Effects Of Cotreatment With Apigenin And Trail On Apoptotic Death Of Human Gastric Cancer Cells

ObjectiveTo investigate whether apigenin(API) enhances apoptosis induced by TNF-related apoptosis-inducing ligand(TRAIL) through depletion of intracellular glutathione(GSH) in human gastric cancer BGC-823 cell line.MethodsHuman gastric cancer BGC-823 cell line and immortalized human gastric mucosa GES-1 cell line were cultured in vitro. MTS assay was used to determine the cell viability. Flow cytometry (FCM) after 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 with the fluorescein isothiocyanate(FITC) tagged antibody against Death receptor 5(DR5) was used to observe the expression level of DR5.ResultsMTS assay showed that treatment with API (10.0, 20.0μmol/L) or TRAIL(10.0, 100.0 ng/mL) alone for 24 h, the inhibitory effecet on the viability of BGC-823 cells was weaker, and the difference has not significance for statistics comparison with the vehicle(0.1% DMSO) group.TRAIL(10.0, 100.0, 1000.0 ng/mL) treatment for 24h pretreated with 20.0μmol/L API for 1 h, the inhibitory effecet on the viability in BGC-823 cells was significantly potentiated(P<0.05), but was weaker in GES-1 cell line. FCM analysis after PI staining indicated that the apoptotic rate of BGC-823 cells by 20.0μmol/L API or 100 ng/mL TRAIL alone and in combination for 24h was 4.46%±0.56%, 5.18%±0.40%, 32.2%±3.82% , and that of GES-1 cells was 4.14%±0.23%, 4.07%±0.23% , 4.50%±0.32%, respectively. In BGC-823 cell line, Caspase-3 activity was significantly increased by treatment with the combination of 20.0μmol/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 these effects(P<0.05). In GES-1 cell line, the treatment with 20.0μmol/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(20.0, 40.0μmol/L) significantly reduced the intracellular GSH level in BGC-823 cells, but had no effect in GES-1 cells. Analysis of FCM with FITC tagged antibody against DR5 showed that API(20.0, 40.0μmol/L) upregulated the expression level of DR5(P<0.05) in BGC-823 cell line , in a concentration-dependent manner, but did not effect in GES-1 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 by API, and induction of apoptosis and activation of caspase-3 by the combination of API and TRAIL in BGC-823 cells (P<0.05).Conclusion1. Apigenin at subtoxic concentrations possess enhancement of TRAIL induced apoptosis of human gastric cancer BGC-823 cells.2. The combination of apigenin at subtoxic concentrations and TRAIL has little effect on apoptosis of immortalized human gastric mucosa GES-1 cells3. One of the mechanisms underling apigenin at subtoxic concentrations enhances apoptosis induced by TRAIL is the depletion of intracellular glutathione(GSH) in human gastric cancer BGC-823 cells.