吐根碱结合PARP-1逆转肝癌细胞HepG2/DDP耐药的作用研究

Reversal of drug resistance of hepatocellular carcinoma cells HepG2/DDP by emetine combined with PARP-1

目的探讨吐根碱逆转耐药肝癌耐药细胞HepG2/DDP的耐药性及机制。方法采用顺铂(DDP)大剂量冲击结合低剂量持续诱导方法构建肝癌耐药细胞株HepG2/DDP;采用反向虚拟筛选、分子对接、表面离子体共振实验和免疫印迹实验寻找吐根碱逆转耐药的靶标;转染实验验证吐根碱作用靶标;细胞毒实验检测吐根碱联合DDP对HepG2/DDP细胞增殖的抑制作用;流式细胞实验验证吐根碱的增敏作用;免疫印迹实验分析凋亡相关蛋白Bcl2、BAX及Cleaved-caspase-3的表达。结果吐根碱能够增强DDP对HEPG2/DDP细胞的敏感性,使HEPG2/DDP对DDP的耐药指数(RI)由3.69降低到0.93;反向虚拟筛选、分子对接、表面离子体共振实验揭示吐根碱和PARP-1有良好的结合作用;免疫印迹实验显示吐根碱在细胞内显著抑制PARP-1活性;siRNA干扰实验显示HepG2/DDP细胞中PARP-1表达抑制后吐根碱增敏DDP细胞毒性的作用基本消失;流式细胞实验显示吐根碱能够增强DDP对HepG2/DDP细胞的促凋亡作用。结论吐根碱能够增强HepG2/DDP细胞对DDP的敏感性,其作用机制可能与其抑制PARP-1的活性有关。

Objective To investigate the reversal of drug resistance of drug-resistant hepatoma cell line HepG2/DDP by emetine and its mechanism.Methods The drug-resistant hepatoma cell line HepG2/DDP was established by high-dose cisplatin(DDP)shock combined with low-dose continuous induction.Reverse virtual screening,molecular docking,surface plasmon resonance(SPR)and Western blot assays were employed to explore the target of emetine.The target was validated by transfection assay in vivo.The inhibitory effect of emetine combined with DDP on the proliferation of HepG2/DDP cells was detected by cytotoxicity assay.The sensitization effect of emetine was verified by flow cytometry,and the expression of the apoptosis related proteins BCL2,Bax and Cleaved-caspase-3 were analyzed by Western blot.Results Emetine enhanced the sensitivity of HepG2/DDP cells to DDP and reduced the resistance index(RI)from 3.69 to 0.93.Reverse virtual screening,molecule docking and SPR results showed that emetine can stably bind to PARP-1.Western blot assays showed that emetine had a potent enzymatic inhibitory activity against PARP-1 in vivo.Furthermore,emetine’s potentiation of DDP in HEPG2/DDP cells nearly disappeared when the cells were transfected with siRNAs against PARP-1.Flow cytometry showed that emetine could enhance the proapoptotic effect of DDP on HepG2/DDP cells.Conclusion Emetine can enhance the sensitivity of HepG2/DDP cells to DDP,and its mechanism may be related to its inhibition of PARP-1.