摘要
目的:探讨染色体区域维持因子1(CRM1)抑制剂莱菔素(LFS-01)通过抑制信号转导及转录激活因子3(STAT3)信号通路杀伤三阴性乳腺癌(TNBC)细胞的作用及其机制。方法:通过分子动力学模拟技术,验证LFS-01是否可与CRM1分子结构上的核输出信号(NES)口袋结合。通过CCK-8法检测LFS-01对4种不同的乳腺癌细胞杀伤活力。用不同浓度的LFS-01处理TNBC细胞MDA-MB-468和MDA-MB-231,免疫荧光法检测CRM1货物蛋白STAT3以及带有NES序列的蛋白在细胞内定位的变化;WB检测LFS-01对STAT-3信号通路以及其下游蛋白表达的影响;WB、细胞免疫荧光和透射电镜法检测自噬的发生;通过流式细胞术检测药物对细胞周期和凋亡的影响。结果:分子动力学模拟结果表明,LFS-01能够与CRM1的NES口袋结合,显示其在结构上影响后者蛋白转运功能的可能性。LFS-01能特异性杀伤TNBC细胞MDA-MB-468和MDA-MB-231。10μmol/L LFS-01处理后TNBC细胞中STAT3和带有NES标签的蛋白均被阻滞于细胞核中,而在对照组中这些蛋白均匀分布在细胞质中。随着LFS-01剂量的提高和处理时间的延长,MDA-MB-468和MDA-MB-231细胞中磷酸化STAT3蛋白、Bcl-xL和Cylin D1表达均降低,细胞内自噬标志蛋白LC3B表达上升;同时出现高密度、多层的团状自噬小体;细胞周期阻滞于S期,并且凋亡率显著升高(P<0.05或P<0.01)。结论:LFS-01可阻断CRM1运载蛋白出核、进而抑制STAT3信号通路的激活,从而促进TNBC细胞MDA-MB-468和MDA-MB-231发生自噬、细胞周期阻滞和凋亡。
Objective :To investigate the role and mechanism of chromosomal region maintenance 1(CRM1) inhibitor sulforaphene(LFS-01) in killing triple negative breast cancer(TNBC) cells by inhibiting signal transducer and activator of transcription 3(STAT3) signaling pathways. Methods: Whether LFS-01 could combine with the NES pocket of CRM1 was verified by molecular dynamics simulation techniques. The killing activity of LFS-01 on four different breast cancer cell lines was detected by CCK-8 method. TNBC MDA-MB-468 and MDA-MB-231 cells were treated with different concentrations of LFS-01, and the intracellular localization of CRM1 cargo protein STAT3 and protein with NES sequence was detected by immunofluorescence;WB was used to detect the effect of LFS-01 on the expression of STAT-3 signaling pathway and its downstream proteins;WB, cellular immunofluorescence and transmission electron microscopy were adopted to detect the occurrence of autophagy;the effect of LFS-01 on cell cycle and apoptosis was detected by flow cytometry. Results: Molecular dynamics simulations showed that LFS-01 can bind to the NES pocket of CRM1, indicating that it may structurally affect the latter’s protein transport function. LFS-01 could specifically kill TNBC MDA-MB-468 and MDA-MB-231 cells. STAT3 and NES-tagged proteins were mainly blocked in the nucleus of TNBC cells after the treatment with 10 μ mol/L LFS-01, while they were evenly distributed in the cytoplasm in the control group. The expressions of phosphorylated STAT3 protein, Bcl-xL and Cylin D1 were decreased in MDA-MB-468 and MDA-MB-231 cells with the increase of LFS-01 dose and the prolongation of treatment time;the expression of autophagy marker protein LC3 B increased, and highdensity, multi-layered autophagosomes appeared at the same time;cell cycle arrest was observed in S phase and apoptosis rate was significantly increased(P<0.05 or P<0.01). Conclusion: LFS-01 blocks the export of CRM1 carrier protein, thereby inhibiting the activation of STAT3 signaling pathway and promoting autophagy, cell cycle arrest and apoptosis in TNBC MDA-MB-468 and MDAMB-231 cells.
作者
高久蕉
张琦
杨永亮
GAO Jiujiao;ZHANG Qi;YANG Yongliang(Center for Molecular Medicine,School of Bioengineering,Dalian University of Technology,Dalian 116023,Liaoning,China)
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
北大核心
2019年第8期837-844,共8页
Chinese Journal of Cancer Biotherapy
基金
国家自然科学基金资助项目(No.81874301)~~
