五株柳珊瑚Anthogorgia caerulea 共生细菌中抗肿瘤活性成分及其作用机制研究

Cancer, known medically as a malignant neoplasm, is a serious threat to human life and health. Medication, as the main treatment for tumors, is always the focus of research by the scientists of biology, medicine and pharmaceutics. The marine environment is being exploited for novel antitumor drugs with unique structure and excellent bioactivity. Marine microorganisms have been becoming an important resource because they are with various species, higher hit rate and can be easily reproduced in large scale in industry..In order to obtain highly efficient and novel lead compounds for developing marine anti-cancer drugs with independent intellectual property, the project plans to further investigate antitumor compounds from the five marine gorgonian-associated bacteria isolated from the Beibu Gulf gorgonian Anthogorgia caerulea. In this study, we aim at 1) isolating pure secondary compounds from the active EtOAc layer of culture broth of the strain by purification process; 2) examining the antitumor activity of these secondary metabolites toward P388, MDA-mB-231, SW480,CNE1, and GRC-1 cancer cell lines by the models of MTT; 3) investigating the antitumor activity of these secondary metabolites against protein tyrosine kinase activity by ELISA method; 4) modifying the structures of the antitumor compounds by biological and chemical modification for obtaining highly efficient and novel lead compounds; 5) studing the mode-of-action of antutimor activity by the models of western blot and immunocytochemistry.

恶性肿瘤是一类严重威胁人类健康和生命的疾病。海洋微生物由于拥有多种优点已成为获得结构新颖和活性显著的抗肿瘤先导化合物的重要源泉。本申请项目拟对中国北部湾柳珊瑚Anthogorgia caerulea的五株共生细菌中抗肿瘤活性成分及其作用机制进行研究，旨在发现具有显著抗肿瘤活性、结构新颖的药用先导化合物。运用多种现代高效分离制备技术和分析技术，紧密结合体外细胞模型和分子模型的抗肿瘤活性筛选以及化学筛选，对五株共生细菌进行系统的化学成分分离与结构鉴定，发现结构新颖的抗肿瘤活性先导化合物。应用流式细胞仪等方法观察活性化合物诱导肿瘤细胞凋亡的作用。运用western blot、免疫组化等方法，初步探讨活性化合物抗肿瘤作用的分子机制和对细胞酪氨酸激酶磷酸化的影响。开展本项目研究，将为研制我国具有原创性和自主知识产权的抗肿瘤海洋创新新药提供药用先导化合物，促进我国海洋微生物资源的利用。

北部湾位于我国南海的西北部，属于亚热带海湾，蕴藏着丰富的海洋微生物资源。从北部湾柳珊瑚Anthogorgia caerulea中分离获得15属50株共生细菌和9属23株放线菌，并测试了其生物毒活性，发现有11株具有较强的生物毒活性。从5株共生微生物中 B. subtilis、 B. methylotrophicus、N. Alba、 B. Amyloliquefaciens、R. Pyridinivorans共分离鉴定了87个化合物，其中新化合物13个。获得的3个新化合物cyclo-(Leu-Pro-Ile-Pro)、cyclo-(Tyr-Pro-Phe-Gly)和Nocarazepine A具有较显著抗HepG2和HeLa活性。另外，3个新化合物cyclo-(Leu-Pro-Ile-Pro)、cyclo-(Tyr-Pro-Phe-Gly)和halomide在10µM和1µM浓度范围对酪氨酸激酶蛋白有较好的抑制率。化合物cyclo-(Leu-Pro-Ile-Pro)的作用机理为调节p38丝裂原活化蛋白激酶(MAPK)通路。本项目发表学术论文10篇，其中SCI收录论文7篇，申请国家发表专利2项，培养硕士研究生5名。本项目研究的开展，丰富柳珊瑚共生菌株的物种和共生菌株代谢产物的化合物结构类型，为研制出具有我国自主知识产权的海洋原创性药物提供结构新颖的活性先导化合物，为促进我国海洋创新药物的研究与开发奠定基础。