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.
ZHU Qingfeng, HU Xiaoli, ZHU Jianyi, LANG Wenjing, ZHONG Jihua, CHEN Fangyuan
. Study on mechanism of synergistic effect of ASP2215 combined with SAHA on FLT3-ITD mutant cell line[J]. Journal of Diagnostics Concepts & Practice, 2018
, 17(05)
: 538
-546
.
DOI: 10.16150/j.1671-2870.2018.05.011
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