内质网应激具有“促进存活”和“诱发死亡”的双重效应，这一效应可调控获得性癌症多药耐药的进程。申请人发现，激活平衡点ATF4，可打破内质网稳态，阻断化疗耐药的发展。滇重楼具有抗癌活性，但对ATF4的调控无人研究。申请人利用硼替佐米骨癌耐药模型筛选本实验室的2123个中药来源单体，发现重楼皂苷Ⅰ能诱导ATF4表达，并显著抑制耐药骨癌细胞的增殖。申请人通过生信学发现临床病人组织中HSPA5、ATF4、CBLC、RET表达具有相关性，但相互间的调控关系并无报道。进一步研究发现，重楼皂苷Ⅰ能通过上调ATF4，促进ATF4第323位精氨酸（ARG323）、LYS335分别与RET激酶域GLU971、ASP974结合，加速模型中突变RET的泛素化降解。本项目将阐明重楼皂苷Ⅰ的活性机制，利用蛋白质谱找出其在模型中结合的靶蛋白；为滇重楼单独或联合硼替佐米，化疗耐药癌症的应用提供新思路和新的理论依据。

Endoplasmic reticulum (ER) stress triggers alive or death, which regulates the progress of acquired drug resistance in cancer. We found that ATF4 activation interrupts ER homeostasis, induces cancer cell apoptosis and blocks the development of drug resistance. We established bortezomib-resistant osteocarcinoma models (OS/BTZ) and found Polyphyllin Ⅰ promotes ATF4 protein levels in OS/BTZ and inhibits OS/BTZ proliferation markedly. We used bioinformatics to analyze 311 bone-related sarcoma counts, and found that HSPA5, ATF4, CBLC, RET gene levels exist correlation but no evidence can be found in STRING. Further investigation demonstrated that Polyphyllin Ⅰ induces ATF4 to combine with GLU971 and ASP974 of tyrosine kinase domain in RET through its ARG323 and LYS335, thereby accelerates mutant RET degradation by ubiquitination pathway. In this project we will illustrate the mechanism of Polyphyllin Ⅰ and find out its target by using protein mass spectrometry. This study will provide new strategy and research foundation for Paris polyphylla Smith var. yunnanensis (Franch.) Hand.-Mazz. in the therapy of drug-resistant cancer.