title: Histone deacetylase inhibitors inducing human cervical cancer cell apoptosis by decreasing DNA-methyltransferase 3B.
abstract: BACKGROUND
  Histone deacetylase (HDAC) inhibitors are a group of small chemical molecules that inhibit histone deacetylase. At cell level, HDAC inhibitors have multiple biological effects such as cell cycle arrest, apoptosis, cell differentiation and auotophagy. At molecular level, HDAC inhibitors cause histone and nonhistone acetylation and induce gene expression. HDAC inhibitors are widely used in cancer therapy because of its function of inducing apoptosis. However, the mechanisms of apoptosis effect are not fully understood. TSA is a classical HDAC inhibitor and widely used in epigenetic and anti-cancer research. In this study, we selected Trichostatin A (TSA) to investigate the mechanisms of HDAC inhibitors apoptotic effect on cancer cells.
  
  
  METHODS
  Cervical cancer cell lines such as Hela, Caski and normal human keratinocyte line HaCaT were treated with various concentrations of TSA. Crystal violent assay and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay were performed to determine cell number. PARP cleavage and FITC-AnexinV were performed to determine apoptosis. DNA-methyltransferase (DNMT)1, DNMT3A and DNMT3B were determined by regular PCR, qPCR and Western Blotting. Small interfering RNA (SiRNAi) was used to knock down DNMT3B.
  
  
  RESULTS
  HDAC inhibitors only induce cervical cancer cell apoptosis. At 1 µmol/L of TSA, 86% of Hela cell and 76% of Caski went apoptosis. For normal cells, HDAC inhibitors have no cytotoxic effect at therapeutic dosage, (90.0 ± 8.4)% of normal cell survive after treated with 1 µmol/L of TSA. We compared 1 µmol/L group with untreated control with t-test. There was no significance between 1 µmol/L group and untreated control for normal cell (P > 0.05). HDAC inhibitors decreased DNMT3B in cancer cell but not in normal cell. Manually knock-down of DNMT3B induced Hela and Caski cell apoptosis. More than 99% of Hela and Caski cell went apoptosis after deprived of DNMT3B.
  
  
  CONCLUSIONS
  DNMT3B was essential to cervical cancer cell survival. Down-regulated DNMT3B by HDAC inhibitors may play an important role in the toxicity of HDAC inhibitors on cervical cancer cells.
authors.affiliation: Department of Obstetrics and Gynecology, Peking University People's Hospital, Beijing, China.
authors.forename: Ning
authors.initials: N
authors.lastname: Liu
authors.forename: Li-jun
authors.initials: LJ
authors.lastname: Zhao
authors.forename: Xiao-ping
authors.initials: XP
authors.lastname: Li
authors.forename: Jian-liu
authors.initials: JL
authors.lastname: Wang
authors.forename: Guo-lin
authors.initials: GL
authors.lastname: Chai
authors.forename: Li-hui
authors.initials: LH
authors.lastname: Wei
mesh_terms: D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D002460:Cell Line; D045744:Cell Line, Tumor; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006367:HeLa Cells; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D002583:Uterine Cervical Neoplasms / Q000201:enzymology* / Q000235:genetics; D000097584:DNA Methyltransferase 3B
chemical_list: D004248:DNA (Cytosine-5-)-Methyltransferases; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D000097584:DNA Methyltransferase 3B; C012589:trichostatin A