目的:观察毛钩藤碱对缺氧诱导的人乳腺癌MCF-7细胞迁移和侵袭能力的影响,并探讨其分子机制.方法:选取MCF-7细胞作为研究对象,采用CCK-8法检测毛钩藤碱的细胞毒性作用;采用细胞划痕实验测量细胞迁移率;采用Transwell侵袭实验检测细胞侵袭力;采用Western blot法检测缺氧诱导因子1α(hypoxia-inducible factor-1α,HIF-1α)、Snail、E-钙黏蛋白(E-cadherin)和基质金属蛋白酶9(matrix metalloproteinase-9,MMP-9)的蛋白表达水平;采用RT-PCR法检测HIF-1α的mRNA水平.结果:自32μmol/L开始,随着毛钩藤碱浓度的升高,MCF-7细胞存活率明显降低(P<0.05),IC50为62.82μmol/L;在缺氧条件下,MCF-7细胞的迁移和侵袭能力显著增强(P<0.05),HIF-α、Snail和MMP-9蛋白表达水平明显升高(P<0.05),E-cadherin蛋白表达水平明显下降(P<0.05),HIF-1α的mRNA水平也显著升高(P<0.05);除缺氧MCF-7细胞中HIF-α的mRNA水平未受影响外,上述效应均被毛钩藤碱(16μmol/L)明显抑制(P<0.05).结论:毛钩藤碱可在体外抑制缺氧诱导的人乳腺癌MCF-7细胞迁移和侵袭,这可能与毛钩藤碱下调HIF-1α、Snail和MMP-9蛋白表达水平,上调E-cadherin蛋白表达水平有关.%AIM:To investigate the effect of hirsutine on hypoxia-induced migration and invasion abilities of human breast cancer MCF-7 cells and its possible mechanism. METHODS:CCK-8 assay was employed to detect the cyto-toxic effect of hirsutine on the MCF-7 cells. Cell migration was observed by wound healing assay,and cell invasion ability was measured by Transwell invasion assay. Western blot was used to analyze the protein levels of hypoxia-inducible factor-1α(HIF-1α),Snail,E-cadherin and matrix metalloproteinase-9 (MMP-9). The mRNA levels of HIF-1α was detected by RT-PCR. RESULTS:Hirsutine remarkably reduced the cell viability from 32 μmol/L(P<0.05),and the IC50value was 62.82 μmol/L. In hypoxia state,MCF-7 cells showed more powerful capabilities of migration and invasion (P<0.05), higher protein levels of HIF-1α,Snail and MMP-9 (P<0.05),lower protein level of E-cadherin(P<0.05),and higher mRNA level of HIF-1α (P<0.05). These hypoxia-induced effects were all inhibited by hirsutine at 16 μmol/L (P<0.05),apart from the mRNA level of HIF-1α. CONCLUSION:Hirsutine inhibits hypoxia-induced migration and inva-sion in human breast cancer MCF-7 cells most likely via down-regulation of the protein levels of HIF-1α,Snail and MMP-9,and up-regulation of the protein level of E-cadherin.
展开▼
