title: Inhibition of VEGF expression through blockade of Hif1α and STAT3 signalling mediates the anti-angiogenic effect of melatonin in HepG2 liver cancer cells.
abstract: BACKGROUND
  Hepatocellular carcinoma (HCC) growth relies on angiogenesis via vascular endothelial growth factor (VEGF) release. Hypoxia within tumour environment leads to intracellular stabilisation of hypoxia inducible factor 1 alpha (Hif1α) and signal transducer and activator of transcription (STAT3). Melatonin induces apoptosis in HCC, and shows anti-angiogenic features in several tumours. In this study, we used human HepG2 liver cancer cells as an in vitro model to investigate the anti-angiogenic effects of melatonin.
  
  
  METHODS
  HepG2 cells were treated with melatonin under normoxic or CoCl2-induced hypoxia. Gene expression was analysed by RT-qPCR and western blot. Melatonin-induced anti-angiogenic activity was confirmed by in vivo human umbilical vein endothelial cells (HUVECs) tube formation assay. Secreted VEGF was measured by ELISA. Immunofluorescence was performed to analyse Hif1α cellular localisation. Physical interaction between Hif1α and its co-activators was analysed by immunoprecipitation and chromatin immunoprecipitation (ChIP).
  
  
  RESULTS
  Melatonin at a pharmacological concentration (1 mM) decreases cellular and secreted VEGF levels, and prevents HUVECs tube formation under hypoxia, associated with a reduction in Hif1α protein expression, nuclear localisation, and transcriptional activity. While hypoxia increases phospho-STAT3, Hif1α, and CBP/p300 recruitment as a transcriptional complex within the VEGF promoter, melatonin 1 mM decreases their physical interaction. Melatonin and the selective STAT3 inhibitor Stattic show a synergic effect on Hif1α, STAT3, and VEGF expression.
  
  
  CONCLUSIONS
  Melatonin exerts an anti-angiogenic activity in HepG2 cells by interfering with the transcriptional activation of VEGF, via Hif1α and STAT3. Our results provide evidence to consider this indole as a powerful anti-angiogenic agent for HCC treatment.
authors.affiliation: Institute of Biomedicine (IBIOMED), University of León, Campus Universitario, 24071 León, Spain.
authors.forename: S
authors.initials: S
authors.lastname: Carbajo-Pescador
authors.forename: R
authors.initials: R
authors.lastname: Ordoñez
authors.forename: M
authors.initials: M
authors.lastname: Benet
authors.forename: R
authors.initials: R
authors.lastname: Jover
authors.forename: A
authors.initials: A
authors.lastname: García-Palomo
authors.forename: J L
authors.initials: JL
authors.lastname: Mauriz
authors.forename: J
authors.initials: J
authors.lastname: González-Gallego
mesh_terms: D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D015687:Cell Hypoxia; D003035:Cobalt; D003499:Cyclic S-Oxides / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic; D056945:Hep G2 Cells; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D008113:Liver Neoplasms / Q000378:metabolism*; D008550:Melatonin / Q000494:pharmacology*; D009389:Neovascularization, Pathologic / Q000188:drug therapy; D011401:Promoter Regions, Genetic; D050796:STAT3 Transcription Factor / Q000378:metabolism*; D015398:Signal Transduction; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D050880:p300-CBP Transcription Factors / Q000378:metabolism
chemical_list: D020533:Angiogenesis Inhibitors; D003499:Cyclic S-Oxides; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C517409:stattic; D003035:Cobalt; D050880:p300-CBP Transcription Factors; C018021:cobaltous chloride; D008550:Melatonin