Proteomics analysis of tumor exosomes reveals vital pathways of Jinfukang inhibiting circulating tumor cells metastasis in lung cancer
Proteomics analysis of tumor exosomes reveals vital pathways of Jinfukang inhibiting circulating tumor cells metastasis in lung cancer
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肿瘤外泌体蛋白质组学分析揭示金复康抑制肺癌循环肿瘤细胞转移的重要通路
DOI：
10.1016/j.jep.2020.112802
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发表时间：
2020
影响因子：
5.4
通讯作者：
Tian Jian-Hui
中科院分区：
文献类型：
--
作者：
Que Zu-Jun;Luo Bin;Wang Chen-Tong;Qian Fang-Fang;Jiang Yi;Li Yan;Han Xiang-Hui;Li He-Gen;Liu Jia-Xiang;Tian Jian-Hui
金妇康长期用于临床治疗肺癌。既往研究表明，金妇康可通过干预ros介导的DNA损伤通路诱导循环肿瘤细胞凋亡。然而，金复康是否能抑制循环肿瘤细胞的转移及其机制尚不清楚。目的从肿瘤外泌体干预的角度进一步探讨金复康抗肺癌转移的作用机制。材料与方法：采用免疫荧光法测定细胞内胚形成。采用Transwell法检测入侵和迁移。采用超离心分离外泌体。通过电镜、纳米颗粒跟踪分析和免疫印迹分析对外泌体进行了表征，并通过蛋白质组学分析对蛋白质谱进行了评估。利用基因本体（GO）和京都基因与基因组百科全书（KEGG）进行分子功能、生物学过程和信号通路富集分析。Western blot验证关键差异表达蛋白。结果金复康能抑制CTC-TJH-01细胞MMP14、cortnn、Tks5的表达，并抑制细胞内侵过体的形成。此外，金复康能明显抑制CTC-TJH-01细胞的侵袭和迁移。经金复康处理的CTC-TJH-01细胞外泌体直径为30-100 nm，外泌体标志物CD63、CD81和TSG101均有表达。我们鉴定了680个递质表达蛋白。基因肿瘤学分析表明，外泌体主要来源于质膜，主要参与蛋白质定位和细胞内信号转导。匠心通路分析显示，EGF通路被显著抑制，而GP6信号通路被显著激活。我们也证实了金复康抑制CTC-TJH-01细胞中EGF通路相关蛋白的表达。此外，当使用EGF激活EGF信号通路时，金妇康对CTC细胞转移的抑制作用被逆转。结论金妇康通过EGF途径抑制CTC-TJH-01细胞的转移。
Ethnopharmacological relevanceJinfukang has long been used for the clinical treatment of lung cancer. Previous studies have shown that Jinfukang can induce the apoptosis of circulating tumor cells by intervening ROS-mediated DNA damage pathway. However, whether Jinfukang can inhibit the metastasis of circulating tumor cells and its mechanism are still unclear.Aim of the studyTo further investigate the mechanism of Jinfukang in anti-metastasis of lung cancer from the perspective of intervention of tumor exosomes.Materials and methodsThe invadopodia formation was determined with immunofluorescence. Invasion and migration were detected using the Transwell assay. Ultracentrifugation was used to isolate exosomes. Exosomes were characterized by electron microscopy, nanoparticle tracking analysis and immunoblotting, and the protein profile was evaluated by proteomic analysis. The molecular functions, biological processes and signaling pathway enrichment analysis were performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Key differentially expressed proteins were verified by Western blot.ResultsJinfukang can inhibit the expression of MMP14, cortactin, Tks5 and the formation of invadopodia of CTC-TJH-01 cells. Furthermore, Jinfukang can significantly inhibit the invasion and migration of CTC-TJH-01 cells. The diameter of exosomes extracted from the CTC-TJH-01 cells treated by Jinfukang was 30–100 nm, and the exosomal markers CD63, CD81 and TSG101 were expressed. We identified 680 deferentially expressed proteins. Gene oncology analysis indicated that exosomes were mostly derived from plasma membrane and mainly involved in protein localization and intracellular signaling. The ingenuity pathway analysis showed that the EGF pathway was significantly inhibited, whereas the GP6 signaling pathway was significantly activated. We also confirmed that Jinfukang inhibited the expression of EGF pathway-related proteins in CTC-TJH-01 cells. Besides, when EGF was used to activate EGF signaling pathway, the inhibition of Jinfukang on CTC cell metastasis was reversed.ConclusionJinfukang inhibits the metastasis of CTC-TJH-01 cells through the EGF pathway.