DNA damage and disruption of cellular bioenergetics contribute to the anti-cancer effects of pharmacological ascorbate

• Main Author: Buranasudja, Visarut
• Other Authors: Buettner, Garry R.
• Format: Others
• Language: English
• Published: University of Iowa 2018
• Subjects: bioenergetics; DNA damage; hydrogen peroxide; pancreatic cancer; pharmacological ascorbate; quantitative redox biology
• Online Access: https://ir.uiowa.edu/etd/6551; https://ir.uiowa.edu/cgi/viewcontent.cgi?article=8050&context=etd

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, which leads to oxidative distress. Here, we use cell culture models of pancreatic cancer, MIA PaCa-2, PANC-1, and 339 cells, 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 (potential 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 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 the potential to improve outcomes of cancer treatment. 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-. OCR and ECAR/PPR together indicate the rate of production of intracellular ATP; therefore, the rate of production is unchanged after challenge with P-AscH-. Thus, the severe decrease in ATP is due solely to increased demand. Genetic deletion and pharmacological inhibition of PARP-1 preserved both NAD+ and ATP; however, the toxicity of P-AscH- remained. These data indicate that loss of NAD+ and ATP are secondary factors in the toxicity of P-AscH-, and damage to DNA is the primary factor. These preclinical findings can guide the best use of P-AscH- as an adjuvant in cancer therapy.