PP242通过下调Akt/mTOR/4EBP1/eIF4E信号通路与柔红霉素存在协同抗急性白血病作用

PP242 synergizing with daunorubicin anti-acute erythromycin by downregulating Akt/mTOR/4EBP1/eIF4E signaling pathway

目的通过研究PP242单药及与柔红霉素(DNR)联合抗急性白血病的效应及作用机制,探寻急性白血病治疗的新方法。方法以NB4、THP-1、SUP-B15三种急性白血病细胞株为研究模型,采用MTT药物敏感试验检测PP242、DNR单药及两药联合的抗细胞增殖作用;Western Blot方法分析药物对Akt/mTOR/4EBP1/eIF4E信号通路关键点蛋白磷酸化的表达水平的影响;7-甲基鸟嘌呤帽亲和分析法分析药物对eIF4F翻译起始复合物中4EBP1、eIF4E、eIF4G表达的影响;流式细胞学检测PP242的促凋亡作用。结果 (1)MTT显示PP242、DNR单药对急性白血病细胞株均有显著的抗细胞增殖作用,而100 nmol/L的PP242与DNR联合使IC_(50)下降明显,计算协同指数均小于1,表明两药存在协同作用。(2)Western Blot显示PP242可有效下调Akt/mTOR/4EBP1/eIF4E轴的关键磷酸化蛋白及Mcl-1表达,而DNR却反馈上调这个通路的蛋白表达,PP242可协同DNR下调这条通路表达。(3)7-甲基鸟嘌呤帽亲和分析法显示PP242可增加eIF4E与4EBP1的结合,减少与eIF4G的结合,从而抑制eIF4F的形成,而DNR单药并没有这个作用。(4)流式细胞学显示PP242可促进细胞凋亡发生。结论 PP242单药可抑制三种急性白血病细胞株的增殖,具有明显的抗急性白血病作用,分析其可能作用机制为:通过抑制Akt/mTOR/4EBP1/e IF4E信号通路活性,抑制eIF4F翻译起始复合物形成以及促进细胞凋亡的发生。PP242与DNR联合有协同抗急性白血病作用。

Objective To explore a new method for the treatment of acute leukemia by studying the effects and mecha- nisms of PP242 monotherapy and combination therapy of PP242 and daunorubicin（DNR） in acute leukemia. Methods NB4, THP-1 and SUP-B15 were used as the research model. MTT drug sensitivity test was used to detect the anti-cell proliferation of PP242, DNR and the combination of the two drugs. The Western Blot method was used to analyze the effect of drugs on expression level of key protein phosphorylation in Akt/Akt/mTOR/4EBP1/eIF4E signaling pathway. 7- methylguanine cap affinity analysis method was used to analyze the effect of drugs on the expression of 4EBP1, eIF4E and eIF4G in eIF4F translation initiation complex. Cytological detection was used to detect the pro-apoptotic effect of PP242. Results （1）MTT showed that PP242 or DNR alone had significant anti-cell proliferation effects on acute leukemia cell lines, and 100 nmol/L PP242 combined with DNR decreased the IC50. The calculated synergy index was less than 1, indicating that there was synergy effect between the two drugs. （2）Western Blot showed that PP242 could effectively down-regulate the key phosphotylation protein and Mcl-1 expression of Akt/mTOR/4EBP1/eIF4E axis, while DNr feedbackly up-regulated the protein expression of this pathway. PP242 could down-regulate this pathway expression in collaboration with DNR. （3）7-methyl guanine cap affinity analysis showed that PP242 could increase the binding of eIF4E to 4EBP1, reduce the binding to eIF4G, and thus inhibit the formation of eIF4F, but DNR alone didn＇t have this effect. （4）Flow cytology showed that PP242 could promote apoptosis. Conclusion PP242 alone can inhibit the proliferation of three acute leukemia cell lines and has obvious anti-acute leukemia effect. The possible mechanism of action is to inhibit the eIF4F translation initiation complex and promote the occurrence of apoptosis by inhibiting the activity of Akt/mTOFU4EBP1/eIF4E signaling pathway. PP242 and DNR have synergistic anti-acute leukemia effects.