The Anticancer Agent Elesclomol Has Direct Effects on Mitochondrial Bioenergetic Function in Isolated Mammalian Mitochondria

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 M 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.

Keywords: anti-cancer; bioenergetics; elesclomol; mitochondria.

Figure 1

Chemical structure of elesclomol (MedChemExpress, https://www.medchemexpress.com/).

Figure 2

Monitoring mitochondrial superoxide production in whole cells. (A) Confocal images of control and elesclomol:copper (E:C)-treated (70 μM) CV-1 cells at various time points after staining with the mitochondria-specific superoxide indicator MitoSOX and the nuclear stain NucBlue Live. (B) Quantification of MitoSOX fluorescence intensity in CV-1 cells at the different time points after E:C treatment. (C) Confocal images of MitoSOX fluorescence intensity in CV-1 cells at the 20-min time point after E:C treatment in the presence and absence of the mitochondrial superoxide scavenger MitoTEMPO. (D) Quantification of MitoSOX fluorescence intensity in CV-1 cells at the 20-min time point after E:C treatment in the presence and absence of the mitochondrial superoxide scavenger MitoTEMPO. Images in the NucBlue Live channel were adjusted to improve visual clarity. This adjustment does not affect the quantification. A minimum of three cells/field and three fields/slide were imaged for each plate at 20× magnification using a Zeiss confocal microscope. The images were analyzed using Image J software, and the change in mean fluorescence among groups was plotted. Normality of distribution of the data was determined using the D-Agostino Omnibus test. A one-way ANOVA analysis was performed followed by a Dunn’s post hoc test using GraphPad Prism software (San Diego, CA, USA) to identify statistical difference among the samples. N = 3 for each condition; N represents one glass-bottomed 35 mm petri dish. * p < 0.05 vs control or T0.

Figure 3

Monitoring the effect of E:Con mitochondrial membrane potential in whole cells at various time points. (A) Confocal images of control and E:C-treated (70 μM) CV-1 were observed at various time points after staining with the membrane potential dependent fluorescent dye tetramethylrhodamine, ethyl ester (TMRE) and the nuclear stain NucBlue Live. The arrows in the merge channel point to representative cells that appear to be undergoing morphological changes, or to cell debris. (B) Quantification of TMRE fluorescence intensity after treatment with E:C at different time points. A minimum of three cells/field and three fields/slide were imaged for each plate at 20× magnification using a Zeiss confocal microscope. The images were analyzed using Image J software, and the change in mean fluorescence among groups was plotted. Normality of distribution of the data was determined using the D-Agostino Omnibus test. A one-way ANOVA analysis was performed followed by a Dunn’s post hoc test using GraphPad Prism software to identify statistical difference among the samples. N = 3 for each condition; N represents one glass-bottomed 35 mm petri dish. * p < 0.05 vs control or T0.

Figure 4

The relationship between E:C-induced superoxide production, membrane potential effects, and cell viability. (A) Confocal images and fluorescence intensity measurements in CV-1 Cells treated for 20 min with various concentrations of E:C after staining with the mitochondria-specific superoxide indicator MitoSOX, the cell viability stain NucGreen Dead, and the nuclear stain NucBlue Live. (B) Confocal images and fluorescence intensity measurements in CV-1 Cells treated for 20 min with various concentrations of E:C after staining with the mitochondrial membrane potential probe TMRE, the cell viability stain NucGreen Dead, and the nuclear stain NucBlue Live. The proton ionophore and mitochondrial uncoupler, FCCP, was included as a positive control. A minimum of 3 cells/field and 3 fields/slide were imaged for each plate at 20× magnification using a Zeiss confocal microscope. The images were analyzed using Image J software and the change in mean fluorescence among groups was plotted. Normality of distribution of the data was determined using the D-Agostino Omnibus test. A one-way ANOVA analysis was performed followed by a Dunn’s post hoc test using GraphPad Prism software to identify statistical difference among the samples. N = 3 for each condition; N represents one glass bottomed 35 mm petri dish. * p < 0.05 vs control or T0.

Figure 5

The effect of elesclomol and CuCl₂, as single agents, on mitochondrial membrane potential in whole cells. Fluorescence intensity measurements of (A) elesclomol and (B) CuCl₂ treated CV-cells observed at various time points after staining with the mitochondrial membrane potential probe TMRE. The proton ionophore and mitochondrial uncoupler, FCCP, was included as a positive control. A minimum of three cells/field and three fields/slide were imaged for each plate at 20× magnification using a Zeiss confocal microscope. The images were analyzed using Image J software, and the change in mean fluorescence among groups was plotted. Normality of distribution of the data was determined using the D-Agostino Omnibus test. A one-way ANOVA analysis was performed followed by a Dunn’s post hoc test using GraphPad Prism software to identify statistical difference among the samples. N = 3 for each condition; N represents one glass-bottomed 35 mm petri dish. * p < 0.05 vs control or T0.

Figure 6

Polarographic measurement of oxygen consumption. Measurements of respiratory rates in intact, isolated rat liver mitochondria were made in the absence or presence of: varying concentrations of the E:C chelate with either (A) glutamate/malate or (B) succinate as the respiratory substrate; varying concentrations of CuCl₂ as a single agent with either (C) glutamate/malate or (D) succinate as the respiratory substrate; or varying concentrations of elesclomol as a single agent with either (E) glutamate/malate or (F) succinate as the respiratory substrate. Data are expressed as mean values ± SE for at least three independent experiments. A one-way ANOVA was performed followed by a Tukey’s multiple comparison test using GraphPad Prism software; * p < 0.05 vs control.

Figure 7

Effect of varying concentrations of E:C on mitochondrial respiratory enzyme complex activity. (A) NADH-ubiquinone oxidoreductase activity, (B) succinate cytochrome c reductase activity, and (C) cytochrome c oxidase activity were measured in freeze-thawed preparations of bovine mitochondria in the absence or presence of varying concentration of E:C. Data presented are the mean of three separate experiments +/− SE.