67 - Dual Oxidase 1 Promotes Crosstalk between Extracellular H2O2 and Mitochondrial Respiration During Pharmacological Ascorbate Induced Toxicity of Pancreatic Cancer Cells

Abstract

Pharmacological ascorbate (P-AscH●–, approximately 300-1000 times nutritional level of 0.2 mM) induces toxicity selectively in cancer vs. normal cells. P-AscH●– produces a high flux of extracellular H2O2 via the formation of ascorbate radical; redox active metals (Fe and Mn) catalyze the oxidation of P-AscH●–. Dual oxidases (DUOX1 and DUOX2) are members of the NADPH oxidase family that are localized in the cell membrane, and are known to produce H2O2. Recent evidence also suggests that H2O2 treatment enhances DUOX expression. This study investigates whether DUOX mediates P-AscH●– induced and extracellular H2O2 dependent toxicity of human pancreatic ductal adenocarcinoma (PDAC) cells by enhancing mitochondrial respiration. P-AscH●– treatments of PDAC cells resulted in a dose (1-20 mM) and duration (24-72 h) dependent increase in DUOX (4-6 fold) and DUOXA1 (DUOX 1 maturation factor; 2-3 fold) expression, which is associated with a significant increase in ROS levels (1.5 fold) and oxygen consumption rate (OCR: 8 ± 2.5 attomoles cell-1 sec-1 in control and 36 ± 5 attomoles cell-1 sec-1 in P-AscH●– treated cells) at 48 h post-treatment. P-AscH●– treatment did not perturb glucose uptake, but significantly decreased expression of lactate dehydrogenase A. This suggests that P-AscH●– treatment may shunt pyruvate more towards mitochondrial respiration resulting in increased OCR. Pretreatment with diphenyleneiodonium inhibited P-AscH●– induced increases in DUOX expression and toxicity. Pancreatic ductal epithelial cells (non-malignant) are resistant to P-AscH●– induced activation of DUOX1 expression, ROS accumulation, increases in OCR, and toxicity. Overall, these results suggest that DUOX1 mediates a feed-forward H2O2-signaling pathway shifting cellular metabolism more towards mitochondrial respiration resulting in P-AscH●– induced toxicity of PDAC cells.