Effects Of Wnt/β-catenin Inhibitor On The Proliferation And Apoptosis Of Human Hepatic Carcinoma Cell Lines Hep3B

Objective1. To investigate the effects of IWR-1 on proliferation and the possible mechanism of human hepatic carcinoma cell lines Hep3B.2. To study the effects of Wnt/β-catenin inhibitor IWP-2 on the proliferation and apoptosis of human hepatic carcinoma cell lines Hep3B,and to elucidate the possible mechanism of such effects.Methods1. Hep3B cells were treated with different concentration of IWR-1 (2,4,8, 16μmol/L). Cell growth inhibition rates were determined by CKK-8 assay. Cell cycle and apoptosis rate were analyzed by flow cytometry. The expression levels of proteins were detected by western blotting, β-catenin and c-myc mRNA expression were determined by real time polymerase chain reaction(RT-PCR).2. Human hepatic carcinoma Hep3B cells were treated with IWP-2 (0、20、40、 80、160、20μmol/L). The inhibitory effect of IWP-2 on cell proliferation was examined by CCK-8. The cell cycle distribution and apoptosis rate of Hep3B cells induced by IWP-2 were detected by flow cytometry(FCM). The expression of apoptotic-related protein were detected by western blotting. The expression levels of Cyclin D1 and survivin proteins were detected by western blotting. Cyclin D1 and survivin mRNA expression were determined by real time polymerase chain reaction(RT-PCR).Results1. The proliferation rate of Hep3B cells in the IWR-1 treated group decreased in a time-concentrated dependent manner(P<0.01). FCM results showed that the proportion of cells in Go/Gi phase were increased while the apoptosis rate of Hep3B cells was decreased in a concentration-dependent manner(P<0.01). IWR-1 could down-regulate β-catenin and c-myc mRNA levels, down-regulate β-catenin and c-myc mRNA protein levels, but significantly up-regulate Axin 1 and p-β-catenin protein levels.2. The proliferation of cells was inhibited in a concentration-and time-dependent manner when it was treated with IWP-2(20μmol/L～320μmol/L). Absorbance values between groups had statistically significant difference(P<0.01). FCM results showed that the proportion of cells in Go/Gi phase were increased while which in S phase were decreased by IWP-2(20μmol/L～320μmol/L) for 48h. The apoptosis rate of Hep3B cells induced by IWP-2 for 48h was incresed in a concentration-dependent manner. Absorbance values between groups had statistically significant difference(P<0.01). The expression levels of caspase-3, caspase-7 proteins could be increased by IWP-2 in a concentration-manner. The expression levels of Cyclin D1 and survivin proteins and mRNA were down-regulated by IWP-2 in a concentration-manner.Conclusion1. IWR-1 can inhibite proliferation of Hep3B cells through inhibiting Wnt/p-catenin signaling pathway.2. IWP-2 can inhibited the proliferation of Hep3B cells and induces apoptosis of Hep3B cells. These effects may related with the down-regulation of Cyclin D1 and survivin.