Pharmacological ascorbate induces sustained mitochondrial dysfunction

Pharmacological ascorbate (P-AscH^-; high dose given intravenously) generates H₂O₂ that is selectively cytotoxic to cancer compared to normal cells. The RAS-RAF-ERK1/2 is a major signaling pathway in cancers carrying RAS mutations and is known to be activated by H₂O₂. Activated ERK1/2 also phosphorylates the GTPase dynamin-related protein (Drp1), which then stimulates mitochondrial fission. Although early generation of H₂O₂ leads to cytotoxicity of cancer cells, we hypothesized that sustained increases in H₂O₂ activate ERK-Drp1 signaling, leading to an adaptive response; inhibition of this pathway would enhance the toxicity of P-AscH^-. Increases in phosphorylated ERK and Drp1 induced by P-AscH^- were reversed with genetic and pharmacological inhibitors of ERK and Drp1, as well as in cells lacking functional mitochondria. P-AscH^- increased Drp1 colocalization to mitochondria, decreased mitochondrial volume, increased disconnected components, and decreased mitochondrial length, suggesting an increase in mitochondrial fission 48 h after treatment with P-AscH^-. P-AscH^- decreased clonogenic survival; this was enhanced by genetic and pharmacological inhibition of both ERK and Drp1. In murine tumor xenografts, the combination of P-AscH^- and pharmacological inhibition of Drp1 increased overall survival. These results suggest that P-AscH^- induces sustained changes in mitochondria, through activation of the ERK/Drp1 signaling pathway, an adaptive response. Inhibition of this pathway enhanced the toxicity P-AscH^- to cancer cells.