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目的探究橙皮素对P-选择素介导的MDA-MB-231乳腺癌细胞转移的影响及其作用机制。方法运用计算机虚拟对接技术体外评价橙皮素与P-选择素的结合能力;采用MDA-MB-231乳腺癌细胞为模型,MTS法观察不同浓度的橙皮素/P-选择素对MDA-MB-231生长能力的影响;ELISA法检测橙皮素对活化的血小板表面P-选择素分泌的影响;黏附实验考察橙皮素对P-选择素介导的MDA-MB-231与内皮细胞黏附的影响;Transwell实验分析橙皮素对P-选择素促进MDA-MB-231乳腺癌细胞迁移的影响;采用Western blotting法考察橙皮素对MDA-MB-231细胞表面糖蛋白(Mucin-1)、整合素(Integrinβ3、β1)及基质金属蛋白酶(MMP-2、MMP-9)蛋白表达的影响;进一步分析橙皮素对MDA-MB-231细胞Integrin-MMP信号通路的影响,阐明橙皮素抗肿瘤转移的作用机制。结果计算机虚拟对接技术证实橙皮素与P-选择素有较好的结合能力;在体外橙皮素能剂量依赖性抑制MDA-MB-231增殖及其与内皮细胞的黏附;橙皮素能降低活化的血小板表面P-选择素的分泌;迁移实验中橙皮素能显著抑制P-选择素介导的MDA-MB-231乳腺癌细胞迁移;降低P-选择素诱导的细胞表面糖蛋白Mucin-1、Integrinβ3、Integrinβ1及MMP-2、MMP-9蛋白的表达。结论橙皮素具有抑制MDA-MB-231乳腺癌细胞生长能力,阻断P-选择素诱导的乳腺癌MDA-MB-231肿瘤细胞迁移以及其与内皮细胞的黏附,其机制为橙皮素通过竞争性结合P-选择素,阻断其与Mucin-1的结合,抑制PI3K-AKT-Paxillin-FAK-Src信号通路,下调P-选择素调控的Integrins及MMP-2、MMP-9表达。
Objective To investigate the effect of hesperetin on P-selectin-mediated MDA-MB-231 breast cancer cell metastasis and its mechanism. Methods The binding ability of hesperetin to P-selectin was evaluated in vitro by computer virtual docking technology. The MDA-MB-231 breast cancer cells were used as the model. The effect of different concentrations of hesperetin / P- -231 growth; the effect of hesperetin on the activity of P-selectin on activated platelets was detected by ELISA; the effect of hesperetin on P-selectin-mediated adhesion of MDA-MB-231 to endothelial cells The effect of hesperetin on the migration of MDA-MB-231 breast cancer cells by P-selectin was analyzed by Transwell assay. The effect of hesperetin on the expression of Mucin-1, Integrinβ1 and MMP-2, MMP-9 and MMP-9 in MDA-MB-231 cells were analyzed. The effects of hesperetin on Integrin-MMP signaling pathway in MDA-MB-231 cells were further analyzed. Mechanism of tumor metastasis. Results The computer virtual docking technology showed that hesperetin and P-selectin had better binding ability. In vitro, hesperetin could inhibit the proliferation of MDA-MB-231 and its adhesion to endothelial cells in a dose-dependent manner; Secretion of P-selectin on the surface of activated platelets; Hesperetin significantly inhibited the P-selectin-mediated migration of MDA-MB-231 breast cancer cells in migration experiments; decreased P-selectin-induced cell surface glycoprotein Mucin- 1, Integrinβ3, Integrinβ1 and MMP-2, MMP-9 protein expression. Conclusion Hesperetin can inhibit the growth of MDA-MB-231 breast cancer cells and block the migration of P-selectin-induced MDA-MB-231 breast cancer cells and its adhesion to endothelial cells. P-selectin was competitively combined to block its binding to Mucin-1, inhibit PI3K-AKT-Paxillin-FAK-Src signaling pathway and down-regulate P-selectin-regulated Integrins and MMP-2, MMP-