Medical School, Kunming University of Science and Technology, Kunming 650500, PR China.
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China.
Phytomedicine. 2020 Mar;68:153187. doi: 10.1016/j.phymed.2020.153187. Epub 2020 Feb 8.
P53 is the most frequently mutated gene in most tumour types, and the mutant p53 protein accumulates at high levels in tumours to promote tumour development and progression. Thus, targeting mutant p53 for degradation is one of the therapeutic strategies used to manage tumours that depend on mutant p53 for survival. Buxus alkaloids are traditionally used in the treatment of cardiovascular diseases. We found that triterpenoid alkaloids extracted from Buxus sinica found in the Yunnan Province exhibit anticancer activity by depleting mutant p53 levels in colon cancer cells.
To explore the anticancer mechanism of action of the triterpenoid alkaloid KBA01 compound by targeting mutant p53 degradation.
Different mutant p53 cell lines were used to evaluate the anticancer activity of KBA01. MTT assay, colony formation assay and cell cycle analysis were performed to examine the effect of KBA01 on cancer cell proliferation. Western blotting and qPCR were used to investigate effects of depleting mutant p53, and a ubiquitination assay was used to determine mutant p53 ubiquitin levels after cells were treated with the compound. Co-IP and small interfering RNA assays were used to explore the effects of KBA01 on the interaction of Hsp90 with mutant p53.
The triterpenoid alkaloid KBA01 can induce G2/M cell cycle arrest and the apoptosis of HT29 colon cancer cells. KBA01 decreases the stability of DNA contact mutant p53 proteins through the proteasomal pathway with minimal effects on p53 mutant protein conformation. Moreover, KBA01 enhances the interaction of mutant p53 with Hsp70, CHIP and MDM2, and knocking down CHIP and MDM2 stabilizes mutant p53 levels in KBA01-treated cells. In addition, KBA01 disrupts the HSF1-mutant p53-Hsp90 complex and releases mutant p53 to enable its MDM2- and CHIP-mediated degradation.
Our study reveals that KBA01 depletes mutant p53 protein in a chaperone-assisted ubiquitin/proteasome degradation pathway in cancer cells, providing insights into potential strategies to target mutant p53 tumours.
P53 是大多数肿瘤类型中最常发生突变的基因，突变型 p53 蛋白在肿瘤中积累水平较高，促进肿瘤的发生和发展。因此，靶向突变型 p53 进行降解是管理依赖突变型 p53 生存的肿瘤的治疗策略之一。黄杨生物碱传统上用于治疗心血管疾病。我们发现，从云南省产的黄杨中提取的三萜生物碱通过降低结肠癌细胞中突变型 p53 的水平表现出抗癌活性。
通过靶向突变型 p53 降解，探索三萜生物碱 KBA01 化合物的抗癌作用机制。
使用不同的突变型 p53 细胞系来评估 KBA01 的抗癌活性。MTT 测定、集落形成测定和细胞周期分析用于检测 KBA01 对癌细胞增殖的影响。Western blot 和 qPCR 用于研究耗尽突变型 p53 的效果，并用泛素化测定法确定细胞用化合物处理后突变型 p53 的泛素水平。免疫共沉淀和小干扰 RNA 测定用于研究 KBA01 对 Hsp90 与突变型 p53 相互作用的影响。
三萜生物碱 KBA01 可诱导 HT29 结肠癌细胞的 G2/M 细胞周期停滞和凋亡。KBA01 通过蛋白酶体途径诱导 DNA 接触突变型 p53 蛋白的稳定性降低，对 p53 突变蛋白构象的影响最小。此外，KBA01 增强了突变型 p53 与 Hsp70、CHIP 和 MDM2 的相互作用，敲低 CHIP 和 MDM2 可稳定 KBA01 处理细胞中的突变型 p53 水平。此外，KBA01 破坏了 HSF1-突变型 p53-Hsp90 复合物，并释放突变型 p53，使其能够被 MDM2 和 CHIP 介导降解。
我们的研究揭示了 KBA01 通过伴侣辅助泛素/蛋白酶体降解途径在癌细胞中耗尽突变型 p53 蛋白，为靶向突变型 p53 肿瘤提供了潜在的策略。
