木黄酮对MCF-7/HER-2细胞uPA表达影响
Effect of genistein on expression of uPA and activity of PTK in MCF7/HER-2 Cells
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摘要:目的 探讨植物化学物质染料木黄酮(genistein)抗HER-2/neu高表达乳腺癌血管生成的分子机制。方法 采用基因转染技术建立HER-2/neu高表达MCF-7乳腺癌细胞(命名为MCF-7/HER-2), 5×10-5mol/L染料木黄酮处理MCF-7/HER-2细胞24, 48, 72 h后, 应用western bolt、免疫沉淀、RT-PCR及激酶活性分析法检测genistein对MCF-7/HER-2乳腺癌细胞尿激酶型纤维蛋白溶酶原激活剂(urokinase-type plasminogen activator, uPA)表达、HER-2/neu受体蛋白磷酸化水平及蛋白酪氨酸激酶(PTK)活性变化。结果 MCF-7/HER-2细胞uPA的mRNA和蛋白表达量比MCF-7细胞uPA的mRNA和蛋白表达量高, HER-2/neu受体蛋白磷酸化水平和PTK活性增加, 染料木黄酮处理MCF-7/HER-2细胞后, uPA的mRNA和蛋白表达量下调, HER-2/neu受体蛋白磷酸化水平降低, PTK活性下降, 且这种作用具有时效性。结论 染料木黄酮能下调MCF-7/HER-2细胞HER-2/neu受体的PTK活性和蛋白磷酸化水平, 抑制uPA表达, 这可能是染料木黄酮抗乳腺癌血管生成的分子机制之一。Abstract:Objective To explore the molecular mechanism of anti-angiogensis in HER-2/neu-overexpressing breast cancer by genistein.Methods HER-2/neu negative expression breast cancer MCF-7 cells were transfected with HER-2/neu to establish HER-2/neu-overexpressing MCF-7 cells(named MCF-7/HER-2).Western blot, immunoprecipitate, reverse transcription-polymerase chain reaction(RT-PCR)and kinase activity analysis technicals were used to observe the expression of uPA and the protein phosphorylation of HER-2/neu receptor and the activity of protein tyrosine kinase(PTK)in MCF-7/HER-2 cells treated by genistein for 24, 48, 72 h.Results The expression of uPA was significantly higher in MCF-7/Her-2 cells than that in MCF-7 cells both in mRNA and protein.The expression of uPA mRNA and the protein, phosphorylation of HER-2/neu receptor and the activity of PTK also significantly were decreased after treated with 5×10-5 mol/L genistein, which had a time-dependence.Conclusion Genistein could down-regulate the activity of PTK and the protein phosphorylation of HER-2/neu receptor, and inhibit the expression of uPA in breast cancer cells, which is possible one of the molecular mechanisms of genistein anti-angiogenesis in breast cancer.
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