title: Vitamin C enhances epigenetic modifications induced by 5-azacytidine and cell cycle arrest in the hepatocellular carcinoma cell lines HLE and Huh7.
abstract: BACKGROUND
  5-Azacytidine (5-AZA), a DNA methyl transferase inhibitor, is a clinically used epigenetic drug for cancer therapy. Recently, we have shown that 5-AZA upregulates ten-eleven translocation (TET) protein expression in hepatocellular carcinoma (HCC) cells, which induce active demethylation. Vitamin C facilitates TET activity and enhances active demethylation. The aim of this study is to investigate whether vitamin C is able to enhance the effect of 5-AZA on active demethylation and to evaluate its consequence in HCC cell lines.
  
  
  METHODS
  HCC cell lines (Huh7 and HLE) were treated with 5-AZA and vitamin C. After 48 h of treatment, viability (resazurin conversion), toxicity (lactose dehydrogenase (LDH) release), and proliferation ((proliferating cell nuclear antigen (PCNA)) of single- and combined-treated cells were assessed. The effect of the treatment on 5-hydroxymethylcytosine (5hmC) intensity (immunofluorescence (IF) staining), TET, Snail, GADD45B, and P21 mRNA (real-time PCR) and protein expression (Western blot) were investigated.
  
  
  RESULTS
  Our results indicated that vitamin C enhances the anti-proliferative and apoptotic effect of 5-AZA in HCC cell lines. By further analyzing the events leading to cell cycle arrest, we have shown for the first time in HCC that the combination of 5-AZA and vitamin C leads to an enhanced downregulation of Snail expression, a key transcription factor governing epithelial-mesenchymal transition (EMT) process, and cell cycle arrest.
  
  
  CONCLUSIONS
  We conclude that when combined with 5-AZA, vitamin C enhances TET activity in HCC cells, leading to induction of active demethylation. An increase in P21 expression as a consequence of downregulation of Snail accompanied by the induction of GADD45B expression is the main mechanism leading to cell cycle arrest in HCCs.
authors.affiliation: Eberhard Karls University Tuebingen, BG Trauma Clinic, SWI, Schnarrenbergstraße 95, 72076 Tuebingen, Germany.
authors.forename: Sahar Olsadat
authors.initials: SO
authors.lastname: Sajadian
authors.affiliation: Eberhard Karls University Tuebingen, BG Trauma Clinic, SWI, Schnarrenbergstraße 95, 72076 Tuebingen, Germany.|CSIR - Centre for Cellular and Molecular Biology, Uppal Road, Habsiguda, Hyderabad India.
authors.forename: Chaturvedula
authors.initials: C
authors.lastname: Tripura
authors.affiliation: Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
authors.forename: Fazel Sahraneshin
authors.initials: FS
authors.lastname: Samani
authors.affiliation: Eberhard Karls University Tuebingen, BG Trauma Clinic, SWI, Schnarrenbergstraße 95, 72076 Tuebingen, Germany.
authors.forename: Marc
authors.initials: M
authors.lastname: Ruoss
authors.affiliation: Department of Medicine II, Section Molecular Hepatology, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany.
authors.forename: Steven
authors.initials: S
authors.lastname: Dooley
authors.affiliation: Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
authors.forename: Hossein
authors.initials: H
authors.lastname: Baharvand
authors.affiliation: Eberhard Karls University Tuebingen, BG Trauma Clinic, SWI, Schnarrenbergstraße 95, 72076 Tuebingen, Germany.
authors.forename: Andreas K
authors.initials: AK
authors.lastname: Nussler
mesh_terms: D001205:Ascorbic Acid / Q000494:pharmacology*; D001374:Azacitidine / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000235:genetics*; D059447:Cell Cycle Checkpoints / Q000187:drug effects*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D019175:DNA Methylation / Q000187:drug effects; D004357:Drug Synergism; D044127:Epigenesis, Genetic / Q000187:drug effects*; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics*
chemical_list: D001374:Azacitidine; D001205:Ascorbic Acid
keywords: 5-Azacytidine; 5hmC; EMT/MET; GADD45B; Snail; TETs; Vitamin C