阿帕替尼对FLT3-ITD突变急性髓系白血病MV4-11和MOLM-13细胞增殖与凋亡的影响及其机制

Effects of apatinib on the proliferation and apoptosis of FLT3-ITD mutant acute myeloid leukemia MV4-11 and MOLM-13 cells and their mechanisms

目的:研究阿帕替尼对FLT3-ITD突变的急性髓系白血病(AML)细胞增殖及凋亡的影响,并探讨相关机制。方法:取对数生长期FLT3-ITD突变的AML细胞株MV4-11和MOLM-13,分别采用不同浓度阿帕替尼处理48 h,采用CCK-8法检测细胞增殖情况,流式细胞术检测细胞凋亡情况,JC-1法检测线粒体膜电位的变化,蛋白质印迹法分析血管内皮生长因子受体2(VEGFR2)通路相关蛋白表达的变化。结果:阿帕替尼对MV4-11和MOLM-13细胞株均具有增殖抑制作用,48 h半数抑制浓度(IC 50)分别为(2.23±0.42)μmol/L、(4.08±2.62)μmol/L。流式细胞术检测细胞凋亡结果显示,10、20、30、40μmol/L阿帕替尼对MV4-11和MOLM-13细胞具有诱导凋亡作用,且作用呈浓度依赖性,48 h细胞凋亡率分别为(81.95±1.15)%、(88.80±0.23)%、(97.46±0.49)%、(99.29±0.05)%及(47.30±0.87)%、(67.00±3.71)%、(82.60±2.89)%、(98.06±5.34)%,差异均有统计学意义(F=6915.0,P<0.01;F=5385.0,P<0.01)。JC-1法检测线粒体膜电位结果显示,10、20、30、40μmol/L阿帕替尼作用MV4-11和MOLM-13细胞24 h后,JC-1多聚体/单体平均荧光强度(MFI)比值分别为0.45±0.06、0.19±0.07、0.12±0.03、0.09±0.01及0.84±0.05、0.66±0.13、0.35±0.11、0.27±0.02,与对照组(0.67±0.15和0.97±0.42)相比,差异均具有统计学意义(F=372.3,P<0.05;F=276.4,P<0.05)。蛋白质印迹法检测不同浓度阿帕替尼(2.5、5.0、10.0μmol/L)作用MV4-11细胞株24 h结果发现,阿帕替尼可下调VEGFR2、Src和STAT3磷酸化水平,且作用呈浓度依赖性。结论:阿帕替尼对FLT3-ITD突变AML细胞具有抑制增殖和诱导凋亡的作用,其机制可能与下调VEGFR2及其下游Src和STAT3磷酸化水平相关。

Objective To explore the effects of apatinib on the proliferation and apoptosis of FLT3-ITD mutant acute myeloid leukemia(AML)cells,and to explore the related mechanisms.Methods The logarithmic growth phase FLT3-ITD mutant AML cell lines MV4-11 and MOLM-13 were treated with different concentration of apatinib for 48 hours.The cell proliferation was detected by CCK-8 method.Flow cytometry was performed to examine the effect of apatinib on apoptosis.The cell mitochondrial membrane potential changes were detected by JC-1.Then the expression changes of vascular endothelial growth factor receptor 2(VEGFR2)pathway-related proteins were examined by Western blot.Results Apatinib had proliferation inhibitory effects on both MV4-11 and MOLM-13 cells,and the half-maximal inhibitory concentration(IC50)at 48 hours was(2.23±0.42)μmol/L and(4.08±2.62)μmol/L,respectively.After exposure to apatinib with increasing concentrations(10,20,30,and 40μmol/L)for 48 h hours,the percentage of apoptotic cells was significantly increased in MV4-11 cells[(81.95±1.15)%,(88.80±0.23)%,(97.46±0.49)%,and(99.29±0.05)%]and MOLM13 cells[(47.30±0.87)%,(67.00±3.71)%,(82.60±2.89)%,and(98.06±5.34)%]in a dose-dependent manner,and the differences were statistically significant(F=6915.0,P<0.01;F=5385.0,P<0.01).Detection of mitochondrial membrane potential by JC-1 method showed that after MV4-11 and MOLM-13 cells were treated by 10,20,30,and 40μmol/L apatinib for 24 hours,the JC-1 aggregate/monomer mean fluorescence intensity(MFI)ratios were 0.45±0.06,0.19±0.07,0.12±0.03,0.09±0.01,and 0.84±0.05,0.66±0.13,0.35±0.11,0.27±0.02,which were different from the control group(0.67±0.15 and 0.97±0.42),and the differences were statistically significant(F=372.3,P<0.05;F=276.4,P<0.05).Western blot was performed to detect different concentration of apatinib(2.5,5.0 and 10.0μmol/L)on the MV4-11 cells for 24 hours,the results showed that apatinib could down-regulate the phosphorylation of VEGFR2,Src and Stat3 in a dose-dependent manner.Conclusions Apatinib can inhibit cell proliferation and induce apoptosis in AML with FLT3-ITD mutation.The possible mechanism is related to the down-regulation of phosphorylation of VEGFR2 and its downstream targets Src and Stat3.