa细胞的化疗敏感性,这种作用部分是通过下调GRP78蛋白实现的.%Objective To observe the effect of metformin′s enhancing chemotherapy sensitivity of cisplatin on human endometrial cancer cell line, Ishikawa cell in vitro and to explore its possible mechanism.Methods There were four groups: control group, metformin group(Ishikawa cells were pretreated by 10 mmol/L metformin), cisplatin group(Ishikawa cells were pretreated by 20 μmol/L cisplatin), metformin/cisplatin group(Ishikawa cells were pretreated by 10 mmol/L metformin and 20 μmol/L cisplatin).After treated for 24 h, the growth and proliferation of Ishikawa cell was detected by CCK-8 assay and clone formation test.Jin′s formula was used to assess the synergistic effects of two drugs.Cell phase and apoptosis were detected by the flow cytometry.Cell invasive ability was measured with Transwell method.The expression of GRP78 protein was detected by Western blotting.Results Metformin, cisplatin alone could inhibit cell proliferation, induce cell apoptosis, decrease cell survival rate and invasive ability and down-regulate the expression of GRP78 in Ishikawa cells(P<0.05).Metformin could arrest cell cycle in G0/G1 phase while csplatin alone or combination with metformin could arrest cell cycle in G2/M phase.The effect above of cisplatin combination with metformin was better than that of cisplatin, metformin alone(P<0.01).Conclusion Metformin could enhance chemotherapy sensitivity of cisplatin on human endometrial cancer Ishikawa cell in vitro, which was partly related to the down-regulation of GRP78 protein in tumor cells.