虫草素及甘草素定向合成产物促人肝癌细胞凋亡调控机制研究

Our previous experiments have already demonstrated that through regulation of different signaling pathways, liquiritigenin or cordycepin significantly inhibited the growth of human hepatocellular carcinoma (HCC) cells. Based on the complementary of molecular mechanisms, a new compound named CLCN was synthesized by using liquiritigenin and cordycepin. CLCN displayed five-fold more suppressive effect on HCC cell viability than both iquiritigenin and cordycepin. In the present study, the security and metabolism of CLCN were investigated in animal models firstly. Further, the suppressive effects of CLCN on liver tumor growth, metastasis and angiogenesis were investigated in both HCC cell line (PLC/PRF/5 and HepG2) and a new orthotopical HCC-implantation mouse model with luciferin expression. Whether mitogen-activated protein kinases (MAPKs), AKT/mammalian target of rapamycin (mTOR) signaling and mitochondrial function contribute to CLCN-induced HCC cells apoptosis were investigated. Moreover, experiments were performed to examine whether intracellular reactive oxygen species (ROS) accumulation served as a link between MAPKs activities and mitochondrial apoptosis. Additionally, the effects of MAPKs and AKT/mTOR on mitochondrial apoptosis, and the relationship between hypoxia inducible factor (HIF-1alpha)/Vascular endothelial growth factor (VEGF) and phosphor-AKT or phosphor-extracellular-signal regulated protein kinase (phosphor-ERKs) were studied. Through this project, we can not only find a candidate compounds for liver cancer treatment with clear mechanisms, but also provide a new idea for natural compounds research--Based on the complementary of molecular pharmacology mechanisms of different natural compounds, new products with better pharmaceutical effects can be synthesized from these materials.

前期实验表明通过调控不同信号通路，甘草素或虫草素可显著抑制人肝癌细胞生长。通过分子机制互补，以两种单体为原料定向合成CLCN，其抑制人肝癌细胞存活的药效可提高五倍以上。本研究将评价CLCN体内安全性并进行初步药物代谢实验。在此基础上，在人肝癌细胞系（PLC/PRF/5和HepG2）及表达荧光素酶的原位肝肿瘤小鼠模型中，考察CLCN抑制肝肿瘤生长、转移及血管生成的效果；探讨MAPKs、AKT/mTOR及线粒体功能在其促人肝癌细胞凋亡中的调控作用；研究各种凋亡通路间的相互关系：胞内ROS积累在MAPKs活性及线粒体凋亡中的桥梁作用、MAPKs及ATK/mTOR活性对线粒体凋亡的影响以及AKT和ERKs对HIF-1alpha/VEGF表达的调控。本项目实施后，不仅可找到机制明确的抗肝癌候选化合物，还将为天然化合物研究提供新的思路—通过分子药理机制互补，以植物单体为原料，定向合成药效更好的化合物。

本课题组前期对甘草及冬虫夏草做了系统性研究，证实虫草素和甘草素均可促人肝癌细胞凋亡，此外，两种天然产物的促凋亡机制存在互补。本项目以虫草素和甘草素为原料定向合成CLCN，在人肝癌细胞系（PLC/PRF/5和HepG2）及荷瘤裸鼠模型中，通过细胞凋亡、细胞形态、线粒体功能、LDH释放、肿瘤抑制率以及Caspase3、PARP活性检测等一系列实验，证实了CLCN的促肝癌细胞凋亡作用；实验中将抗体芯片和Western blot等实验结果相结合，证实氧化应激（Nrf2/HO-1）介导的线粒体凋亡通路在CLCN的促肝癌细胞凋亡中起重要作用。.在CLCN合成过程中，构建了小分子化合物库，筛选获得2种可保护PC12细胞对抗L-Glu诱导凋亡的化合物，同时证实了线粒体凋亡通路在此过程中的主导作用。.利用本项目成熟的细胞及动物模型，本项目系统的研究了蛹虫草提取物、松茸提取物、虫草素以及鼠尾草酸的促癌细胞凋亡作用。证实蛹虫草提取物可通过线粒体相关的Caspase3 途径促肝癌及乳腺癌细胞凋亡；虫草素通过调控线粒体功能促人肝癌细胞凋亡、通过调控Erk/mTOR相关管的Caspase途径促乳腺癌细胞凋亡；鼠尾草酸通过调控线粒体相关的Caspase3 途径促肝癌细胞凋亡。 .项目进行过程中，课题组对氧化应激相关的线粒体凋亡途径在促癌症细胞中的作用进行了系统研究。共发表SCI论文7篇，获授权专利2项，本项目的研究成果不仅找到了机制明确的可用于肝癌和乳腺癌治疗的化合物，同时为天然化合物研究提供新的思路—通过分子药理机制互补，以植物单体为原料，定向合成药效更好的化合物。