达沙替尼对人脐静脉内皮细胞的损伤与PI3K/Akt途径抑制有关

Inhibition of PI3K/Akt pathway involved in damaging of dasatinib to human umbilical vein endothelial cells

目的探索达沙替尼对人脐静脉内皮细胞(HUVEC)增殖、迁移和凋亡等生物学特性的影响,为完善其临床应用提供资料。方法实验分组:达沙替尼组(达沙替尼处理浓度为50 nmol/L)、LY294002组(PI3K抑制剂,处理浓度为20μmol/L)、联合处理组(达沙替尼处理浓度为50 nmol/L、LY294002处理浓度为20μmol/L)及溶媒对照组(DMSO浓度为0.1%)。CCK8法检测细胞活性,划痕法检测细胞迁移,流式细胞术检测细胞凋亡和细胞周期,Western blot检测Akt和p-Akt蛋白水平。结果达沙替尼(1~400 nmol/L)不仅抑制HUVEC增殖,而且诱导其凋亡、抑制其迁移、阻滞细胞周期G1-S期转化。随浓度的升高与处理时间的延长(50 nmol/L,24 h^96 h)达沙替尼对细胞增殖抑制作用和诱导凋亡作用明显,同时HUVEC的迁移能力随达沙替尼浓度(50~100 nmol/L)的升高而降低。达沙替尼和PI3K抑制剂LY294002两者单独处理时均具有细胞增殖抑制作用,两者均明显抑制Akt蛋白磷酸化水平;两者联合处理时虽然细胞增殖抑制作用增强,但对Akt蛋白磷酸化水平的影响与单独处理相比差异不明显。结论达沙替尼可通过PI3K/Akt通路促进HUVEC损伤,抑制HUVEC增殖和迁移,改变细胞形态,阻滞HUVEC G1-S期转化,诱导细胞凋亡。

Dasatinib ; Cell proliferation; Cell migration; Apoptosis ; Cell cycle; PI3K/Akt pathway [ ABSTRACT] Aim To explore the effect of dasatinib on proliferation, migration, cell cycle and apoptosis of human umbilical vein endothelial cells cytologically and molecularly to offer help on clinical application of dasatinib. Methods Experimental grouping： dasatinib group （dasatinib concentration was 50 nmol/L） and LY294002 group （PI3K inhibitor, concentration was 20μmol/L）, combined treatment group （dasatinib treatment concentration was 50 nmol/L, the concen-tration of LY294002 for 20μmol/L） and mock group （DMSO was 0.1%）. Cell viability was measured by CCK8. The migration ability of human umbilical vein endothelial cells was measured by scratch assay. Apoptosis and cell cycle were analyzed by flow cytometry. The expression and phosphorylation of Akt protein were determined by Western blot. Results The cell viability of human umbilical vein endothelial cells decreased gradually following the increasing concentration （ 1- 400 nmol/L） and prolonged exposure to dasatinib （50 nmol/L, 24-96 h）. Dasatinib and LY294002 （an inhib- itor of PI3K）, both inhibited cell viability of human umbilical vein endothelial cells. Dasatinib （50-100 nmol/L） weak-ened the migration capability of human umbilical vein endothelial cells. In addition, compared with control, dasatinib （50 nmol/L） induced apoptosis and cell cycle arrest （P〈0. 05） of human umbilical vein endothelial cells. The phospho-rylation of Akt was inhibited by dasatinib and LY294002, and LY294002 had more powerful inhibition of p-Akt than dasat- inib. Conclusion Dasatinib could facilitate the injures of human umbilical vein endothelial cells via PI3K/Akt path- way, mainly to suppress the proliferation and migration of human umbilical vein endothelial cells, alter the cell morphology, blocked the G1-S transition and induced apoptosis.