双硫仑/铜复合物通过Drp1去磷酸化和线粒体转位抑制肝癌细胞增殖并诱导细胞凋亡

Disulfiram/copper complex inhibits proliferation and induces apoptosis in hepatocellular carcinoma cells through dephosphorylation and mitochondrial translocation of Drp1

目的研究双硫仑/铜复合物二乙基二硫代氨基甲酸铜[copper(Ⅱ)diethyldithiocarbamate,CuET]对肝癌细胞增殖及凋亡的影响,并探讨其作用机制。方法用不同浓度(0、0.01、0.05、0.10、0.50、1.00、2.00μmol/L)的CuET处理,CuET(2.00μmol/L)作用不同时间(0、3、6、9、12、24 h)或Drp1线粒体转位的选择性抑制剂Mdivi-1(1.00μmol/L)预处理肝癌细胞HepG2、SMMC-7721后,MTT实验检测细胞存活率,软琼脂克隆形成实验确定集落形成,流式细胞术分析细胞凋亡,激光共聚焦检测Drp1线粒体转位,Western blot检测细胞凋亡的相关蛋白cleaved-Caspase 9、cleaved-Caspase 3、cleaved-PARP1、细胞色素C(cytochrome C,cyto C),以及线粒体分裂蛋白phospho-Drp1(S637)、phospho-Drp1(S616)和Drp1的表达。结果CuET对HepG2和SMMC-7721细胞增殖和集落形成的抑制作用呈剂量依赖性(P<0.01);对HepG2和SMMC-7721细胞凋亡诱导作用也有明显的剂量和时间依赖性(P<0.01);Western blot检测结果显示,CuET可导致Caspase 3和Caspase 9的裂解激活,PARP的降解激活以及cyto C从线粒体向细胞质的释放。CuET处理可导致Drp1(S637)去磷酸化和Drp1线粒体转位。细胞经Mdivi-1预处理后可明显阻断CuET诱导的Drp1线粒体转位、Caspase 3的裂解/激活、cyto C的释放以及细胞凋亡。结论双硫仑/铜复合物通过Drp1去磷酸化和线粒体转位抑制肝癌细胞增殖并诱导细胞发生凋亡。

Objective To determine the effects of disulfiram/copper complex,copper(Ⅱ)diethyldithiocarbamate(CuET)on the proliferation and apoptosis of hepatocellular carcinoma cells,and investigate the molecular mechanisms.Methods MTT assay was used to detect cell viability in the hepatocellular carcinoma HepG2 and SMMC-7721 cells treated with CuET at the concentrations of 0,0.01,0.05,0.1,0.5,1 or 2μmol/L for 24 h.Then after the HepG2 and SMMC-7721 cells were treated with 2μmol/L CuET for 0,3,6,9,12 or 24 h,MTT assay,soft agar assay and flow cytometry were used to determine cell viability,colony formation and cell apoptosis,respectively.And Western blotting was performed to detect the expression of apoptosis-related proteins,including cleaved-Caspase 9,cleaved-Caspase 3,cleaved-PARP1 and cytochrome C,and mitochondrial mitotic proteins,such as phospho-Drp1(S637),phospho-Drp1(S616),and dynamin-related protein 1(Drp1).Laser confocal scanning microscopy was carried out to observe the effect of 1μmol/L Mdivi-1 pretreatment(a selective inhibitor of Drp1 mitochondrial translocation)for 2 h on the mitochondrial translocation of Drp1.Its effects on the apoptosis and expression levels of above proteins were also studied.Results Compared with the control cells(0μmol/L),CuET treatment showed inhibitory effects on the proliferation and colony formation,and inductive effect on the apoptosis in HepG2 and SMMC-7721 cells in dose-and time-dependent manners(all P<0.01).Western blot assay indicated that CuET treatment led to cleavage/activation of Caspase 3 and Caspase 9,degradation of PARP,as well as release of cytochrome C from the mitochondria into the cytoplasm.CuET treatment also caused dephosphorylation and mitochondrial translocation of Drp1(S637).Mdivi-1 pretreatment obviously blocked the mitochondrial translocation of Drp1,cleavage/activation of Caspase 3,release of cytochrome C,as well as cell apoptosis induced by CuET treatment.Conclusion CuET inhibits the proliferation and promotes apoptosis in hepatocellular carcinoma cells through dephosphorylation and mitochondrial translocation of Drp1.