Ascorbic acid (AA) exhibits significant anticancer activity at pharmacologic doses achievable by parenteral administration that have minimal effects on normal cells. Thus, AA has potential uses as a chemotherapeutic agent alone or in combination with other therapeutics that specifically target cancer-cell metabolism. We compared the effects of AA and combinations of AA with the glycolysis inhibitor 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3-PO) on the viability of three non-small cell lung cancer (NSCLC) cell lines to the effects on an immortalized lung epithelial cell line. AA concentrations of 0.5 to 5 mM caused a complete loss of viability in all NSCLC lines compared to a <10% loss of viability in the lung epithelial cell line. Combinations of AA and 3-PO synergistically enhanced cell death in all NSCLC cell lines at concentrations well below the IC50 concentrations for each compound alone. A synergistic interaction was not observed in combination treatments of lung epithelial cells and combination treatments that caused a complete loss of viability in NSCLC cells had modest effects on normal lung cell viability and reactive oxygen species (ROS) levels. Combination treatments induced dramatically higher ROS levels compared to treatment with AA and 3-PO alone in NSCLC cells and combination-induced cell death was inhibited by addition of catalase to the medium. Analyses of DNA fragmentation, poly (ADP-ribose) polymerase cleavage, annexin V-binding, and caspase activity demonstrated that AA-induced cell death is caused via the activation of apoptosis and that the combination treatments caused a synergistic induction of apoptosis. These results demonstrate the effectiveness of AA against NSCLC cells and that combinations of AA with 3-PO synergistically induce apoptosis via a ROS-dependent mechanism. These results support further evaluation of pharmacologic concentrations of AA as an adjuvant treatment for NSCLC and that combination of AA with glycolysis inhibitors may be a promising therapy for the treatment of NSCLC.

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Purpose An exceptional characteristic of many cancer cells is increased glucose uptake and elevated glycolysis with a concomitant reduction in oxidative respiration, also known as the Warburg effect. The increase in basal levels of reactive oxygen species (primarily, H2O2) and their increased dependence on glycolysis for their survival make tumor cells more susceptible than normal cells to treatment with a combination of pro-oxidant agents and chemotherapy. This common feature of tumor cells has been targeted for the development of new cancer therapeutics. Ascorbic acid (AA) has been shown to have cancer curative potential, at clinically achievable doses that have minimal or no toxic effects on normal cells. We have previously shown that some cancer cell lines are significantly more sensitive to treatment with ascorbic acid at concentrations where it may function as a pro-oxidant. In the present case, we investigated the effects of AA alone or in combinations of AA with the glycolysis inhibitor 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3-PO) on the viability of three non-small lung cancer (NSCLC) cell lines vs immortalized lung epithelial cell line. Results Cell viability assays showed that combined treatment of AA and 3-PO caused a synergistic increase in cell death in all the NSCLC cell lines at concentrations well below the IC50 concentrations for each compound alone, while no synergistic cytotoxic effect was observed in immortalized lung epithelial cells. Key markers such as DNA fragmentation, PARP cleavage, annexin-V binding, and caspase activity indicated that AA-induced cell death is caused via the activation of apoptosis. Metabolomic analysis revealed a decrease in NAD/NADH in the combination treatment that correlated well with the synergistic cell death observed in the viability results. The importance of NAD/NADH depletion in the synergistic response was confirmed by rescue experiments showing addition of NAD to the culture protected cells from the combination treatment. The major significance of these studies is that, we show for the first time that AA selectively synergizes with a glycolytic inhibitor in killing NSCLC cells and that pharmacologic AA treatment (either alone or combined with any other chemotherapeutic agent or glycolytic inhibitors used in the treatment of cancer) may be a promising therapy for non-small lung cancer and other challenging to-treat cancers. Citation Format: Saleha B. Vuyyuri, Jacob Rinkinen, Erin Worden, Keith R. Davis. Ascorbic acid combined with a cytostatic inhibitor of glycolysis synergistically induces apoptosis in non-small lung cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 602. doi:10.1158/1538-7445.AM2013-602

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