title: Regulation of demethylation and re-expression of RASSF1A gene in hepatocellular carcinoma cell lines treated with NCTD in vitro.
abstract: BACKGROUND
  Hepatocellular carcinoma, a lethal malignant neoplasm with poor prognosis, has dismal results of surgical resection and chemoradiotherapy. Norcantharidin (NCTD), the demethylated analog of cantharidin derived from a traditional Chinese medicine, Mylabris, has been used in the treatment of cancer. However, the detailed mechanisms underlying this process are generally unclear.
  
  
  OBJECTIVE
  The aim of this study was to investigate the mechanism of NCTD-induced apoptosis in HepG2 cells.
  
  
  METHODS
  Human HepG2 cell lines were treated with NCTD at different concentrations (2.50, 5.00, 10.00, 20.00, 40.00 μg/mL) for 24 hours. Cell proliferation was evaluated by measurement of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The methylation levels of RASSF1A (Ras-association domain family 1 A) in HepG2 cells were detected by methylation-specific PCR (MSP). The mRNA levels of RASSF1A in HepG2 cells were detected by real-time fluorescent quantitative PCR (RT-PCR). The levels of RASSF1A protein expression of HepG2 cells were detected by Western blotting assay.
  
  
  RESULTS
  The inhibition of cell proliferation was observed when treated with NCTD at concentrations (2.5 μg/mL), and as concentration increased, the proliferation of HepG2 cells was markedly inhibited by NCTD in dose-dependent manners. The levels of methylation of RASSF1A decreased at the increasing concentration of 10, 20 and 40 μg/mL. The levels of RASSF1A mRNA and protein were decreased when treated with NCTD at the concentrations of 10, 20 and 40 μg/mL, which were also in a dose-dependent manner.
  
  
  CONCLUSIONS
  NCTD can reverse the methylation state of RASSF1A gene and induce its re-expression, which will provide the theoretical basis for the clinical practice.
authors.forename: Yong
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Gastroenterology, Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu, P. R. China.
authors.forename: Min
authors.initials: M
authors.lastname: Xu
authors.forename: Zhen-Hai
authors.initials: ZH
authors.lastname: Di
authors.forename: Jian
authors.initials: J
authors.lastname: Zhang
authors.forename: Xue-Qun
authors.initials: XQ
authors.lastname: Mao
authors.forename: Hai-Bo
authors.initials: HB
authors.lastname: Sun
mesh_terms: D015153:Blotting, Western; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000235:genetics* / Q000473:pathology; D019175:DNA Methylation / Q000187:drug effects*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D066298:In Vitro Techniques; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000235:genetics* / Q000473:pathology; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; C411762:RASSF1 protein, human; D025521:Tumor Suppressor Proteins; C069741:norcantharidin