蛋白酶体抑制剂MG132诱导HepG2细胞凋亡及其机制研究

目的观察蛋白酶体抑制剂MG132对人肝癌细胞HepG2的致凋亡作用,并从泛素蛋白酶体途径(UPP)相关基因E1、E2和E3及天冬氨酸特异的半胱氨酸蛋白酶3(Caspase3)表达初步探讨MG132的致细胞凋亡机制。方法采用多个浓度(2、5、10μmol·L-1)的蛋白酶体抑制剂MG132处理HepG2细胞;流式细胞术检测细胞周期和细胞凋亡率,DNA琼脂糖凝胶电泳检测细胞凋亡;逆转录聚合酶链反应(RTPCR)检测UPP相关基因E1、E2、E3和凋亡相关基因Caspase3的转录水平;免疫细胞化学检测Caspase3蛋白表达。结果对照组HepG2细胞凋亡率低于5%,在2、5和10μmol·L-1MG132作用下,细胞凋亡率分别为42.9%、66.1%、72.8%,MG132诱导HepG2细胞凋亡具有量效关系;RTPCR检测发现细胞内UPP相关基因E1、E2、E3mRNA表达下降,而凋亡相关基因Caspase3mRNA表达上调;免疫组化检测Caspase3蛋白表达水平升高。结论蛋白酶体抑制剂MG132能够诱导HepG2细胞凋亡,其机制可能与MG132抑制UPP活性,使细胞内Caspase3蛋白降解减少,同时上调Caspase3基因转录,促进细胞凋亡。

Mechanism involved in the apoptosis of human hepatocelluar cancer cell line HepG2 induced by proteasome inhibitor MG132

Aim To study the effect of proteasome inhibitor MG132 on expression of Caspase-3 and UPP associated genes in the apoptosis of human hepatocelluar cancer cells.Methods HepG2 cells were treated with MG132 (2,5,10 μmol·L -1) for 24 h. The apoptotic cells were determined with flow cytometric analysis. The levels of E1, E2, E3 and Caspase-3 mRNA expression were detected with RT-PCR. Caspase-3 protein expression was detected with immunohistochemistry. Results The results showed that the increase of the degree of HepG2 cell apoptosis was concentrationly dependent. RT-PCR showed that E1, E2 and E3 gene expressions were decreased in the treatment group. MG132 down-regulated the gene expression of E1, E2, E3 and up-regulated the gene/protein expression of Caspase-3. Conclusion Proteasome inhibitor MG132 may induce HepG2 cells apoptosis by inhibitting UPP activity,up-regulating the gene/protein expression of Caspase-3.