Effect And Mechanism Of EGCG On Papillary Thyroid Cancer Cells

Objective: The aim of this study was to explore the effect of EGCG on TPC-1 papillary thyroid cancer cells,and its related-molecular mechanism such as cell apoptosis,autophagy and thyroid redox balance.Methods: 1.After treated with EGCG with different concentrations(0,5,10,20,40 ?mol/L)and different times(24,48 h),MTT assay was used to detect the proliferation of TPC-1 papillary thyroid cancer cells and Nthy-ori 3-1 normal thyroid epithelial cells,in order to determine the best work concentration and time of EGCG according to the value of IC50.The value of IC50 was about 17.2 ?mol/L caculated by Graphpad softwere.2.After treated with EGCG(0 ?mol/L),3-MA(0.3 mmol/L),EGCG(17.2 ?mol/L),EGCG(17.2 ?mol/L)combined with 3-MA(0.3 mmol/L)for 48 h,the the proliferation of TPC-1 papillary thyroid cancer cells was assessed by the MTT assay.And flow cytometry and the accumulation of Hoechst33342 were used to detect the apoptosis of TPC-1 papillary thyroid cancer cells.3.After treated with EGCG(0,17.2 ?mol/L)for 24 h,the cells were then stained with PI(Propidium Iodide),and the distribution of cell cycle in TPC-1 papillary thyroid cancer cells was determined by flow cytometery method.4.After treated with EGCG(0,17.2 ?mol/L)for 48 h,the expression levels of ROS in TPC-1 papillary thyroid cancer cells were detected by flow cytometery method.5.After treated with EGCG(0,17.2 ?mol/L)for 48 h,Western blot was used to measure the expression apoptosis-related protein Bax,Bcl-2,Cyt c,cleaved-caspase-9,cleaved-caspase-3,cleaved-PARP and autophagy associated protein LC3B-II and p62 in TPC-1 papillary thyroid cancer cells.6.After treated with EGCG(0,17.2 ?mol/L)for(0,15,30,60 min),Western blot was used to measure the autophagy related-protein expression of p-AKT,p-m TOR,t-AKT,t-m TOR in TPC-1 papillary thyroid cancer cells.Results: 1.EGCG inhibited the growth of TPC-1 papillary thyroid cancer cell in a time-dependent in a dose-dependent manner(P < 0.001),and EGCG had no effect on Nthy-ori 3-1 normal thyroid epithelial cells.2.Compared with the control group,after treated with EGCG(17.2 ?mol/L)for 48 h,the apoptosis of TPC-1 papillary thyroid cancer cells was distinctly increased(P < 0.01),and the relative expression level of ROS in TPC-1 papillary thyroid cancer cells was significantly decreased(P < 0.05).In addition,the apoptosis-associated protein Bax was significantly increased(P < 0.01),and the anti-apoptosis protein Bcl-2 was significantly decreased(P < 0.01).And the expressions of apoptosis associated proteins Cyt C,cleaved-caspase-9,cleaved-caspase-3 and cleaved-PARP were distinctly increased in TPC-1 papillary thyroid cancer cells(P < 0.05).3.Compared with the control group,after treated with EGCG(17.2 ?mol/L),an appreciable arrest of cell cycle at S phase was observed in TPC-1 papillary thyroid cancer cells(P < 0.01).And the S phase cycle-related proteins cyclin A and CDK2 were significantly decreased(P < 0.01).4.Compared with the control group,after treated with EGCG(17.2 ?mol/L)for 48 h,autophagy-associated protein LC3B-II was significantly increased(P < 0.01),while p62 was significantly decreased(P < 0.05)and the expressions of p-AKT,p-m TOR significantly decreased in TPC-1 papillary thyroid cancer cells(P < 0.05).In addition,after treated with EGCG(17.2 ?mol/L)or EGCG(17.2 ?mol/L)combined with 3-MA(0.3mmol/L)for 48 h,compared with EGCG(17.2 ?mol/L)treated group,the proliferation of TPC-1 papillary thyroid cancer cells was apparently decreased(P < 0.05);while the apoptosis of TPC-1 papillary thyroid cancer cells was distinctly increased in EGCG(17.2 ?mol/L)combined with 3-MA(0.3mmol/L)treated group(P < 0.01).Conclusion: 1.EGCG inhibited the growth and induced apoptosis in TPC-1 papillary thyroid cancer cells.And EGCG had no influence on Nthy-ori 3-1 normal thyroid epithelial cells.2.EGCG inhibited the relative expression levels of ROS in TPC-1 papillary thyroid cancer cells and induced apoptosis of TPC-1 papillary thyroid cancer cells.3.EGCG induced TPC-1 papillary thyroid cancer cells arrest at S phase.4.EGCG increased the autophagy activation in TPC-1 papillary thyroid cancer cells through the down-regulation of AKT/m TOR signaling pathway.