The Inhibitory Effects And Molecular Mechanisms Of FICD Alone Or In Combination With Sorafenib On Hepatoma

Objective: To investigate the inhibitory effects of FICD alone or in combination with Sorafenib on the growth of cultured human hepatoma HepG2 cells and H22 tumor bearing mice,and to explore its molecular mechanisms.Methods: Tetrazolium salt(MTT)reduction method was used to determine the inhibitory effects of FICD alone or combined with Sorafenib on the growth of tumor cells in vitro.The tumor model of H22 bearing solid liver tumor in mice was adopted to evaluate the inhibitory effects of FICD alone or combined with Sorafenib on tumor growth in vivo.Flow cytometry was done to detect the effects of FICD alone or combined with Sorafenib on apoptosis and cell cycle of HepG2 cells.The morphology of apoptosis was observed by laser scanning confocal microscopy.β-Catenin,C-Myc,Vimentin,CyclinD1 and Ki-67 protein expressions in HepG2 cells were determined using Western blotting assay.Results:MTT assay results showed that FICD concentration-dependently inhibited the growth of human hepatocellular carcinoma HepG2 and SMMC-7721,glioma U251,cervical cancer HeLa,gastric cancer MGC-231,and breast cancer MAD-MB-231 cells.After treatment with FICD 48 h,the IC50 values were 10.1μg/mL,14.2 μg/mL,16.8 μg/mL,17.3 μg/mL,18.6 μg/mL,and 15.6 μg/mL,respectively.According to the IC50 values,FICD has the strongest effects on human hepatoma HepG2 and SMMC-7721 cells.Further studies showed that FICD also inhibited the growth of HepG2 cells in time-and concentration-dependent manner.After treatment with FICD for 24 h,48 h and 72 h,the IC50 values were 16.8 μg/mL,9.8 μg/mL and 6.7 μg/mL,respectively.Flow cytometry and confocal microscopy assay showed that FICD induced HepG2 cell apoptosis and arrested cell cycle in G2/M.Meanwhile,FICD decreased β-Catenin,C-Myc,CyclinD1 and Ki-67 protein expressions while increased Vimentin proteinin expression in HepG2 cells.In vivo experiments confirmed that FICD ig or ip had the same inhibitory effects on the growth of hepatocellular carcinoma H22 cells.Inhibitory ratios of FICD 50,100,150mg/kg ip were 40.3%,46.3% and 53%,and those of intragastric administration were40.1%,48.2% and 53.4%,respectively.The combined use of FICD and Sorafenib synergistically inhibited the growth of HepG2 cells in vitro,and also had a synergistic inhibitory effects on the growth of H22 hepatoma cells in vivo.Combined use of FICD and Sorafenib also promoted apoptosis of HepG2 cells.FICD 10 μg/mL arrested HepG2 cell cycle in G2/M phase but reduced G0/G1 phase and S phase.Sorafenib 4 μg/mL decreased S and G2/M phase but increased G0/G1 phase in HepG2 cells.When combined use of FICD and Sorafenib,G0/G1 phase cells was decreased but S and G2/M phase was increased as compared with Sorafenib 4 μg/m L alone.FICD 10 μg/mL and Sorafenib 4 μg/mL alone or in combination,significantly decreased beta-Catenin,C-Myc,CyclinD1 and Ki-67 protein expressions.However,Vimentin expression was significantly increased by FICD or Sorafenibthe alone,and was significantly decreased by combined FICD with Sorafenibthe treatment.Conclusion: FICD has a better anti hepatoma effects both in vivo and in vitro,and has synergistic effects with Sorafenib.The mechanisms may be related to the induction of apoptosis via Wnt/ beta-Catenin signaling pathway.