以Snail调控界面为靶标解析紫珠属中草药的抗乳腺癌转移作用物质基础及其作用机制

Breast cancer is the first leading cause of death in women patients with cancer, and most of them were dead because of the distance spread of cancer cells. Although it is the most vicious aspect in breast cancer, but the effective strategies to control metastasis still lack. However, Chinese Medicine has certain advantages in this field, and some traditional Chinese remedies can help with these patients. Snail is an important transcription factor, which contains a highly conserved domain. This protein plays a critical role in the process of formation of metastatic lesions from primary site in breast cancer. In our previous study, it was found that iridoids, isolated from callicarpa nudiflora inhibited activation of Snail and metastasis of breast cancer cells. Led by these concepts, we here hypothesize that iridoids from Callicarpa suppress metastasis of breast cancer through downregulating activities of Snail. The specific aim of this program is evaluation of anti-metastatic effects of herbs in Callicarpa and clarification of their mechanisms by targeting interaction between Snail and its target genes in breast cancer. To test our hypothesis, we will: (1) extract iridoids from callicarpa nudiflora, Callicarpa kwangtungensis Chun and Callicarpa kochiana, which have tight relationship, and get several candidate compounds; (2) evaluate anti-metastatic and Snail-inhibitive properties of these candidates in vivo and in vitro, and confirm the leading compounds; (3) analyze the mechanisms of Snail inhibition of the leading compounds. These investigations will provide a foundation for elucidation of effects and the material base of herbs from Callicarpa to prevent distant spread of breast cancer cells.

清热解毒药历来是中药控制癌转移与复发的重要组成部分。紫珠属中药多具有清热解毒、散瘀止血的功效，在江西民间广泛使用，其抗癌活性日益受到关注。Snail是一种高度保守的转录因子，在调控癌细胞转移信号传递与转移行为中发挥重要作用，已成为抗转移药物设计的新靶点。前期研究证实裸花紫珠乙酸乙酯部位具有抗转移作用，其中一个环烯醚萜具有较强的体外抗转移活性，作用机制涉及Snail 抑制。本研究拟在此基础上，以Snail调控界面为靶标，解析紫珠属中草药的抗转移物质基础及作用机制。首先从裸花紫珠、广东紫珠和枇杷叶紫珠等生缘关系接近的紫珠中分离环烯醚萜，筛选其Snail抑制活性；其次采用多种体内外模型，验证其抗转移和Snail抑制活性，确定先导物。最后，从Snail上游信号转导、snail基因自身及下游靶基因表达调控入手，探索先导物的Snail抑制机制。本研究将为从中草药中发现新型抗转移药物提供理论与实践基础。

中医认为热毒蕴结是肿瘤发生、发展最常见的病因病机。因此，清热解毒药历来是中药控制癌转移与复发的重要组成部分。紫珠属中草药具有清热解毒、散瘀止血的功效，在江西民间广泛使用，其抗癌活性日益受到关注。Snail是一种高度保守的转录因子，在癌细胞转移信号传递与转移行为中发挥重要的整合作用，已成为抗转移新药设计的靶点之一。本研究在前期研究基础上，以Snail调控界面为靶标，解析紫珠属中草药的抗肿瘤转移物质基础及作用机制。.化学方面，对几种紫珠属植物进行了系统分离，从中总计完成了黄酮类、环烯醚萜类在内的近60个化合物的提取分离。.活性筛选方面，我们采用乳腺癌细胞的体外迁移、侵袭和增殖实验，对分离得到的化合物进行活性筛选，确定环烯醚萜类化合物2-14和2-24对乳腺癌细胞MDA-MB-231和MDA-MB-468具有较强的体外抗转移活性。.药效学研究方面，细胞横向迁移侵袭实验结果表明，2-14和2-24的各给药组的迁移、侵袭细胞率与模型组相比显著减少（P<0.01）；纵向迁移侵袭实验结果表明，2-14和2-24的各给药组的迁移、侵袭细胞数与模型组相比显著减少（P<0.01）；另外，与模型组相比，2-14和2-24可显著干扰细胞骨架蛋白F-Actin排列，使其排列紊乱，细胞伪足减少；体外粘附实验表明，不同浓度的2-14和2-24给药组可降低乳腺癌细胞与细胞外基质蛋白之间的粘附（P<0.05）。.机制研究方面，化合物2-14和2-24可通过抑制Snail的活化，从而下调EGF所介导的上皮间质转化。进一步的研究表明，化合物2-14和2-24可降低PI3K/Akt通路上PI3K110β、PDPK1、P-PDPK1（Ser241）、pan-akt、P-Akt(Ser473)、GSK3β(Tyr216) 等蛋白的表达；免疫荧光实验结果显示，与模型组相比，胞浆蛋白PI3K110β的蛋白表达下调。表明，环烯醚萜类化合物2-14和2-24的抗转移作用与PI3K/Akt信号通路有关。.本项目完成了几种紫珠属植物中化学成分的系统分离与抗乳腺癌转移活性研究，并对其中活性较好的环烯醚萜类化合物2-14和2-24的药效进行了系统研究，并深入探讨了这些化合物的作用机理，为新型抗肿瘤转移药物的创制奠定了基础。