摘要
目的:研究去氢骆驼蓬碱体外诱导HepG2细胞凋亡过程中c-Jun N末端激酶(JNK)途径的作用,并观察其联合化疗药物对HepG2细胞的影响。方法:CCK-8法检测联合或不联合使用JNK特异性抑制剂SP600125对细胞增殖的抑制作用;克隆形成实验观察不同浓度药物对细胞克隆形成的影响;Hoechst 33258染色法观察细胞形态变化;PI单染检测细胞亚二倍体率;Annexin V-PI双染测定细胞早期凋亡水平;Western blotting检测细胞聚(ADP-核糖)聚合酶(PARP)、JNK和p-JNK蛋白表达的改变;联合5-氟尿嘧啶(5-FU)或顺铂(DDP)观察细胞对化疗药物的敏感性。结果:去氢骆驼蓬碱随药物浓度的升高而抑制作用增强,48 h后IC50为9.80 mg/L;去氢骆驼蓬碱能显著抑制细胞克隆形成,并且导致细胞凋亡形态学变化;PI单染检测发现细胞周期的G1期前有亚二倍体凋亡峰,Annexin V-PI双染检测细胞出现明显的早期凋亡细胞群;Western blotting检测到随着去氢骆驼蓬碱浓度增加PARP及p-JNK蛋白表达增加,JNK蛋白表达减少;联合使用SP600125对细胞增殖的抑制作用减弱;联合5-FU或顺铂明显增强化疗药物对肿瘤细胞的抑制作用,增敏倍数分别为1.47和5.78倍。结论:去氢骆驼蓬碱对HepG2细胞有增殖抑制作用,并诱导其凋亡,激活JNK信号通路可能是其主要机制之一;同时去氢骆驼蓬碱可以增强HepG2细胞对5-FU和顺铂的敏感性。
AIM ： To investigate the proliferation-inhibitory and apoptosis-inducing effects of harmine on human hepatocarcinoma HepG2 cells. METHODS ： The proliferation of HepG2 cells was determined in the absence or presence of a JNK inhibitor SP600125 by Cell Counting Kit-8 （ CCK-8 ） assay and colony formation test. The morphology of HepG2 cells was observed by Hoechst 33258 staining under fluorescence microscope. The cell apoptosis was analyzed by Annexin V-PI staining. The expression of apoptosis-regulated proteins, poly （ADP-ribose） polymerase （PARP） , c-Jun N-terminal ki- nase （JNK） and p-JNK, was detected by Western blotting. The sensitizing effects of harmine on HepG2 cells to chemother- apeutic drugs 5-fluorouracil （5-FU） and cisplatin were determined by CCK-8 assay. RESULTS： Harmine inhibited the proliferation of HepG2 cells and induced apoptosis in a dose-dependent manner. After the JNK signaling pathway was blocked by SP600125, the cell apoptotic rate decreased significantly. Hoechst 33258 staining revealed that the nuclear frag- mentation, chromosomal condensation, cell shrinkage and attachment loss appeared in the HepG2 cells treated with harmine. The percentage of the sub-G~ fraction was increased and the population of early apoptotic cell death was observed. Apoptosis of HepG2 cells with harmine treatment was associated with the activation of JNK. Combined with harmine, the IC5o values of 5-FU and cisplatin for the tumor cells were 1.47 and 5.78 times sensitized as compared with the correspon- ding single drug treatment groups, respectively. CONCLUSION： Harmine exhibits an anti-proliferative effect on HepG2 cells by inducing apoptosis. The JNK signaling pathway is involved in the apoptosis of HepG2 cells. Harmine enhances the chemosensitivity of the cells to 5-FU and cisplatin.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2013年第2期284-289,共6页
Chinese Journal of Pathophysiology
基金
广东省科技计划(No.2010B031600248)
广东省中医药管理局基金资助项目(No.20122114)
