title: Effect of trichostatin A on anti HepG2 liver carcinoma cells: inhibition of HDAC activity and activation of Wnt/β-Catenin signaling.
abstract: OBJECTIVE
  To investigate the effect of deacetylase inhibitory trichostatin A (TSA) on anti HepG2 liver carcinoma cells and explore the underlying mechanisms.
  
  
  METHODS
  HepG2 cells exposed to different concentrations of TSA for 24, 48, or 72h were examined for cell growth inhibition using CCK8, changes in cell cycle distribution with flow cytometry, cell apoptosis with annexin V-FTIC/PI double staining, and cell morphology changes under an inverted microscope. Expression of β-catenin, HDAC1, HDAC3, H3K9, CyclinD1 and Bax proteins was tested by Western blotting. Gene expression for β-catenin, HDAC1and HDAC3 was tested by q-PCR. β-Catenin and H3K9 proteins were also tested by immunofluorescence. Activity of Renilla luciferase (pTCF/LEF-luc) was assessed using the Luciferase Reporter Assay system reagent. The activity of total HDACs was detected with a HDACs colorimetric kit.
  
  
  RESULTS
  Exposure to TSA caused significant dose-and time-dependent inhibition of HepG2 cell proliferation (p<0.05) and resulted in increased cell percentages in G0/ G1 and G2/M phases and decrease in the S phase. The apoptotic index in the control group was 6.22±0.25%, which increased to 7.17±0.20% and 18.1±0.42% in the treatment group. Exposure to 250 and 500nmol/L TSA also caused cell morphology changes with numerous floating cells. Expression of β-catenin, H3K9and Bax proteins was significantly increased, expression levels of CyclinD1, HDAC1, HDAC3 were decreased. Expression of β-catenin at the genetic level was significantly increased, with no significant difference in HDAC1and HDAC3 genes. In the cytoplasm, expression of β-catenin fluorescence protein was not obvious changed and in the nucleus, small amounts of green fluorescence were observed. H3K9 fluorescence protein were increased. Expression levels of the transcription factor TCF werealso increased in HepG2 cells following induction by TSA, whikle the activity of total HDACs was decreased.
  
  
  CONCLUSIONS
  TSA inhibits HDAC activity, promotes histone acetylation, and activates Wnt/β-catenin signaling to inhibit proliferation of HepG2 cell, arrest cell cycling and induce apoptosis.
authors.affiliation: Laboratory of Stem Cell and Tissue Engineering, Department of Histology and Embryology, Chongqing Medical University, Chongqing, China E-mail : lijingyangyang@126.com.cn; chendilong@21cn.com.
authors.forename: Qing-Qiang
authors.initials: QQ
authors.lastname: Shi
authors.forename: Guo-Wei
authors.initials: GW
authors.lastname: Zuo
authors.forename: Zi-Qiang
authors.initials: ZQ
authors.lastname: Feng
authors.forename: Lv-Cui
authors.initials: LC
authors.lastname: Zhao
authors.forename: Lian
authors.initials: L
authors.lastname: Luo
authors.forename: Zhi-Mei
authors.initials: ZM
authors.lastname: You
authors.forename: Dang-Yang
authors.initials: DY
authors.lastname: Li
authors.forename: Jing
authors.initials: J
authors.lastname: Xia
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.forename: Di-Long
authors.initials: DL
authors.lastname: Chen
mesh_terms: D000107:Acetylation; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D056284:Histone Deacetylase 1 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006655:Histone Deacetylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D060449:Wnt Signaling Pathway / Q000187:drug effects*; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism*; D000099186:Histone Deacetylase 3
chemical_list: D056284:Histone Deacetylase 1; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases; D006877:Hydroxamic Acids; D012333:RNA, Messenger; D051176:beta Catenin; D000099186:Histone Deacetylase 3; C012589:trichostatin A; C110550:HDAC1 protein, human