Abstract In 2014, 76,100 new cases of melanoma were diagnosed in the United States. Treatment consists of surgery and chemotherapy, but efficacy is low. Indeed, five-year survival rates for patients with metastatic melanoma remain below 25%, indicating the need for new therapeutic strategies. In parallel, some studies indicate that vitamin C (ascorbic acid, ascorbate) only in pharmacologic doses may have a beneficial anti-tumor effect. Although ascorbate is an essential nutrient for humans, who are unable to synthesize it, pharmacologic ascorbate concentrations are achieved by parenteral administration. Although pharmacologic ascorbate in cancer treatment in humans is unproven, many studies report a selective cytotoxic effect in cancer cells in vitro. Cytotoxicity is thought to be mediated by hydrogen peroxide formation. To investigate the effect of high dose ascorbate in melanoma, we first used a murine melanoma cell line B16F10. We confirmed that ascorbate induced cell death in a dose-dependent manner (up to 40mM) in this cell line which is mostly reversed by adding catalase into the media, reinforcing the hydrogen peroxide dependent effect. We next investigated the role of ascorbic acid in vivo using a murine B16 melanoma model. Tumor-bearing C57BL/6 mice were treated with pharmacological ascorbic acid (4g/kg mice/twice a day) or hypertonic NaCl solution. We observed that the tumor size was decreased by 63% in the vitamin C treated group compared to the control group. We also used this model to analyze collagen expression in B16 tumors from ascorbate or saline-treated mice by qPCR and ELISA. Vitamin C treated tumors showed increased expression of various types of collagen. Indeed, we observed a five-fold increase in Col1A1, Col1A2, Col1A3, Col8A1 and Col28A1 mRNA expression and we also observed an increase in the Prolyl 4-hydroxylase, alpha peptide III (P4HA3) expression, an enzyme involved in collagen formation. No side effects were evident from administration of high dose ascorbate, which is fully consistent with previously published clinical data. We are currently investigating the role of hydrogen peroxide in the murine model by generating tumors with B16F10 cells that overexpress catalase. Taken together, these results showed that pharmacologic ascorbate is cytotoxic to B16F10 melanoma cells, with cytotoxicity in vitro mediated by hydrogen peroxide. Ascorbate is effective in reducing melanoma tumor size without apparent host toxicity. These data support the idea that vitamin C should be studied in humans both for combination chemotherapy and also as a stand-alone treatment. Citation Format: Pierre-Christian Violet, Nermi L. Parrow, Serrano L. Oscar, Jacqueline Yang, Mark A. Levine. Pharmaceutical effect of vitamin C (ascorbate) on B16 melanoma in vitro and in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5134. doi:10.1158/1538-7445.AM2015-5134