Effects Of Casticin On Production Of Reactive Oxygen Species And Induction Of Apoptosis In Human Cervical Cancer HeLa Cells

ObjectiveTo investigate whether casticin (CAS) induces apoptosis of human cervical cancer HeLa cell line through promoting generation of reactive oxygen species (ROS).MethodsHuman cervical cancer HeLa cells were cultured in vitro. The Histone/DNA fragments and caspase-3 activity were detected using enzyme linked immunosorbent assay (ELISA). The apoptotic rate (the percentage of cells in sub-G1 phase) was determined by flow cytometry (FCM) using propidium iodide (PI) fluorescence staining. The generation of ROS was analyzed by flow cytometry using the fluorescent probe 2'7'-dichlorofluoresein diacetate (DCFH-DA).ResultsELISA assay showed that the Histon/DNA fragment level of HeLa cells were significantly increased (P <0.05) after treatment with 0.5, 1.0, 2.0μmol/L CAS for 24 h, in a concentration-dependent manner. FCM analysis using PI staining indicated that the apoptotic rate by treatment with 0.5, 1.0, 2.0μmol/L CAS for 24 h was 3.76%±0.59%, 11.80%±0.79% and 23.97%±1.33%,respectively, and the difference has statistical significance comparison with the vehicle group(0.1% DMSO, 0.4%±0.05% ). The results of ELISA assay indicated that Caspase-3 activity in HeLa cells treated with 0.5, 1.0, 2.0μmol/L CAS for 24 h was 2.18, 11.16 and 20.51 fold in comparison with the vehicle group(0.1% DMSO, 1.00).Analysis by FCM using the fluorescent probe DCFH-DA indicated that CAS elevated ROS generation level in HeLa cells(P <0.05), in a concentration-dependent manner. N-Acetylcysteine (NAC) can block generation of ROS and attenuate induction of apoptosis, increase of the Histone/DNA fragments, and activation of caspase-3 by CAS(P <0.05).Conclusion1. CAS possesses the apoptosis-induced effect in human cervical cancer HeLa cells.2. The induction of apoptosis in HeLa cells by casticin is associated with promoting generation of ROS.