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

摘要:

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G1 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；G2/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G2/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。

Abstract:

Objective To investigate the effect of Metformin on proliferation, cell cycle and apoptosis of pancreatic adenocarcinoma Panc-1 cells in vitro, and appraise the possible mechanism. Methods Panc-1 cells were treated with 0.5, 2.0 and 8.0 mmol 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 After 48 h, the inhibition rates of Metformin at dosage of 0.5, 2.0 and 8.0 mmol were (7.20 ± 5.92)%, (18.35 ± 4.77)% and (33.45 ± 4.10)%, respectively. After 72 h, the inhibition rates of Metformin at dosage of 0.5, 2.0 and 8.0 mmol were (24.81 ± 4.04)%, (53.42 ± 4.18)% and (61.36 ± 2.00)%,respectively. The proliferation of Panc-1 cells was inhibited by Metformin in a dose- and time-dependent manner.After 48 h, the percentage of G1 phase cells in the 8.0 mmol Metformin group was significantly lower than that in the controlled group and the 0.5 mmol Metformin group (P < 0.05). The percentage of G2/M phase cells in the 8.0 mmol Metformin group was remarkably higher than that in the other groups (P < 0.05). After 48 h, 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 the 0.5 mmol Metformin group to (12.64 ± 2.74)% in the 8.0 mmol Metformin group (P < 0.05).After 48 h, compared with the controlled group and the 0.5 mmol Metformin group, the expression of PTEN was activated and the expressions of p-Akt (Ser473) and mTOR were reduced in the 2.0 and 8.0 mmol Metformin groups (P < 0.05). Conclusions Metformin can suppress the proliferation of human pancreatic adenocarcinoma Panc-1 cells, 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.