吉祥草活性成分RCE-4选择性抗宫颈癌的分子作用机制研究

Reineckia carnea, also known as “Guanyin” herbs, was reputed as the miracle drug in Tujia minority area and have specified in local pharmacopoeia of Hubei Province. RCE-4, a spirostanol saponin, was isolated from the Reineckia Carnea by our laboratory and has been chosen as the symbolic component of Reineckia carnea because of the highest content and superior activity. In our previous studies, we found that RCE-4 exhibited a broad spectrum of anti-tumor activity in vitro against several cancer cell lines, especially for cervical Ca Ski cells, and limited toxicity against two normal cell lines. Further research found that: (1) RCE-4 could significantly inhibit the growth of tumor in xenograft nude mice with very low tissue toxicity. These results indicated that RCE-4 has an excellent selectivity; (2) RCE-4 could significantly induced apoptotic cell death of Ca Ski cells via mitochondria-dependent activation of caspase cascade in a dose-dependent manner; (3) RCE-4 could significantly induced autophagy cell death; (4) RCE-4 could inhibit the release of inflammatory cytokines and mediators and exhibited significantly anti-inflammatory activity. So, based on this, our study will take the critical signaling pathway of “the conversion of inflammation to cancer” as the breakthrough point, focusing on the multiple cross regulatory network of apoptosis and autophagy, to systematically study the selectively anti-cervical cancer molecular mechanism in the Ca Ski cells and xenograft mice. In conclusion, our studies will provide theoretical and experimental basis for developing superior anti-cervical cancer drugs and for the future clinical application of RCE-4.

吉祥草是本地土家族的一味名药，已收载入湖北省地方中药材标准，资源十分丰富。RCE-4是本实验室从中分离得到的一个含量最高、活性最好的甾体皂苷，是吉祥草的标志性成分。前期研究发现RCE-4对宫颈癌Ca Ski细胞具有显著的细胞毒活性(IC50为3.71μM)和对正常细胞的选择性，能诱导Ca Ski细胞发生凋亡和自噬。裸鼠移植瘤试验发现RCE-4具有较好的体内抗宫颈癌活性（抑瘤率69.1%）和极低的组织毒性。另外能通过抑制多种炎性介质和细胞因子的释放而发挥显著的抗炎活性。目前尚未见其他研究者关于其药理活性和机制的报道。本课题拟在前期工作的基础上，以“炎-癌”转化的关键信号通路为切入点，以Ca Ski细胞程序性死亡-凋亡和自噬的互反馈调控为中心，从细胞和实验动物两个层面系统解析RCE-4的选择性抗宫颈癌分子作用机制，为开发更为高效低毒的抗宫颈癌药物和RCE-4将来的临床应用奠定基础。

本项目对吉祥草活性成分RCE-4选择性抗宫颈癌分子作用机制进行了系统和深入的研究。首先，阐明了RCE-4对Ca Ski细胞自噬和凋亡的诱导作用以及分子机制；其次，在分子层面全面解析了RCE-4对凋亡和自噬的互反馈调控机制，并找到了增强RCE-4抗宫颈癌活性的途径：与自噬抑制剂Baf A1联用或者与Beclin 1或Bcl-2的Si RNA质粒联用；随后，揭示了RCE-4调控凋亡和自噬互作的关键靶点：Bcl-2-Beclin 1复合体，并首次揭示了RCE-4对Bcl-2-Beclin 1复合体形成的抑制作用和途径，并进一步发现了自噬相关因子ATG4B新的生物学功能：ATG4B分子具有增强Bcl-2-Beclin 1复合体的稳定性阻止其解离的独特作用，抑制其活性可显著增强化疗药物的抗肿瘤作用，这对研发新的抗肿瘤药物具有重大意义；再次，以“炎-癌”转化为切入点，系统阐明了RCE-4对关键的炎症介质COX-2和旁路蛋白NAG-1分子的作用，以及对主要炎症信号通路NF-κB/PI3K/AKT/mTOR和Ras/Raf/MEK/Erk的影响，并在此基础上首次发现了非甾体抗炎药塞来昔布对RCE-4显著而独特的减毒增效作用以及分子作用机制；另外，本研究还阐明了RCE-4抗宫颈癌Ca Ski细胞迁移和侵袭的活性及其分子机制：RCE-4通过调控Wnt/β-catenin及Hippo/YAP信号通路，抑制相关通路核心蛋白β-catenin和YAP/TAZ分子的核转运，进而减弱下游靶蛋白的表达，从而抑制了肿瘤的迁移和侵袭。此外，还解析了Ca Ski细胞逃避RCE-4化疗的途径和机制：在RCE-4的刺激下，Ca Ski细胞通过上调PERK/eIF2α/ATF4/CHOP信号通路主动激活细胞内质网应激，并以P21、P27、MDM2、GADD45α等分子为介导，调控ATM/Chk2/cdc25C信号通路，诱导细胞阻滞于G2/M期从而为其存活赢得机会；最后，在动物层面研究了RCE-4的抗宫颈癌活性以及作用机制。以上研究为RCE-4将来的临床应用奠定了坚实的基础。