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.
| Identifer | oai:union.ndltd.org:uiowa.edu/oai:ir.uiowa.edu:etd-8050 |
| Date | 01 December 2018 |
| Creators | Buranasudja, Visarut |
| Contributors | Buettner, Garry R. |
| Publisher | University of Iowa |
| Source Sets | University of Iowa |
| Language | English |
| Detected Language | English |
| Type | dissertation |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | Copyright © 2018 Visarut Buranasudja |
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