Pharmacologic ascorbate induces DNA damage, alters the bioenergetic profile, but does not influence cellular respiration or glycolysis in human pancreatic cancer cells

We have investigated the effects of high dose ascorbate, (pharmacological ascorbate, P-AscH-) as an adjuvant to common cancer treatment modalities. We have proposed that P-AscH- acts as a pro-drug for delivery of extracellular H2O2 to tumors. This H2O2 enhances or exacerbates DNA damage. Many cancer treatment modalities, whether biophysical (ionizing radiation) or chemical (drug), are believed to involve direct damage to DNA or inhibit DNA repair processes. We have observed that DNA damage occurs in both nuclear as well as mitochondrial DNA in human pancreatic cancer cell lines upon exposure to P-AscH- with concomitant decreases in NAD+, NADH, and ATP. After removal of ascorbate, all the above resolve with time suggesting a link to cellular bioenergetics. To address steady-state changes in these redox-related species, we used Seahorse XF analysis. In media as ascorbate oxidizes it consumes oxygen in a concentration-dependent manner; this could lead to misinterpretation of data on cellular bioenergetics. To avoid this we incubated cells (0.5 to 1 h) with lethal doses of P-AscH- and then rapidly removed it before initiating bioenergetic experiments. We detected no significant changes in cellular bioenergetics following lethal doses (clonogenic) of P-AscH- at 0.5 h and up to 24 h after exposure. These results indicate that the decreased levels of NAD+, NADH, and ATP observed following short-term exposure to P-AscH- is not due to decreased production but rather due solely to increased demand. In additional experiments we observed no changes in metabolism upon exposure to dehydroascorbate (DHA) or ascorbate 2-phosphate, forms that load cells with ascorbate, but produce no extracellular H2O2. These data indicate that P-AscH- generates extracellular H2O2 leading to DNA damage resulting in depletion (demand) of cellular NAD+, NADH, and ATP. There appears to be no bioenergetic crisis due to a loss (production) in bioenergetic capacity.