title: Effects of bufalin on up-regulating methylation of Wilm's tumor 1 gene in human erythroid leukemic cells.
abstract: OBJECTIVE
  To explore the effects of bufalin on inhibiting proliferation, up-regulating methylation of Wilm' tumor 1 gene (WT1) as well as its possible mechanisms in human erythroid leukemic (HEL) cells.
  
  
  METHODS
  The HEL cells were treated with bufalin at various concentrations to observe cellular morphology, proliferation assay and cell cycle. The mRNA and protein expression levels of WT1 were detected by reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunocytochemistry, DNA methylation of WT1 and protein expression levels of DNA methyltransferase 3a (DNMT3a) and DNMT3b were analyzed by methylation-specific PCR, and Western blot respectively.
  
  
  RESULTS
  The bufalin was effective to inhibit proliferation of HEL cells in a dose-dependent manner, their suppression rates were from 23.4%±2.1% to 87.2%±5.4% with an half maximal inhibit concentration (IC50) of 0.046 μmol/L. Typical apoptosis morphology was observed in bufalin-treated HEL cells. The proliferation index of cell cycle decreased from 76.4%±1.9% to 49.7%±1.3%. The expression levels of WT1 mRNA and its protein reduced gradually with increasing doses of bufalin, meanwhile, the methylation status of WT1 gene changed from unmethylated into partially or totally methylated. While, the expression levels of DNMT3a and DNMT3b protein gradually increased by bufalin treatment in a dose-dependent manner.
  
  
  CONCLUSIONS
  Bufalin can not only significantly inhibit the proliferation of HEL cells and arrest cell cycle at G0/G1phase, but also induce cellular apoptosis and down-regulate the expression level of WT1. Our results provide the evidence of bufalin for anti-leukemia, its mechanism may involve in increasing WT1 methylation status which is related to the up-regulation of DNMT3a and DNMT3b proteins in erythroid leukemic HEL cells.
authors.affiliation: College of Basic Medical Sciences, Zhejiang Chinese Medical University, Hangzhou, 310053, China.
authors.forename: Li-Pei
authors.initials: LP
authors.lastname: Wang
authors.affiliation: College of Basic Medical Sciences, Zhejiang Chinese Medical University, Hangzhou, 310053, China.
authors.forename: Yan-Na
authors.initials: YN
authors.lastname: Zhao
authors.affiliation: Institution of Oncology, Zhejiang Provincial People's Hospital, Hangzhou, 310006, China. fallingyezi@126.com.
authors.forename: Xin
authors.initials: X
authors.lastname: Sun
authors.affiliation: College of Basic Medical Sciences, Zhejiang Chinese Medical University, Hangzhou, 310053, China.
authors.forename: Rui-Lan
authors.initials: RL
authors.lastname: Gao
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D002018:Bufanolides / Q000494:pharmacology*; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects* / Q000235:genetics; D000090205:DNA Methyltransferase 3A; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects* / Q000235:genetics; D025721:WT1 Proteins / Q000235:genetics* / Q000378:metabolism; D000097584:DNA Methyltransferase 3B
chemical_list: D002018:Bufanolides; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D012333:RNA, Messenger; D025721:WT1 Proteins; D000097584:DNA Methyltransferase 3B; C000717967:DNMT3A protein, human; C583751:WT1 protein, human; C022777:bufalin
keywords: DNA methyltransferase 3a; DNA methyltransferase 3b; Wilm’s tumor 1 gene; bufalin; human erythroid leukemic cells; methylation