Abstract 229: Restoring physiological levels of ascorbate alleviates HIF-1 activation and reduces tumour growth in Gulo-/- mice

Abstract

Abstract Background: Hypoxia-inducible factor-1 (HIF-1) upregulates the expression of hundreds of genes involved in adaption of tumours to the hypoxic microenvironment. HIF-1 levels and transcriptional activity are regulated by hydroxylase enzymes, which require ascorbate as a cofactor. Adequate supplementation of cells with ascorbate has been shown to reduce HIF-1 activation in vitro, and we hypothesise that a similar activity could affect tumour growth in vivo. In this study we investigated the effect of dietary ascorbate on tumour growth in Gulo-/- mice, a model of the human ascorbate deficiency condition. Methods: C57/Bl6 Gulo-/- mice were supplemented with 3300 mg/L, 330 mg/L or 33mg/L of ascorbate in their drinking water for one month before subcutaneous, syngeneic tumour implantation with either B16-F10 melanoma, or Lewis lung carcinoma (LL/2), and then for duration of the experiment. Levels of HIF-1α, and its targets, carbonic anhydrase-IX (CA-IX), and glucose transporter-1 (GLUT-1) were analysed by western blotting, and vascular endothelial growth factor (VEGF) levels by ELISA. Ascorbate concentrations of tissue, plasma and tumours were measured using high pressure liquid chromatography with electrochemical detection (HPLC-EC). Results: Across all organs and plasma samples, ascorbate content was in the physiological range (up to levels seen in wild-type mice) and was correlated with dietary ascorbate intake. Tumour ascorbate also reflected ascorbate intake and optimal ascorbate levels (3300 mg/L) significantly increased the time for LL/2 tumours to reach 200 mm3 (lag growth) (13.3±2.6 days), compared to these same tumours in mice supplemented with either 330 mg/L (9.1±2.6 days) or 33 mg/L (9.8±1.39 days) of ascorbate (p<0.001). Similarly, tumours took significantly longer to grow from 200-800 mm3 (log phase) when mice were supplemented with 3300 mg/L compared to lower supplementation (p<0.05). B16-F10 tumours showed no difference in the lag growth period between ascorbate treatments (p=0.49). However, supplementing mice with 3300 mg/mL of ascorbate lead to a significant reduction in the tumour growth rate during log phase (3.9±1.1 days), compared to mice supplemented with either 330 mg/L (2.0±0.90 days) or 33mg/L (2.1±0.71 days) (p<0.001). Levels of HIF-1α protein in tumours decreased as dietary ascorbate supplementation increased for both LL/2 (p<0.001) and B16F10 (p<0.001). Tumour ascorbate was significantly correlated with low levels of CA-IX, GLUT-1 and VEGF levels in both LL/2 and B16 tumours (all p<0.05). Conclusions: The data from the current study support the hypothesis that restoration of optimal intracellular ascorbate levels is associated with reduced HIF-1 levels and slower tumour growth. This data may have implications for the management of cancer, although clinical trials are required to assess whether human tumour levels of ascorbate can similarly be manipulated. Citation Format: Elizabeth J. Campbell, Stephanie M. Bozonet, Bridget A. Robinson, Margreet CM Vissers, Gabi U. Dachs. Restoring physiological levels of ascorbate alleviates HIF-1 activation and reduces tumour growth in Gulo-/- mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 229. doi:10.1158/1538-7445.AM2014-229