ASP2215联合SAHA对FLT3-ITD突变细胞株体外协同机制研究

目的：探究FLT3急性髓细胞性白血病（acute myeloid leukemia, AML）抑制剂ASP2215联合组蛋白去乙酰化酶（histone deacetylase inhibitor,HDAC）抑制剂SAHA,对伴FLT3-ITD突变的细胞株MV4-11的协同作用及其作用机制。方法：用不同浓度的ASP2215、SAHA单药或两药联合处理白血病细胞株MV4-11,观察细胞形态变化,采用CCK-8试剂盒检测其细胞活力,用流式细胞仪检测细胞凋亡率,蛋白免疫印迹检测FLT3及下游信号分子STAT5的磷酸化,分析凋亡调控蛋白Mcl-1、Bcl-xL、Bcl-2、Bax和caspase-9、caspase-3的蛋白含量。结果：①相较于FLT3野生型细胞株THP-1,ASP2215能够特异性地抑制FLT3-ITD突变AML细胞株MV4-11细胞的增殖。②ASP2215和SAHA单药处理均能抑制MV4-11细胞的活性,这种抑制作用呈浓度和时间依赖性,且两药联合使用时能够协同抑制MV4-11细胞株的活性,不同药物浓度联合的联合指数（combination index,CI）值皆小于1。③ASP2215和SAHA呈浓度和时间依赖性诱导MV4-11细胞凋亡,两者联合能够进一步增加MV4-11细胞凋亡。MV4-11细胞凋亡的过程中,伴caspase-3和caspase-9蛋白剪切活化,且活化程度随细胞凋亡增加而上升。相较于单药作用,两药联合能够引起更多的caspase-3和caspase-9剪切活化。形态学上,细胞凋亡和坏死改变随着药物浓度增加依次增多,且两药物联合能够引起细胞更明显的凋亡和坏死改变。④FLT3抑制剂ASP2215能够降低FLT3受体及下游分子STAT5的磷酸化水平,SAHA对FLT3及下游STAT5分子磷酸化也有轻度抑制作用。ASP2215和SAHA都能引起Mcl-1和Bcl-xL抗凋亡蛋白水平的降低,轻度下调Bcl-2蛋白和轻度上调Bax蛋白表达水平。两药联合相较于单药能够进一步下调Mcl-1、Bcl-xL的蛋白表达水平和Bcl-2/Bax蛋白比例。结论：ASP2215联合SAHA能够协同抑制伴FLT3-ITD突变MV4-11细胞株增殖,促进细胞凋亡,其机制涉及两药对FLT3-STAT5通路不同程度抑制作用,以及对凋亡相关蛋白Mcl-1、Bcl-xL及Bcl-2/Bax蛋白比例的调控。

Study on mechanism of synergistic effect of ASP2215 combined with SAHA on FLT3-ITD mutant cell line

Objective: To study the synergistic effect and mechanism of FLT3 inhibitor ASP2215 combined with HDAC inhibitor SAHA on FLT3-ITD mutated cell line MV4-11. Methods: MV4-11cells were treated with ASP2215, SAHA or ASP2215 combined with SAHA at different concentrations, and then cell morphological changes were observed, cell viability was detected by CCK-8 method and apoptosis rate measured by flow cytometry. Phosphorylation of FLT3 and downstream signaling molecule STAT5, as well as the levels of apoptosis regulated proteins Mcl-1, Bcl-xL, Bcl-2, Baxand Caspase-9/3 were detected by Western blot. Results: ① Compared with wild-type FLT3 cell line THP-1, ASP2215 could specifically inhibit the proliferation of FLT3-ITD mutated AML cell line MV4-11. ② ASP2215 or SAHA alone could inhibit the viability of MV4-11 cells in a dose and time dependent manner. Moreover, the combination of the two drugs could synergistically inhibit the viability of MV4-11cells, and the CI values of combination of the two drugs at different concentrationswere all less than 1. ③ASP2215 or SAHA alone could induce apoptosis of MV4-11 cells in a dose and time dependent manner, and there was a synergistic effect for the two drugs combined to induce apoptosis of MV4-11 cells. Apoptosis was accompanied by cleaved activation of caspase-3 and caspase-9. Combination of the two drugs caused more cleaved activation of caspase-3 and caspase-9 than the effect of single-drug. Morphologically, the changes of apoptosis and necrosis increased with the increase of drug concentration, and combination of the two drugs could lead to more obvious cell apoptosis and necrosis changes. ④The FLT3 inhibitor ASP2215 could reduce the phosphorylation level of FLT3 receptor and the downstream molecule STAT5, and SAHA also had a slight inhibitory effect on the phosphorylation of FLT3 and the downstream molecule STAT5. Both ASP2215 and SAHA induced a decrease in McL-1 and Bcl-xL anti-apoptotic proteins, with a slight down-regulation of Bcl-2 protein and a slight up-regulation of Bax protein expression. Combination of the two drugs further reduced the Mcl-1, Bcl-xL expression level and Bcl-2/Bax protein ratio when compared with the effect of single-drug. Conclusions: ASP2215 combined with SAHA can synergistically inhibit the proliferation and enhance the apoptosis of MV4-11 cell line with FLT3-ITD mutation. The mechanism involves the inhibition of FLT3-STAT5 pathway and the regulation of apoptosis-related proteins Mcl-1, Bcl-xL and Bcl-2/Bax protein ratio.