Complex I inhibition augments dichloroacetate cytotoxicity through enhancing oxidative stress in VM-M3 glioblastoma cells

<div><p>The robust glycolytic metabolism of glioblastoma multiforme (GBM) has proven them susceptible to increases in oxidative metabolism induced by the pyruvate mimetic dichloroacetate (DCA). Recent reports demonstrate that the anti-diabetic drug metformin enhances the damaging oxidative stress associated with DCA treatment in cancer cells. We sought to elucidate the role of metformin’s reported activity as a mitochondrial complex I inhibitor in the enhancement of DCA cytotoxicity in VM-M3 GBM cells. Metformin potentiated DCA-induced superoxide production, which was required for enhanced cytotoxicity towards VM-M3 cells observed with the combination. Similarly, rotenone enhanced oxidative stress resultant from DCA treatment and this too was required for the noted augmentation of cytotoxicity. Adenosine monophosphate kinase (AMPK) activation was not observed with the concentration of metformin required to enhance DCA activity. Moreover, addition of an activator of AMPK did not enhance DCA cytotoxicity, whereas an inhibitor of AMPK heightened the cytotoxicity of the combination. Our data indicate that metformin enhancement of DCA cytotoxicity is dependent on complex I inhibition. Particularly, that complex I inhibition cooperates with DCA-induction of glucose oxidation to enhance cytotoxic oxidative stress in VM-M3 GBM cells.</p></div

DCA promotes superoxide production and dissipation of ΔΨ_m in VM-M3 cells.

<p>(a) Western blot analysis of p-PDH-E1α (Ser293) and PDH-E1α in VM-M3 lysates following 4-hour treatment with DCA. Densitometric ratio of p-PDH to PDH was determined for each treatment relative to PBS control. (b) Quantification of lactate concentration in culture medium following 24-hour incubation with indicated treatment. (c) Quantification of average MitoSox Red fluorescence intensity as an indication of VM-M3 superoxide production following 1-hour incubation with DCA. (d) Quantification of average tetramethylrhodamine (TMRE) fluorescence intensity as an indication of mitochondrial membrane potential following 4-hour DCA treatment. (b) Error bars represent standard error of the mean (SEM) of three experimental replicates. (c-d) Error bars represent SEM of a single experiment replicated in triplicate; * p<0.05, and ***p<0.001.</p

Metformin enhances DCA cytotoxicity.

<p>(a) Western blot analysis of p-PDH-E1α (Ser293) and PDH-E1α in VM-M3 cell lysates following 4-hour treatment with 5mM DCA and 100μM metformin. Densitometric ratio of p-PDH to PDH was determined for each treatment relative to PBS control. (b) Quantification of superoxide production with MitoSox Red following 1-hour treatment with DCA and metformin. (c) Quantification of BOPIDY® 581/591 oxidation as an indicator of lipid peroxidation in VM-M3 cells following 4-hour treatment with DCA and metformin ± NAC. Determination of VM-M3 cell viability following 24-hour treatment with (d) DCA and metformin ± NAC or (e) combinatorial treatment with DCA and metformin in increasing concentrations. (b, c) Error bars represent SEM of a single experiment replicated in triplicate (d, e) Error bars represent SEM of three experimental replicates; *p<0.05, **p<0.01, and ***p<0.001.</p

Complex I inhibition, but not AMPK activation enhances DCA cytotoxicity.

<p>(a) Average VM-M3 superoxide production following 1-hour treatment with DCA and rotenone. (b) Ratiometric detection of BOPIDY® 581/591 oxidation as an indicator of lipid peroxidation in VM-M3 cells following 4-hour treatment with DCA and rotenone ± NAC. (c-e) Analysis of rotenone, AICAR, and metformin ± compound C modulation of DCA cytotoxicity towards VM-M3 cells. (f) In-cell ELISA analysis of p-AMPKα (Thr172), and AMPKα in VM-M3 cells following 4-hour treatment with modulators of AMPK activation. (a, b, f) Error bars represent SEM of a single experiment replicated in triplicate (c-e) Error bars represent SEM of three experimental replicates; **p<0.01 and ***p<0.001.</p

DCA cytotoxicity is dependent on oxidative stress.

<p>(a) Ratiometric detection of BOPIDY® 581/591 oxidation as an indicator of lipid peroxidation in VM-M3 cells following 4-hour treatment with DCA ± NAC. (b) Quantification of average TMRE fluorescence intensity following 4-hour DCA treatment ± N-acetylcysteine (NAC). (c) Analysis of VM-M3 viability following 24-hour treatment with DCA. Bars represent fraction of cells stained positively for ethidium homodimer-I (Ethd-1). (d) Evaluation of VM-M3 viability following 24-hour DCA treatment in the presence of modulators of glutathione availability. (a-b) Error bars represent SEM of a single experiment replicated in triplicate (c-d) Error bars represent SEM of three experimental replicates; *p<0.05, **p<0.01, and ***p<0.001.</p