二甲双胍联合顺铂对孕激素耐药子宫内膜癌Ishikawa细胞增殖、凋亡的影响及机制

Effects of metformin combined with cisplatin on proliferation and apoptosis of progesterone resistant endometrial carcinoma cells Ishikawa

目的探讨二甲双胍联合顺铂对孕激素耐药子宫内膜癌Ishikawa细胞增殖、凋亡的影响及机制。方法将Ishikawa细胞体外培养,取对数期细胞,应用甲羟孕酮诱导建立孕激素耐药体系。取耐药成功的Ishikawa细胞,随机分为5组,二甲双胍组:加入稀释后不同浓度(终浓度为1、2.5、5、10、15 mmol/L)的二甲双胍;顺铂组:加入浓度为5、12.5、25、50、75μmol/L的顺铂;联合用药组:二甲双胍联合顺铂以各自相应的终浓度按1∶200进行给药;对照组:未添加任何药物的无血清培养液处理细胞;空白组:无药物及细胞的培养液。MTT法检测各组细胞增殖抑制率及联合指数(CI),计算二甲双胍及顺铂的半数抑制浓度(IC_(50)),根据计算的IC_(50),后续研究中分别用10 mmol/L二甲双胍(二甲双胍组)、60μmol/L顺铂(顺铂组)、10 mmol/L二甲双胍+60μmol/L顺铂(联合用药组)处理细胞48 h。流式细胞仪检测各组细胞凋亡率,实时荧光定量PCR法分析相关凋亡基因表达;同时,应用Western blotting法检测各组mTOR信号通路相关蛋白表达。结果二甲双胍在2.5~15 mmol/L、顺铂在12.5~70μmol/L浓度内孕激素耐药的Ishikawa细胞增殖受到抑制,且呈剂量依赖性效应,二者抑制作用随药物浓度的增高而逐渐增大,差异有统计学意义(P均<0.05)。二甲双胍的IC_(50)为(10.44±0.57)mmol/L、顺铂为(60.22±0.78)μmol/L。联合用药组二甲双胍终浓度为10、15 mmol/L时联合顺铂(对应终浓度为50、75μmol/L)对肿瘤细胞增殖的抑制程度高于二甲双胍组及顺铂组(P均<0.05)。流式细胞仪结果显示,联合用药组的细胞凋亡率高于顺铂组、二甲双胍组,差异有统计学意义(P均<0.05);实时荧光定量PCR及Western blotting结果表明联合用药组Bcl-2相对表达产物含量最低,其p-AMPK蛋白表达较二甲双胍组、顺铂组增加,而p-mTOR含量则降低(P均<0.05)。结论二甲双胍联合顺铂对孕激素耐药的子宫内膜癌细胞有协同作用,可抑制肿瘤细胞增殖,促进其凋亡;机制可能与AMPK/mTOR通路相关。

Objective To observe the effect and mechanism of metformin combined with cisplatin on the proliferation and apoptosis of progesterone resistant endometrial carcinoma ishikawa cells. Methods Ishikawa cells were cultured in vitro. We chose the cells in the logarithmic phase to establish the progesterone resistance system through progesterone induction. The successfully progesterone resistant resistant Ishikawa cells were divided into five groups. The cells in the metformin group were added with different concentrations of metformin after dilution（ final concentrations of 1,2. 5,5,10,15 mmol/L）; cells in the cisplatin group were added with 5,12. 5,25,50,and 75 μmol/L cisplatin; cells in the combination group were added with the metformin combined with cisplatin at a respective final concentration of 1： 200; cells in the control group were cultured in the serum-free culture solution; cells in the blank group were not treated with drug or culture solution. MTT was used to detect cell proliferation inhibition rate and combination index（ CI） in each group,and we calculated the 50% inhibitory concentration（ IC-（50）） of metformin and cisplatin. Based on the calculated IC-（50）,in the following study,we selected 10 mmol/L metformin（ metformin group）,60 μmol/L cisplatin（ cisplatin group）,and 10 mmol/L metformin ＋ 60 μmol/L cisplatin（ combination group） to treat cells for 48 h. Flow cytometry was used to detect the apoptosis rate of each group; the real-time fluorescent quantitative PCR was used to analyze the expression of apoptosis-related genes; the expression of mTOR signaling pathway-related proteins in each group was detected by Western blotting. Results The proliferation of progesterone resistant Ishikawa cells treated with metformin at 2. 5-15 mmol/L and cisplatin at 12. 5-70 μmol/L was inhibited in a dose-dependent manner,and the inhibitory effects increased with the increasing concentrations,with statistically significant difference（ all P〈0. 05）. The IC-（50） of metformin was（ 10. 44 ± 0. 57） mmol/L,and cisplatin was（ 60. 22 ± 0. 78） μmol/L. The inhibitory effect of the combination group（ 10,15 mmol/Lmetformin and 50,75μmol/Lcisplatin） on tumor cells was higher than that of metformin group and cisplatin group（ all P〈0. 05）. Flow cytometry results showed that the apoptosis rate of the combination group was higher than that of the cisplatin group and metformin group,and the differences were statistically significant（ P〈0. 05）; real-time fluorescent quantitative PCR and Western blotting showed that the relative expression of Bcl-2 was the lowest in the combination group and the expression of p-AMPK protein significantly increased as compared with that of the metformin group and cisplatin group,while the expression of pmTOR significantly decreased（ all P〈0. 05）. Conclusions Metformin combined with cisplatin has synergistic effect on progesterone resistant endometrial cancer cells and can inhibit tumor cell proliferation and promote apoptosis,which The mechanism may be related to AMPK/mTOR pathway.