Ascorbic acid and ascorbate-2-phosphate decrease HIF activity and malignant properties of human melanoma cells

Abstract

Background: Hypoxia inducible factor-1 alpha (HIF-1aα) is thought to play a role in melanoma carcinogenesis. Posttranslational regulation of HIF-1aα is dependent on Prolyl hydroxylase (PHD 1-3) and Factor Inhibiting HIF (FIH) hydroxylase enzymes, which require ascorbic acid as a co-factor for optimal function. Depleted intra-tumoral ascorbic acid may thus play a role in the loss of HIF-1aα regulation in melanoma. These studies assess the ability of ascorbic acid to reduce HIF-1aα protein and transcriptional activity in metastatic melanoma and reduce its invasive potential. Methods: HIF-1aα protein was evaluated by western blot, while transcriptional activity was measured by HIF-1 HRE-luciferase reporter gene activity. Melanoma cells were treated with ascorbic acid (AA) and ascorbate 2-phosphate (A2P) to assess their ability to reduce HIF-1aα accumulation and activity. siRNA was used to deplete cellular PHD2 in order to evaluate this effect on AA's ability to lower HIF-1aα levels. A2P's effect on invasive activity was measured by the Matrigel invasion assay. Data was analyzed by One-way ANOVA with Tukey's multiple comparisons test, or Student-T test as appropriate, with p