Pan Yan, Li Xin, Duan Jianhui, Yuan Lan, Fan Shengjun, Fan Jingpu, Xiaokaiti Yilixiati, Yang Haopeng, Wang Yefan, Li Xuejun
State Key Laboratory of Natural and Biomimetic Drugs, Department of Pharmacology, School of Basic Medical Sciences, Health Science Center and Beijing Key Laboratory of Tumor Systems Biology, Peking University, Beijing, People's Republic of China (Y.P., X.L., J.D., S.F., J.F., Y.X., H.Y., Y.W., X.L.); and Medical and Healthy Analytical Center, Peking University Health Science Center, Beijing, People's Republic of China (L.Y.).
State Key Laboratory of Natural and Biomimetic Drugs, Department of Pharmacology, School of Basic Medical Sciences, Health Science Center and Beijing Key Laboratory of Tumor Systems Biology, Peking University, Beijing, People's Republic of China (Y.P., X.L., J.D., S.F., J.F., Y.X., H.Y., Y.W., X.L.); and Medical and Healthy Analytical Center, Peking University Health Science Center, Beijing, People's Republic of China (L.Y.)
Mol Pharmacol. 2015;87(3):378-90. doi: 10.1124/mol.114.094425. Epub 2014 Dec 8.
Gefitinib is widely used for the treatment of lung cancer in patients with sensitizing epidermal growth factor receptor mutations, but patients tend to develop resistance after an average of 10 months. Low molecular weight heparins, such as enoxaparin, potently inhibit experimental metastasis. This study aimed to determine the potential of combined enoxaparin and gefitinib (enoxaparin + gefitinib) treatment to inhibit tumor resistance to gefitinib both in vitro and in vivo. A549 and H1975 cell migration was analyzed in wound closure and Transwell assays. Akt and extracellular signal-related kinase 1/2 signaling pathways were identified, and a proteomics analysis was conducted using SDS-PAGE/liquid chromatography-tandem mass spectrometry analysis. Molecular interaction networks were visualized using the Cytoscape bioinformatics platform. Protein expression of dedicator of cytokinesis 1 (DOCK1) and cytoskeleton intermediate filament vimentin were identified using an enzyme-linked immunosorbent assay, Western blot, and small interfering RNA transfection of A549 cells. In xenograft A549-luc-C8 tumors in nude mice, enoxaparin + gefitinib inhibited tumor growth and reduced lung colony formation compared with gefitinib alone. Furthermore, the combination had stronger inhibitory effects on cell migration than either agent used individually. Additional enoxaparin administration resulted in better effective inhibition of Akt activity compared with gefitinib alone. Proteomics and network analysis implicated DOCK1 as the key node molecule. Western blot verified the effective inhibition of the expression of DOCK1 and vimentin phosphorylation by enoxaparin + gefitinib compared with gefitinib alone. DOCK1 knockdown confirmed its role in cell migration, Akt expression, and vimentin phosphorylation. Our data indicate that enoxaparin sensitizes gefitinib antitumor and antimigration activity in lung cancer by suppressing DOCK1 expression, Akt activity, and vimentin phosphorylation.
吉非替尼广泛用于治疗具有敏感表皮生长因子受体突变的肺癌患者，但患者平均在10个月后往往会产生耐药性。低分子量肝素，如依诺肝素，能有效抑制实验性转移。本研究旨在确定联合使用依诺肝素和吉非替尼（依诺肝素 + 吉非替尼）治疗在体外和体内抑制肿瘤对吉非替尼耐药性的潜力。通过伤口愈合实验和Transwell实验分析A549和H1975细胞的迁移情况。鉴定Akt和细胞外信号调节激酶1/2信号通路，并使用SDS-PAGE/液相色谱 - 串联质谱分析进行蛋白质组学分析。使用Cytoscape生物信息学平台可视化分子相互作用网络。使用酶联免疫吸附测定、蛋白质免疫印迹法以及对A549细胞进行小干扰RNA转染来鉴定胞质分裂 dedicator 1（DOCK1）和细胞骨架中间丝波形蛋白的蛋白质表达。在裸鼠的异种移植A549-luc-C8肿瘤中，与单独使用吉非替尼相比，依诺肝素 + 吉非替尼抑制肿瘤生长并减少肺集落形成。此外，与单独使用任何一种药物相比，联合用药对细胞迁移的抑制作用更强。与单独使用吉非替尼相比，额外给予依诺肝素能更有效地抑制Akt活性。蛋白质组学和网络分析表明DOCK1是关键节点分子。蛋白质免疫印迹法证实，与单独使用吉非替尼相比，依诺肝素 + 吉非替尼能有效抑制DOCK1的表达和波形蛋白的磷酸化。敲低DOCK1证实了其在细胞迁移、Akt表达和波形蛋白磷酸化中的作用。我们的数据表明，依诺肝素通过抑制DOCK1表达、Akt活性和波形蛋白磷酸化，使吉非替尼在肺癌中的抗肿瘤和抗迁移活性敏感化。
