雷公藤红素依赖ROS升高介导DNA损伤诱导肝癌细胞凋亡

Celastrol induced hepatocellular carcinoma cell death via ROS-mediated DNA damage

目的探究雷公藤红素(celastrol)诱导细胞内活性氧(reactive oxygen species,ROS)产生在介导细胞DNA损伤中的作用及对肝癌细胞凋亡的影响。方法人肝癌细胞经雷公藤红素处理后,镜下观察细胞形态变化;MTT法测定肝癌细胞的存活率;Hoechst 33258染色观察肝癌细胞核变化;流式细胞术检测细胞死亡;CM-H2DCFDA探针检测细胞内ROS水平;免疫荧光方法检测细胞内DNA损伤标志蛋白γ-H2AX的荧光强度;Western blot检测γ-H2AX、caspase-3、PARP等蛋白表达水平。结果雷公藤红素可显著抑制肝癌细胞的存活,并可升高细胞内ROS水平,呈浓度依赖性关结论雷公藤红素可诱导肝癌细胞内ROS聚集,高水平的ROS引起细胞DNA损伤,最终诱导细胞凋亡。

Aim To investigate the role of celastrol in reactive oxygen species(ROS)accumulation and DNA damage in hepatocellular carcinoma cells,and further investigate its effect on apoptosis induction in cancer cells.Methods Human liver cancer HepG2 and Huh7 cells were cultured with celastrol,then the morphological changes of cells were observed under microscope.MTT assay was employed to detect the proliferation of hepatocellular carcinoma cells,CM-H2DCFDA probe to detect intracellular ROS levels,and immunofluorescence to detect the expression level ofγ-H2AX in celastrol-treated cells.Hoechst 33258 staining was used to observe nuclear condensation and fragmentation;Flow cytometry was used to evaluate cell death.Western blot was applied to measure the expression levels ofγ-H2AX,caspase-3,PARP and other proteins.Results Celastrol had a significant inhibitory effect on the proliferation of liver cancer cells in dose-dependent manner.Compared with the control group,the cell viability was reduced and intracellular ROS level also increased significantly after celastrol treatment in a dose-dependent manner(P<0.05).With Hoechst staining,typical apoptotic characteristics such as nuclear chromatin condensation and fragmentation were observed in celastrol-treated cells.Western blot results showed that pro-form of caspase-3 significantly decreased,and the cleavage of PARP markedly increased by celastrol.After pretreatment with ROS inhibitor NAC,celastrol-mediated caspase-3 activation and PARP cleavage were significantly reversed(P<0.05).Conclusions Celastrol can induce apoptosis in hepatocellular carcinoma cells,and its anti-cancer effect is dependent on ROS-mediated DNA damage.