Effects Of The Combination Of Apigenin And Trail On Apoptosis In Human Hepatocellular Carcinoma Hep G2 Cell Line

ObjectiveTo investigate whether apigenin(API) enhance apoptosis induced by TNF-related apoptosis-inducing ligand(TRAIL) through depletion of intracellular glutathione(GSH) in human hepatocellular carcinoma Hep G2 cell line.MethodsHuman hepatocellular carcinoma Hep G2 cell line and human embryo liver L-02 cell line were cultured in vitro. MTS assay was used to determine the cell viability. Flow cytometry (FCM) using propidium iodide (PI) staining was used to analyse the apoptotic rate of cells. Enzyme linked immunosorbent assay(ELISA) was used to detect the caspase-3 activity of cells. The intracellular GSH level was measured using spectrophotometry. FCM using the fluorescein isothiocyanate (FITC) tagged antibody against Death receptor 5 (DR5) was used to observe the expression level of DR5.ResultsMTT assay showed that the treatment with 5.0μmol/L API or TRAIL(10～1000ng/mL) alone for 24 h, the inhibitory effecet on the viability of Hep G2 cells was weaker, and the difference has not significance for statistics comparison with the vehicle(0.1% DMSO) group.TRAIL treatment for 24h pretreated with API(5.0μmol/L) for 1 h, the inhibitory effecet on the viability in Hep G2 cells was significantly potentiated(P<0.05), but was weaker in L-02 cell line. FCM analysis using PI staining indicated that the apoptotic rate of Hep G2 cells by 5.0μmol/L API or 100ng/mL TRAIL alone and in combination for 24h was 2.44%±0.34%, 2.36%± 0.08% and 30.23%±4.89% and that of L-02 cells was 0.36±0.07%, 0.37±0.03% and 0.37±0.03% , respectively. In Hep G2 cell line, Caspase-3 activity was significantly increased by treatment with the combination of 5.0μmol/L API and 100ng/mL TRAIL for 24 h(P<0.05), and 10μmol/L Ac-DEVD-CHO, a specific inhibitor for caspase-3 could efficaciously abrogate this effect (P<0.05). In L-02 cell line, the treatment with 5.0μmol/L API or 100 ng/mL TRAIL or both for 24 h have little effect on caspase-3 activity. Data by the spectrophotometry demonstrated that API(5.0, 10.0, 20.0μmol/L) significantly reduced the intracellular GSH level in Hep G2 cells, but had no effect in L-02 cells. Analysis of FCM using FITC tagged antibody against DR5 showed that API(5.0μmol/L,10μmol/L,20.0μmol/L) upregulated the expression level of DR5(P<0.05) in Hep G2 cell line , in a concentration-dependent manner, but did not affective in L-02 cell line. To add to the ectogenic GSH(pretreatment with 500μmol/L GSH for 1 h) could agonisticed the reduction of the intracellular GSH level and upregulation of DR5 expression in Hep G2 cells by API, and induction of apoptosis and activation of caspase-3 by the combination of API and TRAIL(P<0.05).Conclusion1. Apigenin at subtoxic concentrations possess enhancement of TRAIL induced apoptosis of human hepatocellular carcinoma Hep G2 cells.2. The combination of Apigenin and TRAIL at subtoxic concentrations has little effect on apoptosis of human embryo liver L-02 cells.3. One of the mechanisms underlying apigenin at subtoxic concentrations enhances apoptosis induced by TRAIL is depletion of intracellular glutathione(GSH) in human hepatocellular carcinoma Hep G2 cell line.