索拉非尼抑制肝癌细胞增殖中自噬的作用及其机制

Effect and mechanism of sorafenib in inhibiting autophagy of HepG_2 cells proliferation

目的:研究分子靶向药物索拉非尼在体外对人肝癌细胞株HepG_2增殖抑制过程中自噬的表达及作用,并探讨其可能机制。方法:以吖啶橙染色荧光显微镜对自噬进行定性观察;cell counting kit-8检测活性氧(reactiveoxygen species,ROS)抑制前后HepG_2细胞成活率的变化;RT-PCR检测自噬基因Beclin-1表达的变化。Western印迹检测自噬相关蛋白Beclin-1的变化:荧光分光光度计检测胞内二氯荧光素DCF的荧光强度。结果:索拉非尼对肝癌细胞HepG_2具有显著的抑制作用;索拉非尼可诱导肝癌细胞HepG_2产生自噬及ROS,自噬在基因及蛋白水平表达均增加;抑制ROS的产生可减少索拉非尼诱导的肝癌细胞HepG_2自噬的表达量,自噬的抑制增强了索拉非尼对肝癌细胞的抑制作用。结论:ROS参与索拉非尼诱导肝癌细胞HepG_2的自噬表达,索拉非尼在抑制肝癌细胞自噬过程中自噬可能起到保护作用,抑制自噬可能为提高进展期肝癌病人索拉非尼分子靶向治疗敏感性提供新的思路。

Objective This study was designed to investigate the effects and mechanism of autophagy on the viability change of human hepatoma cell line HepG_2 induced by sorafenib.Methods The acidic vesicular organelles were observed using fluorescent microscope by acriding orange staining.The viability change of the HepG_2 cell line induced by sorafenib was analyzed by using cell counting kit-8 in the presence or absence of reactive oxygen species（ROS） scavenger. RT-PCR and Western blot were used to examine Beclin-1（autophagy-specific protein） expression at the gene and protein level.The intracellular fluorescence of dichlorofluorescein was measured by fluorescence spectrometry.Results Sorafenib has a significant inhibitory effect towards the hepatocellar carcinoma（HCC） cell line HepG_2.Sorafenib could induce autophagy and ROS in HepG_2,the inhibition of ROS lead to a reduction in autophagic activity which could enhance the inhibitory effect of Sorafenib in HepG_2.Conclusions ROS is involved in the induction of autophagy by Sorafenib in the HepG_2 cell line,autophagy is hepatoprotective during the inhibitory effect of sorafenib in HepG_2.Inhibition of autophagy can lead to a novel target of molecular targeted therapy in advanced HCC.