Abstract
Elesclomol ((N-malonyl-bis(N′-methyl-N′-thiobenzoylhydrazide)); formerly STA-4783) is a mitochondria-targeted chemotherapeutic agent that has demonstrated efficacy in selective cancer cell killing in pre-clinical and clinical testing. The biologically active form of elesclomol is a deprotonated copper chelate (elesclomol:copper; E:C), which has been shown to enhance reactive oxygen species (ROS) production and induce a transcriptional gene profile characteristic of an oxidative stress response in vitro. Previous studies suggest that E:C interacts with the electron transport chain (ETC) to generate high levels of ROS within the organelle and ultimately induce cell death. The purpose of this study was to further explore the mechanism of cellular and mitochondrial toxicity of E:C by examining its direct effect on mitochondrial bioenergetic function. The results obtained indicate that E:C treatment in whole cells of non-tumorigenic origin at high concentrations (40 μM and higher) induces a rapid and substantial increase in mitochondrial superoxide levels and dissipation of mitochondrial membrane potential. Furthermore, similar higher concentrations of E:C act as a direct uncoupler of oxidative phosphorylation and generalized inhibitor of electron transport activity in isolated, intact mitochondria, and induce a dose-dependent inhibition of mitochondrial NADH-ubiquinone oxidoreductase activity in freeze-thawed mitochondrial preparations. The results of this study are important in that they are the first to demonstrate a direct effect of the E:C chelate on bioenergetic function in isolated mammalian mitochondria, and suggest the possibility that the increase in ROS production and cytotoxicity induced by E:C may in part be due to uncoupling of mitochondrial oxidative phosphorylation and/or inhibition of electron transport activity. These results also provide important information about the mechanisms of mitochondrial and cellular toxicity induced by E:C and will ultimately contribute to a better understanding of the therapeutic potential of elesclomol as an anticancer compound.
Highlights
The results suggest that E:C interacts with the electron transport chain (ETC), a major component of the process of oxidative phosphorylation, to generate high levels of reactive oxygen species (ROS) within the organelle and induce cell death
As expected, when CV-1 cells were treated with 70 μM E:C and MitoSOX in the presence of the mitochondria-targeted antioxidant MitoTEMPO, no increase in fluorescence was detected (Figure 2C,D), confirming that the observed increase in fluorescence with E:C treatment was due to an increase in mitochondrial superoxide production
The results of this study indicate that E:C treatment in non-tumorigenic cells induces a rapid and substantial increase in mitochondrial superoxide levels (Figure 2)
Summary
The biologically active form of elesclomol is a deprotonated copper chelate [8] Upon therapeutic administration, this chelate forms when elesclomol acquires Cu2+ in the bloodstream. In vitro studies investigating the mechanism of action of elesclomol have demonstrated that the addition of a pre-formed elesclomol:copper (E:C) complex is necessary to induce cytotoxicity [8]. In one such in vitro study, comparative growth assays using deletion mutants of a yeast model yield evidence that E:C works through a biologically coherent set of processes occurring in the mitochondrion [9]
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