title: Ascorbic acid and ascorbate-2-phosphate decrease HIF activity and malignant properties of human melanoma cells.
abstract: BACKGROUND
  Hypoxia inducible factor-1 alpha (HIF-1α) is thought to play a role in melanoma carcinogenesis. Posttranslational regulation of HIF-1α 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-1α regulation in melanoma. These studies assess the ability of ascorbic acid to reduce HIF-1α protein and transcriptional activity in metastatic melanoma and reduce its invasive potential.
  
  
  METHODS
  HIF-1α 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-1α accumulation and activity. siRNA was used to deplete cellular PHD2 in order to evaluate this effect on AA's ability to lower HIF-1α 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 < .05 considered significant.
  
  
  RESULTS
  Supplementation with both AA and A2P antagonized normoxic as well as cobalt chloride- and PHD inhibitor ethyl 3, 4-dihydroxybenzoate induced HIF-1α protein stabilization and transcriptional activity. Knockdown of the PHD2 isoform with siRNA did not impede the ability of AA to reduce normoxic HIF-1α protein. Additionally, reducing HIF-1α levels with A2P resulted in a significant reduction in the ability of the melanoma cells to invade through Matrigel.
  
  
  CONCLUSIONS
  These studies suggest a positive role for AA in regulating HIF-1α in melanoma by demonstrating that supplementation with either AA, or its oxidation-resistant analog A2P, effectively reduces HIF-1α protein and transcriptional activity in metastatic melanoma cells. Our data, while supporting the function of AA as a necessary cofactor for PHD and likely FIH activity, also suggests a potential non-PHD/FIH role for AA in HIF-1α regulation by its continued ability to reduce HIF-1α in the presence of PHD inhibition. The use of the oxidation-resistant AA analog, A2P, to reduce the ability of HIF-1α to promote malignant progression in melanoma cells and enhance their response to therapy warrants further investigation.
authors.affiliation: Department of Biochemistry and Microbiology, Joan C. Edwards School of Medicine, Marshall University, One John Marshall Drive, Huntington, WV, 25755, USA. kittlaus1@marshall.edu.
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Miles
authors.affiliation: Department of Biochemistry and Microbiology, Joan C. Edwards School of Medicine, Marshall University, One John Marshall Drive, Huntington, WV, 25755, USA. fischer35@marshall.edu.
authors.forename: Adam P
authors.initials: AP
authors.lastname: Fischer
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 655 W. Baltimore Street, Baltimore, MD, 21201, USA. SJoshi@som.umaryland.edu.
authors.forename: Sandeep J
authors.initials: SJ
authors.lastname: Joshi
authors.affiliation: Department of Biochemistry and Microbiology, Joan C. Edwards School of Medicine, Marshall University, One John Marshall Drive, Huntington, WV, 25755, USA. Niles@marshall.edu.
authors.forename: Richard M
authors.initials: RM
authors.lastname: Niles
mesh_terms: D001205:Ascorbic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology*; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D015870:Gene Expression; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D017930:Genes, Reporter; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D008545:Melanoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D009362:Neoplasm Metastasis; D055550:Protein Stability / Q000187:drug effects; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C011669:ascorbate-2-phosphate; D001205:Ascorbic Acid