二甲双胍联合2-脱氧-D-葡萄糖对肝癌细胞增殖与凋亡的影响及其机制

目的 研究二甲双胍(Met)和2-脱氧-D-葡萄糖(2DG)联合用药对肝癌HepG2、Hep3B细胞增殖及凋亡的影响及其机制.方法 Wst-1试剂检测Met和2DG单独及联合应用对HepG2、Hep3B细胞增殖的影响;光学显微镜下观察Met、2DG单独及联合作用后HepG2、Hep3B细胞形态上的改变;流式细胞术观察各组药物处理后细胞凋亡的变化;Western blotting测定HepG2细胞的Caspase-3、PARP、Mcl-1的表达情况.结果 药物联合组HepG2细胞的存活率为(22.48 ±0.51)％,与对照组(100.00±5.05)％、Met组(80.68±5.10)％和2DG组(72.56±4.34)％比较,差异具有统计学意义(P ＜0.001;P＜0.001;P=0.001).联合组Hep3B细胞的存活率为(29.16±1.34)％,与对照组(100.00±1.23)％、Met组(59.58±1.92)％和2DG组(33.87±1.95)％比较,差异具有统计学意义(P ＜0.001;P＜0.001;P=0.001).显微镜下,Met和2DG联合用药组HepG2细胞数量明显减少,而漂浮的死亡细胞增多;而联合组虽然也造成Hep3B细胞密度减少,但并未见显著增加脱落细胞.流式细胞术检测HepG2细胞联合用药组的凋亡率为(39.63±0.21)％,与对照组(7.12±0.14)％、Met组(12.56±0.35)％和2DG组(15.16±1.93)％比较,差异具有统计学意义(P ＜0.001;P ＜0.001;P=0.001);Hep3B细胞联合用药组的凋亡率为(12.58±1.03)％,与对照组(2.82±0.51)％和Met用药组(8.98±0.86)％比较,差异具有统计学意义(P ＜0.001;P=0.007),与2DG用药组(12.40±1.78)％比较,差异不具有统计学意义(P=1.000).Western blotting进一步显示,联合用药组可以观察到Caspase-3激活、多腺苷二磷酸核糖聚合酶(PARP)切割以及Mcl-1表达降低.结论 Met和2DG联合用药可以有效抑制HepG2、Hep3B细胞的增殖,并诱导HepG2细胞凋亡,其机制可能与激活Caspase-3、切割PARP底物、降低Mcl-1蛋白表达有关.

Effect and mechanism of metformin combined with 2-deoxy-D-glucose on proliferation and apoptosis of liver cancer cells

Objective To investigate the combined effect and mechanism of metformin (Met) and 2-deoxy-D-glucose (2DG) on cell proliferation and apoptosis in liver cancer cells HepG2 and Hep3B.Methods Wst-1 reagent was used to determine the anti-proliferation effects after treatments with Met and 2DG alone or combined in HepG2 and Hep3B cells.Microscopy was used to observe cell morphological changes after treatments with Met and 2DG alone or combined in HepG2 and Hep3B cells.Cell apoptosis was observed by flow cytometry after treatment of different kinds of drugs.Western blotting was used to analyze the protein expressions of Caspase-3,PARP,Mcl-1 of HepG2.Results The survival rate of HepG2 cells in the combination group was (22.48 ± 0.51)％,and compared with the control group (100.00 ± 5.05)％,Met group (80.68 ±5.10)％ and 2DG group (72.56 ±4.34)％,the differences were statistically significant (P ＜ 0.001;P ＜ 0.001;P =0.001).The survival rate of Hep3B cells in the combination group was (29.16 ± 1.34) ％,and compared with the control group (100.00 ± 1.23) ％,Met group (59.58 ± 1.92) ％ and 2DG group (33.87 ± 1.95) ％,the differences were statistically significant (P ＜ 0.001;P ＜ 0.001;P =0.001).Microscopy observation showed that combined treatment of Met and 2DG caused less viable adherent cells of HepG2,but more floating dead cells.While the combination group also caused a decrease in the density of Hep3B cells,but did not significantly increase the shedding of cells.The apoptosis of HepG2 cells in the combination group was (39.63 ± 0.21) ％,and compared with the control group (7.12 ± 0.14) ％,Met group (12.56 ± 0.35) ％ and 2DG group (15.16 ± 1.93) ％,the differences were statistically significant (P ＜0.001;P ＜ 0.001;P =0.001).The apoptosis of Hep3B cells in the combination group was (12.58 ± 1.03) ％,and compared with the control group (2.82 ± 0.51) ％ and Met group (8.98 ± 0.86) ％,the differences were statistically significant (P ＜ 0.001;P =0.007),but compared with the 2DG group (12.40 ± 1.78) ％,the difference was not statistically significant (P =1.000).Furthermore,Western blotting demonstrated that the combined treatment induced evident Caspase-3 activation and poly ADP-ribose polymerase (PARP) cleavages,and decreased expression of Mcl-1.Conclusion The combination of Met and 2DG can effectively inhibit cell proliferation of HepG2 and Hep3B,and induce apoptosis of HepG2 cells.The mechanism may be involved with Caspase-3 activation,cutting PARP substrate and decreasing Mcl-1 protein.