Abstract
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.
Highlights
Cancers 2019, 11, research x 2 ofreactiveCumulative suggests a causative involvement of altered redox homeostasis and oxygen species (ROS)-dependent signaling in the control of cancer cell survival, proliferation
Even at lower concentrations of Phenazine methosulfate (PMS) (1 μM; a concentration not associated with the induction of A375 cell death; Figure 2A), a significant reduction in GSH levels by approximately 20% occurred within 6 h exposure time (Figure 5B)
Preliminary flow cytometric viability analysis assessing the differential cytotoxicity of PMS, comparing sensitivity of malignant and non-transformed cultured human skin cells, revealed that Hs27 human diploid dermal fibroblasts maintained full viability at PMS concentrations (5–10 μM; 24 continuous exposure; that are effective in eliminating malignant melanoma cells contained in our test panel (A375, A375R, G361, LOX; Figures 2 and 6), an observation suggesting a potential therapeutic window associated with PMS-apoptogenicity, awaiting further elucidation in future follow-up studies
Summary
Cumulative suggests a causative involvement of altered redox homeostasis and oxygen species (ROS)-dependent signaling in the control of cancer cell survival, proliferation. It has been suggested that constitutively elevated levels of oxidative research suggests aand causative involvement of altered redox homeostasis and stress andCumulative dependence on mitogenic anti-apoptotic reactive oxygen species (ROS) signaling reactive oxygen species (ROS)-dependent signaling thecan control of cancer cell survival,by proliferation represent a specific vulnerability of malignant cells in that be selectively targeted redox-directed and invasiveness[1,2,3]. We present novel experimental evidence supporting activity of PMS as a prooxidant and mitochondriotoxic redox reactant, suggesting feasibility of repurposing this superoxide generating mitochondriotoxic redox reactant, suggesting feasibility of repurposing this superoxide generating pharmacophore for therapeutic apoptogenic ofmalignant malignant melanoma cells.
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