Effects Of Apigenin On Trail-induced Apoptosis In Human Ovarian Cancer CoC1 Cells

ObjectiveTo investigate whether apigenin(API) enhance apoptosis induced by TNF-related apoptosis-inducing ligand(TRAIL) via inhibiting the activity of NF-κB in human ovarian cancer CoC1 cell line.MethodsHuman ovarian cancer CoC1 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 colorimetric method. The characteristic features of cell apoptosis was examined using DNA agarose gel electrophoresis. The expressions of protein were analyzed by Western blot.ResultsThe Cytotoxicity assay based on a LDH release showed that EC₅₀ of cytotoxicity in human ovarian cancer CoC1 cells by API and TRAIL alone for 48h were 340μmol/L and 795ng/mL, respectively. However, IC50 by TRAIL treatment for 48h pretreated with 20μmol/L API for 30 minute was 29ng/mL, and the CI value for API and TRAIL was 0.1273. The results by trypan blue exclusion confirmed that EC₅₀ of cell death in human ovarian cancer CoC1 cells by API and TRAIL alone for 48h were 134μmol/L and 234ng/mL, respectively. However, IC50 by TRAIL treatment for 48h pretreated with 20μmol/L API for 30 minute was 37 ng/mL, and the CI value for API and TRAIL was0.4322. FCM analysis with PI stainning indicated that the percentage of sub-G1 cells in human ovarian cancer CoC1 cells by 20μmol/L API and 20ng/mL TRAIL alone for 48h were 8.83%±2.33% and 8.32%±2.80%, respectively. However, the sub-G1 cell percentage after 20ng/mL TRAIL treatment for 48h pretreated with 20μmol/L API for 30 minute was 69.50%±4.65%. Caspase-3 activity in CoC1 cells treated with 0.1%DMSO or 20μmol/L API or 20ng/mL TRAIL or both for 48h was 1.1, 1.3, 1.4, 6.5 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. Western blot analysis indicated that API inhibited expression of NF-κB(p65) protein and reduced the phosphorylation level of IκBαprotein in CoC1 cells, in a time- and concentration- dependent manner.Conclusion1. Apigenin at subtoxic concentration possess enhancement of TRAIL induced apoptosis of human ovarian cancer CoC1 cells.2. The sensitization of TRAIL induced apoptosis of human ovarian CoC1 cells by API is associated with inhibiting expression of NF-κB(p65) protein and reducing the phosphorylation level of IκBαprotein...