Effects Of Myricetin On Proliferation Of Human Bladder Carcinoma T24cells In Vitro And In Vitro

Part 1Objective The present study was undertaken to examine the effects of Myricetin on growth inhibition, cell cycle and corresponding molecular mechanism of T24 cells.Methods The cytotoxic effect of Myricetin on human bladder cancer T24 cells was determined with varying concentration of Myricetin (10, 20, 40, 60, 80, and 100μM) treatment for 12, 24, 36 and 48 hours by MTT assay. The cell cycle modulating protein Cyclin B1 and CDC2 was determined by immunoblotting.Results1. Using the T24 human bladder cancer cell line, we found that Myricetin treatment resulted in dose-dependent (10-100μM) and time-dependent (12-48 hours) inhibition of cellular proliferation and cell viability.2. As shown by flow cytometry, Myricetin treatment ( 0, 20, 40, and 80μM for 24 h) of the T24 cells results in significant G2/M-phase cell arrest.3. As shown by immunoblot analysis, Myricetin treatment (0, 20, 40, and 80μM for 24 h) of the T24 cells resulted in dose-dependent downmodulation of the protein expression of Cyclin B1, CDC2.Conclusions Our data suggested Myricetin treatment resulted in a significant dose- and time-dependent Inhibition in the Growth of T24 cells. Our study also suggests that Myricetin causes a decrease in the protein levels of the Cyclin B1 and CDC2 thereby causing a G2/M-phase arrest of the cell cycle.Part 2 Effects of Myricetin on apoptosis of human bladder carcinoma T24 cellsObjective The present study was undertaken to examine the effects of Myricetin on apoptosis of human bladder carcinoma T24 cells and to identify the altered signaling pathway(s) underlying the response to Myricetin exposure.Methods We determined whether Myricetin-mediated loss of T24 cell viability was the result of the induction of apoptosis. The induction of apoptosis by Myricetin (20-80μM) was measured by DAPI assay, and the proapoptotic effect of Myricetin was confirmed by analyzing cell morphology and by PI staining and the annexin V method. The estent of apoptosis was quantified by flow-cytometric analysis of Myricetin-treated cells labeled with PI and annexin V. The role of caspase-3, PARP-1, Bcl-2 family in apoptosis was analyzed by Western blotting.Results1. Using the T24 human bladder cell line, we found that Myricetin treatment resulted in induction of apoptosis in dose-dependent manner (20-80μM).2. Phase-contast photomicrographs taken 24 h after Myricetin 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 Myricetin caused a dosage dependent increase in T24 cell apoptosis.4. Western blot analysis indiceted that treatment of T24 cells with Myricetin could not activate caspase-3 and PARP.5. Myricetin treatment of T24 cell lines resulted in a decrease in antiapoptotic Bcl-2 and a concomitant increase in proapoptotic Bax proteins. Part 3 Effects of Myricetin on invasion and metastasis of human bladder carcinoma T24 cellsObjective The present study was undertaken to examine the effects of Myricetin on invasion and metastasis of human bladder carcinoma T24 cells and to identify the possible mechanisms.Methods Wound healing assay and the in vitro invasion assay were used to investigate the antimetastatic activities of Myricetin against T24 cells. The effect of Myricetin on the gene expression of MMPs was examined by treating human bladder carcinoma T24 cells with Myricetin (20-80μM). The transcription levels of MMP-2 and MMP-9 were assessed by reverse transcription-polymerase chain reaction (RT-PCR).Results1. The cellular motility was evidently inhibited in dose-dependent (20-80μM) and time-dependent (6-24 hours) manner by Myricetin.2. The results of the in vitro invasion assay also displayed that Myricetin was able to inhibit invasion ability of T24 cells dose-dependently.3. RT-PCR analysis indicated that treatment of T24 cells with Myricetin resulted in a dose-dependent decrease in MMP-9 mRNA levels.Conclusions Myricetin elicited a significant inhibition of in vitro cell migration and invasion in human bladder carcinoma T24 cells. The inhibition of invasion ability of T24 cells by Myricetin was shown to may be attributed to decreases of the expression of MMP-9. Thus, clinical application of Myricetin may contribute to the potential benefit for suppression of bladder cancer invasion and metastasis. Part 4 Effects of Myricetin on xenografted tumor of human bladder carcinoma T24 cell line on nude miceObjective The present study was undertaken to examine the effects of Myricetin on xenografted tumor of human bladder carcinoma T24 cell line on nude mice.Methods Twelve nude mice which had been inoculated with T24 cells by subcutaneous injection into the right legs were divided into the PBS control group (6 mice) and the 100μg/mouse Myricetin group (6 mice). They were given PBS or Myricetin into the tumor once every 2 days for one week, starting on the 14th day after tumor cell transplantation. Tumor volumes were measured every 2 days for drawing the growth curve.Results Myricetin inhibited the growth of xenograft tumor, and extended the life span of mice. Myricetin was shown to slow down the decreases of body weight of mice.Conclusion Myricetin can inhibite the growth of xenografted tumor of the human bladder cancer T24 cell line on nude mice.