ABSTRACT
Objective:To investigate the effects and mechanisms of Wnt pathway inhibitor IWR-1-endo on the biological behaviors of human hepatocarcinoma cell Huh7.Methods:Human hepatocellular carcinoma cell Huh7 was cultured in vitro, and Huh7 cells were treated with IWR-1-endo at different concentrations (0, 20, 40, 80, 160, 320 μmol/L). Scratch test was used to detect changes in cell migration ability at diffe-rent drug concentrations, plate cloning was used to detect changes in cell proliferation, Western blotting was used to detect changes in the expression of Wnt pathway related protein β-catenin, and immunofluorescence staining was used to detect the expression of β-catenin in cytoplasm and nucleus. Results:The results of the scratch test showed that the 24 h scratch healing rates of Huh7 cells treated with 0, 20, 40, 80, 160, 320 μmol/L IWR-1-endo were (20.55±0.05)%, (12.10±0.08)%, (9.36±0.10)%, (3.62±0.09)%, (0.62±0.04)% and (0.23±0.02)%, respectively, and there was a statistically significant difference ( F=230.87, P<0.001). Further pair comparison showed that there were statistically significant differences in 24 h scratch healing rates among different concentrations (all P<0.001). The 48 h scratch healing rates were (34.77±0.08)%, (17.69±0.05)%, (11.60±0.04)%, (5.68±0.07)%, (2.66±0.04)% and (1.75±0.02)%, respectively, and there was a statistically significant difference ( F=589.68, P<0.001). Further pair comparison showed that there were statistically significant differences in 48 h scratch healing rates among different concentrations (all P<0.001). After treatment with IWR-1-endo at the concentration of 0, 20, 40, 80, 160, 320 μmol/L, the clone formation rates of Huh7 cells were (61.67±0.21)%, (57.33±0.11)%, (50.00±0.25)%, (36.67±0.28)%, (23.33±0.12)% and (15.00±0.08)%, respectively, and there was a statistically significant difference ( F=403.56, P<0.001). Further pair comparison showed that there were statistically significant differences in clone formation rates among different concentrations (all P<0.001). After treatment with 0, 20, 40, 80, and 160 μmol/L IWR-1-endo for 24 h, the relative expression levels of β-catenin in Huh7 cells were 0.30±0.08, 0.25±0.07, 0.22±0.05, 0.15±0.01 and 0.06±0.02, respectively, and there was a statistically significant difference ( F=247.00, P<0.001). Compared with 0 μmol/L, the relative expression levels of β-catenin treated with 80 and 160 μmol/L had statistical significance ( P=0.014; P=0.008). Compared with 0 mol/L, immunofluorescence showed that the expressions of β-catenin in cytoplasm and nucleus were reduced after 80 μmol/L IWR-1-endo treatment. Conclusion:Wnt pathway inhibitor IWR-1-endo can inhibit the migration and proliferation of hepatocarcinoma cells Huh7 by inhibiting the activity of Wnt pathway. The above inhibitory effects are dose-dependent.
ABSTRACT
Objective: To investigate the effects of Wnt inhibitor IWR-1-endo (IWR-1) on the proliferation and migration of human osteosarcoma MG-63 cells, and to explore the possible mechanism. Methods: MG-63 cells were treated with different concentrations of IWR-1 (2, 4, 8 and 16 μmol/L). Then the growth inhibitory rates of MG-63 cells was determined by CCK-8 assay. The cell cycle and apoptosis rate of MG-63 cells were detected by FCM method. The migration ability of MG-63 cells was abserved by scratch wound healing assay. The expression levels of β-catenin and cyclin D1 mRNAs were detected by real-time fluorescent quantitative PCR. The expression levels of Axis inhibition protein 1 (AXIN1), β-catenin, phospho-β-catenin (p-β-catenin) and cyclin D1 proteins were detected by Western blotting. Results: The proliferation inhibitory rate of MG-63 cells after IWR-1 treatment increased in a time-and concentration-dependent manner (all P < 0.01). The proportion of MG-63 cells treated with IWR-1 in G0/G1 phase were increased (P < 0.05), and the apoptosis rate of MG-63 cells increased significantly with the increase of IWR-1 concentration (P < 0.01). IWR-1 reduced significantly the scratch healing rate of MG-63 cells (P < 0.01), decreased the mRNA (both P < 0.01) and protein (both P < 0.05) expression levels of β-catenin and cyclin D1, and increased the expression levels of AXIN 1 and p-β-catenin proteins (both P < 0.05). Conclusion: IWR-1 can inhibit the proliferation and migration of MG-63 cells by blocking Wnt/β-catenin signaling pathway.