Effects of Asiaticoside on Epithelial-Mesenchymal Transition and Immune Escape in Lung Cancer Cells by Regulating the TIM3/Gal9 Axis
YANG Xuepeng1*, WANG Lan2, CHAI Jutang2, YANG Weiqi3
This study aims to investigate the effects of ATS (Asiaticoside) on epithelial-mesenchymal transition and immune escape in lung cancer cells by regulating the TIM3 (T cell immunoglobulin domain and mucin domain-3)/Gal9 (galectin-9) signaling axis. Cells were treated with different concentrations of ATS (0, 5,10, 20, 40, 80 μmol/L) to screen for the optimal concentration. The cells were separated into control group, ATS group (25 μmol/L ATS), ATS+pcDNA-NC group (25 μmol/L ATS+pcDNA-NC), and ATS+pcDNA-TIM3 group (25 μmol/L LATS+pcDNA-TIM3). The CCK-8 assay kit and flow cytometry were used to detect cell proliferation and apoptosis. Transwell chamber was used to detect cell invasion and migration. ELISA was applied to detect the levels of TNF-α, IFN-γ, and IL-2 in cells. Western blot was applied to detect the expression of E-cadherin, Vimentin, PD-L1, TIM3, and Gal9 proteins in cells. BALB/c nude mice were used to construct NSCLC (nonsmall cell lung cancer) models, which were separated into control group, ATS group, ATS+AAV-NC group, and ATS+AAV-TIM3 group. Tumor volume and mass were measured. Immunohistochemical staining was applied to observe the expression of PD-L1 and Gal9 proteins. Compared with the control group, the cell survival rate, cell migration number, cell invasion number, Vimentin, PD-L1, TIM3, and Gal9 protein expression in the ATS group were lower (P<0.05), the apoptosis rate, TNF-α, IFN-γ, IL-2 levels, and E-cadherin protein expression were higher (P<0.05). Compared with the ATS+pcDNA-NC group, the cell survival rate, cell migration number, cell invasion number, Vimentin, PD-L1, TIM3, and Gal9 protein expression were higher in the ATS+pcDNATIM3 group (P<0.05), the apoptosis rate, TNF-α, IFN-γ, IL-2 levels, and E-cadherin protein expression were lower (P<0.05). In vivo tumor experiments showed that compared with the control group, the tumor volume andmass of mice in the ATS group were lower (P<0.05), and PD-L1 and Gal9 protein expression levels were lower. Compared with the ATS+AAV-NC group, the tumor volume and mass of mice in the ATS+AAV-TIM3 group were higher (P<0.05), and PD-L1 and Gal9 protein expression levels were higher. ATS may inhibit epithelialmesenchymal transition and immune escape in lung cancer cells by suppressing the TIM3/Gal9 signaling axis.
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