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（Integrin β3、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、Integrin β3、Integrin β1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 study the effect of hesperetin on the migration of P-selectin mediated MDA-MB-231 breast cancer cells and its mechanism. Methods Using computer virtual docking to evaluate the capacity of hesperetin binding to P-selectin in vitro; MTS test was observed with different concentration of hesperetin or P-selectin on the growth capacity of MDA-MB-231; The effect of hesperetin on P-selectin secretion by activated platelet was detected by Elisa kit; Adhesion experiments examined hesperetin on P-selectin-mediated MDA-MB-231 and endothelial cell adhesion; Transwell experiment was performed to analyze the effect of P-selectin on MDA-MB-231 breast cancer cell migration affected by hesperetin; Western blotting investigated MDA-MB-231 cell surface glycoprotein Mucin-1, Integrin β3, β1 and matrix metalloproteinase expression of MMP-2 and MMP-9 protein expression influenced by hesperetin; impact of hesperetin on MDA-MB-231 cell integrin-matrix metalloproteinase signaling pathway was analyzed to clarify the anti-tumor metastasis mechanism of hesperetin. Results Hesperetin inhibited P-selectin-induced MDA-MB-231 cell migration and reduced HUVEC-breast cancer cell adhesion. Hesperetin down-regulated the expression of β1 and β3 integrins, and MMP-2 and MMP-9 at protein levels in MDA-MB-231 cells. Conclusion Hesperetin can inhibit the growth capacity of MDA-MB-231 breast cancer cells, block P-selectin-induced breast cancer MDA-MB-231 tumor cell migration and adhesion, and the mechanism for hesperetin is through competitive P-selectin binding to Mucin-1. Subsequently, hesperetin could block PI3K/AKT/Paxillin/FAK/Src signaling pathway and down-regulate P-selectin mediated Integrins, MMP-2, and MMP-9 expression.

江苏省高校自然科学基金资助项目（15KJB310018）