Copper-containing agents are promising antitumor pharmaceuticals due to the ability of the metal ion to react with biomolecules. In the current study, we demonstrate that inorganic Cu2+ in the form of oxide nanoparticles (NPs) or salts, as well as Cu ions in the context of organic complexes (oxidation states +1, +1.5 and +2), acquire significant cytotoxic potency (2-3 orders of magnitude determined by IC50 values) in combinations with N-acetylcysteine (NAC), cysteine, or ascorbate. In contrast, other divalent cations (Zn, Fe, Mo, and Co) evoked no cytotoxicity with these combinations. CuO NPs (0.1-1 µg/mL) together with 1 mM NAC triggered the formation of reactive oxygen species (ROS) within 2-6 h concomitantly with perturbation of the plasma membrane and caspase-independent cell death. Furthermore, NAC potently sensitized HCT116 colon carcinoma cells to Cu-organic complexes in which the metal ion coordinated with 5-(2-pyridylmethylene)-2-methylthio-imidazol-4-one or was present in the coordination sphere of the porphyrin macrocycle. The sensitization effect was detectable in a panel of mammalian tumor cell lines including the sublines with the determinants of chemotherapeutic drug resistance. The components of the combination were non-toxic if added separately. Electrochemical studies revealed that Cu cations underwent a stepwise reduction in the presence of NAC or ascorbate. This mechanism explains differential efficacy of individual Cu-organic compounds in cell sensitization depending on the availability of Cu ions for reduction. In the presence of oxygen, Cu+1 complexes can generate a superoxide anion in a Fenton-like reaction Cu+1L + O2 → O2-. + Cu+2L, where L is the organic ligand. Studies on artificial lipid membranes showed that NAC interacted with negatively charged phospholipids, an effect that can facilitate the penetration of CuO NPs across the membranes. Thus, electrochemical modification of Cu ions and subsequent ROS generation, as well as direct interaction with membranes, represent the mechanisms of irreversible membrane damage and cell death in response to metal reduction in inorganic and organic Cu-containing compounds.
Keywords: cell death; copper; drug resistance; reactive oxygen species; redox potential; tumor cells.