2-脱氧葡萄糖联合二甲双胍抗人肝癌HepG2细胞作用及与AMPK、mTOR相关性研究

Effect of 2-deoxyglucose combined with metformin on human hepatocellular carcinoma HepG2 cells and its correlation with AMPK and mTOR

目的考察2-脱氧葡萄糖(2-DG)联合二甲双胍(Met)协同抗人肝癌HepG2细胞作用并探索其机制。方法刃天青法测定2-DG及Met单独及联合应用对HepG2细胞的生长抑制作用,利用金氏公式计算联合用药Q值;利用高内涵细胞成像系统考察单独及联合用药对细胞线粒体膜电位及活性氧产生的影响;Western blotting检测Cleave-Caspase 3、p-AMPK及p-mTOR的蛋白变化;考察AMPK及mTOR抑制剂对联合用药后细胞生长抑制作用的影响。结果 2-DG、Met单独应用的IC50值分别为2.45及16.35 mmol/L,联合用药对细胞生长抑制具有协同作用,Q值均大于1.15;联合用药能显著降低细胞内线粒体膜电位及p-mTOR蛋白表达,显著增加细胞内活性氧产生及Cleave-Caspase3、p-AMPK蛋白表达;抑制AMPK或mTOR能显著增加联合用药对细胞的生长抑制作用(P<0.05、0.01)。结论 2-DG联合Met具有协同抑制HepG2细胞增殖作用,其机制与降低细胞线粒体膜电位,促进活性氧产生、细胞凋亡,以及非AMPK依赖性的抑制mTOR活化有关。

Objective To investigate whether 2-deoxyglucose(2-DG)combined with metformin(Met)has synergistic antitumor effect on HepG2 cells,and explore the underling mechanisms.Methods The cell growth inhibitory ratio of HepG2 cells treated by 2-DG,Met,and the combination was determined by the resin azure method,and the synergistic antitumor effects of the combined application was also examined by calculating the Q values of Jin's formula;Mitochondrial membrane potential and reactive oxygen species generation were detected by high conformation cell imaging system;The expression level of Cleave-Caspase 3,p-AMPK,and p-mTOR were examined by Western blotting;The cell growth inhibitory ratio was detected by the resin azure method after AMPK and mTOR inhibitors administrating.Results The IC50 values of 2-DG and Met alone in HepG2 cells were 2.45 mmol/L and 16.35 mmol/L,respectively.Combined application has synergistic antitumor effects,and the Q values of Jin's formula was more than 1.15.Combined application significantly reduced mitochondrial membrane potential and increased intracellular reactive oxygen species production and Cleave-Caspase3 expression.Combined application significantly increased the expression of P-AMPK,decreased the expression of p-mTOR.Moreover,inhibiting AMPK and mTOR by the inhibitors significantly enhanced the cell growth inhibitory effect of combination group(P<0.05 and 0.01).Conclusion 2-DG combined with Met has synergistic antitumor effect on HepG2 cells.The underlying mechanism is related to decreasing mitochondrial membrane potential,promoting reactive oxygen production and apoptosis,and non-AMPK dependent inhibition of mTOR activation.