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Effects of Bufalin on Up-regulating Methylation of wilm's tumor 1 gene in Human Erythroid Leukemic Cells

Chinese Journal of Integrative Medicine 2017年第4期23卷 288-294页

作者： WANG Li-pei ZHAO Yan-na sUN Xin GAO Rui-lan College of Basic Medical sciences Zhejiang Chinese Medical University Institution of Oncology Zhejiang Provincial People's Hospital

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 (ICsub>50sub>) 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 Gsub>0sub>/Gsub>1sub> phase, 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.

关键词： bufalin human erythroid leukemic cells wilm’s tumor 1 gene methylation DNA methyltransferase 3a DNA methyltransferase 3b

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