Disulfiram causes selective hypoxic cancer cell toxicity and radio-chemo-sensitization via redox cycling of copper

Therapy for lung cancer patients includes surgery, radiation, and chemotherapy. While treatments initially illicit desirable responses, the presence of hypoxia and drug resistant cells within tumors ultimately lead to treatment failure. Disulfiram (DSF) is an FDA approved, copper chelating agent that can target oxidative metabolic frailties in cancer versus normal cells and be repurposed as an adjuvant to cancer therapy. Clonogenic survival assays showed that DSF (50-150 nM) combined with physiological levels of Cu (15 µM CuSO4) is selectively toxic to lung cancer cells versus normal bronchial epithelial cells. Furthermore, cancer cell toxicity is exacerbated at 1% O2, relative to 4 or 21% O2. This selective toxicity of DSF/Cu can be attributed to its differential Cu ionophore capabilities. DSF/Cu treatment significantly increased total cellular Cu levels, with higher Cu present in tumor versus normal cells and in cancer cells at 1% O2 versus 21% O2. DSF toxicity was shown to be dependent on Cu by inhibiting toxicity with a Cu chelator, bathocuproinedisulfonic acid (BCS), which inhibits redox cycling. Toxicity was also shown to be dependent on oxidative stress mechanisms that are initiated by Cu, including the production of superoxide and peroxide, which were inhibited by a superoxide dismutase (SOD) mimetic, GC4419, and overexpression of catalase. DSF/Cu treatment also induced lipid oxidation, increased mitochondrial superoxide production, and decreased mitochondrial membrane potential, which were detected by C11-BODIPY, MitoSox, and JC-1. In addition to its selective toxicity to hypoxic cancer cells, DSF/Cu also enhanced radio-chemotherapy-induced cancer cell killing and reduced therapy resistance at hypoxia. DSF also decreased xenograft tumor growth in vivo when combined with radiation and carboplatin. These results support the hypothesis that DSF is a promising adjuvant for cancer therapy based on its apparent ability to selectively target fundamental differences in cancer cell oxidative metabolism.