基于BAX/BAK非依赖细胞凋亡机制的特境真菌抗肿瘤活性物质研究

Cancer is one of the leading causes of death. Cancer cells have developed drug resistence by lowering the expression of apoptotic protein of BAX/BAK. In our early work , the applicant screened the apoptosis-inducing effect of two hundred culture extracts from the fungi collected in the Tibet Plateau using the BAX/BAK deficient MEF cells. As a result, the culture extracts of three stains were found to be active in inducing the cell apoptosis of BAX/BAK deficient MEFs and contain the rich secondary metabolites by HPLC profile analysis. In this project, the applicant will isolate and identify the bioactive compounds with apoptosis-inducing effect on BAX/BAK deficient cells by using chromatographic and spectroscopic techniques. Furthermore, the anti-cancer mechanism mainly concerning about the cell apoptosis and its related signaling pathway will be explored. Combined with the in vivo animal experiments, the efficacy and safety evaluation will be carried out as well. The fungal strains producing bioactive compounds will be identified by morphorlogical traits and DNA molecular analysis. The fermentation process will be optimized in order to maximize the production rate of new bioactive compounds. This project is to discover novel anti-cancer leading compounds for drug development and new chemcial probes with new action mechanism for chemical biology investigation.

癌症是危害人类生命的主要疾病之一。肿瘤细胞可以通过降低凋亡蛋白BAX/BAK的表达水平产生耐药。本课题在前期研究中利用BAX/BAK双缺失耐药细胞株对200余株青藏高原特境真菌发酵提取物进行诱导细胞凋亡活性筛选，并结合HPLC指纹图谱发现了3株活性较好、次级代谢产物丰富的菌株。在此基础上，将应用现代分离及光谱技术发现结构新颖的不依赖BAX/BAK基因的抗肿瘤活性化合物；并针对分离到的强活性次级代谢产物围绕细胞凋亡相关蛋白及其信号通路开展作用机制研究；结合体内动物实验开展体内药效、安全性初步评价；在此基础上通过培养基优化、菌种改造等技术提高活性次级代谢产物的产率，为深入研究提供足量样品。研究工作充分挖掘青藏高原特境真菌中新结构的抗肿瘤活性化合物，为抗肿瘤新药研发提供先导化合物，为凋亡机制研究提供工具分子。

恶性肿瘤严重威胁人类健康，肿瘤细胞耐药已成为临床重大挑战。本课题利用BAX-/-/BAK-/-耐药细胞株和胃癌、肝癌等细胞系对青藏高原来源真菌发酵提取物进行细胞毒和诱导细胞凋亡活性筛选，获得6株活性较好、次级代谢产物丰富的菌株。通过对6株目标活性真菌进行放大发酵及深入的化学研究，构建包含97个化合物（31个新结构化合物）的次级代谢产物库；利用多种肿瘤细胞细胞系评价了化合物的抗肿瘤作用，发现26个化合物对不同肿瘤细胞株具有中等以上的抗肿瘤作用；并对抗肿瘤活性显著的西藏白肉灵芝三萜GL22和西藏冷杉褐褶菌甾醇化合物G10-10A进行了诱导细胞凋亡的作用机制研究，揭示了西藏白肉灵芝活性三萜“饿死”肿瘤细胞新机制。GL22显著降低肝癌细胞Huh 7.5细胞质中脂肪酸结合蛋白(FABPs)的表达水平，通过PPAR-FABPs信号通路发挥抗肿瘤作用。西藏白肉灵芝抗肿瘤活性成分的发现和作用机制研究极大地促进了西藏特色药用真菌的开发，西藏白肉灵芝2020年入选全国扶贫产品目录。“科技日报”和“人民日报”撰文介绍了本课题在西藏白肉灵芝抗肿瘤研究方面的新成果。本课题执行期间在Cell Death & Disease、RSC advance、Journal of Antibiotics、Planta Medica等期刊共发表SCI论文8篇，申请专利1项。