Disulfiram/copper-disulfiram Damages Multiple Protein Degradation and Turnover Pathways and Cytotoxicity is Enhanced by Metformin in Oesophageal Squamous Cell Carcinoma Cell Lines

ABSTRACT Disulfiram (DSF), used since the 1950s in the treatment of alcoholism, is reductively activated to diethyldithiocarbamate and both compounds are thiol‐reactive and readily complex copper. More recently DSF and copper‐DSF (Cu‐DSF) have been found to exhibit potent anticancer activity. We have previously shown that the anti‐diabetic drug metformin is anti‐proliferative and induces an intracellular reducing environment in oesophageal squamous cell carcinoma (OSCC) cell lines. Based on these observations, we investigated the effects of Cu‐DSF and DSF, with and without metformin, in this present study. We found that Cu‐DSF and DSF caused considerable cytotoxicity across a panel of OSCC cells, and metformin significantly enhanced the effects of DSF. Elevated copper transport contributes to DSF and metformin‐DSF‐induced cytotoxicity since the cell‐impermeable copper chelator, bathocuproinedisulfonic acid, partially reversed the cytotoxic effects of these drugs, and interestingly, metformin‐treated OSCC cells contained higher intracellular copper levels. Furthermore, DSF may target cancer cells preferentially due to their high dependence on protein degradation/turnover pathways, and we found that metformin further enhances the role of DSF as a proteasome inhibitor. We hypothesized that the lysosome could be an additional, novel, target of DSF. Indeed, this acid‐labile compound decreased lysosomal acidification, and DSF‐metformin co‐treatment interfered with the progression of autophagy in these cells. In summary, this is the first such report identifying the lysosome as a target of DSF and based on the considerable cytotoxic effects of DSF either alone or in the presence of metformin, in vitro, and we propose these as novel potential chemotherapeutic approaches for OSCC. J. Cell. Biochem. 116: 2334–2343, 2015. © 2015 Wiley Periodicals, Inc.