Identification of differential anti-neoplastic activity of copper bis(thiosemicarbazones) that is mediated by intracellular reactive oxygen species generation and lysosomal membrane permeabilization

Bis(thiosemicarbazones) and their copper (Cu) complexes possess unique anti-neoplastic properties. However, their mechanism of action remains unclear. We examined the structure–activity relationships of twelve bis(thiosemicarbazones) to elucidate factors regarding their anti-cancer efficacy. Importantly, the alkyl substitutions at the diimine position of the ligand backbone resulted in two distinct groups, namely, unsubstituted/monosubstituted and disubstituted bis(thiosemicarbazones). This alkyl substitution pattern governed their: (1) CuII/I redox potentials; (2) ability to induce cellular 64Cu release; (3) lipophilicity; and (4) anti-proliferative activity. The potent anti-cancer Cu complex of the unsubstituted bis(thiosemicarbazone) analog, glyoxal bis(4-methyl-3-thiosemicarbazone) (GTSM), generated intracellular reactive oxygen species (ROS), which was attenuated by Cu sequestration by a non-toxic Cu chelator, tetrathiomolybdate, and the anti-oxidant, N-acetyl-l-cysteine. Fluorescence microscopy suggested that the anti-cancer activity of Cu(GTSM) was due, in part, to lysosomal membrane permeabilization (LMP). For the first time, this investigation highlights the role of ROS and LMP in the anti-cancer activity of bis(thiosemicarbazones). Bis(thiosemicarbazones) and their copper complexes possess unique anti-neoplastic properties. However, their mechanism of action remains unclear. We examined twelve bis(thiosemicarbazones) to elucidate mechanisms behind their anti-cancer efficacy. For the first time, this investigation highlights the role of reactive oxygen species and lysosomal membrane permeabilization in the anti-cancer activity of bis(thiosemicarbazones). [Display omitted] •Bis(thiosemicarbazones) and their Cu complexes have unique anti-cancer activity.•We examined twelve bis(thiosemicarbazones) to elucidate their mechanisms of action.•For the first time, we show they mediate lysosomal membrane permeabilization.