Analysis Of Theafalvins And The Inhibitory Effects And Mechanisms Of Theaflavins On Human Ovarian Carcinoma Cells

Black tea is widely consumed all around the world.Epidemiological studies have demonstrated that black tea consumption is connected with a reduced risk of ovarian cancer.Theaflavins are unique black tea polyphenols which exert multiple anti-tumor activities.Ovarian cancer is the most fatal gyneco logical cancers.Ovarian cancer recurrence after first-linre therapy is believed to be induced mainly by ovarian cancer stem cells.Ovarian cancer stem cells can cause chemotherapy resistance and stronger invasion which lead to low 5-year survival rate for patients with advanced ovarian cancer.The main results are as follows:A reliable and rapid UPLC method was establish to analyze GA,caffeine,8 catechine and 4 theaflavins in black tea within 26min.The UPLC method was able to separate the 14 analytes with good resolutions(>1.96).All the analytes showed good linearity with coefficient of determination(R2)ranging from 0.9992 to 0.9999.Good recoveries of 95.81%-104.48%were found for all analytes.The precision was within 15%,and accuracy was between-15%and 15%.The UPLC method was accurate,reliable and reproducible.Compared to previous liquid chromatography methods,the UPLC method can be applied to analyze for the 14 analytes in black tea simultaneously in shorter time with better chromatographic resolution attained.Treatment with TF2a and TF2b exhibited a potent growth inhibitory effect in cisplatin-resistant ovarian cancer A2780/CP70 cells and were less cytotoxic to normal ovarian IOSE-364 cell line.Flow cytometry analysis and western blotting indicated that TF2a and TF2b induced apoptosis and G1 cell cycle arrest in ovarian cancer A2780/CP70 cells.Hoechst 33342 staining was used to confirm the apoptotic effect Downregulation of CDK2 and CDK4 for TF2a and CDK2 and cyclin El for TF2b led to the accumulation of cells in G1 phase.Treatment with TF2a and TF2b induced apoptosis and G1 through p53-dependent pathways.Treatment with TF2a and TF2b induced DNA damage through ATM/Chk/p53 pathway.Treatment with TF2a and TF2b also induced inhibition of A2780/CP70 cells through Akt and MAPK pathways.Our study contributes to elucidate the mechanisms by which TF2a and TF2b may help prevent and treat platinum-resistant ovarian cancer.Treatment with TF3 inhibited cisplatin-resistant ovarian cancer cells synergistically with cisplatin.The effects of TF3 alone,CDDP alone and the combination on A2780/CP70 and OVCAR3 cell viability were analyzed using MTS assay.Combined TF3 and CDDP treatment showed a synergistic cytotoxic effect in both cell lines with C1 values less than 1.0.Colony formation assay was carried out to further confirm the synergistic effect of TF3 and CDDP against both cell lines.Treatment with TF3 and CDDP showed a synergistic pro-apoptotic effect against both cell lines by Hoechst 33342 staining and flow cytometry analysis.Treatment with TF3 and CDDP synergistically induced Gl/S phase cell cycle arrest in both cell lines.Apoptosis induced by combination treatment was accompanied by regulating protein expression of cleaved caspase 3,cleaved caspase 7,cytochrome c,Bax and Bc1-2.Combination treatment induced G1/S phase cell cycle arrest via regulating protein expression of cyclin A2,cyclin Dl,cyclin El,CDK2 and CDK4.Combination treatment also regulated Akt and Erk phosphorylation in A2780/CP70 and OVCAR3 cells.TF3 and CDDP combination treatment may be effective for advanced ovarian cancer.Treatment with TF3 inhibited ovarian CSCs through Wnt/?-Catenin pathway.The inhibitory effect of TF3 against A2780/CP70 and OVCAR3 CSCs from spheres was evaluted using MTS assay,colony formation assay and sphere formation assay.The cell viability,number of colony and phere formation capacity of both CSCs were inhibited after treatment with TF3.The flow cytometre analysis showed the percent of A2780/CP70 CSCs from spheres decreased and the percent of OVCAR3 CSCs from spheres increased after treatment with TF3 which indicated a different inhibitory effect of TF3 against CSCs and non-CSCs.The inhibitory effect of TF3 against A2780/CP70 and OVCAR3 ALDH+ and ALDH-cells sorted from spheres were evaluted using MTS assay.Treatment with TF3 showed a stronger inhibitory effect against A2780/CP70 ALDH+ cells than ALDH-cells.TF3 showed a stronger inhibitory effect against OVCAR3 ALDH-cells than ALDH+ cells.The inhibitory effect of TF3 against A2780/CP70 and OVCAR3 ALDH+ cells was confirmed by western blot.TF3 down-regulated the protein levels of ?-Catenin,LEF-1,c-Myc and cyclin Dl in A2780/CP70 and OVCAR3 ALDH+ cells which indicated that TF3 might inhibit ALDH+ cells through Wnt/?-Catenin pathway.P-Catenin overexpression in ALDH+ cells was observed by western blot after transfection with pcDNA3-S33Y P-Catenin plasmid.?-Catenin overexpression attenuated the inhibitory effect of TF3 on the cell viavility and sphere formation capacity of both ALDH+ cells.Taken together,TF3 could inhibit ovarian CSCsthrough Wnt/?-Catenin pathway.In conclusion,a rapid UPLC method was establish to analyze 4 theaflavins in black tea.We proved theaflavins reduced the viability,induced apoptosis and caused cell cycle arrest in ovarian cancer cells.Theaflavins inhibited cisplatin-resistant ovarian cancer cells synergistically with cisplatin and inhibited ovarian CSCs.This study provides novel perspectives and potential targets for the anti-cancer activity of theaflavins,suggesting that theaflavins might serve as a potent anti-cancer agent.