基于STAT-3靶点的泽兰属两种外来入侵植物抗肝癌药效物质基础及作用机理研究

Liver cancer is a common malignant tumor. The target of STAT-3 can be highly expressed in hepatocellular carcinoma, and plays a key role in tumor tissue of hepatocellular carcinoma, invasion and metastasis, which is a new direction in the research of liver cancer drugs. The Eupatorium odoratum and E. adenophorum, as two kinds of genus Eupatorium, have becomed the invasion of poisonous weeds. According to the "chemical weapons" hypothesis, invasive species can produce strong biological toxins or allelochemicals, suggesting that this phenomenon may be the development of new resources of cytotoxic drugs. We found that the two invasive plants of cyclohexane extract and ethyl acetate extract in hepatocellular carcinoma cell lines and STAT-3 protein,and showed strong activity in the early stage. We then focus on cyclohexane extracts of E. odoratum, 13 terpenes and flavonoids were isolated and purified. These compounds have been used MTT and STAT-3 technology for activity screening test, and they were found to have strong inhibitory activity of HepG2.. The project's research program, first of all, will be separated more compounds, and analysis of the relationship between the structure and efficacy of these compounds. Second, to analyze the relationship between the key parts of the active fractions and the STAT-3 protein, to better find two kinds of plant anti liver cancer active ingredients. Third, the project intends to use computer aided drug design technology, study on the relationship between the structure and effect of the compounds. Finally, an experimental animal model will be used to evaluate the effect of anti liver cancer in vivo. The research purpose is through from molecules to the cell level, from the cell to the animal level, to clarify the material basis and the effect of two kinds of plant invasion mechanism, promote the level of prevention and treatment of liver cancer, promote the development of new drugs from invasive plants.

肝癌是常见恶性肿瘤，STAT-3靶点在肝癌中高表达，在肿瘤组织产生、侵袭和转移中起关键作用，是研究肝癌药物新方向。泽兰属飞机草和紫茎泽兰两植物是入侵我国的毒草，据“化学武器”假说，入侵物种产生强生物毒素或化感物质，提示可能将是开发细胞毒性药物新资源。前期发现其环己烷和乙酸乙酯提取物对肝癌细胞株和STAT-3显强活性，从飞机草环己烷提取物中分离的13个萜类与黄酮类，采用MTT和STAT-3初筛显示化合物均对HepG2有强增殖抑制性。本研究拟分离更多化合物，阐明抗肝癌构效关系，发现关键药效团与STAT-3蛋白作用程度，更好发现两植物抗肝癌活性成分，并对活性提取物放大制备，采用MTT、STAT-3靶点筛选抗肝癌活性成分，应用计算机辅助药物设计研究构效关系及其与靶蛋白的作用，运用动物模型评价体内抗肝癌效果，从分子-细胞-动物水平上阐明两种植物的物质基础和作用机理，为肝癌防治和入侵植物开发提供依据。

课题首先开展两种外来入侵植物紫茎泽兰、飞机草不同萃取部位的抗肝癌活性研究。发现紫茎泽兰石油醚部位对肝癌细胞的抑制作用呈时间-剂量依赖关系，能抑制肝癌细胞迁移，阻滞细胞周期并引起凋亡。体内外Western blot结果显示，石油醚部位能对线粒体与内质网相关蛋白进行调节，下调抑凋亡蛋白Bcl-2与上调促凋亡蛋白Bax，提高Caspase-3与Caspase-7的剪切活化，降低Grp78的表达，提高Caspase-12与Caspase-7的剪切活化，通过线粒体与内质网凋亡途径，抑制STAT3与AKT磷酸化的机制诱导肝癌细胞凋亡；飞机草石油醚部位能阻滞肝癌细胞周期，引起凋亡，具有良好的总抗氧化能力、清除超氧阴离子能力、羟自由基能力，并且能抑制黄嘌呤氧化酶活力。体外Western blot结果显示，能对线粒体与内质网相关蛋白进行调节，下调抑凋亡蛋白Bcl-2与上调促凋亡蛋白Bax，并提高Caspase-3与Caspase-7的剪切活化，通过线粒体凋亡途径诱导肝癌细胞凋亡。. 接着重点对两种外来入侵植物的石油醚强活性部位进行化学成分研究，共分离80个化合物，鉴定了66个单体化合物，其中萜类和黄酮化合物46个，新化合物7个。研究化合物与STAT3靶点蛋白相互作用程度，初步阐明抗肝癌活性构效关系。部分化合物具有较好的抗肝癌活性，其中尤其以飞机草中分离得到的1个倍半萜α-humulene活性最强。课题组接着对α-humulene进行大量制备，发现α-humulene 对肝癌细胞具有细胞毒作用，并能诱导肝癌细胞线粒体凋亡，促进 Caspase-3激活和PARP切除。机制上，α-humulene可抑制 Akt 激活，随后降低 GSK-3和不良磷酸化，促进凋亡诱导，还能抑制肝癌移植瘤的细胞增殖和促进细胞凋亡。该化合物成为本课题迄今发现最重要的先导化合物。以上研究表明，紫茎泽兰与飞机草活性部位和部分化合物具有良好的深入研究和应用开发潜力。. 课题目前取得成果为1件授权专利，发表刊出7篇学术论文，其中SCI论文2篇（中科院二区期刊），国外普通英文期刊论文1篇，北大中文核心论文4篇。已经正式接收的学术论文2篇，其中1篇SCI论文，1篇北大中文核心论文；课题培养3个硕士研究生，其中2人考上博士研究生。