见血封喉苷 H 诱导肝癌细胞凋亡与自噬的机制研究

Toxicarioside H (ToxH) is a new cardenolide chemical compound isolated from the latex of Antiaris toxicaria in tropical Hainan Island. Its structure was elucidated by our research group and showed significant cytotoxicity against K562, SGC-7901, SMMC-7721 and HeLa cell lines in vitro by MTT method. In addition, we also found that ToxH can induce strong apoptosis and autophagy in hepatoma carcinoma cells. However, there are not any mechanism research done till now except for our group. The detail molecular mechanism of apoptosis and autophagy, the relationship between apoptosis and autophagy, the shared molecular signnaling pathway and the target protein moleculs between apoptosis and autophagy in the tumor cells induced by ToxH remains unknowed, it is thus need further more studies. In our current study, we will in vitro culture hepatoma carcinoma cells and establish in vivo tumor models to in invetigate whether apoptosis and autophagy are surely existed in hepatoma carcinoma cells induced by ToxH. In addition, we will use molecular methods to vertify the apoptotic and autophagic signaling pathways and the relationship between apoptosis and autophagy induced by ToxH. Otherwise, we will also use quantum dot (QD) method to locate the ToxH's binding-site and screen its target protein molecules in the tumor cells. Lastly, we will also use molecular blockers, RNA interference, or even gene knockout to verify the target protein molecules to ultimately prove the detail apoptitic and autophagic mechanism induced by ToxH. Our study will offer experimental data for ToxH to be developed as a novel anti-tumor compound.

见血封喉苷H（Toxicarioside H，ToxH）是我们从海南热带植物见血封喉乳汁中分离并进行了结构鉴定的新化学物。我们前期研究发现，ToxH对肝癌等多种肿瘤细胞具有较强的细胞毒活性，能够同时诱导肝癌细胞凋亡和自噬，但详细凋亡和自噬的机理、凋亡和自噬之间关系、凋亡和自噬二者是否存在共同的分子诱导机制、ToxH结合靶蛋白分子等问题仍需要深入研究。因此，本项目拟通过体外细胞培养和小鼠体内模型二个层面开展研究，利用分子生物学技术和方法了解ToxH诱导肝癌细胞凋亡和自噬的分子信号通路，确定凋亡和自噬二者之间的关系，应用磁性量子点标记技术对ToxH进行细胞定位并筛选ToxH结合的靶蛋白分子，最终利用靶分子功能阻断剂和RNA干扰等技术确认靶分子的功能，确定ToxH诱导肿瘤细胞凋亡和自噬的分子机制，为把ToxH更进一步开发成临床高效、低毒和具有自主知识产权的抗肝癌创新药物提供实验和理论依据。

见血封喉苷H（ToxH）是我们课题组从海南热带植物见血封喉提取的一种新型强心苷，前期研究发现强心苷类化合物具有很好的抗肿瘤作用。本项目从自噬和凋亡层面了解强心苷类化合物抗肿瘤作用的机理。1、ToxH处理后细胞数量逐渐变少，细胞间连接消失，细胞逐渐脱落，死亡增多，MTT和EDU检测结果ToxH 对肺癌细胞的生长和增殖有明显的抑制作用，凋亡和剂量正相关；线粒体膜电位和细胞色素C（Cyt c）下降均呈剂量效应，同时裂解（活化）的PARP、Caspase-3和Caspase-9和细胞质Cyt c则相应增高，但Caspase-8则没有改变，这此结果说明ToxH通过线粒体算什么诱导肿瘤细胞调亡；2、我们还发现ToxH处理的细胞中Tom20、Tim23、Hsp60等分子的表达明显下调，细胞中LC3斑点的数量明显增多，并且这些LC3斑点和Mito-Red标记的线粒体以及P62分子均有明显的共定位现象，线粒体中含有更多的P62、LC3-II和Parkin，表明ToxH通过Parkin通路诱导产生了线粒体自噬；3、进一步检测还发现ToxH处理的细胞中Sirt3 表达明显增高，但Tom20表达明显减少；RNA干扰Sirt3（siSirt3）表达后Tom20表达恢复正常， LC3斑点不再和Mito-Red有明显的共聚现象，RNA干扰Parkin或者用自噬阻断剂抑制线粒体自噬后，发现ToxH处理的肺癌细胞的线粒体中也没有检测到明显的P62、LC3-II和Parkin，说明ToxH通过Sirt3通路诱导产生线粒体自噬；4、我们的研究结果还发现，ToxH并不能促使Hex II表达，而是促使主要分布在线粒体中的Hex II向胞质转移；5、干扰Sirt3表达后则出现了和未干扰相反的结果，Parkin和DVAC1共沉淀消失而Hex II和VDAC1发生共沉淀；6、发现干扰或阻断线粒体自噬后，ToxH抑制肺癌细胞生长增殖更加明显，通过线粒体途径凋亡的细胞明显增加。我们的实验结果表明：ToxH通过线粒体途径诱导肺癌细胞凋亡并能产保护性线粒体自噬；ToxH通过上调 Sirt3，同时下调Hex II 与 VDAC1 结合，进而促使VDAC1和Parkin结合，这可能是激活线粒体自噬经典Parkin通路的一种机制；联合线粒体自噬抑制剂或新型自噬阻断技术有可能是增强ToxH 抗肿瘤类药物疗效的重要策略。