Periostin-avβ3-FAK-PI3K通路在褐藻糖胶抗乳腺癌转移中的作用机制研究

The malignance and invasiveness of triple-negative breast cancer is high, the prognosis is poor. The effect of endocrine and target therapy was poor. There is very necessary to find a natural active drug with less toxicity. Fucoidan is a complex sulfated polysaccharide that is found in brown algae. Recently, the diverse biological activities of fucoidan have been studied intensively. In particular, the antitumor activity has recently attracted considerable attention. Our recent studies have demonstrated that fucoidan has an anti-tumor effect on breast cancer（Plos One，2012; Nutrition and Cancer, 2013）. We found that fucoidan could strongly inhibit the metastatic of breast cancer, and this effect might be related to the Periostin-αvβ3-FAK-PI3K pathway. The goal of this experiment is to investigate the effect and mechanism of fucoidan on the metastatic of MDA-MB-231 breast cancer in vivo and in vitro. The signal molecules involved in Periostin-αvβ3-FAK-PI3K pathway are analyzed by fluorescence quantity RT-PCR and Western Blot. The siRNA and FAK inhibitor will be applied to determine the effect of fucoidan on Periostin-αvβ3-FAK-PI3K pathway. The targeting molecules of fucoidan will be further identified by the affinity labeling and proteins flight mass spectrometry technology. Thereby, the study may determine the molecular mechanism of anti-tumor activity of fucoidan and provide theory and experimental evidence for prevent and therapy of fucoidan on breast cancer.

三阴性乳腺癌恶性程度高、侵袭性强，对内分泌及靶向治疗的效果均欠佳。褐藻糖胶（Fucoidan）是从海带中提取纯化的天然活性分子。申请者前期研究证实Fucoidan能减少乳腺癌组织内新生血管，抑制其肺转移。在乳腺癌的转移中起重要作用的Periostin蛋白通过αvβ3-FAK-PI3K信号通路调控肿瘤细胞的增殖、凋亡及血管生成，而我们前期检测的survivin、bcl-2、cyclinD1等均是该通路的下游靶基因，据此推测Fucoidan可能通过该通路抗乳腺癌转移，但还缺乏实验依据。本项目拟利用三阴性乳腺癌细胞系和动物模型明确Fucoidan抗乳腺癌转移的分子作用，探讨其对Periostin-αvβ3-FAK-PI3K通路的影响，并应用siRNA和通路抑制剂下调通路进行验证；采用亲和标记法结合蛋白飞行质谱技术探究其作用靶点，筛选膜上受体,为Fucoidan抗乳腺癌转移的分子机制提供实验依据。

褐藻糖胶（Fucoidan）是从海带中提取纯化的天然活性分子，其化学成分是以硫酸岩藻糖为主的多糖体，前期研究表明，褐藻糖胶对小鼠乳腺癌细胞有抑制作用，并减少其肺转移。本研究利用人三阴性乳腺癌细胞系明确Fucoidan抗乳腺癌转移的分子作用，探讨其对Periostin-αvβ3-FAK-PI3K通路的影响；同时应用二甲基苯蒽建立大鼠乳腺癌动物模型，观察长期褐藻糖胶干预治疗对大鼠生理病理改变及肿瘤发生率、潜伏期及肿瘤生长的抑制作用及其免疫调节作用，并且收集大鼠粪便用于检测褐藻糖胶对肠道菌群的影响。体外实验结果发现，褐藻糖胶能够抑制MD-MBA-231细胞增殖，下调PI3K/AKT/GSK3β通路，减少β-catenin的核转位，诱导乳腺癌细胞发生caspase依赖和非caspase依赖的线粒体凋亡；褐藻糖胶作用后细胞上清液中Periostin的水平明显下降，Western blot结果显示，褐藻糖胶可以降低FAK、PI3K和AKT的磷酸化，并减少下游靶蛋白MMP-2等的表达，下调间质分子Vimentin等的水平，上调上皮分子E-cadherin等的表达；褐藻糖胶还可以下调HIF-1α通路抑制癌细胞迁移。血清药理学实验显示，褐藻糖胶可显著抑制人乳腺癌MCF-7细胞增殖，并且能发挥促凋亡和抗迁移作用。在体内实验中，应用二甲基苯蒽诱发乳腺癌建立大鼠乳腺癌动物模型，膳食补充褐藻糖胶4个月后发现褐藻糖胶可以抑制肿瘤发生和生长，通过下调PD1/PDL1通路，抑制肿瘤组织Foxp3的表达，下调CD3+Foxp3+的调节性T细胞（Treg细胞），增加NK细胞活性，增强乳腺癌大鼠的免疫功能。另外，褐藻糖胶干预改变了乳腺癌大鼠的肠道菌群结构，增加肠道菌群多样性，并上调小肠粘膜Occludin等紧密连接蛋白水平，改善乳腺癌大鼠肠粘膜屏障功能。.本项目完成了预期目标，阐明了褐藻糖胶对乳腺癌细胞凋亡诱导和抑制转移作用的具体机制，明确了其下调Periostin-αvβ3-FAK和HIF-1α通路抑制癌细胞的侵袭和转移，并且通过PI3K/AKT/GSK3β通路下调β-catenin的作用；体内实验证实膳食补充褐藻糖胶可以抑制大鼠乳腺癌的发生、增强抗肿瘤免疫，改善粪便微生物群组成、修复肠道屏障功能，揭示了膳食褐藻糖胶防治乳腺癌的作用机制，为开发以褐藻糖胶为功效因子的保健食品和药物提供理论支持。