肿瘤囊泡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途径。
Proteomics analysis of tumor exosomes reveals vital pathways of Jinfukang inhibiting circulating tumor cells metastasis in lung cancer.
机构信息
Institute of Traditional Chinese Medicine Oncology, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 200032, China; School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China.
Institute of Traditional Chinese Medicine Oncology, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 200032, China.
出版信息
J Ethnopharmacol. 2020 Jun 28;256:112802. doi: 10.1016/j.jep.2020.112802. Epub 2020 Mar 30.
ETHNOPHARMACOLOGICAL RELEVANCE
Jinfukang 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 STUDY
To further investigate the mechanism of Jinfukang in anti-metastasis of lung cancer from the perspective of intervention of tumor exosomes.
MATERIALS AND METHODS
The 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.
RESULTS
Jinfukang 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.
CONCLUSION
Jinfukang inhibits the metastasis of CTC-TJH-01 cells through the EGF pathway.
民族药理学相关性
金复康长期以来一直用于肺癌的临床治疗。先前的研究表明，金复康可以通过干预 ROS 介导的 DNA 损伤途径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。
研究目的
从干预肿瘤外泌体的角度进一步探讨金复康抑制肺癌转移的机制。
材料和方法
用免疫荧光法测定侵袭小体的形成。用 Transwell 测定法检测侵袭和迁移。超速离心法分离外泌体。用电镜、纳米颗粒跟踪分析和免疫印迹法对其进行鉴定，并通过蛋白质组学分析评估其蛋白质谱。利用基因本体论（GO）和京都基因与基因组百科全书（KEGG）进行分子功能、生物过程和信号通路富集分析。通过 Western blot 验证关键差异表达蛋白。
结果
金复康可抑制 CTC-TJH-01 细胞中 MMP14、cortactin、Tks5 的表达和侵袭小体的形成。此外，金复康能显著抑制 CTC-TJH-01 细胞的侵袭和迁移。金复康处理后的 CTC-TJH-01 细胞提取的外泌体直径为 30-100nm，表达外泌体标志物 CD63、CD81 和 TSG101。我们鉴定了 680 种差异表达蛋白。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。创意通路分析显示，EGF 通路明显受到抑制，而 GP6 信号通路明显被激活。我们还证实，金复康抑制了 CTC-TJH-01 细胞中 EGF 通路相关蛋白的表达。此外，当 EGF 用于激活 EGF 信号通路时，金复康对 CTC 细胞转移的抑制作用被逆转。
结论
金复康通过 EGF 通路抑制 CTC-TJH-01 细胞的转移。
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