三氧化二砷对T急淋白血病及DNA转甲基酶1的影响

Effect of Arsenic Trioxide on Acute T-Lymphocytic Leukemia and DNA methyltransferase 1

【目的】本研究旨在探索三氧化二砷(ATO)对急性T淋巴细胞白血病(T-ALL)细胞中DNA转甲基酶1(DNMT1)表达的影响及其抗白血病作用机制。【方法】体外培养T-ALL细胞株(Jurkat、CCRF-CEM、Molt-4),依据不同ATO干预浓度,分为对照组(0μmol/L)、低浓度组(3μmol/L)、高浓度组(6μmol/L),应用RT-qPCR和western blot研究ATO(0、3、6μmol/L)干预24 h后细胞内DNMT1和cleaved-caspase-3表达的变化;应用流式细胞术测定T ALL细胞系(Jurkat、CCRF-CEM、MOLT-4)的细胞死亡率;在ATO的干预下,应用Z-DEVD-FMK后检测DNMT1和cleaved-caspase-3表达和细胞死亡率的变化;在ATO的干预下,过表达DNMT1检测T-ALL细胞死亡率的变化。【结果】随着ATO浓度的增加,T-ALL细胞DNMT1表达水平降低,cleaved-caspase-3蛋白表达水平增加,细胞死亡率增加,差异具有统计学意义(P<0.05);应用Z-DEVD-FMK后可特异性抑制cleaved-caspase-3,减弱ATO对DNMT1表达的抑制作用,降低细胞死亡率,差异具有统计学意义(P<0.05);在T-ALL细胞中过表达DNMT1可显著降低ATO处理后诱导的细胞死亡,差异具有统计学意义(P<0.05)。【结论】在一定浓度范围内,ATO可通过激活caspase-3有效抑制DNMT1的表达从而诱导T-ALL细胞死亡,为未来ATO作为去甲基化药物应用于提高T-ALL的临床治疗提供了一定的理论依据。

【Objective】To explore the effect of arsenic trioxide(ATO)on the expression of DNA methyltransferase 1(DNMT1)and its anti-leukemia mechanism in acute T-lymphocytic leukemia(T-ALL)cells.【Methods】T-ALL cell lines(Jurkat,CCRF-CEM,Molt-4)were cultured in vitro and divided into control(0μmol/L),low concentration(3μmol/L)and high concentration(6μmol/L)groups according to the dose of ATO,and the expression of DNMT1 and cleaved-caspase-3 were investigated by RT-qPCR and western blot after ATO treatment for 24 h(0,3 and 6μmol/L)in⁃tervention;Flow cytometry was applied to detect cell death in T-ALL cell lines(Jurkat,CCRF-CEM,MOLT-4);The ex⁃pression of DNMT1 and cleaved-caspase-3 and cell death were detected after applying ATO and Z-DEVD-FMK(caspase 3 specific inhibitor);T-ALL cell death was detected after overexpressing DNMT1 under ATO intervention.【Results】With the dose of ATO increasing,the expression level of DNMT1 in T-ALL cells decreased,the expression level of cleaved-cas⁃pase-3 protein increased,and the cell mortality increased(P<0.05);The application of Z-DEVD-FMK specifically inhib⁃ited cleaved-caspase-3,diminished the inhibitory effect of ATO on DNMT1 expression,and decreased the cell mortality(P<0.05);Overexpression of DNMT1 in T-ALL cells significantly reduced cell death induced by ATO treatment(P<0.05).【Conclusion】Within a certain concentration range,ATO effectively down-regulates the expression of DNMT1 via the acti⁃vation of caspase-3 in a dose-dependent manner,thus inducing cell death in T-ALL cells,which provides a theoretical basis for the future application of ATO as a demethylating drug to improve the clinical treatment of T-ALL.