The Inhibition Of Chakasaponin ? And ? Extracted From Tea Flowers On The Proliferation Of Human Ovarian Cancer Cells And Its Mechanisms

Tea(Camellia sinensis(L.)O.Kuntze)flowers,as one of new resource foods,are very rich in our country,but the current utilization rate is quite low.Saponins,as one of important functional and characteristic components in tea plants,have attracted more attention nowadays for the bioactivities.However,the antitumor activities of tea flower saponins have been seldom studied yet due to the great difficulty in isolation.Ovarian cancer is a malignant tumor with high mortality and poor prognosis in the world.In this thesis,on the basis of predecessors'research,purified tea flower saponins(PTFSs)were separated and identified.In this thesis,ovarian cancer cells were used as in vitro model to investigate the inhibitory effects and the mechanisms of PTFSs on their proliferation and growth.The main results are as follows:1.Purified tea flower saponins(PTFSs)was successfully saparated and characterized by UPLC-Q-TOF/MS/MS analysis.Three saponins were identified as Chakasaponin I,Chakasaponin IV,and Floratheasaponin A(allocating 80.1%,8.1%,and 1.4%,respectively),based on published fragmentation data and nominal massed calculated from known structures.2.The cytotoxicity of PTFSs on A2780/CP70,OVCAR-3 and IOSE-364 cells was analyzed using the MTS assay.PTFSs inhibited the growth of all three cell lines in a dose-dependent manner,but had lower cytotoxic effect against normal epithelial ovarian cell line IOSE-364 cells constrast to ovarian cancer cell lines.The cell viability decreased to 20.28%for A2780/CP70,to 6.52%for OVCAR-3,and to 53.64%for IOSE-364 cells after treated with PTFSs for 24 h.3.The effects of PTFSs on cell cycle of ovarian cancer cells were clarified.As evidenced by the results of flow cytometry,the proportion of S phase cells increased significantly after treated with PTFSs.PTFSs downregulated the protein levels of Cylin A2,CDK2 and CyclinD1,which led to the decrease in CyclinE/A-CDK2 complexes,and following the cells were arrested at S phase.4.The effects and mechanisms of PTFSs on apoptosis in ovarian cancer cells were clarified.PTFSs treatment induced mitochondrial membrane potential collapsing and apoptosis in ovarian cancer cells and it is related to the activation of caspase-3,-7 and-9 but not related to the activation of caspase-8.At the mean time,the expressions of intrinsic apoptosis related protein were regulated while that of extrinsic apoptosis related protein were not regulated.All these results indicated PTFSs may induce apoptosis in A2780/CP70 cells through the intrinsic pathway rather than extrinsic pathway.5.PTFSs-induced intrinsic apoptosis and S-phase arrest are dependent on p53 in ovarian cancer cells.We pre-incubated cells with p53 specific inhibitor pifithrin-?(PFT-?)(20?M)for 24 h and performed the Hoechst 33342 and JC-1 assays after the PTFSs treatment to check the cell apoptosis.Moreover,we knocked out p53 gene by p53 siRNA and then checked intrinsic apoptotic related proteins.All the results suggested that p53 is a pivotal mediator of PTFSs-induced intrinsic apoptosis and S-phase arrest in ovarian cancer cells.6.PTFSs could inhibit the proliferation and cancer stem cells traits of ovarian cancer stem-like cells(OCSLCs),which was associated with inducing apoptosis and inhibiting the Wnt/?-catenin signaling pathway.OCSLCs was obtained by serum-free suspension culture.The inhibition of PTFSs on the OCSLCs proliferation was determined by MTS assay andcell clone formation assay.The inhibition on the stem cell traits of OCSLCs by PTFSs was determined by tumor ball formation assay,flow cytometry detection of stem cell marker ALDH activity and Western blot analysis.PTFSs could suppress cell activity,reduce colony formation ability and inhibit the tumor sphere formation.The ALDH~+population and protein expressions of Oct-4 were decreased in OCSLCs treated with PTFSs.PTFSs could also induce apoptosis of OCSLCs.Besides,PTFSs could up-regulate the protein expression of?-catenin and GSK-3?,down-regulate the protein expression of p-GSK-3?and?-catenin,which indicated that suppression of the Wnt/?-catenin signaling pathway might be one of the mechanisms associated with inhibiting OCSLCs.Taken together,PTFSs mainly containing Chakasaponin I and IV,were isolated and purified from tea flowers.PTFSs could induce p53-dependent intrinsic apoptosis and S phase arrest in ovarian cancer cells and inhibit the proliferation and cancer stem cells traits of ovarian cancer stem-like cells.The strong anticancer activities of PTFSs were demonstrated.This thesis provides a new theoretical basis for the study of antitumor efficacy of tea flower saponins.