阻断PI3K/Akt信号通路对低氧微环境中BCSCs微球体细胞增殖的影响  

Effect of blocking the PI3K/Akt signalling pathway on cell proliferation of the aggregated breast cancer stem cells in microspheres under hypoxic microenvironment

作　　者：屈洪波[1] 韩明利[1] 范原铭[1] 曾妮[1] 朱之坤[1] 吴诚义[1] 汤为学[2] 

机构地区：[1]重庆医科大学附属第一医院内分泌外科,重庆400016 [2]重庆医科大学病理生理学教研室,重庆400016

出　　处：《肿瘤》2013年第1期36-41,共6页Tumor

摘　　要：目的:探讨LY294002特异性阻断磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)/Akt信号通路对低氧条件下乳腺癌干细胞(breast cancer stem cells,BCSCs)微球体细胞增殖的影响。方法:通过无血清悬浮培养技术从人乳腺癌MCF-7细胞中筛选BCSCs微球体,应用免疫荧光染色检测BCSCs相关标志物CD44及CD133的表达,MTT法检测不同浓度LY294002处理后BCSCs微球体细胞的增殖情况,RT-PCR法检测BCSCs微球体细胞中缺氧诱导因子-2α(hypoxia inducible factor-2α,HIF-2α)mRNA的表达,蛋白质印迹法检测BCSCs微球体细胞中HIF-2α、PI3K和磷酸化Akt(phospho-Akt,p-Akt)蛋白的表达。结果:筛选获得的微球体细胞高表达BCSCs相关标志物CD44和CD133。LY294002能有效抑制低氧条件下BCSCs微球体细胞的增殖,抑制效应在药物作用后72～96h最为明显(P<0.05)。与低氧组比较,低氧+LY294002组微球体细胞中HIF-2αmRNA及PI3K、p-Akt和HIF-2α蛋白表达水平均明显下调,差异有统计学意义(P<0.05)。结论:LY294002通过特异性阻断PI3K/Akt信号通路而有效抑制低氧条件下BCSCs微球体细胞的体外增殖能力。Objective： To investigate the effect of LY294002, a P13K （phosphatidylinositol 3-kinase）- specific inhibitor that can block the P13K/Akt signalling pathway, on cell proliferation of aggregated human BCSCs （breast cancer stem cells） in microspheres under hypoxic microenvironment, lvlethods： Stem cell microspheres were screened from human breast cancer MCF-7 cells by serum-free suspension culture. The expressions of cancer stem cell markers -- CD44 and CD133 were detected by immunofluorescence staining method. The proliferative abilities of the aggregated BCSCs in microspheres exposed to different concentrations of LY294002 were detected by MTT method. The expression of HIF-2α （hypoxia-inducible factor-2α） mRNA in BCSCs in microspheres was detected by RT-PCR. The expressions of HIF-2α, P13K and phospho-Akt proteins were detected by Western blotting. Results： The aggregated BCSCs in microspheres expressed high levels of CD44 and CD133. The cell proliferation of BCSCs in microspheres was effectively inhibited by LY294002 under hypoxic microenvironment, and this effect was most apparent at 72-96 h after treatment with LY294002 （P 〈 0.05）. The expression levels of HIF-2α mRNA and P13K, phospho-Akt and HIF-2α proteins in BCSCs in microspheres exposed to hypoxic conditions and LY294002 were significantly lower than those only exposed to hypoxic conditions （P 〈 0.05）. Conclusion： P13K-specific inhibitor LY294002 can suppress the proliferative ability of aggregated BCSCs in microspheres by blocking P13K/Akt signalling pathway in vitro.

关 键 词：乳腺肿瘤 细胞低氧 微球体 乳腺癌干细胞 

分 类 号：R737.9[医药卫生—肿瘤]