3-epi-12β-hydroxyfroside诱导胃癌细胞自噬凋亡的分子机制研究

3-epi-12β-hydroxyfroside (HyFS) is a new cardenolide chemical compound isolated from Calotropis gigantea in tropical Hainan Island. Its structure was elucidated by our research group and showed significant cytotoxicity against SGC-7901, K562, HeLa and SMMC-7721 cell lines in vitro by MTT assay. In addition, we also found that HyFS can induce strong autophagy and apoptosis in gastric carcinoma cells. However, there are not any mechanism research done till now except for our group. The detail molecular mechanism of autophagy and apoptosis，the relationship between autophagy and apoptosis, the shared molecular signnaling pathway and the target protein moleculs between autophagy and apoptosis in the tumor cells induced by HyFS remains unknowed, it is thus need further more studies. In this study, we will in vitro culture gastric carcinoma cells and establish in vivo tumor models to in invetigate whether autophagy and apoptosis are surely existed in gastric carcinoma cells induced by HyFS. In addition, we will use molecular methods to study the autophagic and apoptotic signaling pathways and the relationship between autophagy and apoptosis induced by HyFS. Moreover, we will also use quantum dot (QD) method to locate the HyFS’s binding-site and screen its target protein molecules in the gastric carcinoma cells. At last, we will also use RNA interference, molecular blockers or even gene knockout technique to comfirm the target protein molecules to ultimately prove the detail autophagic and apoptitic mechanism induced by HyFS. Our study will offer experimental data for HyFS to be developed as a novel anti-tumor compound.

3-epi-12β-hydroxyfroside（HyFS）是我们合作团队从热带植物牛角瓜中分离并结构鉴定证实为新的强心苷类化合物。我们前期研究发现，HyFS对多种胃癌细胞有较强的细胞毒活性，能够同时诱导胃癌细胞自噬和凋亡，但详细的分子机理、凋亡和自噬间的关系、凋亡和自噬二者之间是否有共同的分子机制、HyFS结合的靶点分子等问题仍需要进行深入的研究。因此，本项目拟从体外细胞培养和肿瘤小鼠体内模型二个层面进行研究，利用免疫学、分子生物学等技术了解HyFS诱导胃癌细胞凋亡和自噬的信号通路，阐明凋亡和自噬之间的关系，应用磁性量子点标记技术对HyFS进行细胞定位，筛选并确定HyFS结合的靶蛋白分子，最终利用RNA干扰和功能阻断剂等方法确定靶分子，争取最终阐明HyFS诱导肿瘤细胞自噬凋亡的分子机制，寻求一种高效、低毒并具有自主知识产权治疗胃癌的新药，具有良好的应用前景。

3-epi-12β-hydroxyfroside (HyFS) 是从热带植物牛角瓜中分离并被鉴定为新的强心苷类化合物，我们前期发现HyFS有诱导凋亡及自噬的作用，本项目进一步研究其分子机制。MTT和EDU检测以及克隆形成实验结果表明，HyFS显著抑制A549和H460两种NSCLC细胞的增殖；Western Blot检测凋亡相关蛋白、流式细胞术及TUNEL实验结果表明HyFS促进NSCLC细胞死亡；Western Blot检测自噬相关蛋白表达、荧光显微镜检测LC3-II的累积、吖啶橙检测OVA和电镜结果表明HyFS能诱导自噬体生长并呈剂量和时间效应；联合应用自噬抑制剂或siRNA干扰阻断自噬生长，经Western Blot和荧光免疫自噬相关蛋白和自噬底物蛋白的表达，证明HyFS能有效诱导整个自噬流；应用自噬抑制剂或siRNA干扰阻断自噬后观测MTT、EDU和克隆形成试验结果发现HyFS诱导自噬促进NSCLC细胞增殖。Western Blot检测发现HyFS抑制Akt/mTOR信号相关分子表达，过表达Akt抑制HyFS诱导的自噬生成，抑制Akt表达则结果相反。此外，Western Blot检测发现HyFS促进Hsp90表达下调，Hsp90过表达或干扰后，自噬相关蛋白和LC3斑点表达增加，说明 HyFS通过下调Hsp90诱导NSCLC细胞产生自噬。Hsp90过表达或干扰后，Western Blot检测结果Hsp90下调并抑制Akt的活性，而免疫共沉淀实验结果表明HyFS降低Hsp90与Akt之间的相互作用，这些结果表明HyFS通过下调Hsp90抑制Akt/mTOR通路从而诱导自噬的产生。RT-PCR检测发现HyFS不影响Hsp90的转录，联合使用CHX或MG132并后Hsp90蛋白水平下降，共转染Flag-Ub和HA-Hsp90并联合使用MG132后进行免疫共沉淀检测结果发现HyFS能促使Hsp90泛素化，说明HyFS通过泛素/蛋白酶体途径降解Hsp90。综合这些结果，说明HyFS诱导自噬的机理是通过泛素/蛋白酶体降解途径下调Hsp90，从而促使其客户蛋白Akt去磷酸化，进而抑制Akt/mTOR通路导致导自噬的产生；同时还提示，联合应用自噬抑制剂对HyFS治疗NSCLC可能会取得更好的效果。