二甲双胍对人胰腺癌细胞增殖、细胞周期和凋亡的影响及机制

Influence of Metformin on proliferation, cell cycle and apoptosis in human pancreatic adenocarcinoma cells and mechanisms

目的探索不同浓度二甲双胍对人胰腺癌Panc-1细胞增殖、细胞周期和凋亡的影响及其可能的分子机制。方法用不同浓度的二甲双胍处理人胰腺癌Panc-1细胞后,采用MTT法检测其对癌细胞增殖能力的影响,流式细胞术检测其对细胞凋亡和细胞周期的影响,Western blot观察PTEN、p-Akt(Ser473)、mTOR蛋白表达水平的变化。结果干预48 h时,不同浓度二甲双胍(0.5、2.0和8.0 mmol)对细胞生长的抑制率依次为(7.20±5.92)%、(18.35±4.77)%和(33.45±4.10)%;72 h时对细胞生长的抑制率分别为(24.81±4.04)%、(53.42±4.18)%和(61.36±2.00)%。该抑制作用随着药物浓度增加和干预时间延长而增强,呈药物浓度依赖性和时间依赖性。流式细胞术检测结果显示,二甲双胍干预48 h时,8.0 mmol组G_1期细胞比例与对照组和0.5 mmol组比较,差异有统计学意义(P<0.05),8.0 mmol组低于对照组和0.5 mmol组;G_2/M期细胞比例与对照组和0.5 mmol组比较,差异有统计学意义(P<0.05),8.0 mmol组高于对照组和0.5 mmol组。二甲双胍作用48 h时8.0 mmol组细胞的中晚期凋亡率为(12.64±2.74)%,与对照组(7.01±1.14)%和0.5 mmol组(6.19±0.32)%比较,差异有统计学意义(P<0.05),8.0 mmol组高于对照组和0.5 mmol组。Western blot检测结果显示二甲双胍作用48 h时,2.0 mmol组和8.0 mmol组与对照组和0.5 mmol组比较,PTEN蛋白表达量差异有统计学意义(P<0.05),2.0 mmol组和8.0 mmol组升高,而p-Akt(Ser473)和mTOR的表达水平降低。结论二甲双胍能抑制人胰腺癌Panc-1细胞的增殖能力,引起G_2/M细胞周期阻滞,同时诱导胰腺癌细胞的凋亡,其可能的机制是通过激活PTEN的表达,抑制PI3K/Akt/mTOR通路。

Objective To investigate the effect of Metformin on proliferation,cell cycle and apoptosis of pancreatic adenocarcinoma Panc-1cells in vitro,and appraise the possible mechanism.Methods Panc-1cells were treated with0.5,2.0and8.0mmol Metformin in vitro.Cell proliferation was measured by MTT assay.Cell apoptosis and cell cycle distribution were detected by flow cytometery.The expression of PTEN,p-Akt(Ser473)and mTOR were dected by Western blot.Results After48h,the inhibition rates of Metformin at dosage of0.5,2.0and8.0mmol were(7.20±5.92)%,(18.35±4.77)%and(33.45±4.10)%,respectively.After72h,the inhibition rates of Metformin at dosage of0.5,2.0and8.0mmol were(24.81±4.04)%,(53.42±4.18)%and(61.36±2.00)%,respectively.The proliferation of Panc-1cells was inhibited by Metformin in a dose-and time-dependent manner.After48h,the percentage of G1phase cells in the8.0mmol Metformin group was significantly lower than that in the controlled group and the0.5mmol Metformin group(P<0.05).The percentage of G2/M phase cells in the8.0mmol Metformin group was remarkably higher than that in the other groups(P<0.05).After48h,the percentage of cells in the middle and late stages of apoptosis was increased from(7.01±1.14)%in the controlled group and(6.19±0.32)%in the0.5mmol Metformin group to(12.64±2.74)%in the8.0mmol Metformin group(P<0.05).After48h,compared with the controlled group and the0.5mmol Metformin group,the expression of PTEN was activated and the expressions of p-Akt(Ser473)and mTOR were reduced in the2.0and8.0mmol Metformin groups(P<0.05).Conclusions Metformin can suppress the proliferation of human pancreatic adenocarcinoma Panc-1cells,cause G2/M phase checkpoint arrest and induce cell apoptosis in vitro at moderate to high dosage.The mechanism may be inhibition of the PI3K/Akt/mTOR pathway by activating the expression of PTEN.