Ou Hong-Ling, Wu Hui, Ren Yu-Liang, Si Yuan, Duan Zhong-Qi, Liu Xue-Wen
School of Basic Medical Sciences,Hubei University of Medicine Shiyan 442000,China Hubei Key Laboratory of Wudang Local Chinese Medicine Research,Hubei University of Medicine Shiyan 442000,China.
School of Basic Medical Sciences,Hubei University of Medicine Shiyan 442000,China.
Zhongguo Zhong Yao Za Zhi. 2023 Aug;48(16):4483-4492. doi: 10.19540/j.cnki.cjcmm.20230510.706.
This study aims to investigate the effect and mechanism of hydnocarpin(HC) in treating triple negative breast cancer(TNBC). Cell counting kit-8(CCK-8), xCELLigence real-time cellular analysis(RTCA), and colony formation assay were employed to determine the effects of HC on the proliferation of two TNBC cell lines: MDA-MB-231 and MDA-MB-436. The effects of HC on the migration and invasion of TNBC cells were detected by high-content analysis, wound-healing assay, and Transwell assay. The changes in the epithelial-mesenchymal transition(EMT) and the expression of invasion-and migration-associated proteins [E-cadherin, vimentin, Snail, matrix metalloproteinase-2(MMP-2), and MMP-9] were detected by Western blot. Western blot and RT-qPCR were employed to determine the protein and mRNA levels of Yes-associated protein(YAP) and downstream targets(CTGF and Cyr61). TNBC cells were transfected with Flag-YAP for the overexpression of YAP, and the role of YAP as a key target for HC to inhibit TNBC malignant progression was examined by CCK-8 assay, Transwell assay, and wound-healing assay. The pathway of HC-induced YAP degradation was detected by the co-treatment of proteasome inhibitor with HC and ubiquitination assay. The binding of HC to YAP and the E3 ubiquitin ligase Ccr4-not transcription complex subunit 4(CNOT4) was detected by microscale thermophoresis(MST) assay and drug affinity responsive target stability(DARTS) assay. The results showed that HC significantly inhibited the proliferation, colony formation, invasion, and EMT of TNBC cells. HC down-regulated the protein and mRNA levels of CTGF and Cyr61. HC down-regulated the total protein level of YAP, while it had no effect on the mRNA level of YAP. The overexpression of YAP antagonized the inhibitory effects of HC on the proliferation, migration, and invasion of TNBC cells. HC promoted the degradation of YAP through the proteasome pathway and up-regulated the ubiquitination level of YAP. The results of MST and DARTS demonstrated direct binding between HC, YAP, and CNOT4. The above results indicated that HC inhibited the malignant progression of TNBC via CNOT4-mediated degradation and ubiquitination of YAP.
本研究旨在探讨大风子素（HC）治疗三阴性乳腺癌（TNBC）的效果及机制。采用细胞计数试剂盒-8（CCK-8）、xCELLigence实时细胞分析（RTCA）和集落形成试验来确定HC对两种TNBC细胞系（MDA-MB-231和MDA-MB-436）增殖的影响。通过高内涵分析、划痕愈合试验和Transwell试验检测HC对TNBC细胞迁移和侵袭的影响。采用蛋白质免疫印迹法检测上皮-间质转化（EMT）的变化以及侵袭和迁移相关蛋白[E-钙黏蛋白、波形蛋白、Snail、基质金属蛋白酶-2（MMP-2）和MMP-9]的表达。运用蛋白质免疫印迹法和RT-qPCR来测定Yes相关蛋白（YAP）及其下游靶点（结缔组织生长因子和Cyr61）的蛋白质和mRNA水平。用Flag-YAP转染TNBC细胞以过表达YAP，并通过CCK-8试验、Transwell试验和划痕愈合试验检测YAP作为HC抑制TNBC恶性进展的关键靶点的作用。通过蛋白酶体抑制剂与HC共同处理和泛素化试验检测HC诱导YAP降解的途径。采用微量热泳动（MST）试验和药物亲和力响应靶点稳定性（DARTS）试验检测HC与YAP及E3泛素连接酶Ccr4-Not转录复合物亚基4（CNOT4）的结合。结果表明，HC显著抑制TNBC细胞的增殖、集落形成、侵袭和EMT。HC下调CTGF和Cyr61的蛋白质和mRNA水平。HC下调YAP的总蛋白水平，而对YAP的mRNA水平无影响。YAP的过表达拮抗了HC对TNBC细胞增殖、迁移和侵袭的抑制作用。HC通过蛋白酶体途径促进YAP的降解并上调YAP的泛素化水平。MST和DARTS试验结果表明HC、YAP和CNOT4之间存在直接结合。上述结果表明，HC通过CNOT4介导的YAP降解和泛素化抑制TNBC的恶性进展。
