Redox-directed pharmacophores have shown potential for the apoptotic elimination of
cancer cells through chemotherapeutic induction of oxidative stress.
Phenazine methosulfate (PMS), a N-alkylphenazinium
cation-based redox cycler, is used widely as an electron transfer reactant coupling
NAD(P)H generation to the reduction of
tetrazolium salts in biochemical cell viability assays. Here, we have explored feasibility of repurposing the redox cycler PMS as a
superoxide generating chemotherapeutic for the
pro-oxidant induction of
cancer cell apoptosis. In a panel of malignant human
melanoma cells (A375, G361, LOX), low micromolar concentrations of PMS (1-10 μM, 24 h) displayed pronounced apoptogenicity as detected by
annexin V-ITC/
propidium iodide flow cytometry, and PMS-induced cell death was suppressed by
antioxidant (NAC) or pan-
caspase inhibitor (
zVAD-fmk) cotreatment. Gene expression array analysis in A375
melanoma cells (PMS, 10 µM; 6 h) revealed transcriptional upregulation of heat shock (HSPA6, HSPA1A), oxidative (HMOX1) and genotoxic (EGR1, GADD45A) stress responses, confirmed by immunoblot detection demonstrating upregulation of redox regulators (NRF2, HO-1, HSP70) and modulation of pro- (BAX, PUMA) and anti-apoptotic factors (Bcl-2, Mcl-1). PMS-induced oxidative stress and
glutathione depletion preceded induction of apoptotic cell death. Furthermore, the mitochondrial origin of PMS-induced
superoxide production was substantiated by
MitoSOX-Red live cell fluorescence imaging, and PMS-induced mitochondriotoxicity (as evidenced by diminished transmembrane potential and oxygen consumption rate) was observable at early time points. After demonstrating
NADPH-driven (SOD-suppressible)
superoxide radical anion generation by PMS employing a chemical NBT reduction assay, PMS-induction of oxidative genotoxic stress was substantiated by quantitative Comet analysis that confirmed the introduction of formamido-
pyrimidine DNA glycosylase (Fpg)-sensitive oxidative DNA lesions in A375
melanoma cells. Taken together, these data suggest feasibility of repurposing the biochemical reactant PMS as an experimental
pro-oxidant targeting mitochondrial integrity and redox homeostasis for the apoptotic elimination of
malignant melanoma cells.