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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。
AIM: To investigate the effect of LY294002 on the proliferation of microspheres in breast cancer stem cells (BCSCs) under hypoxic conditions by specifically blocking the phosphorylation of PI3K / Akt signaling pathway. Methods: BCSCs microspheres were screened from human breast cancer MCF-7 cells by serum-free suspension culture. The expression of CD44 and CD133, a marker of BCSCs, was detected by immunofluorescence staining. The expression of BCSCs microspheres after treated with different concentrations of LY294002 The expression of hypoxia inducible factor-2α (HIF-2α) mRNA in BCSCs microspheres was detected by RT-PCR. The expression of HIF-2α, PI3K and phosphorylated Akt (phospho-Akt, p-Akt) protein expression. Results: The microspheres obtained by screening highly expressed BCSCs related markers CD44 and CD133. LY294002 can effectively inhibit the proliferation of BCSCs microspheres under hypoxic conditions. The inhibitory effect of LY294002 was the most obvious at 72-96h (P <0.05). Compared with hypoxia group, the expression of HIF-2αmRNA and PI3K, p-Akt and HIF-2α protein in hypoxia + LY294002 group were significantly decreased (P <0.05). CONCLUSION: LY294002 effectively inhibits the proliferation of BCSCs microspheres in vitro by blocking the PI3K / Akt signaling pathway.