和厚朴酚诱导乳腺癌细胞RIP1非依赖的程序性坏死启动的分子机制研究

A variety of "classical" initiation mechanisms are known to be different in the mode of cell programmed necrosis. On this basis, we also have reported for the first time that honokiol (HNK) induces a programmed necrotic cell death in breast cancer through the mitochondrial permeability transition pore. And we further found that there are several significant differences between the honokiol-induced necrosis and the other types of “classical” necrotic cell death which were reported recently: The necrotic process was RIP1-independent and RIP3-dependent; In some previously known as “necrotic resistance” cell lines, honokiol still could induce the initiation of necrosis etc. So our present study will focus on the research of the composition of the necrotic initiation complex and HNK specific binding proteins. We plan to search and identify the detailed composition of the initiation complex by co-immunoprecipitation and tandem affinity purification; To scan and identify the HNK specific binding proteins by idTarget structure prediction and siRNA pool searching; Further to investigate the binding parterner and function of the above identified proteins. At present, there is no associated report about the HNK specific binding proteins and composition of the initiation complex of "non-classical" RIP1-independent programmed necrotic death inducing by a single small molecule drug-honokiol. This project will be helpful in improving the initiation mechanisms of programmed cell necrosis in breast cancer.

至今已报道肿瘤细胞存在多种不同的程序性坏死分子机制，本课题组曾首次报道：和厚朴酚（HNK）可诱导乳腺癌细胞发生线粒体相关的程序性坏死，且该坏死模式与已知的几类经典程序性坏死有明显不同：具有坏死相关蛋白RIP1非依赖而RIP3依赖性；对于 “坏死耐受”的肿瘤细胞仍能诱导坏死发生等。本项目拟对这一“非经典性”程序性坏死启动的分子机制，包括起始复合物组成以及HNK特异结合蛋白进行深入研究。我们拟运用免疫共沉淀及串联亲和纯化方法寻找并鉴定坏死起始复合物的具体蛋白组成；运用idTarget模拟、siRNA pool筛选并鉴定HNK潜在的细胞内靶蛋白；并进行蛋白间结合位点、功能鉴定等相关实验。目前国内外尚无报道HNK诱导的RIP1非依赖的程序性坏死中，起始复合物的具体组成以及HNK特异性结合蛋白。本项目有助于完善HNK诱导的乳腺癌细胞程序性坏死启动阶段的分子机制，为抗肿瘤治疗提供新的思路。

至今已报道肿瘤细胞存在多种不同的程序性坏死分子机制，我课题组前期研究发现和厚朴酚可诱导肿瘤细胞发生Cyclophilin D（CypD）介导的程序性坏死。且该坏死模式与已知的几类经典程序性坏死有明显不同：具有坏死相关蛋白RIP1非依赖而RIP3依赖性；对于 “坏死耐受”的肿瘤细胞仍能诱导坏死发生等。本研究在国家自然科学基金支持下利用免疫共沉淀（IP）联合液相色谱‐串联质谱联用（LC-MS/MS）分析鉴定和厚朴酚诱导乳腺癌细胞非经典性程序性坏死启动阶段“坏死复合物”。利用靶点稳定性的药物亲和反应（DARTS）用于寻找Honokiol主要作用靶点；首先我们构建完成 Flag-RIP3 融合表达稳定转染的细胞系。后用和厚朴酚40µg/ml作用细胞6小时发生程序性坏死（AV-FITC/PI双染流式细胞仪检测显示坏死）。我们进一步在MCF-7细胞中验证得到和厚朴酚可诱导MCF-7细胞发生RIP3-MLKL通路相关的程序性坏死。同时我们利用免疫共沉淀（IP）联合液相色谱‐串联质谱联用（LC-MS/MS）发现热休克蛋白HSP90及其伴侣蛋白CDC37参与了坏死复合物的组成。进一步我们利用靶点稳定性的药物亲和反应（DARTS）也发现HSP90是和厚朴酚的潜在靶点之一。综上HSP90将是今后我们坏死复合物及和厚朴酚潜在靶点的主要研究目标分子。本项目首次对和厚朴酚诱导的“非经典性”程序性坏死的起始复合物组成及和厚朴酚特异性结合靶蛋白进行研究；有助于丰富与完善目前知之甚少的细胞程序性坏死的分子机制，并有可能揭示全新的特殊程序性坏死的起始与调控机制。在课题执行阶段，发表标注基金资助的SCI论文2篇；另有1篇已经接收，研究进行中培养博士研究生1名。