Anti-Cancer Effects of Ascorbic Acid and Hyperbaric Oxygen Therapy in vitro by Janine M. DeBlasi, Nathan P. Ward, Angela M. Poff, Andrew P. Koutnik, Christopher Q. Rogers, and Dominic P. D’Agostino

Cancer is the second leading cause of death in the U.S., projected to take 595,690 lives in 2016 and cost the nation over 125 billion dollars. To effectively reduce these detrimental losses, non-toxic, low-cost therapies should be further examined to supplement the standard of care. An anti-carcinogenic, non-toxic therapy currently under investigation is high dose ascorbic acid (AA). AA can function as a pro-oxidant at pharmacological levels, delivering H2O2 to tumorous tissue upon oxidation and initiating cell death. At pharmacological concentrations (achieved i.v.), AA has shown significant anticancer effects in vitro, in vivo, and in small-scale human reports at concentrations nontoxic to normal cells, thus having great potential as an adjuvant to the standard of care. Hyperbaric oxygen therapy (HBOT) is another non-toxic, pro-oxidant metabolic therapy that delivers 100% oxygen at elevated barometric pressure, elevating tissue pO2 and oxygenating hypoxic tumor cells, which, coupled with high levels of reactive oxygen and nitrogen species present in cancer cells, can further augment oxidative stress (OxS) and lead to cell death. This study’s aims are as follows (1) to examine the anticancer effect of AA in vitro, (2) to evaluate the mechanism of AA-induced OxS, (3) to investigate the potential synergy between AA and HBOT. We anticipate that this approach will yield significant insight into and further investigate the hypothesis that AA and HBOT can supplement the current standard of care.

To characterize the anticancer effects of AA in vitro, we measured cell viability and proliferation following treatment with varying concentrations of AA in mouse brain tumor-derived VM-M3 cells. It was found that AA mediates cell death in a concentration-dependent manner, with all tested concentrations ≥ 0.3mM AA significantly inducing cell death compared to control, specifically 0.3mM (p<0.05) and 0.5– 5mM (p=0.0001). Preliminary results also indicate that concentrations ≥ 0.05mM AA inhibit cell proliferation compared to control and 0.01mM AA.

To investigate the role of OxS in AA-induced cytotoxicity, we measured VM-M3 cell viability in the presence of AA and antioxidant N-Acetylcysteine (NAC) and found that treatment with 5mM NAC attenuates the cytotoxic effect of AA (p<0.0001). To evaluate the role of H2O2 in AA-induced cytotoxicity, we will measure VM-M3 cell viability in the presence of AA and the antioxidant enzyme catalase. Consistent with the literature, we anticipate that treatment with 300 units/mL of catalase will attenuate the cytotoxic effect of AA in vitro.

To determine if HBOT can enhance the therapeutic effect of AA, we measured measure VM-M3 cell viability following treatment with HBOT and AA. We found that HBOT significantly enhanced the cytotoxic effect of 0.3mM AA (p<0.001). To complete this aim, we will measure VM-M3 cell proliferation following treatment with HBOT, HBOT pre-treatment, and AA.

This data indicates that AA exhibits anti-cancer effect in vitro through an OxS mechanism and that HBOT can enhance this therapeutic effect. These non-toxic, pro-oxidant metabolic therapies should be further investigated as adjuvants to the current standard of care.