title: Melatonin induces transcriptional regulation of Bim by FoxO3a in HepG2 cells.
abstract: BACKGROUND
  Melatonin induces apoptosis in many different cancer cell lines, including hepatocellular carcinoma cells. However, the responsible pathways have not been clearly elucidated. A member of the forkhead transcription factors' family, FoxO3a, has been implicated in the expression of the proapoptotic protein Bim (a Bcl-2-interacting mediator of cell death). In this study, we used human HepG2 liver cancer cells as an in vitro model to investigate whether melatonin treatment induces Bim through regulation by the transcription factor FoxO3a.
  
  
  METHODS
  Cytotoxicity of melatonin was compared in HepG2 hepatoblastoma cells and primary human hepatocytes. Proapoptotic Bim expression was analysed by reverse transcriptase-polymerase chain reaction and western blot. Reporter gene assays and chromatin immunoprecipitation assays were performed to analyse whether FoxO3a transactivates the Bim promoter. Small interfering RNA (siRNA) was used to study the role of FoxO3a in Bim expression. Immunofluorescence was performed to analyse FoxO3a localisation in HepG2 cells.
  
  
  RESULTS
  Melatonin treatment induces apoptosis in HepG2 cells, but not in primary human hepatocytes. The proapoptotic effect was mediated by increased expression of the BH3-only protein Bim. During melatonin treatment, we observed increased transcriptional activity of the forkhead-responsive element and could demonstrate that FoxO3a binds to a specific sequence within the Bim promoter. Furthermore, melatonin reduced phosphorylation of FoxO3a at Thr(32) and Ser(253), and induced its increased nuclear localisation. Moreover, silencing experiments with FoxO3a siRNA prevented Bim upregulation.
  
  
  CONCLUSIONS
  This study shows that melatonin can induce apoptosis in HepG2 hepatocarcinoma cells through the upregulation of proapoptotic Bim mediated by nuclear translocation and activation of the transcription factor FoxO3a.
authors.affiliation: Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd) and Institute of Biomedicine, University of León, León, Spain.
authors.forename: S
authors.initials: S
authors.lastname: Carbajo-Pescador
authors.forename: C
authors.initials: C
authors.lastname: Steinmetz
authors.forename: A
authors.initials: A
authors.lastname: Kashyap
authors.forename: S
authors.initials: S
authors.lastname: Lorenz
authors.forename: J L
authors.initials: JL
authors.lastname: Mauriz
authors.forename: M
authors.initials: M
authors.lastname: Heise
authors.forename: P R
authors.initials: PR
authors.lastname: Galle
authors.forename: J
authors.initials: J
authors.lastname: González-Gallego
authors.forename: S
authors.initials: S
authors.lastname: Strand
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000096:biosynthesis / Q000235:genetics*; D000072224:Bcl-2-Like Protein 11; D001665:Binding Sites; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects; D006801:Humans; D008550:Melatonin / Q000378:metabolism / Q000494:pharmacology*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering; D014158:Transcription, Genetic / Q000187:drug effects*; D015533:Transcriptional Activation
chemical_list: D051017:Apoptosis Regulatory Proteins; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; C499517:FOXO3 protein, human; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D008550:Melatonin