富马酸二甲酯对急性T淋巴细胞白血病影响的研究

Effect of Dimethyl Fumarate(DMF) on T-cell Acute Lymphoblastic Leukemia

目的:探讨富马酸二甲酯(DMF)对急性T淋巴细胞白血病的影响及潜在的作用机制,为临床治疗急性T淋巴细胞白血病提供实验基础和理论依据。方法:用不同浓度DMF处理Jurkat细胞24 h,然后采用流式细胞术检测Ki67^(+)的Jurkat细胞比例和绝对计数;同时采用Western blot方法检测DMF处理24 h之后,Jurkat细胞内核因子E2相关因子2(Nrf2)和E3泛素连接酶HACE1的蛋白水平变化;利用免疫共沉淀方法富集Jurkat细胞内Nrf2蛋白,然后采用Western blot方法检测HACE1蛋白水平;在HEK293T细胞中过表达Flag-Nrf2和不同梯度的Flag-HACE1质粒,48 h后采用Western blot检测Flag-Nrf2蛋白水平变化。结果:DMF能显著抑制Ki67^(+)的Jurkat细胞比例和绝对计数,即DMF抑制Jurkat细胞增殖,且呈剂量依赖性(r=0.9595,r=0.9054)。DMF能显著上调Jurkat细胞内Nrf2和E3泛素连接酶HACE1蛋白水平(P<0.01,P<0.01)。Jurkat细胞内Nrf2和HACE1蛋白存在相互作用。过表达Flag-HACE1显著上调Flag-Nrf2的蛋白水平,且呈剂量依赖性(r=0.9771)。结论:DMF抑制急性T淋巴细胞白血病细胞的增殖,其机制可能是DMF显著上调Nrf2和E3泛素连接酶HACE1蛋白水平,HACE1与Nrf2相互作用并正向调控Nrf2蛋白水平。

Objective:To explore the effect and possible mechanism of dimethyl fumarate(DMF) on T-cell acute lymphoblastic leukemia(T-ALL),and provide experimental and theoretical basis for the clinical treatment of T-ALL.Methods:Jurkat cells were treated with different concentrations of DMF for 24 hours,and then the proportion and absolute count of Ki67-positive Jurkat cells were analyzed by flow cytometry.Meanwhile,the protein levels of nuclear factor-erythroid 2-related factor 2(Nrf2) and E3 ubiquitin ligase HACE1 in Jurkat cells treated with DMF for 24 hours were evaluated by Western blot.Nrf2 proteins were co-immunoprecipitated in Jurkat cells,and then HACE1 protein was assessed by Western blot.Plasmids of Flag-Nrf2 and different gradients of Flag-HACE1 were transfected into HEK293 T cells,and the levels of Flag-Nrf2 were detected by Western blot after 48 hours.Results:DMF could significantly inhibit the proportion and absolute count of Ki67-positive Jurkat cells,and DMF inhibited the proliferation of Jurkat cells in a dose-dependent manner(r=0.9595,r=0.9054).DMF could significantly up-regulate the protein levels of Nrf2 and E3 ubiquitin ligase HACE1 in Jurkat cells(P<0.01,P<0.01).HACE1 physically interacted with Nrf2 in Jurkat cells.Overexpression of Flag-HACEl significantly increased the protein level of Flag-Nrf2 in a dose-dependent manner(r=0.9771).Conclusion:DMF inhibits the proliferation of T-cell acute lymphoblastic leukemia cell.The mechanism may be that,DMF significantly up-regulates the protein levels of Nrf2 and E3 ubiquitin ligase HACE1,and HACE1 interacts with Nrf2 and positively regulates Nrf2 protein level.