Abstract: 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.

Key words: Hepatoma, Hep3B cells, Disulfiram, Cuproptosis, Proliferation, Migration, Invasion