Penicilazaphilone C诱导胃癌细胞凋亡的机制研究

Penicilazaphilone C (PAC) is a new azaphilone chemical compound isolated from the marine fungus Penicillium sclerotiorum M-22. Its structure was elucidated by our research group and shows significant cytotoxicity against gastric carcinoma cell MGC-803, SGC-7901 melanoma B-16 and HeLa cell lines in vitro by MTT method. In addition, we also found that PAC can induce strong apoptosis in gastric carcinoma cells. However, there are not any mechanism research done till now except for our group. The detail molecular mechanism of apoptosis, the share molecular signaling pathway and the target protein molecules in the tumor cells induced by PAC remains unknown, it is thus need further more studies. In our current study, we will in vitro culture gastric carcinoma cells and established in vivo tumor models to in investigate whether apoptosis are surely existed in gastric carcinoma cells induced by PAC. In addition, we will use molecular methods such as Gene chip, protein chip technology, Western blot and flow cytometry technology to verify the apoptotic signaling pathways induce by PAC. Otherwise, we will also use quantum dot (QD) method to locate the PAC’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 apoptotic mechanism induce by PAC. Our study will offer experimental data for PAC to be developed as a novel anti-cancer compound.

Penicilazaphilone C（PAC）是我们从海洋真菌Penicillium sclerotiorum中分离并鉴定的新Azaphilone类化合物。在前期研究中发现，PAC对多种胃癌细胞具有较强的细胞毒活性，并能够同时诱导胃癌细胞凋亡发生，但详细的诱导细胞凋亡机理，PAC结合靶蛋白分子，以及诱导细胞凋亡的信号通路等问题仍需要进一步研究。因此，本项目拟通过体外细胞培养和小鼠体内模型二个层次开展研究，利用现代分子生物学技术如基因芯片、蛋白芯片、Western blot、流式细胞技术等手段来了解PAC诱导胃癌细胞凋亡的分子信号通路，应用磁性量子点标记技术对PAC进行细胞定位及筛选PAC结合的靶蛋白分子，最终利用靶分子功能阻断剂和RNA干扰等技术确定靶分子的功能，确定PAC诱导胃癌细胞凋亡的分子机制，为把PAC更进一步开发成临床高效、低毒和具有自主知识产权的抗肿瘤新药提供实验和理论依据。

Penicilazaphilone C是我们从海洋真菌Penicillium sclerotiorum中分离并鉴定的新Azaphilone类化合物。在前期研究中发现，PAC对多种胃癌细胞具有较强的细胞毒活性，并能够同时诱导胃癌细胞凋亡发生。本项目利用体内外实验进一步深入研究Penicilazaphilone C的抗肿瘤活性，通过现代细胞、分子生物学技术去发现Penicilazaphilone C诱导胃癌细胞凋亡作用机制。随着研究的深入开展，我们发现Penicilazaphilone C是通过阻断Notch/PTEN/AKT信号轴来诱导胃癌细胞凋亡的，Penicilazaphilone C能够抑制α-secretase酶水解激活的Notch受体-配体复合物水解从而阻止Notch受体释放NICD进入细胞核，导致不启动其下游应靶基因Hes 1表达，Hes 1表达可以使核PETN蛋白磷酸化，而未磷酸化的PETN蛋白阻止AKT磷酸化，磷酸化的AKT可以激活抑制促凋亡因子（例如Bax，Bad和caspase-9），并激活抗凋亡因子（例如Bcl-2和Bcl-xL），促凋亡因子可以剪切Pro-caspase-9成具有活性的caspase-9，caspase-9从而剪切Pro-caspase-3从而激活caspase-3导致细胞凋亡。当我们用质粒转染过表达的Notch（NTM）进入的两种胃癌细胞系中检测Penicilazaphilone C对细胞凋亡的影响发现，发现过表达Notch1和（或）Notch2可以逆转Penicilazaphilone C诱导细胞凋亡，用Notch1（NTM）和（或）Notch2（NTM）过表达质粒转染2种胃癌细胞可显著减少了Penicilazaphilone C诱导的细胞凋亡的发生。本项目研究表明Penicilazaphilone C是通过抑制激活的Notch受体的蛋白水解裂解来阻断Notch受体释放NICD到细胞质，从而诱导胃癌细胞凋亡的，导致抑制肿瘤的生长，因此Penicilazaphilone C可以作用一种潜在的治疗肿瘤药物的先导物。