Objective To investigate the effects of astragaloside Ⅳ (AS-Ⅳ) on the proliferation, migration, invasion, and apoptosis of human tongue squamous cell carcinoma CAL-27 cells and its mechanism.
Methods The CAL-27 cells were cultured in vitro.The cells were treated with different concentrations of AS-Ⅳ (0, 5, 10, 20, 40, 80, 160 μmol/L) for 24, 48, 72 hours, and cell proliferation was detected by CCK-8 method.After treatment with 0, 20, 40, and 80 μmol/L AS-Ⅳ for 24 hours, cell migration and invasion abilities of CAL-27 cells were evaluated by Transwell assay, and apoptosis was analyzed by flow cytometry.The CAL-27 cells were divided into the control group, transforming growth factor-β1 (TGF-β1) group (10 ng/mL TGF-β1), and combination group (40 μmol/L AS-Ⅳ+10 ng/mL TGF-β1), and the cell migration and invasion abilities of each group were detected by Transwell assay, the protein expression levels of transforming growth factor-β receptor Ⅱ (TGF-β RⅡ) and Smad4 were analyzed by Western blotting.
Results The results of CCK-8 method showed that compared with the 0 μmol/L AS-Ⅳ group, the optical density values of 20, 40, and 80 μmol/L AS-Ⅳ treatment for 24, 48, and 72 hours were reduced (P < 0.05 to P < 0.01).The results of flow cytometry indicated that compared with the 0 μmol/L AS-Ⅳ group, 20, 40, and 80 μmol/L AS-Ⅳ treatment for 24 hours could significantly induce apoptosis in CAL-27 cells (P < 0.01);compared with the 20 μmol/L AS-Ⅳ group and 40 μmol/L AS-Ⅳ group, the apoptosis rate in the 80 μmol/L AS-Ⅳ group was significantly increased (P < 0.01).The results of Transwell assay found that compared with the 0 μmol/L AS-Ⅳ group, 20, 40, and 80 μmol/L AS-Ⅳ could significantly inhibit the migration and invasion abilities of CAL-27 cells (P < 0.01), and the number of migrating and invading cells gradually decreased with the increase of AS-Ⅳ concentration (P < 0.01);the number of migrating and invading cells in the transforming growth factor-β1 (TGF-β1) group was significantly higher than that in the control group (P < 0.01), the number of migrating and invading cells in the combination group was higher than that in the control group (P < 0.05 and P < 0.01), but lower than that in the TGF-β1 group (P < 0.05).The results of Western blotting implied that the levels of TGF-β RⅡ and Smad4 proteins in the TGF-β1 group and combination group were significantly higher than those in the control group (P < 0.01), while the levels of TGF-β RⅡ and Smad4 proteins in the combination group were significantly lower than those in the TGF-β1 group (P < 0.01).
Conclusions AS-Ⅳ can induce apoptosis and inhibit proliferation, migration, and invasion of tongue squamous cell carcinoma CAL-27 cells.AS-Ⅳ may regulate the migration and invasion abilities of CAL-27 cells by blocking the TGF-β/Smad signaling pathway mediated by TGF-β1.
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