二甲双胍联合顺铂抑制神经胶质瘤细胞增殖及其机制初步探讨

摘要

Objective To observe the effect of metformin on U251 cell proliferation,apoptosis,and the growth of glioma in nude mice.Methods Metformin in combination with cisplatin were used to treated the U251 cells,and the cell viability was assayed by MTT methods.Cell cycle,apoptosis rate were assayed by flow cytometry.The expression of apoptotic related proteins (Bcl-2,Bcl-xl,Bax,PARP,et al) were detected by western-blotting.Nude mice were transplanted with U251 cells,and tumor growth inhibition rate by metformin combined with or without cisplatin were detected.Results Compared with the control group,metformin combined with or without cisplatin could inhibit the proliferation of U251 cells in a dose-and time-dependent manner.After U251 cells were treated with 10 mM metformin for 48 h,the growth cycle was arrested in S phase,the apoptosis rate was (20.57±3.16)％.The expression of anti-apoptotic protein Bcl-2,Bcl-xl were down-regulated after metformin treatment,while the pro-apoptotic protein Bax was significantly increased.In vivo studies,the inhibition rates in the metformin-treated group,cisplatin-treated group,and cisplatin combined with metformin-treated group were 39.22％,52.97％,and 71.55％,respectively.The inhibition rates in the above three groups were all higher than that in the control group.The difference between the two groups was statistically significant (metformin-treated group vs.control group,cisplatin combined with metformin-treated group vs.cisplatin-treated group,P＜0.01).However,metformin could not inhibit the glioma metastasis in vivo.Conclusion Metformin inhibits the proliferation of U251 cells,enhances the sensitivity of glioma U251 cells to cisplatin,and induces apoptosis via the mitochondria mediated apoptotic signaling pathway in vitro.%目的 观察二甲双胍对人神经胶质瘤U251细胞增殖、凋亡及及裸鼠荷胶质瘤生长的影响.方法 噻唑蓝(MTT)比色法检测二甲双胍单用或联合顺铂对胶质瘤细胞存活率的影响;流式细胞术检测二甲双胍作用于胶质瘤细胞后其细胞周期时相及细胞凋亡率的变化;免疫印迹法(Western blot)检测二甲双胍对肿瘤细胞凋亡相关蛋白Bcl-2、Bcl-xl、Bax、PARP等表达的影响,将U251细胞接种于裸鼠,观察二甲双胍单用或联合顺铂对裸鼠荷胶质瘤体内生长的影响.结果 与对照组相比,二甲双胍单用或联合顺铂能够显著抑制胶质瘤细胞U251的增殖活性,且表现为一定的浓度-时间依赖性;10 mmol/L二甲双胍作用细胞48 h时,细胞生长周期阻滞于S期,U251细胞凋亡率为(20.57±3.16)％;Western blot检测显示Bcl-2、Bcl-xl等抗凋亡蛋白表达随着药物浓度的增加而减少,促凋亡蛋白Bax蛋白表达随着药物浓度增加而增加;体内试验部分:二甲双胍组、顺铂组及联合用药组裸鼠皮下瘤体积抑瘤率分别为39.22％、52.97％和71.55％,均显著高于对照组,组间比较差异有统计学意义(二甲双胍组VS对照组,联合用药组VS顺铂组,均P＜0.01),裸鼠体内试验证实二甲双胍对胶质瘤细胞的转移没有明显的抑制作用.结论 二甲双胍能够显著抑制体外培养的胶质瘤细胞的增殖及增强胶质瘤U251细胞对顺铂的敏感性,其机制可能与激活线粒体介导的细胞凋亡信号通路有关.