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

Abstract

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 2 μmol/L for 24 h. Then after the HepG2 and SMMC-7721 cells were treated with 2 μmol/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 1 μmol/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 (0 μmol/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.

Keywords

• disulfiram
• copper(ⅱ) diethyldithiocarbamate
• liver neoplasms
• cell apoptosis
• dynamin-related protein