The Experimental Study Of The Effects And Mechanism Of (-)-epigallocatechin Gallate On Human Bladder Carcinoma T24 Cells

Part 1 Effects of EGCG on proliferation of human bladdercarcinoma T24 cellsObjective The present study was undertaken to examine the effects of (-)-epigallocatechin gallate (EGCG-the major phytochemical in green tea) on growth inhibition, cell cycle and corresponding molecular mechanism of T24 cells. Methods The cytotoxic effect of EGCG on human bladder cancer T24 cells was determined with varying concentration of EGCG(10, 20, 40, and 80μg/mL) treatment for 12, 24, 36, and 48 hours by MTT assay. The antiproliferative or anticarcinogenic effect of EGCG in T24 cells was determined by anchorage-dependent colony-forming assay. In colony-forming assay, the cells were tested for their proliferation potential and formation of an individual colony. The cell cycle arrest effect of EGCG on human bladder cancer T24 cells was determined with varying concentration of EGCG treatment for 24 hours by flow cytometry. The cell cycle modulating protein Cyclin D1and CDK4 was determined by immunoblotting. Results1. Using the T24 human bladder cancer cell line, we found that EGCG treatment resulted in dose-dependent (10-80 μg/mL) and time-dependent (12-48 hours) inhibition of cellular proliferation and cell viability.2. Reduction in cell viability with EGCG treatment at concentrations of 20-100μg/mL after 12 hours ranged from 1% to 26%, whereas after 24, 36, and 48 hours ranged from 4% to 61%, 9% to 83% and 10% to 91% respectively.3. There was a drastic decrease in the ability of the T24 cells to form colonies with increasing doses of EGCG (10-40 μg/mL). EGCG at dosages of 20 and 40 μg/mL completely inhibited the proliferation of cells with no colonies formed by the end of 7 days.4. As shown by flow cytometry, EGCG treatment (10, 20,40, and 80μg/mL for 24 h) of the T24 cells resulted in significant G0/G1-phase cell cycle arrest.5. As shown by immunoblot analysis, EGCG treatment (10, 20, 40, and 80μg/mL for 24 h) of the T24 cells resulted in significant dose-dependent downmodulation of the protein expression of cyclin D1, CDK4.Conclusions Our data suggested EGCG Treatment Resulted in a significant dose- and time-dependent Inhibition in the Growth and Colonogenic Survival of T24 Cells. Our study also suggests that EGCG causes a decrease in the protein levels of the cyclin D1 and cdk 4 thereby causing a G0/G1-phase arrest of the cell cycle.Part 2Effects of EGCG on apoptosis of human bladder carcinoma T24 cellsObjective The present study was undertaken to examine the effects of (-)-epigallocatechin gallate (EGCG-the major phytochemical in green tea) on apoptosis of human bladder carcinoma T24 cells and to identify the altered signaling pathway(s) underlying the response to EGCG exposure.Methods We determined whether EGCG-mediated loss of T-24 cell viability was the result of the induction of apoptosis. The induction of apoptosis by EGCG (10-80 μg/mL) was measured by acridine orange/ethidium bromide assays, and the proapoptotic effect of EGCG was also confirmed by analyzing cell morphology and by PI staining and the annexin V method. The extent of apoptosis was quantified by flow-cytometric analysis of EGCG-treated cells labeled with PI and annexin V. Alterations in signaling events were determined in Western blot analysis probing for phosphorylated MAPK and Akt proteins, indicative of activation. The role of caspase3, PARP-1, Bcl-2 family in apoptosis was analyzed by Western blotting. Results1. Using the T24 human bladder cancer cell line, we found that EGCG treatment resulted in induction of apoptosis in dose-dependent manner (10-80 μg/mL).2. Phase-contrast photomicrographs taken 24 h after EGCG treatment revealed a dose-dependent decrease in cell density. Changes in cell morphology and cell membrane blebbing, which are characteristics of apoptosis, were also detected.3. As shown by PI staining and the annexin V method, we found that EGCG caused a dosage dependent increase in T24 cell apoptosis. It was observed that treatment of T24 cells with 10 and 80μg/mL of EGCG for 24 hours increased the number of early apoptotic cells (LR) from 0.7% to 13.1% respectively, in a dose-dependent manner compared to 0.3% in untreated control cells. The number of late apoptotic cells (UR) increased from 0.9% to 26.0% compared with 0.5% in non-EGCG treated cells. The total percent of apoptotic cells (UR + LR) increased from 0.8% in untreated T24 cells to 39.1% with 80μg/mL of EGCG treatment for 24 hours.4. Western blot analysis indicated that treatment of T24 cells with EGCG resulted in a dose-dependent activation of caspase-3 and PARP proteins 24 hours after EGCG treatment.5. EGCG treatment resulted in an appreciable down-regulation of protein expression of phospho-PDK1, phospho-Thr308-Akt, and phospho-Ser473-Akt without an effect on total Akt expression in T24 cells.6. Western blot analysis indicated that treatment of T24 cells with EGCG resulted in a dose-dependent increase in pErk1/2 protein levels.7. EGCG treatment of T24 cell lines resulted in a decrease in antiapoptotic Bcl-2 and a concomitant increase in proapoptotic Bax proteins.8. The ratio of Bax/Bcl-2 was significantly increased in a dose-dependent manner with EGCG treatment.9. EGCG caused an increase in Bad and a decrease in Bcl-xL protein levels. Conclusions Our data suggested that EGCG causes an inhibition of phosphatidylinositol 3'-kinase/Akt activation that, in turn, results in modulations inBcl-2 family proteins in such a way that the apoptosis of T24 cells is promoted. Based on these studies, we suggest that EGCG could be developed as an agent for the management of bladder cancer.Part 3 Effects of EGCG on invasion and metastasis of human bladdercarcinoma T24 cellsObjective The present study was undertaken to examine the effects of (-)-epigallocatechin gallate (EGCG-the major phytochemical in green tea) on invasion and metastasis of human bladder carcinoma T24 cells and to identify the possible mechanisms.Methods To evaluate the antimetastatic activity of EGCG, we first assessed the inhibitive effect of EGCG on the adhesion of T24 cells to fibronectin with MTT assay. Wound healing assay and the in vitro invasion assay were used to investigate the antimetastatic activities of EGCG against T24 cells. The effect of EGCG on the gene expression of MMPs was examined by treating human bladder carcinoma T24 cells with EGCG (10-80 μg/mL). The transcription levels of MMP-2 and -9 were assessed by reverse transcription-polymerase chain reaction (RT-PCR). The role of MMP-9 in antimetastatic activities was analyzed by Western blotting. Results1. Using the T24 human bladder cancer cell line, we found that EGCG treatment resulted in dose-dependent (10-80 μg/mL) inhibition of the adhesion activities.2. The cellular motility was evidently inhibited in dose-dependent (10-80 μg/mL) and time-dependent (6-24 hours) manner by EGCG.3. Similarly, the results of the in vitro invasion assay also displayed that EGCG was able to inhibit invasion ability of T24 cells dose-dependently.4. RT-PCR analysis indicated that treatment of T24 cells with EGCG resulted in a dose-dependent decrease in MMP-9 mRNA levels.5. Western blot analysis indicated that treatment of T24 cells with EGCG resulted in a dose-dependent decrease in MMP-9 protein levels.Conclusions EGCG elicited a significant inhibition of in vitro cell adhesion, migration, and invasion in human bladder carcinoma T24 cells. The inhibition of invasion ability of T24 cells by EGCG was shown to may be attributed to decreases of the expression of MMP-9. Thus, clinical application of EGCG may contribute to the potential benefit for suppression of bladder cancer invasion and metastasis.