基于细胞热转移分析技术的蔓荆呋喃诱导宫颈癌细胞凋亡分子靶点研究

Cervical cancer is one of common gynecological malignant tumors seriously harmful to the health of women, incidence rate of global female malignant tumor second, second only to breast cancer. The abnormal properties of cancer cells for proliferation and apoptosis play a vital role in the formation and development of cervical cancer, the process of which can be stopped by promoting cell apoptosis. Our previous research displays that rotundifuran (RTF), one of active components isolated from the mature fruits of Vitex trifolia L. var. simplicifolia Cham., promoted apoptosis of cervical cancer cells at a lower dose，and bound to Bim protein by high through-put screening and MTT assay for gene silencing cells. In this project, we intend to screen and substantiate definitely the binding target of RTF for induction of apoptosis in human cervical carcinoma cells, and to investigate the regulative mechanism of apoptosis based on this target. Furthermore, the effects of RTF will be investigated on the target and proteins and genes expression associated with cell apoptosis. The following research methods will be applied in the investigation, including cell thermal shift assay, surfacep plasmon resonance technology, analysis of confocal fluorescence, gene transfection, RNA interference, western blot, Q-RT-PCR, ELISA, TUNEL, flow analysis, and immunohistochemistry based on the knowledge of molecular pharmacology, biochemistry, cell and molecular biology and other disciplines. The goals of this study are to provide novel technologies and methods for the research of molecular binding targets of compounds, to supply new thought and strategy for the prevention and treatment of cervical cancer, and also to lay a solid foundation further for the development of new traditional Chinese drugs with clear composition, specific targets, safety and effectiveness, and stability and controllability, which will have the demonstrating and promoting effects on the modernization and internationalization of traditional Chinese medicine.

宫颈癌是妇科常见恶性肿瘤，癌细胞异常增殖凋亡在宫颈癌形成和发展过程中起着至关重要的作用。前期通过高通量筛选和基因干扰研究发现，Bim蛋白可能是中药蔓荆子有效成分蔓荆呋喃诱导宫颈癌细胞凋亡的分子结合靶点。本课题拟综合运用分子药理学、生物化学和细胞分子生物学等学科知识，采用最新细胞热转移分析技术，结合表面等离子体共振、激光共聚焦分析、报告基因检测、基因转染、RNA干扰、western blot、Q-RT-PCR、ELISA、TUNEL、流式分析和免疫组化等方法，确证蔓荆呋喃诱导宫颈癌细胞凋亡的分子结合靶点，以及基于该靶点的凋亡调控机制。通过本项目的研究，以期为中药单体化合物分子作用靶点的研究提供新技术和新方法，为中药防治宫颈癌提供新思路和新策略，同时也为进一步开发成分清楚、靶点明确、安全有效、稳定可控的新型中药新药奠定基础，对中医药现代化和国际化具有示范和促进作用。

宫颈癌是妇科常见恶性肿瘤，发病率居女性恶性肿瘤第2位。本课题对中药蔓荆子有效成分蔓荆呋喃诱导宫颈癌细胞凋亡的作用靶点及机制进行了研究。结果显示，蔓荆呋喃明显抑制细胞活力，降低细胞线粒体膜电位，阻滞细胞在G2/M期，增加细胞凋亡率。蛋白质组学分析发现蔓荆呋喃引起 PI3K/AKT以及MAPK信号通路发生显著改变。Western blot分析发现蔓荆呋喃明显降低p-PI3K、p-AKT、p-ERK、Bcl-2、Bcl-xL表达，升高p-JNK、Apaf-1、cleaved-PARP、cleaved-Caspase 3、cleaved-Caspase 8、cleaved-Caspase 9、Bax和Bim蛋白表达。采用DARTS、蛋白质谱分析、CETSA、MST、基因干扰等技术发现CYR61为蔓荆呋喃作用靶点。动物实验结果表明，蔓荆呋喃明显降低荷瘤裸鼠肿瘤体积和重量，促进肿瘤细胞凋亡；但并不影响CYR61基因敲除细胞株裸鼠荷瘤体积、重量及凋亡细胞数量。实验结果提示，蔓荆呋喃能明显减少宫颈癌细胞增殖和诱导凋亡，抑制裸鼠荷瘤生长，作用机制为通过靶向CYR61，抑制PI3K/AKT和ERK信号通路，激活JNK信号通路，上调Bim和Bax，下调Bcl-2和Bcl-xL，从而激活线粒体凋亡通路，引起细胞凋亡。