Wang Li-Pei, Zhao Yan-Na, Sun Xin, Gao Rui-Lan
College of Basic Medical Sciences, Zhejiang Chinese Medical University, Hangzhou, 310053, China.
Institution of Oncology, Zhejiang Provincial People's Hospital, Hangzhou, 310006, China.
Chin J Integr Med. 2017 Apr;23(4):288-294. doi: 10.1007/s11655-017-2404-1. Epub 2017 Apr 1.
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.
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.
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 (IC) 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.
Bufalin can not only significantly inhibit the proliferation of HEL cells and arrest cell cycle at G/G 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.
探讨蟾毒灵对人红白血病（HEL）细胞增殖的抑制作用、上调威尔姆斯瘤1基因（WT1）甲基化水平及其可能机制。
用不同浓度的蟾毒灵处理HEL细胞，观察细胞形态、进行增殖实验及细胞周期分析。采用逆转录聚合酶链反应（RT-PCR）、蛋白质免疫印迹法和免疫细胞化学法检测WT1的mRNA和蛋白表达水平，分别用甲基化特异性PCR和蛋白质免疫印迹法分析WT1的DNA甲基化以及DNA甲基转移酶3a（DNMT3a）和DNMT3b的蛋白表达水平。
蟾毒灵能有效抑制HEL细胞增殖，呈剂量依赖性，抑制率为23.4%±2.1%至87.2%±5.4%，半数最大抑制浓度（IC）为0.046 μmol/L。在经蟾毒灵处理的HEL细胞中观察到典型的凋亡形态。细胞周期增殖指数从76.4%±1.9%降至49.7%±1.3%。随着蟾毒灵剂量增加，WT1 mRNA及其蛋白表达水平逐渐降低，同时WT1基因的甲基化状态从未甲基化转变为部分或完全甲基化。而经蟾毒灵处理后，DNMT3a和DNMT3b蛋白表达水平呈剂量依赖性逐渐升高。
蟾毒灵不仅能显著抑制HEL细胞增殖并使细胞周期阻滞于G/G期，还能诱导细胞凋亡并下调WT1表达水平。我们的结果为蟾毒灵抗白血病提供了证据，其机制可能涉及提高WT1甲基化水平，这与红白血病HEL细胞中DNMT3a和DNMT3b蛋白上调有关。
