Effects Of The Combination Of Apigenin And TRAIL On Apoptosis Of Human Cervical Cancer HeLa Cell Line

ObjectiveTo investigate whether apigenin(API) enhance apoptosis induced by TNF-related apoptosis-inducing ligand(TRAIL) through promoting generation of reactive oxygen species (ROS) in human cervical cancer HeLa cell line.MethodsHuman cervical cancer HeLa cells were cultured in vitro. The lactate dehydrogenase activity of cell medium was evaluated by automatic biochemisitry analyzer. The cell death was detected by trypan blue exclusion. The percentage of sub-G1 cells was determined by flow cytometry(FCM) using PI fluorescence staining. Caspase-3 activity was determined using ELISA assay. The characteristic features of cell apoptosis was examined using DNA agarose gel electrophoresis. The generation of ROS was analyzed by flow cytometry using the fluorescent probe DCFH-DA.ResultsThe Cytotoxicity assay based on a LDH release showed that EC₅₀ for cytotoxicity in human cervical cancer HeLa cells by API or TRAIL or both for 48h were 104μmol/L, 236ng/mL, and 23 ng/mL, respectively. The CI value for API and TRAIL was 0.3186. The results by trypan blue exclusion confirmed that EC₅₀ for cell death in human cervical cancer HeLa cells by API and TRAIL alone for 48h were 109μmol/L and 163ng/mL, respectively. However, EC₅₀ by TRAIL treatment for 48h pretreated with 20μmol/L API for 30 minute was 43ng/mL, and the CI value for API and TRAIL was 0.4562. FCM analysis with PI staining indicated that the percentage of sub-G1 cells in human cervical cancer HeLa cells by 20μmol/L API or 20ng/mL TRAIL alone and in combination for 48h were 3.56%±0.20%, 6.69%±0.40%, and 59.87%±4.20% respectively. Caspase-3 activity in HeLa cells treated with 20μmol/L API or 20ng/mL TRAIL or both for 48h was 1.4, 1.5, 39 fold in comparison with medium group. The ladder band could be shown in DNA agarose gel electrophoresis after treatment with 20ng/mL TRAIL for 44h pretreated with 20μmol/L API for 4h. The flow cytometry using the fluorescent probe DCFH-DA analysis indicated that API elevated ROS level in HeLa cells, in a concentration-dependent manner. N-Acetylcysteine (NAC) could attenuate the potentiation of TRAIL-induced apoptosis by API in HeLa cells.Conclusion1. Apigenin at subtoxic concentration possess enhancement of TRAIL induced apoptosis of human cervical cancer HeLa cells.2. The sensitization of TRAIL induced apoptosis of HeLa cells by API is associated with promoting generation of reactive oxygen species.