The Molecular Mechanism Of Disulfiram/copper Complex(CuET)inhibiting Proliferation And Inducing Apoptosis In Hepatocellular Carcinoma Cells

BackgroundHepatocellular carcinoma（HCC）remains one of the most common malignances in globe with increasing incidence and mortality over the past decade.Chemotherapy,as one of the classic anticancer therapies,others including transplantation,resection,as well as radiation,is recommended for patients who are not candidates for operations or with advanced HCC.Sorafenib is a first-line chemotherapy agent for HCC patients.Contrary to escalating survival benefits,its drug resistance has still harassed a great number of patients.Therefore,all of the above problems and shortcomings strongly call for innovative remedies.Apparently compared with traditional routes,using drugs approved for the treatment of diverse diseases as candidate chemotherapeutics has incomparable advantages——short testing periods,abundant clinical data and low cost.Among promising anticancer drugs,disulfiram（DSF）is a representative drug for alcohol abuse approved by FDA in 1960s,with well-established pharmacokinetics,safety and tolerance at recommended doses.Recently,it has been reported that disulfiram/copper complex（CuET）,a metabolite of DSF in vivo,has shown good anticancer activities in clinical and preclinical studies.Given lack of research on CuET in hepatoma and liver as the main metabolic organ of DSF,we would like to explore the molecular mechanism of CuET inhibiting proliferation and inducing apoptosis in HCC,in order to link important features of DSF with tumors,as well as to provide a new idea for clinical treatment of CuET in HCC.ObjectivesTo elucidate the molecular mechanism of CuET inhibiting proliferation and inducing apoptosis in human hepatoma Hep G2 and SMMC 7721 cells,and to provide theoretical basis for the application of CuET in treatment of HCC.MethodsMTT colorimetric assay and clone formation assay were used to detect cell proliferation.Flow cytometry assay was used to determine cell apoptosis and ROS levels.Western blot assay was employed to examine relevant proteins in apoptosis and mitochondrial fission.Laser confocal microscopy was employed to observe mitochondrial fission and the co-localization of Drp1 and mitochondria.Transmission electron microscopy was performed to observe mitochondrial morphology.Results1.CuET inhibits proliferation and induces apoptosis in human hepatoma cellsThough MTT assay,we found that CuET effectively inhibited proliferation in different types of cancer cells,which was more obvious in Hep G2 and SMMC-7721 cells（P<0.01）,conversely invisible in normal hepatic THLE cells（P<0.01）.Treating with CuET also contributed to decreases in clone formation（P<0.01）.Furthermore,disposing cells with CuET leaded to increases in apoptosis（P<0.01）,mobilization of cytochrome c from mitochondria to cytoplasm,stimulation of caspase 9 and caspase 3,as well as degradation of PARP,in dose-and time-dependent manners.2.CuET impairs mitochondria and induces mitochondrial fragmentationTransmission electron microscopy（TEM）showed that the number of mitochondria in Hep G2 and SMMC-7721 cells treated with CuET decreased（P<0.01）,while the remaining showed typical morphological changes:swollen,broken and distortion of mitochondrial cristae.Immunofluorescence analysis revealed that treated with CuET led to mitochondrial division,broken into"punctate".3.CuET induces mitochondrial fission and apoptosis through dephosphorylation of Drp1（Ser637）and its mitochondrial translocation.Though Western blot analysis,we found that treated with CuET resulted in a reduction of phosphor-Drp1（Ser637）and Drp1 in cytosol,plus an increase of Drp1 in mitochondria,in dose-and time-dependent manner.Pretreatment with Mdivi-1,a Drp1 mitochondrial translocation inhibitor,abrogated CuET-induced mitochondrial fission and apoptosis（P<0.01）.We also used sh RNA to knock down Drp1 protein in Hep G2 cells.Knockdown of Drp1with si RNA abrogated CuET-induced mitochondrial fission and apoptosis（P<0.01）.Furthermore,we constructed the plasmids of Drp1 mutants,Drp1 S637A and Drp1 S637D,which mimiced the dephosphorylated and phosphorylated forms of Drp1（Ser637）,respectively.Our results showed that overexpression of Drp1 S637A enhanced CuET-mediated mitochondrial translocation of Drp1,mitochondrial fission,and apoptosis（P<0.01）.In contrast,overexpression of Drp1 S637D abrogated CuET-mediated mitochondrial translocation of Drp1,mitochondrial fission,and apoptosis（P<0.01）.4.CuET-induced the production of mitochondrial superoxide contributes to dephosphorylation and mitochondrial translocation of Drp1,mitochondrial fission,as well as apoptosisFlow cytometry showed that CuET treatment increase the ROS level in HCC cells（P<0.01）,and the ROS type was superoxide anion（O₂）,mainly came from mitochondria.Pretreatment of Mito Q,a mitochondrial targeted antioxidant,abrogated ROS augment,dephosphorylation of Drp1（Ser637）and its mitochondrial translocation,ultimately restricting mitochondrial fission and apoptosis（P<0.01）,all of the above triggered by CuET.ConclusionCuET induced mitochondrial fission and apoptosis in human hepatoma cells.Mechanistically,CuET-induced mitochondrial superoxide represents a primary event resulting in mitochondrial fragmentation,as a consequence of dephosphorylation at Drp1（Ser637）and its mitochondrial translocation,culminating in cytochrome c release,Csaspase 3/9 activation and apoptosis.