Ascorbate availability has an impact on the hypoxic response in breast cancer cells Author: Citra Praditi(1), Gabi Dachs(2), Stephanie Bozonet(1), Margreet Vissers(1) 1. Centre for Free Radical Research, Dept of Pathology and Biomedical Science, University of Otago Christchurch 2. MacKenzie Cancer Research Group, Dept of Pathology and Biomedical Science, University of Otago Christchurch Background: Ascorbate (vitamin C) is an essential cofactor for the 2-oxoglutarate dependent dioxygenase (2-OGDD) class of enzymes. The HIF (hypoxia-inducible factor)-hydroxylases belong to the 2-OGDD family. Increased ascorbate availability has been shown to up-regulate the activity of the HIF hydroxylases and down-regulate the hypoxic response, which could be beneficial to patients with cancers that use the hypoxic response pathway to support cancer progression. We have investigated this relationship in breast cancer cells and aim to determine whether ascorbate availability could affect the hypoxic response in this cancer. Methods: Human breast cancer cell lines MDA-MB231 and MCF-7 were exposed to ascorbate through media supplementation and uptake monitored. Following exposure to hypoxic conditions induced by the hypoxia-mimetic agent CoCl2 or decreased oxygen tension, cell lysates were collected and analysed by western blotting to measure HIF-1α protein stabilisation and expression of the downstream marker BNIP3 protein. Results: Both breast cancer cell lines expressed the sodium-dependent vitamin C transporter SVCT2 and readily accumulate ascorbate from the medium. Ascorbate pre-treatment was able to inhibit HIF-1a stabilisation and BNIP3 expression under CoCl2 treatment in MDA-MB231 and MCF-7. However, the response to ascorbate varied under hypoxia. In MDA-MB231 cell line at 1% O2 and MCF-7 cells at 0.1% O2, ascorbate did not inhibit HIF-1α protein stabilisation, but BNIP3 protein expression was decreased in the ascorbate-treated cells. Discussion: Our results show that increased ascorbate availability is able to decrease HIF-1a protein stabilisation and expression of its target protein BNIP3 in human breast cancer cell lines. Modulation of the hypoxic response by ascorbate likely reflects capacity for ascorbate to maintain the activity of the HIF-hydroxylases (prolyl hydroxylases 1-3 (PHDs) and factor inhibiting HIF (FIH). The efficiency of this regulation may be moderated under hypoxic conditions. Conclusion: BNIP3, and other proteins expressed as a result of HIF-1 activation, contribute to cancer cell survival under stress. The activation of HIF-1 is marked in breast cancer and is closely associated with patient survival. Our results indicate that uptake of ascorbate by breast cancer cells may be a factor influencing breast cancer outcomes.