二甲双胍对人胰腺癌细胞增殖与凋亡的影响

Effects of metformin on proliferation and apoptosis of human pancreatic cancer cell

背景与目的:近年研究发现二甲双胍对多种肿瘤具有抑制作用,部分临床研究也发现二甲双胍可以降低糖尿病并发胰腺癌的风险和降低胰腺癌的死亡危险。本研究从细胞水平着手研究二甲双胍对胰腺癌细胞增殖和凋亡的影响,并初步探讨其可能机制。方法:体外培养人胰腺癌细胞株Bxpc-3,予二甲双胍进行干预作为二甲双胍干预组,无药物组作为对照组。以MTT检测二甲双胍对Bxpc-3细胞存活率的影响,流式细胞术检测二甲双胍对Bxpc-3细胞周期的影响,流式细胞术及Hoechst 33258荧光染色法检测细胞凋亡,RT-PCR检测AMPKα1、Bax、Bcl-2、Caspase-3、Cyclin D1 mRNA的表达。蛋白质印迹法(Western blot)检测Cyclin D1、Bax、Bcl-2、Caspase-3蛋白的表达。结果:与对照组相比,MTT检测结果显示,二甲双胍可以抑制人胰腺癌细胞株Bxpc-3的增殖,并呈时间-浓度依赖性(F=8.99、124和114.61,P<0.01);流式细胞术检测结果显示,二甲双胍干预组与对照组相比,G0/G1期细胞所占百分比增加(t=-8.71,P<0.01),S期(t=7.54,P<0.01)及G2/M期(t=7.00,P<0.01)细胞所占百分比减少;流式细胞术(早期凋亡率t=-2.68,晚期凋亡率t=1.29,总凋亡率t=-0.85)及Hoechst 33258荧光染色法(t=-0.46)结果显示,两组细胞凋亡率差异无统计学意义(P>0.05);RT-PCR结果显示,二甲双胍干预组Cyclin D1 mRNA表达明显降低(t=4.96,P<0.01),AMPKα1、Bax、Bcl-2、Caspase-3 mRNA表达与对照组相比,差异无统计学意义(t=1.68、-0.56、-1.80、0.67,P>0.05);Westernblot结果显示,二甲双胍干预组Cyclin D1蛋白表达明显降低(t=7.02,P<0.01),Bax、Bcl-2、Caspase-3蛋白表达与对照组相比,差异无统计学意义(t=-0.11、-0.20,0.11,P>0.05)。结论:二甲双胍能显著抑制人胰腺癌细胞株Bxpc-3的增殖,机制主要与其阻滞细胞周期、下调Cyclin D1表达有关;二甲双胍对Bxpc-3细胞的凋亡无明显诱导作用。

Background and purpose： In recent years, studies found that metformin could inhibit various kinds of tumor. And a few clinical studies also found that metformin could reduce the risk of diabetes combined with pancreatic cancer and could reduce the death risk of pancreatic cancer. So we proceed from a cellular level to investigate the effects of metformin on proliferation and apoptosis in human pancreatic cancer cell line Bxpc-3, and to appraise the possible potential mechanism. Methods： Human pancreatic cancer cell line Bxpc-3 were cultured in vitro, and were treated with metformin as trial group or without metformin as control group. Survival rate of Bxpc- 3 cells was determined by Methyl thiazolyl tetrazolium （MTT） assay, and the cell cycle changes were analyzed by flow cytometry （FCM）. Apoptosis was determined by FCM and Hoechst 33258 fluorescence staining. Expressions of AMPK ctl, Bax, Bcl-2, Caspase-3, Cyclin D1 mRNA were determined by RT-PCR. Protein expression of Cyclin D1, Bax, Bcl-2, Caspase-3 were determined by Western blot. Results： Compared with the control, metformin decreased the proliferation of Bxpc-3 cells in a dose- and time-dependent manner （F=8.99, 124, 114.61, P〈0.01）. Compared withthe control, the proportion of the cells at G0/G1 stage in the metformin-treated was increased （t=-8.71, P〈0.01）, and the proportion of the cells at S （t=7.54, P〈0.01） and GJM （t=7.00, P〈0.01） were decreased. The apoptosis rate of control group and metformin-treated group were not difference significantly （early apoptosis rate t=-2.68, late apoptosis rate t=1.29, total apoptosis rate t=-0.85）. Expression of Cyclin D1 mRNA was down-regulated （t=-4.96, P〈0.01 ）. Expression of AMPK al, Bax, Bcl-2 and Caspase-3 mRNA were not altered significantly （t=-1.68, -0.56, -1.80, 0.67, P〉0.05）. Protein expression of Cyclin D 1 was down-regulated （t=-7.024, P〈0.01）. Protein expression of Bax, Bcl-2 and Caspase-3 were not altered significantly （t=-0.11, -0.20, 0.11, P〉0.05）. Conclusion： Metformin can inhibit the proliferation of human pancreatic cancer cell line Bxpc-3 mainly by blocking the cell cycle at G0/G1 and down-regulating the expression of Cyclin D 1. Metformin could not induce significantly apoptosis of human pancreatic cancer cell line Bxpc-3.