DNA Damage and Energy Crisis are Central in the Mechanism for the Cytotoxicity of Pharmacological Ascorbate in Cancer Treatment

Abstract

The clinical application of ascorbate in cancer treatment has a long and controversial history. The clinical potential of pharmacological ascorbate (P-AscH‒; IV delivery achieving mM concentrations in blood) as an adjuvant in cancer therapy is being re-evaluated. At mM concentrations, P-AscH‒ is thought to exhibit anti-cancer activity via generation of a flux of H2O2 in tumors. However, a complete understanding of mechanism for P-AscH‒ has not yet been established. Here, we use a cell culture model of pancreatic cancer, MIA PaCa-2 and PANC-1, to examine the effects of P-AscH‒ on DNA damage, and downstream consequences, including changes in bioenergetics. We have found that the high flux of H2O2 produced by P-AscH‒ induces both nuclear and mitochondrial DNA damage. In response to this DNA damage, we observed that poly (ADP-ribose) polymerase-1 (PARP-1) is hyperactivated, as determined by increased formation of poly (ADP-ribose) polymer. Using our unique absolute quantitation, we found that the P-AscH‒-mediated the overactivation of PARP-1, which results in consumption of NAD+, and subsequently depletion of ATP (energy crisis) leading to mitotic cell death. Time-course studies with MIA PaCa-2 cells showed that the level of NAD+ and ATP were reduced by 80% immediately after a 1-h exposure to P-AscH‒ (4 mM; 14 pmol cell-1); both species returned to near basal levels within 24 h. In parallel with these metabolic and energetic restorations, the lesions in nuclear DNA were removed within 3 h; however, even after 24 h, lesions in mitochondrial DNA were only partially repaired. We have also found that the ATR-Chk1 pathway has a major role in the maintenance of genomic integrity following treatment with P-AscH‒. Hence, combinations of P-AscH‒ and Chk1 inhibitors could have potential in the treatment of cancer. Hyperactivation of PARP-1 and DNA repair are ATP-consuming processes. Using a Seahorse XF96 Analyzer, we observed no changes in OCR or ECAR/PPR following treatment with P-AscH‒, indicating that the severe decrease in ATP is due solely to increased demand, not changes in the rate of production. These results clearly indicate that the combination of DNA damage coupled with compromised ATP availability could be a key aspect of the anti-cancer activity of P-AscH‒. These preclinical findings can guide the best use of P-AscH‒ as an adjuvant in cancer therapy.