EVn-50Increase The Sensitivity Of Human Ovarian Cancer Cell Line COC1/DDP To Cisplatin In Vitro

Objective:To investigate the effects of the acetoacetate extract of Vitex negundoseed(EVn-50) or cisplatin(DDP) or both on the apoptosis of ovarian carcinoma cellline COC1/DDP cultured in vitro and the mechanism underlying EVn-50sensitizesCOC1/DDP cells to DDP-induced apoptosis.Methods:To evaluate EVn-50increasing the inhibitory effect of DDP on the proliferationof COC1/DDP by the MTT assay and the apoptotic effect by Hoechst33258stainingand flow cytometric analysis. The expressions of Caspase-3and proliferating cellnuclear antigen(PCNA)were analyzed by Western blot.Results:MTT assay showed that EVn-50inhibited the growth of COC1/DDP cells, in adose-and time-dependent manner, and the difference of EVn-50(20,40,60,80,100μg/mL) groups and control group had statistical significance(P<0.05). The IC50ofEVn-50for48h suppressed COC1/DDP cells growth was56.34μg/mL.The IC50of DDP for48h suppressed COC1/DDP cells growth was39.04μg/mL;The IC50of DDP for48h suppressed COC1/DDP cells growth was26.39μg/mL whenuniting EVn-50(20μg/mL).The fold-reversal of MDR was1.5.Flow cytometry analysis revealed that treatment COC1/DDP cells with control、EVn-50(20μg/mL)、DDP(30μg/mL)、EVn-50(20μg/mL) and DDP(30μg/mL) for48h, the percentage of apoptotic cells were4.50%±1.30%,9.80%±1.70%, 20.50%±3.70%,50.90%±2.90%respectively, and the difference had statisticalsignificance(P<0.05).The typical morphological change of apoptosis, such as nuclear fragmentation,nuclear karyopycnosis, edge set of nuclear chromosomes, the formation of apoptoticbodies, were observed using fluorescence microscope after exposure of COC1/DDPcells to EVn-50(20μg/mL) and(or) DDP(30μg/mL).The more apoptosis cells wereobserved when EVn-50(20μg/mL) and DDP(30μg/mL).There were difference among COC1/DDP cells treated with control, EVn-50(20μg/mL), DDP(30μg/mL), EVn-50(20μg/mL) and DDP(30μg/mL) for48h in themean relative density of expression of Caspase-3protein (P<0.05). The relativedensity were0.984±0.028,1.710±0.150,2.433±0.151,2.825±0.153respectively;There were all PCNA expression in the four groups, The relative density of PCNAprotein of the EVn-50(20μg/mL) group was lower than control group (P<0.05), andThe relative density of PCNA protein of the EVn-50(20μg/mL)+DDP(30μg/mL)group was lower than the DDP(30μg/mL) group(P<0.05); There were Ubi-PCNAexpression in the DDP(30μg/mL) groups and the EVn-50(20μg/mL)+DDP(30μg/mL) group, The relative density of Ubi-PCNA protein of the the EVn-50(20μg/mL)+DDP(30μg/mL) group was lower than the DDP(30μg/mL) group (P<0.05).Conclusions:1.EVn-50can inhibite the growth of COC1/DDP cells, in a dose-andtime-dependent manner.2.EVn-50can reverse the resistance of COC1/DDP cells with DDP, Thefold-reversal of MDR is1.5fold.3.EVn-50can potentiate apoptosis of COC1/DDP cells induced by DDP, theaugmentation of EVn-50on DDP-induced apoptotic cell death in COC1/DDP cell lineis associated with reduced PCNA expression and activated Caspase-3.