CDK1/PLK1双靶点介导天然产物glucocappasalin诱导宫颈癌细胞自噬和凋亡分子机制研究

Previous studies have indicated single-target drugs cannot efficiently block the signal pathways that are related to the tumorigenesis and progress of cervical cancer. Therefore, identification of novel multi-target agents would provide fundamental to the therapeutic strategies of cervical cancer. According to our previous studies, natural product glucocappasalin (GCP), originally from Descurainia sophia seeds, was identified to induce autophagy and apoptosis by targeting both CDK1 and PLK1 in HeLa cells. However, the molecular mechanism is need to be urgently clarified. In this proposal, optical in vivo imaging, flow cytometry and etc. are going to employed to evaluate the efficacy of GCP against cervical cancer. Then, crispr/cas9 and etc. are further used to verify the specificity of the drug targets. Additionally, the roles and relationships between CDK1 and PLK1 in the induction of cervical cancer cell death by GCP will illustrate by western blot and etc. Lastly, the project will continue to elucidate whether GCP could block the signaling pathways by targeting CDK1 and PLK1 to induce cell cycle arrest and apoptosis. And, to verify whether GCP could regulate Beclin1 by targeting CDK1 and regulate mTOR1 signaling pathways by inhibition of PLK1 to induce autophagy. Taken together, our findings might provide experimental evidence for exploring the multi-target therapy for future cervical cancer therapy.

针对单靶点药物在治疗宫颈癌时存在疗效不佳等问题，研发新型多靶点药物成为当前药物研究的热点领域。课题组前期发现播娘蒿种子中天然产物glucocappasalin（GCP）可以同时靶向CDK1/PLK1并有效诱导宫颈癌细胞自噬和凋亡，但具体分子调控机制亟待阐明。为此，项目拟首先利用活体生物发光成像、细胞流式术等方法从体内、体外两方面明确GCP抗宫颈癌药理作用；然后，采用crispr/cas9等方法验证GCP双靶点作用的特异性；接着，采用western blot等手段阐明双靶点在GCP诱导宫颈癌细胞死亡中的关系和作用机制；最后，结合siRNA等技术揭示GCP协同靶向CDK1、PLK1阻断下游信号通路诱导细胞周期阻滞及凋亡、以及通过调控Beclin1及mTOR1信号通路诱导自噬的分子机制。本研究将为宫颈癌的靶向治疗提供新策略和新思路。

在全球范围内，宫颈癌仍然是女性最常见的癌症之一。针对单靶点药物在治疗宫颈癌时存在疗效不佳等问题，研发新型多靶点药物成为当前药物研究的热点领域。本项目主要阐明了播娘蒿种子中天然产物glucocappasalin（GCP）通过同时靶向CDK1/PLK1并有效诱导宫颈癌细胞自噬和凋亡的分子调控机制。课题组通过体内和体外实验均证明GCP具有良好的抗宫颈癌作用。在体外实验中发现GCP通过同时靶向CDK1和PLK1的表达，抑制宫颈癌细胞增殖，诱导细胞周期阻滞在G2/M期；GCP显著上调了caspase-3，caspase-9，cleaved-PARP，下调Bcl-2和Bcl-XL的表达，揭示GCP通过线粒体途径诱导细胞凋亡；GCP处理后，HeLa细胞中自噬相关蛋白Beclin-1、LC3B、p62和Atg5的表达均呈剂量依赖性增加，phospho-AKT和phospho-mTOR蛋白表达量呈剂量依赖性降低，表明GCP通过调控PI3K/AKT/mTOR信号通路来诱导HeLa细胞发生自噬，并进一步证明GCP通过抑制自噬减弱细胞的凋亡；体内研究发现GCP显著抑制了移植瘤小鼠的肿瘤生长，且无明显毒性。本研究表明天然产物GCP可以作为一种候选的有效治疗宫颈癌的候选药物，本研究同时为宫颈癌的多靶点药物研发提供理论支撑。