Effects Of Chrysin On TRAIL-induced Apoptosis In Human Hepatocellular Carcinoma Bel-7402 Cells

ObjectiveTo investigate whether chrysin(ChR) enhance apoptosis induced by TNF-related apoptosis-inducing ligand(TRAIL) via upregulating the expression of death receptor 5(DR5) in human hepatocellular carcinoma (HCC) Bel-7402 cell line.MethodsHuman hepatocellular carcinoma Bel-7402 cell line was cultured in vitro. The cell viability was evaluated by MTT assay. The percentage of sub-G1 cells was determined by flow cytometry(FCM) using PI fluorescence staining. The characteristic features of cell apoptosis was examined using DNA agarose gel electrophoresis. The expressions of DR5 were analyzed by Western blot.ResultsThe MTT assay showed that IC50 for cell viability inhibition in human hepatocellular carcinoma Bel-7402 cells by ChR and TRAIL alone at 48h were 252μmol/L and 664ng/mL, respectively. However, IC50 by TRAIL treatment for 48h pretreated with ChR(40μmol/L) for 30 minute was 91ng/mL, and the CI value for ChR and TRAIL was 0.497. FCM analysis with PI stainning indicated that the percentage of sub-G1 cells in human hepatocellular carcinoma Bel-7402 cell line by 40μmol/L ChR and 100ng/mL TRAIL alone for 48h were 4.92%±0.38% and 6.32%±0.39%, respectively. However, sub-G1 cell percentage after 100ng/mL TRAIL treatment for 48h pretreated with 40μmol/L ChR for 30 minute was 31.3%±2.50%. Furthermore, human recombinant DR5/Fc chimera protein can result in descend the cell death rate from 32.4%±0.47% to 15.2%±0.38%(P<0.05) in human hepatocellular carcinoma Bel-7402 cell line by 100ng/mL TRAIL treatment for 48h pretreated with 40μmol/L ChR for 30 minute. The ladder band could be shown in DNA agarose gel electrophoresis after treatment with 100ng/mL TRAIL for 48h pretreated with 40μmol/L ChR for 30 minute. Western blot analysis indicated that ChR upregulated expression of DR5 in Bel-7402 cells, in a time- and concentration- dependent manner. Human recombinant DR5/Fc chimera protein could effectively attenuate the up-regulation expression of DR5 by ChR.Conclusion1. Chrysin at subtoxic concentration possess enhancement of TRAIL induced apoptosis of human hepatocellular carcinoma Bel-7402 cells.2. The sensitization of TRAIL induced apoptosis of human hepatocellular carcinoma Bel-7402 cells by ChR is associated with up-regulating the expression of DR5 protien.