达沙替尼通过上调p53表达和下调AKT磷酸化抑制肾癌细胞系786-O及769-P体外活力与迁移

Dasatinib inhibits the viability and migration of renal carcinoma cell lines 786-O and 769-P by up-regulating p53 expression and down-regulating AKT phosphorylation in vitro

目的:探讨达沙替尼(dasatinib)体外对人肾癌细胞系786-O及769-P增殖、凋亡及迁移的影响及其分子机制。方法:不同浓度(0~2μmol/L)达沙替尼处理786-O细胞及769-P细胞(0μmol/L为空白对照组),采用MTT法检测细胞活力,划痕实验检测细胞迁移能力,Hoechst 33258染色观察细胞凋亡,流式细胞术检测细胞周期,Western blot法检测细胞周期和细胞凋亡相关蛋白的表达水平。结果:达沙替尼对786-O及769-P两种细胞均有活力抑制和迁移阻滞的作用,随着浓度增大抑制效果越显著(P<0.05)。达沙替尼对786-O细胞和769-P细胞的IC50分别为(0.9587±0.0288)μmol/L和(0.7843±0.0660)μmol/L。达沙替尼处理24 h后,促凋亡蛋白cleaved caspase-3和cleaved caspase-9的表达显著增加(P<0.01),细胞周期蛋白D1的表达下降(P<0.05),细胞周期相关通路蛋白p53和p21表达量均显著增加(P<0.05),p-AKT蛋白水平显著下降(P<0.05)。结论:达沙替尼通过上调p53表达并下调AKT磷酸化水平抑制人肾癌细胞系786-O及769-P的活力和迁移能力。

AIM:To explore the effect of dasatinib on the viability,apoptosis and migration of human renal carcinoma cell lines 786-O and 769-P,as well as the molecular mechanism in vitro.METHODS:786-O cells and 769-P cells were treated with different concentrations(0~2μmol/L)of dasatinib,and 0μmol/L dasatinib was used as blank control group.MTT method was used to detect cell viability.Wound healing assay was used to detect the effect of dasatinib on migration.Hoechst 33258 staining was used to observe the effect of dasatinib on apoptosis.Flow cytometry was used to detect the effect of dasatinib on cell cycle.Western blot method was used to detected cell cycle-and apoptosis-related protein levels.RESULTS:Dasatinib inhibited viability and migration of 786-O and 769-P cells,and the inhibitory effect of dasatinib increased with the concentration of dasatinib(P<0.05).The IC50 values of dasatinib against 786-O and 769-P cell lines were(0.9587±0.0288)μmol/L and(0.7843±0.0660)μmol/L,respectively.After treatment with dasatinib for 24 h,the expression of pro-apoptotic proteins cleaved caspase-3 and cleaved caspase-9 increased significantly(P<0.01),while the expression of cyclin D1 decreased(P<0.05).The cycle-related pathway proteins p53 and p21 increased(P<0.05),while the level of p-AKT was decreased(P<0.05).CONCLUSION:Dasatinib impaired the viability and migration ability of human renal carcinoma cell lines 786-O and 769-P by up-regulating p53 expression and downregulating AKT phosphorylation.