F. K. Houwen1, B. R. O’Leary1, C. L. Johnson1, B. G. Allen1, G. Buettner1, J. J. Cullen1, D. R. Spitz1, J. J. Mezhir1 1University Of Iowa,Surgery,Iowa City, IA, USA
Introduction: Gastric adenocarcinoma (GAC) is a lethal disease due to resistance to current available therapies and its propensity for metastasis. Pharmacological Ascorbate has been shown to increase the cytotoxicity of chemotherapy and radiation in solid tumors secondary to generation of toxic hydrogen peroxide (H2O2). We therefore hypothesize that Ascorbate, when combined with current multimodality therapies, will increase cytotoxicity in tumor growth and metastasis cell models of GAC.
Methods: Human GAC cell lines MKN-45 and AGS, and normal human stomach/intestinal cell line, Hs738st/Int, were evaluated for clonogenic survival after treatment with clinically relevant doses of Ascorbate (25pmol/cell) and pertinent multimodality therapies including 5-flurouracil (5FU, 5uM), and Ionizing Radiation (IR, 1.8Gy). Cytotoxicity of AGS cells was evaluated with Ascorbate both with and without cisplatin (0.5uM) and irinotecan (2.5uM). GAC cell lines were treated with bovine catalase and deferoxamine (DFO) plus diethylene triamine pentaacetic acid (DTPA) to confirm the mechanisms of H2O2-mediated cell death following Ascorbate exposure in the model systems. Tumor cell invasive capacity was measured using matrigel migration and invasion assays.
Results: Ascorbate reduced GAC clonogenic survival by 50% (p <0.0001) compared to control. Ascorbate when combined with 5-FU or IR resulted in significant increases in cell death compared to 5-FU or IR alone. Triple therapy with Ascorbate, 5FU, and IR showed enhancement in cytotoxicity that was significantly increased over the standard multimodality therapy regimen (Figure, p <0.01). Ascorbate increased cell death when combined with irinotecan and cisplatin compared to either chemotherapy drug alone. Ascorbate combined with catalase or DFO+DTPA yielded a full rescue of cytotoxicity from the toxic effects of Ascorbate treatment (p<0.0001) thereby confirming the dependence on H2O2 levels and catalytic metal ions for cellular death to occur. Ascorbate significantly reduced tumor cell invasion by 57% (p < 0.003) and tumor cell migration by 61% (p < 0.002) compared to control. Normal gastric cell clonogenic survival was not altered by treatment with Ascorbate, suggesting treatment selectivity for malignant cells and safety in normal cells.
Conclusion: Pharmacologic Ascorbate is highly toxic to GAC cells at pharmacologic concentrations while having no effect on normal cells; Ascorbate also reduces tumor cell invasive capacity. Our data confirm that Ascorbate exerts its cytotoxicity via generation of H2O2 and is dependent upon the presence of catalytic metal ions. These highly translational data suggest that Pharmacological Ascorbate is a potential novel therapy for the treatment of GAC.
