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 ( P_EC109<0.001; P_TE⁃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