自噬的特异性抑制对肝癌细胞凋亡的调控及其相关机制

目的:研究自噬特异性抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)对奥沙利铂(oxaliplatin,OX)诱导的肝癌细胞系HepG2存活率的影响,并探讨其机制。方法:MDC与DAPI双重染色后,采用荧光显微镜对自噬进行定性观察;以CCK8检测3-MA抑制剂自噬前后,经OX诱导的HepG2细胞存活率;并用RT-PCR检测自噬特异性基因LC3表达的变化,以Western印迹法分别检测自噬特异性蛋白LC3及凋亡活化蛋白Caspase-3的变化。结果:OX可诱导肝癌细胞HepG2产生自噬,且自噬在基因及蛋白水平的表达均增加;3-MA与OX联合作用可明显增强HepG2细胞的凋亡。结论:OX诱导肝癌细胞系HepG2凋亡的过程中自噬起到保护性作用;抑制自噬可明显增强OX诱导的HepG2细胞凋亡。3-MA可能为提高肝癌对化疗敏感性提供新思路。

A study on autophagy inhibited by 3-MA and oxaliplatin induce HepG2 apoptosis

Objective This study was designed to investigate the effects and mechanism of 3-methyladenine（3-MA,an autophagy inhibitor） on the viability change of human hepatoma cell line HepG2 induced by oxaliplatin.Methods The autophagy was observed using fluorescent microscope by monodansylcadaverin（MDC） and DAPI staining.The viability change of HepG2 cell induced by L-OHP were analyzed using CCK8 before and after autophagy inhibited by 3-MA.PT-PCR was used to examine the microtubule-associated protein 1 light chain 3（LC3,the autophagy-specific protein） expression.The LC3 and caspase-3 protein（apoptosis activating protein） were detected by Western blot.Results The increasing expression of autophagy at the gene and protein levels indicated that L-OHP could induce autophagy in HepG2 cell.The combined effect of 3-MA and L-OHP may enhance the apoptosis of the HepG2 cell line.Conclusions Autophagy is a protective phenomenon during the HepG2 cell line apoptosis induced by L-OHP,and the process could apparently be enhanced by the autophagy inhibitor,3-MA.3-MA may enhance the sensitivity of hepatocellular carcinoma towards chemotherapy.