Study On Inhibitory Effect And Relative Mechanisms Of Digitoxin Combined With Sorafenib On Hepatocellular Carcinoma Cells

Study on the synergism and relative mechanisms of digitoxin combined with sorafenib on hepatocellular carcinoma cellsHepatocellular carcinoma(HCC) has high degree of malignancy, fast growth rate and strong ability of invasion and distant metastases. The molecule mechanism of Hepatocellular carcinoma, development, recurrence and metastasis are very complicated. Angiogenesis and apoptosis of HCC were considered play an important role in development, recurrence and metastasis of Hepatocellular carcinoma. Although the survival early cancer patients have increased by chemoembolization, surgery and other means, it still lack of effective treatment for patients with advanced cancer. With the development technology of molecular biology, Recent studies about apoptosis related proteins and gene research and the study of HCC angiogenesis factor is increasingly becoming the focus of research of Hepatocellular carcinoma.Sorafenib is a multikinse inhibitor, Sorafenib strongly inhibits Raf isoforms. it has shown to block tumor cell angiogenesis and proliferation by inhibiting the RAF/MEK/ERK signal transduction pathway, and the inhibitory actions of sorafenib on tumor angiogenesis con tribute to its antitumor activity in murine models. Sorafenib potently inhibits the VEGFR-2,VEGFR-3and FLT3.Objective:To investigate the inhibitory proliferation effect of digitoxin combined with sorafenib on BEL-7402and HepG-2hepatocellular carcinoma cell, to study the possible molecular mechanisms by detecting the moleculars associated of antiangiogenesis and signaling pathway. Our study aimed at further improving efficacy of digitoxin by combination with sorafenib in Hepatocellular carcinoma and providing preclinical important clue for bio-chemotherapy of HCC.Methods:1、The inhibitory of BEL-7402and HepG-2cells were detected by MTT assay after treatment24h,48h and72h. Calculate the inhibitory of BEL-7402and HepG-2cells according OD value. The interaction between digitoxin and sorafenib was assessed using the q value;2、cell cycle distribution of the BEL-740and HepG-2BEL-740and HepG-2cells in different groups were analyzed by flow cytometry;3、The scratch wound assay was used to determine BEL-7402and HepG-2cell migratory activity. The gap width were computed and were compared to determine the possible effect of digitoxin and sorafenib alone or inconbination on the migratory ability of HCC cells;4、The expression of ERK、p-ERK、HIF-1α、HIF-2αand VEGF protein levels in cytoplasm was evaluated by western blot, and the relative expression level of protein was represented with the ratio between produce of ERK、p-ERK、HIF-1α HIF-2α and VEGF and that of p-actin.Results:MTT assay showed that digitoxin and sorafenib could significantly inhibit the proliferation of BEL-7402and HepG-2cell lines in a dose-and time-dependent manner(P<0.05).There was a synergistic effect in digitoxin (10nmol/L)combined with sorafenib(8μmol/L) at48h on BEL-7402and HepG-2. AO/EB staining indicated dramatic apoptosis morphological changes in BEL-7402and HepG-2cell lines after digitoxin combined with sorafenib treatment48h. digitoxin and sorafenib alone can induced accumulation of cells in S phase compared with control, and combination group showed greater increase of S phase and decrease of Gl phase than single agent group. As western-blot shows that the expression of ERK showed no difference in all groups (P>0.05). The significant decrease of p-ERK, HIF-lα HIF-2αand VEGF protein expression were seen after the treatment of digitoxin alone and in combination with sorafenib for24h and48h(P<0.05) and it showed a time-dependent.Conclusion:digitoxin and sorafenib alone inhibited BEL-7402and HepG-2cell proliferation dose-and time-dependently.digitoxin combined with sorafenib showed synergistic effect on cell cycle arrest、 apoptosis and migratory activity of BEL-7402and HepG-2cells. Digitoxin and sorafenib can synergistically inhibitor BEL-7402and HepG-2cells by cooperatively down regulating the expression of p-ERK、 HIF-1α、 HIF-2aand VEGF protein levels and have no difference of the ERK protein expression.