Effect Of Epigallocatechin Gallate In Papillary Thyroid Carcinoma Cells

Objective:To explore the effect of EGCG on TPC-1 papillary thyroid carcinoma cells mechanism such as apoptosis,tumor migration and invasion.Methods:1.CCK-8 Kit was used to detect the inhibiton of TPC-1 after treated with EGCG different concentrations(0,20,40,80,160,320mg/mL)for 24 h and 48 h.2.Flow cytometry was used to detect the apoptosis of TPC-1 cells after treated with EGCG different concentrations(0,40,80mg/mL)for 24 h and 48 h.3.Cell scratch assay and transwell assay was used to detect the effects of the migration and invasion of TPC-1 after treated with EGCG different concentrations(0,40,80mg/mL).4.Flow cytometry was used to detect the cell cycle of TPC-1 cells after treated with EGCG different concentrations(0,40,80mg/mL)for 24 h and 48 h.5.Immunofluorescence was used to detect the expression of protein Survivin,Bax and Bcl-2 in TPC-1 after treated with EGCG different concentrations(0,40,80mg/mL)for 48 h.6.Real-Time PCR was used to detect the expression of Survivin mRNA in TPC-1 after treated with EGCG different concentrations(0,40,80mg/mL)for 48 h.Results:1.CCK-8 Kit showed that the proliferation of TPC-1 were significantly inhibited by EGCG treatment,the differences have statistic significance.2.The results of flow cytometry indicated that EGCG was able to induce apoptosis of TPC-1 cells.Moreover,the number of the apoptotic cells increased with the drug concentration.After concentrations of 0,40,80mg/mL EGCG treatment for 24 h,the number of apoptotic cells of TPC-1 were(0.27±0.15)%?(17.67±1.46)%?(28.33±3.65)%,respectively,one-way analysis of variance,F=116.8,P<0.001.Similarly,the apoptotic cells number of TPC-1 at the different concentrations of EGCG treatment for 48 h were(7.53±2.59)%?(40.53±4.97)%?(59.77±5.54)%,respectively,one-way analysis of variance,F=101.12,P<0.001.3.Cell scratch assay showed that the migration of TPC-1 cells was inhibited by EGCG.Moreover,the inhibition of migration ability increased with the drug concentration.After concentrations of 0,40,80mg/mL EGCG treatment for 48 h,the cell migration distance measured at 0h,24 h and 48 h was statistically significant.Transwell assay showed that the invasion of TPC-1 cells was inhibited by EGCG.After concentrations of 0,40,80mg/mL EGCG treatment for 48 h,the number of TPC-1 cells passing through the chamber was found to have significant differences by statistical analysis.4.The results of flow cytometry indicated that EGCG was able to induce cell cycle arrest in S phase of TPC-1 cells.Moreover,the proportion of the cell cycle arrest in S phase increased with the drug concentration.After concentrations of 0,40,80mg/mL EGCG treatment for 24 h,the proportion of the cell cycle arrest in S phase were(11.42±0.96)%,(22.50±0.73)%,(44.96±2.56)%,respectively,one-way analysis of variance,F=327.80,P<0.001.Similarly,the cell cycle arrest in S phase at the different concentrations of EGCG treatment for 48 h were(12.93±1.32)%,(25.17±2.14)%,(58.09±3.43)%,respectively,one-way analysis of variance,F=272.35,P<0.001.5.Immunofluorescence results: After concentrations of 0,40,80mg/mL EGCG treatment for 48 h,the expression of Survivin decreased with the increase of concentration,and the nucleus showed positive staining reaction;the positive expression of Bcl-2 decreased with the increase of concentration,and positive staining in cytoplasm and nucleus;however,the expression of Bax increased with the increase of concentration,and positive staining in cytoplasm and nucleus.6.The mRNA expression of Survivin in EGCG 40 mg/mL group and 80 mg/mL group was significantly lower than that in 0 mg/mL group,and it was dependent on the increase of the drug concentration.The differences have statistic significance(0.697±0.069,0.507±0.057 vs 1.002±0.045,F=55.41,P<0.05).Conclusion:1.EGCG inhibit the proliferation of TPC-1 cells and induce its apoptosis,which possibly related to decrease the expression of Survivin.2.EGCG may inhibit the expression of Survivin to regulate cell cycle of TPC-1 cells.3.EGCG inhibit the ability of migration and invasion of TPC-1 cells,which possibly related to decrease the expression of Survivin.