title: Ascorbic acid, but not dehydroascorbic acid increases intracellular vitamin C content to decrease Hypoxia Inducible Factor -1 alpha activity and reduce malignant potential in human melanoma.
abstract: BACKGROUND
  Accumulation of hypoxia inducible factor-1 alpha (HIF-1α) in malignant tissue is known to contribute to oncogenic progression and is inversely associated with patient survival. Ascorbic acid (AA) depletion in malignant tissue may contribute to aberrant normoxic activity of HIF-1α. While AA supplementation has been shown to attenuate HIF-1α function in malignant melanoma, the use of dehydroascorbic acid (DHA) as a therapeutic means to increase intracellular AA and modulate HIF-1α function is yet to be evaluated. Here we compared the ability of AA and DHA to increase intracellular vitamin C content and decrease the malignant potential of human melanoma by reducing the activity of HIF-1α.
  
  
  METHODS
  HIF-1α protein accumulation was evaluated by western blot and transcriptional activity was evaluated by reporter gene assay using a HIF-1 HRE-luciferase plasmid. Protein expressions and subcellular localizations of vitamin C transporters were evaluated by western blot and confocal imaging. Intracellular vitamin C content following AA, ascorbate 2-phosphate (A2P), or DHA supplementation was determined using a vitamin C assay. Malignant potential was accessed using a 3D spheroid Matrigel invasion assay. Data was analyzed by One or Two-way ANOVA with Tukey's multiple comparisons test as appropriate with p<0.05 considered significant.
  
  
  RESULTS
  Melanoma cells expressed both sodium dependent vitamin C (SVCT) and glucose (GLUT) transporters for AA and DHA transport respectively, however advanced melanomas responded favorably to AA, but not DHA. Physiological glucose conditions significantly impaired intracellular vitamin C accumulation following DHA treatment. Consequently, A2P and AA, but not DHA treated cells demonstrated lower HIF-1α protein expression and activity, and reduced malignant potential. The ability of AA to regulate HIF-1α was dependent on SVCT2 function and SVCT2 was not significantly inhibited at pH representative of the tumor microenvironment.
  
  
  CONCLUSIONS
  The use of ascorbic acid as an adjuvant cancer therapy remains under investigated. While AA and A2P were capable of modulating HIF-1α protein accumulation/activity, DHA supplementation resulted in minimal intracellular vitamin C activity with decreased ability to inhibit HIF-1α activity and malignant potential in advanced melanoma. Restoring AA dependent regulation of HIF-1α in malignant cells may prove beneficial in reducing chemotherapy resistance and improving treatment outcomes.
authors.affiliation: Department of Biomedical Sciences, Joan C. Edwards School of Medicine, Marshall University, One John Marshall Drive, Huntington, WV 25755, United States. Electronic address: fischer35@marshall.edu.
authors.forename: Adam P
authors.initials: AP
authors.lastname: Fischer
authors.affiliation: Department of Biomedical Sciences, Joan C. Edwards School of Medicine, Marshall University, One John Marshall Drive, Huntington, WV 25755, United States. Electronic address: kittlaus1@marshall.edu.
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Miles
mesh_terms: D001205:Ascorbic Acid / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology*; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D045744:Cell Line, Tumor; D003683:Dehydroascorbic Acid / Q000494:pharmacology*; D005947:Glucose / Q000378:metabolism; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D008545:Melanoma / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D012964:Sodium / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D059016:Tumor Microenvironment / Q000187:drug effects
chemical_list: C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C011669:ascorbate-2-phosphate; D012964:Sodium; D005947:Glucose; D001205:Ascorbic Acid; D003683:Dehydroascorbic Acid
keywords: Ascorbic acid; Dehydroascorbic acid; Hypoxia inducible factor-1 alpha; Melanoma; SVCT2