The Inhibitory Effects And Mechanisms Of Saponins Isolated From Tea (Camellia Sinensis) Flowers On Human Ovarian Cancer Cells And Its Stem Like Cells

Tea(Camellia sinensis)flowers are rich agricultural resources but low utilization in China.Saponins are functional components of tea flowers which have various biological activities.Recently,more and more attention has been paid to the biological activities of saponins,however there are few studies on anti-tumor activity of saponins.Ovarian cancer is regarded as one of the most severe malignancies for women in the world,which is the fifth leading cause of women cancer deaths.Previous studies have shown that saponins from different plants had well inhibitory effect on ovarian cancer.In the present study,the effect and mechanism of tea flower saponins on ovarian cancer and its stem cells were investigated.The main results are as follows:1.The high purity saponin compounds BTFS 3,contained Floratheasaponin A and Floratheasaponin D,was extracted and isolated from tea(Camellia sinensis cv.Baiye 1)flowers by macroporous resin,preparative liquid chromatography,and detected by UPLC-Q-TOF/MS.BTFS 3 had a significant inhibitory effect on the proliferation of A2780/CP70 cells in vitro.Through MTS experiment,cell clone formation assay and Western blot analysis,we found that BTFS 3 could inhibit the cell viability of A2780/CP70 cells,reduce colony formation ability and suppress the PCNA protein expression.2.BTFS 3 could induce S phase cell cycle arrest of A2780/CP70 cells.Through flow cytometry,reactive oxygen species(ROS)detection and Western blot analysis,it was found that BTFS 3 could up-regulate the protein expression of p21 and Cyclin E1,and down-regulate the protein expression of CyclinA2?CDK2 and Cdc25A,which inhibit in CyclinE1/Cdk2 and CyclinA2/Cdk2 formation,and thus induced A2780/CP70 cells arrested at S phase.In addition,BTFS3 could promote the generation of ROS,cause DNA damage,and BTFS 3-induced S phase arrest was associated with ATM-Chk2-Cdc25A-CDK2 signaling pathway.3.BTFS 3 could induce apoptosis of A2780/CP70 cells by activating the death receptor pathway and mitochondrial pathway.Hoechst 33342 staining,mitochondrial membrane potential detection,flow cytometry,Caspase activity detection and Western blot analysis,which showed that BTFS 3 could up-regulate the expression of death receptor-related proteins Fas,DR5 and FADD,enhance the activity of Caspase-8 and thereby activate the death receptor pathway of apoptosis.BTFS 3 could also increase the ratio of pro-apoptotic/anti-apoptotic proteins in Bcl-2 family,improve the activity of Caspase-9 enzyme,and thus activate the mitochondrial pathway.Meanwhile,BTFS 3 could regulate AKT-MDM2-p53 signaling pathway to promote cell apoptosis.4.Found that BTFS 3 could inhibit the proliferation and cancer stem cells traits of ovarian cancer stem like cells(OCSLCs),which was associated with G0/G1 phase arrest and Wnt/?-catenin signaling pathway inhibition.OCSLCs was obtained by serum-free suspension culture.It was found BTFS 3 could inhibit OCSLCs using MTS assay,cell clone formation and tumor ball formation experiment,flow cytometry detection of stem cell marker ALDH activity and Western blot analysis.BTFS 3 could suppress cell activity,reduce colony formation ability and inhibit the tumor sphere formation.The ALDH~+population and expressions of Oct-4 and Nanog protein were decreased in OCSLCs treated with BTFS 3.And BTFS 3 could also induce OCSLCs arrested at G0/G1 phase.Besides,BTFS 3 could up-regulate the protein expression of?-catenin,down-regulate the protein expression of p-AKT?p-GSK-3???-catenin and c-Myc,which indicated that suppression of the Wnt/?-catenin signaling pathway might be one of the mechanisms associated with inhibiting OCSLCs.