Effects of Bufalin on Up-regulating Methylation of Wilm＇s Tumor I Gene in Human Erythroid Leukemic Cells

• Published in: 中国结合医学杂志：英文版 Vol. 23; no. 4; pp. 288 - 294
• Main Author: WANG Li-pei ZHAO Yan-na SUN Xin GAO Rui-lan
• Format: Journal Article
• Language: English
• Published: 2017
• Subjects: bufalin; cells; DNA; erythroid; gene; human; leukemic; methylation; methyltransferase; methyltransferase3a; tumor; Wilm＇s
• ISSN: 1672-0415; 1993-0402

Objective： To explore the effects of bufalin on inhibiting proliferation, up-regulating methylationof 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 reversetranscription polymerase chain reaction （RT-PCR）, Western blot and immunocytochemistry, DNA methylationof WT1 and protein expression levels of DNA methyltransferase 3a （DNMT3a） and DNMT3b were analyzed bymethylation-specific PCR, and Western blot respectively. Results： The bufalin was effective to inhibit proliferationof 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 （ICso） of 0.046 μmol/L. Typical apoptosis morphology was observed inbufalin-treated HEL cells. The proliferation index of cell cycle decreased from 76.4%± 1.9% to 49.7% ± 1.3%. Theexpression 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, theexpression levels of DNMT3a and DNMT3b protein gradually increased by bufalin treatment in a dose-dependentmanner. Conclusions： Bufalin can not only significantly inhibit the proliferation of HEL cells and arrest cell cycle atGo/G1 phase, but also induce cellular apoptosis and down-regulate the expression level of WTI. Our results providethe evidence of bufalin for anti-leukemia, its mechanism may involve in increasing WT1 methylation status which isrelated to the up-requlation of DNMT3a and DNMT3b proteins in ervthroid leukemic HEL cells.