Wortmannin对人髓母细胞瘤细胞系Daoy增殖和凋亡的影响

Effects of Wortmannin on proliferation and apoptosis of medulloblastoma cell line Daoy

目的:探讨PI3K抑制剂Wortmannin在体外对人髓母细胞瘤细胞系Daoy增殖和凋亡的影响。方法:Wortmannin作用髓母细胞瘤细胞系Daoy后,CCK-8检测细胞增殖情况,流式细胞仪分析细胞凋亡,Westernblot检测细胞Akt、p-Akt、Cleaved-caspase3的表达,Caspase3活性检测试剂盒观察其活性。结果:不同浓度(0.1、0.25、0.5μmol/L)Wortmannin在体外均能抑制Daoy细胞的增殖,并呈浓度时间依赖性。各浓度Wort-mannin处理细胞48h后,流式细胞仪检测到细胞凋亡率分别为(21.16±2.49)%、(31.02±0.71)%、(44.61±1.47)%,与非处理组比较具有显著差异,并呈剂量依赖;Western blot观察到Wortmannin可使p-Akt表达水平降低,而Cleaved-caspase3明显增高;Caspase3活性检测提示其活性加强。结论:Wortmannin通过抑制PI3K/Akt信号通路活性,激活Caspase3,在体外抑制髓母细胞瘤增殖和诱导凋亡来发挥其抗肿瘤活性。

Objective：To investigate the effects of proliferation and apoptosis induced by Wortmannin in human medulloblastoma cell line Daoy.Methods：Human medulloblastoma cell line Daoy were treated by Wortmannin at different concentration（0.1,0.25,0.5μmol/L） for 24,48,72h,the inhibition rates were determined by CCK-8 assay.Then,the apoptosis rates were evaluated by flow cytometry via Annexin-V/PI labeling.The protein expressions of Akt,p-Akt,Cleaved-caspase3 were detected by western blot analysis.The activity of caspase3 was measured by caspase3 activity assay kit.Results：Wortmannin could inhibit the proliferation of Daoy cells at different concentration（0.1,0.25,0.5μmol/L）,and which was in time and dose-dependent manner.After treatment for 48h,flow cytometry found that the apoptosis rates of Daoy cells treated by 0.1,0.25,0.5μmol/L Wortmannin were（21.16 ± 2.49）%,（31.02 ± 0.71）%,（44.61 ± 1.47）%,respectively.It was significantly different,compared with control group.The results of western blot showed that the protein expression of p-Akt was down-regulated,but up-regulation of Cleaved-caspase3 was obvious,which was confirmed by caspase3 activity detection.Conclusion：Wortmannin could inhibit proliferation and induce apoptosis of human medulloblastoma cells via down-regulation of the activity of PI3K/Akt signaling pathway,then up-regulation of caspase3 in vitro.