Effects of disulfiram combined with Cu2+induced cuproptosis on proliferation,migration and invasion of hepatoma Hep3B cells
Objective To investigate the effects of disulfiram(DSF)combined with Cu2+on the proliferation,migration and invasion of hepatoma Hep3B cells and the underlying mechanism.Methods Hepatoma Hep3B cells were cultured in vitro.DSF(30 nmol/L)solution,Cu2+(1 μmol/L)solution and copper chelating agent ammonium tetrathiomolybdate Ⅵ(ATTM)(30 nmol/L)solution were used individually or combinedly for intervention.The cells treated with dimethyl sulfoxide(DMSO)(30 nmol/L)were used as the control group.CCK-8,scratch test and Transwell cell test were used to detect the proliferation,migration and invasion ability of cells.The expression of copper death related proteins dihydrolipoamide S-acetyltransferase(DLAT)and ferredoxin 1(FDX1)were detected by immunofluorescence assay.Results Compared with the control group,the proliferation,migration and invasion ability of Hep3B cells were significantly decreased after DSF or Cu2+or DSF+Cu2+combined intervention(all P＜0.05),and the decrease was more significant in DSF+Cu2+combined intervention(all P＜0.0001).After addition of DSF combined with Cu2+to ATTM,the inhibitory effects of DSF combined with Cu2+on the proliferation,migration and invasion of Hep3B cells were reversed,and the proliferation,migration and invasion ability cells were enhanced compared with DSF+Cu2+group(all P＜0.05).Compared with the control group,the fluorescence intensity of copper death related proteins DLAT and FDX1 showed no significant increase after DSF or Cu2+intervention,but the fluorescence intensity of DLAT protein increased while that of FDX1 protein decreased after DSF+Cu2+combined intervention,and the expression trend of DLAT and FDX1 proteins was reversed after the addition of ATTM.Conclusions DSF combined with Cu2+can inhibit the proliferation,migration and invasion of Hep3B cells,probably through the induction of cuproptosis.
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