148 - Pharmacological ascorbate enhances expression of dual oxidase which mediates sustained toxicity via alterations in cellular metabolism

Abstract

Pancreatic Ductal Adenocarcinoma is a leading cause of cancer related death in the United States that is in critical need of adjuvant therapies to enhance patient survival. One such adjuvant is pharmacological ascorbate treatment (P-AscH-, high-dose, intravenous vitamin C). P-AscH- treatment results in a short-term increased flux of H2O2 that is preferentially cytotoxic to cancer cells vs. normal cells. Treatment with P-AscH- results in sustained increases in oxygen consumption (25-30 attomoles cell-1 sec-1) and reactive oxygen species production (1.5 fold as DCFH-DA oxidation), with no change in glucose metabolism in tumor cells compared to non-tumorigenic pancreatic ductal epithelial cells. These results lead to the hypothesis that there is a sustained effect (> 24 h) of P-AscH- that contributes to cytotoxicity. Seahorse XF96 mito stress test (48 h) demonstrated that P-AscH- results in a significant and sustained increase in non-mitochondrial OCR (~15 attomoles cell-1 sec-1). A potential source of this sustained non-mitochondrial OCR is the NADPH oxidase family of enzymes Dual Oxidase 1 and 2 (DUOX) which are up-regulated after P-AscH-. P-AscH- treatment of PDAC cell lines resulted in a dose (1-10 mM; 4-6 fold) and duration (24-72 h; 5-20 fold) dependent increase in DUOX. Catalase pretreatment reverses the P-AscH--induced increases in DUOX, while DUOX inhibition partially rescues P-AscH- toxicity. Additionally, nutritional ascorbate (100 mm; Ascorbate-2-Phosphate) is unable to mediate an increase in DUOX expression. P-AscH- also induces DUOX protein expression in pancreatic tumor xenografts. These results suggest that P-AscH--induced toxicity may be enhanced by a sustained metabolic shift in tumor cells that is related to the H2O2 produced from P-AscH-. We suggest that P-AscH- results in a feed-forward mechanism of H2O2 generation and induction of metabolic stress via enhanced DUOX expression and OCR.