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中国癌症防治杂志

中国癌症防治杂志 ›› 2023, Vol. 15 ›› Issue (5): 516-524.doi: 10.3969/j.issn.1674-5671.2023.05.08

• 基础研究 • 上一篇    下一篇

二甲双胍调控Gas6/Axl信号通路抑制食管鳞癌细胞增殖及迁移

  

  1. 空军军医大学特色医学中心消化内科
  • 出版日期:2023-10-25 发布日期:2023-11-03
  • 通讯作者: 范崇熙 E-mail：fcx329@fmmu.edu.cn
  • 基金资助:
    国家自然科学基金青年基金项目（81702731）；空军特色医学中心博士助推项目（21ZT16）

Metformin inhibits proliferation and migration of esophageal squamous cancer cells by regulating Gas6/Axl signaling pathway

  • Online:2023-10-25 Published:2023-11-03

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摘要： 目的 探讨二甲双胍对食管鳞癌细胞增殖及迁移的抑制作用，以及生长停滞特异性蛋白6（growth⁃arrest⁃specific protein 6，Gas6）与其配体Axl的调控机制。方法 食管鳞癌EC109和TE⁃1细胞用不同浓度二甲双胍处理后，采用CCK⁃8法或克隆形成实验测定细胞增殖情况，划痕实验和Transwell实验检测细胞迁移能力，TUNEL实验标记细胞凋亡比例，Mito⁃SOX探查线粒体氧化应激状态，JC⁃1显示线粒体膜电位（mitochondrial membrane potential，MMP）水平，免疫荧光实验检测Gas6表达，Western blot法分析Gas6/Axl通路和凋亡相关蛋白变化。此外，联合Axl高效抑制剂（bemcentinib，R428）处理后，观察二甲双胍对EC109细胞增殖、迁移及裸鼠皮下成瘤能力的影响。结果 相较于对照组，二甲双胍显著降低EC109和TE⁃1细胞的活性（PEC109<0.001；PTE⁃1<0.001），而对Het⁃1A细胞活性的影响并不显著（P=0.38）；同时二甲双胍抑制EC109细胞克隆形成能力（P=0.002）和迁移能力（P<0.001），增加凋亡率（P=0.002），上调线粒体活性氧水平（P<0.001），下调MMP水平（P<0.001）。此外，二甲双胍可降低EC109细胞Gas6荧光强度，阻断EC109和TE⁃1细胞中Gas6表达，下调EC109细胞内p⁃Axl、Axl和Bcl2的水平，并增加Bax以及细胞色素C和Caspase3活性。二甲双胍联合R428较单独二甲双胍能更有效抑制EC109细胞增殖、迁移及裸鼠皮下成瘤能力。结论 二甲双胍能够调节线粒体稳态，激活氧化应激诱导的凋亡反应，进而发挥抗EC109细胞增殖及迁移作用，该作用可能与下调Gas6/Axl信号通路相关。

关键词: 食管鳞状细胞癌, 二甲双胍, Gas6/Axl信号通路, 线粒体稳态, 凋亡

Abstract: Objective To investigate the inhibitory effect of metformin on the proliferation and migration of esophageal squamous cell carcinoma, and the regulatory mechanism of the growth⁃arrest⁃specific 6 (Gas6) and its ligand Axl. Methods The EC109 and TE⁃1 cells of esophageal squamous cell carcinoma were treated with different concentrations of metformin. The cell proliferation was measured by the CCK⁃8 assay or the plate clone formation. The migration ability was detected by wound healing and transwell migration assays. Apoptosis was detected by TUNEL assay and the mitochondrial oxidative stress state was detected by Mito⁃SOX. The revealed mitochondrial membrane potential (MMP) was measured by JC⁃1 staining. The expression of Gas6 was detected by immunofluorescence and by the changes of the Gas6/Axl pathway and apoptosis⁃related proteins were analyzed using Western blot. In addition, after treatment with Axl inhibitor (bemcentinib, R428), the effect of metformin on the proliferation, migration of EC109 cells, and subcutaneous tumor formation in nude mice were observed. Results Metformin significantly decreased the cell viability of EC109 and TE⁃1 cells compared to the control group ( PEC109<0.001; PTE⁃1<0.001) , while the effect was not significant on the activity of Het⁃1A cells (P=0.380). Metformin inhibited the colony formation (P=0.002) and migration (P<0.001) of EC109 cells, and increased the apoptosis rate (P=0.002), inducing a dose⁃dependent increase in mitochondrial reactive oxygen species (P<0.001) and decreasing MMP levels (P<0.001). Furthermore, Metformin reduced Gas6 fluorescence intensity in EC109 cells, blocked Gas6 expression in EC109 and TE⁃1 cells, down⁃regulated the levels of p⁃Axl, Axl and Bcl2 in EC109 cells, and increased the activities of Bax, cytochrome C and Caspase3 in EC109 cells. In addition, compared with metformin alone, metformin combined with R428 demonstrated superior efficacy in inhibiting tumor growth, migration and subcutaneous tumor formation in nude mice. Conclusions Metformin can regulate mitochondrial homeostasis, activate an oxidative stress⁃induced apoptotic response, and exert anti⁃proliferative and anti⁃migrative effects on EC109 cells, which may be associated with the down⁃regulation of Gas6/Axl signaling pathway.

Key words: Esophageal squamous cell carcinoma, Metformin, Gas6/Axl sigmaling pathway, Mitochondrial oxidative stress, Apoptosis

中图分类号: 

  • R735.1