基于RAC1/NADPH信号通路对蒽醌化合物GXHSWAQ-3增强鼻咽癌辐射敏感性的时空调控机制

The domestic and foreign research and our studies supported RAC1/ NADPH pathway was involved in cell oxidative damage by reactive oxygen species(ROS) during radiation process. But the study on radiosensitizer regulation of the RAC1/ NADPH pathway is very little. GXHSWAQ-3 , a radiosensitizing activity compound ,was synthesised by our preliminary work. To explore the potential mechanisms involved of GXHSWAQ-3 enhanced different differentiation nasopharyngeal carcinoma cells on X-ray sensitive，the expression of RAC1 and the subunites of NADPH in the RAC1/NADPH pathway and the cell viability are detected using Western blotting, co-immunoprecipitation , flow cytometry . FRET combine with confocal microscopy will be used to dynamic monitoring the distribution of RAC1 activation in cells location, distribution and NADPH oxidase complex formation in different differentiation nasopharyngeal carcinoma cells, to reveal the key role that GXHSWAQ-3 targeting RAC1/NADPH pathway in the regulation of RAC1 interaction with subunites of NADPH oxidase. Over-expressing RAC1 and RAC1 RNAi cell will be established and inoculated in nude mice to investigate RAC1/NADPH pathway in the regulation of nasopharyngeal carcinoma cellular radiosensitivity in vivo. The research results will provide a good platform for dynamic monitoring signal transduction pathway of small molecular medicine, screening the lead compound of radiosensitizer and a novel molecular therapeutic target in nasopharyngeal carcinoma.

国内外研究及本课题组发现，RAC1/NADPH通路与辐射产生活性氧致细胞氧化性损伤密切相关。但RAC1/NADPH通路的调控与辐射敏感关系的研究甚少。本课题以具有放射增敏活性GXHSWAQ-3和不同分化能力的鼻咽癌细胞为对象，采用蛋白印迹、免疫共沉淀、流式细胞术检测GXHSWAQ-3联合射线作用细胞后， RAC1/NADPH信号通路上各亚基的表达和细胞存活情况。FRET结合激光共聚焦显微镜动态监测GXHSWAQ-3作用后不同时点RAC1与NADPH酶亚基结合、转位、分布及蛋白复合物形成的情况，明确GXHSWAQ-3调控不同分化鼻咽癌细胞RAC1与NADPH酶亚基相互作用的时空关系。构建RAC1低表达和过表达的细胞并接种于裸鼠，明确体内RAC1/NADPH信号通路调控与鼻咽癌辐射敏感的关系，建立分子成像研究小分子药物与调控靶蛋白信号通路的新方法，开创一个筛选鼻咽癌放射增敏剂全新的研究平台。

本课题以蒽醌化合物GXHSWAQ-3和不同分化能力的鼻咽癌细胞为研究对象，采用Western blotting、流式细胞仪、激光共聚焦显微镜检测GXHSWAQ-3联合射线作用细胞后，对不同分化鼻咽癌细胞RAC1与NADPH酶亚基相互作用调控的时空关系。结果发现 RAC1/NADPH信号通路上各亚基RAC1、P47、P67的表达均升高，细胞内的活性氧浓度升高，细胞凋亡率增加。对于不同分化能力的鼻咽癌细胞，RAC1激活剂PMA在照射前后均能增加细胞内的活性氧浓度，GXHSWAQ-3联合照射后表现为增加细胞内的ROS浓度；RAC1抑制剂DPI联合照射后对细胞内ROS浓度的影响均表现为抑制作用。激活RAC1/NADPH信号通路联合照射后，NADPH氧化酶下游蛋白如NFκB-p50、P-AP-1、P-P38，JNK等的表达均上调。这种作用对于高分化的CNE-1细胞更为明显。构建沉默RAC1和过表达RAC1的鼻咽癌细胞并接种于裸鼠，结果发现：RAC1(+)组和RAC1(-)组的瘤体生长速度略快于对照组。经4Gy 照射后，各组瘤体体积均比相应未照射组有不同程度的缩小，其中RAC1(+)和RAC1(-)组的瘤体与照射前相比急剧缩减。瘤重下降率RAC1(+)组最为明显，高达68%。过表达RAC1联合照射处理的瘤体组织出现核固缩，核碎裂，呈现胞质红染、细胞坏死形态学改变。电镜下可见肿胀的线粒体并呈空泡样，部分细胞溶解坏死，细胞膜破裂，核碎裂，细胞器损坏消失及大量细胞碎片。P67，P47蛋白表达水平均明显上调，凋亡细胞增多。体内外实验均提示RAC1为蒽醌化合物的作用靶点，RAC1/NADPH信号通路可能是与放射敏感相关的重要通路。激光共聚焦显微镜下动态监测GXHSWAQ-3作用鼻咽癌细胞后,随着时间推移，RAC1与NADPH酶亚基P47结合、转位并发生能量共振，出现FRET现象，从分子水平上阐明了NADPH酶各亚基间的相互作用。成功构建含RAC1启动子荧光素酶报告系统，建立了RAC1/NADPH 信号转导途径为基础的新型放射增敏药物的筛选可视化细胞模型。