Delphinidin 联合自噬抑制剂抗HER-2 阳性乳腺癌效应及分子机制研究

It has been demonstrated that delphinidin not only has potential anti-cancer effects, but also induce tumor cells autophagy which is a self protective response under stress conditions. However, it is still unknown whether delphinidin synergistically inhibit HER-2 positive breast cancer with autophagy inhibitor. Our previous studies have been suggested that delphinidin can combine with HER-2 receptor, and the preliminary experiment suggested that delphinidin also can inhibit the proliferative ability of MDA-MB-453 breast cancer cell, down-regulate the ratio of LC3-Ⅰ/LC3-Ⅱ. In the present study, we will first utilize HER-2 positive breast cancer cells (in vitro) and xenograft model (in vivo) to further verify the inhibitory effect in breast cancer by blocking the MAPK and NF-κB pathways mediated by HER-2 receptor. Then, we investigate the delphinidin-induced autophagy by blocking mTOR and activating MAPK pathway. Finally, we explore the autophagy inhibition in vitro and in vivo to increase the sensitivity of HER-2 positive breast cancer on delphinidin. The results will not only further clarify the inhibitory effect and the underlying molecular mechanism of delphinidin in HER-2 positive breast cancer, but also provide a new strategy in tumor treatment through targeting autophagy inhibition.

业已证实，飞燕草素（delphinidin）不但具有较强的抗癌作用，而且能诱导肿瘤细胞产生保护性自噬反应。但delphinidin联合自噬抑制剂能否增强抗HER-2阳性乳腺癌效应？尚未证实。项目组前期发现，delphinidin能与HER-2受体结合，预实验提示delphinidin抑制MDA-MB-453细胞增殖能力，降低LC3-I/LC3-Ⅱ比值。本项目在此基础上，首先运用移植瘤模型和细胞模型，体内体外进一步证实delphinidin通过阻断MAPK和NF-κB通路抗HER-2阳性乳腺癌的分子机制；然后体外探讨delphinidin通过阻断mTOR通路以及活化MAPK通路诱导自噬；最后体内体外研究靶向自噬抑制，增加HER-2阳性乳腺癌对delphinidin的敏感性。研究结果不但明确delphinidin抗HER-2阳性乳腺癌的分子机制，而且从细胞应答角度为肿瘤化疗增敏提供新措施。

本项目研究了花青素主要活性单体Delphinidin抑制HER-2受体阳性乳腺癌的效应及自噬在其中的作用。项目通过动物实验及体外研究，在动物、细胞、分子水平上证实了Delphinidin能够抑制HER-2乳腺癌细胞的增殖及转移，并诱导其保护性自噬。而抑制自噬则可以增强Delphinidin抗HER-2阳性乳腺癌效应，同时确定了Delphinidin的抗肿瘤作用与调节HER-2受体下游信号通路有关。.本项目研究过程中，首先以HER-2受体阳性乳腺细胞株MDA-MB-453建立了Balb/c裸小鼠异种移植瘤模型，证实了Delphinidin能有效抑制HER-2阳性乳腺癌细胞MDA-MB-453异种移植瘤生长，抑制异种移植瘤、肺转移瘤并促进裸鼠肺转移瘤凋亡。同时在体外证实了Delphinidin通过阻断ERK和NF-κB通路，激活JNK通路诱导乳腺癌细胞MDA-MB-453（ER-/HER-2+）、BT-474（ER+/HER-2+）和MCF-7（ER+/HER-2-）线粒体凋亡从而发挥抗增殖作用。.之后，以MDA-MB-453细胞为研究对象，通过电镜观察、免疫荧光、免疫印迹等试验，体外证实Delphinidin在抗 HER-2阳性乳腺癌的同时，可以诱发MDA-MB-453细胞自噬。其作用机制是通过抑制mTOR和激活AMPK通路产生的。.最后，以MDA-MB-453细胞为研究对象，证实 Delphinidin可以诱导MDA-MB-453细胞的保护性自噬。与Delphinidin单独处理组相比，自噬抑制剂联合处理组与Delphinidin单独处理组相比，细胞活性显著降低，提示阻断自噬能促进Delphinidin诱导MDA-MB-453细胞凋亡的作用，增加抗 HER-2阳性乳腺癌效应。.本项目按计划完成了预期研究目标。受项目资助，项目组在国内外期刊公开发表论文18篇，其中SCI收录9篇。课题负责人以通讯作者发表论文13篇，其中SCI收录5篇，中文核心期刊8篇，交流会议论文12篇。培养研究生5名。.该研究将进一步解释日常膳食中富含花青素类食物的抗癌作用，扩展花青素类植物天然产物在慢性病疾病防治和健康促进领域的应用，为安全有效地辅助治疗HER-2阳性乳腺癌转移提供了新的思路和作用靶点。有利于从膳食营养角度探讨肿瘤“化学预防”的新途径。