[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。10 μmol/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。

[Abstract] 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 μmol/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 LC3B increased, and high-density, 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.

国家自然科学基金资助项目（No. 81874301）