AZD5363诱导肝癌细胞凋亡与自噬的实验研究

目的:研究新型Akt抑制剂AZD5363对人肝癌HepG2和Huh7细胞活力、凋亡和自噬的影响,并探讨其抗肿瘤活性的分子机制。方法:采用不同浓度AZD5363作用于体外培养的HepG2和Huh7细胞,MTT法检测细胞活力;TUNEL标记法检测肝癌细胞凋亡的变化;Western blot实验分析细胞凋亡相关蛋白多腺苷二磷酸核糖聚合酶[poly(ADP-ribose) polymerase,PARP]及自噬标志蛋白LC3-II的表达水平;细胞转染GFP-LC3绿色荧光蛋白融合表达质粒检测细胞自噬。结果:AZD5363能够剂量依赖性地抑制HepG2和Huh7细胞活力,并通过促进PARP的切割而诱导肝癌细胞发生凋亡;肝癌细胞给予AZD5363后,细胞内GFP-LC3融合蛋白斑点增多,同时LC3-II的表达水平增加(P <0.05);当用氯喹阻断自噬后,AZD5363对肝癌细胞凋亡的诱导作用明显增强。结论:AZD5363可促进HepG2和Huh7细胞发生凋亡和保护性自噬。抑制自噬促进了AZD5363诱导的肝癌细胞凋亡。

Effect of AZD5363 on apoptosis and autophagy in hepatocellular carcinoma cells

AIM:To investigate the effect of AZD5363, an inhibitor of Akt, on the viability, apoptosis and autophagy in human hepatocelluar carcinoma cells and the molecular mechanisms. METHODS:MTT assay was used to detect the cell viability. TUNEL assay was used to analysis the apoptosis. The expression of PARP and LC3-Ⅱ proteins was examined by Western blot analysis. The autophagy was characterized by the expression and distribution of GFP-LC3. RESULTS:The results of MTT assay indicated that AZD5363 suppressed the cell viability in a dose-dependent manner (P<0.05). High doses of AZD5363 triggered apoptosis via activating the cleavage of PARP. AZD5363 treatment induced autophagy both in HepG2 cells and Huh7 cells by increasing the level of LC3-Ⅱ. Blockage of autophagy by chloroquine promoted AZD5363-induced apoptosis in the hepatocellular carcinoma cells. CONCLUSION:AZD5363 increased apoptosis and autophagy in HepG2 cells and Huh7 cells. Blockage of autophagy magnified AZD5363-induced apoptosis in hepatocellular carcinoma cells.