Studies On Inhibitory Effect Of Resveratrol On Bladder Cancer T24 Cells And Its Mechanism Of Action

Part 1 Effects of resveratrol on proliferation of human bladder cancer T24 cellsObjective The present study was undertaken to examine the effects of resveratrol on growth inhibition,cell cycle and corresponding molecular mechanism of T24 cells.Methods The effect ofresveratrol on human bladder cancer T24 cells was evaluated with varying concentration of resveratrol(0,50,100,150,200 and 250μM) treatment for 12,24 and 48 hours by MTT assay.The cell cycle arrest effect of resveratrol on human bladder cancer T24 cells was determined with the indicated concentration ofresveratrol treatment for 24 hours by flow cytometry.The cell cycle modulating protein WAF-1/p21,cyclin D1 and CDK4 was determined by western blotting analysis.Results1.Using the T24 human bladder cancer cell line,we found that resveratrol treatment exerted a significant cytotoxic effect upon T24 cells in a dose-dependent(0-250μM)and time-dependent(0-24 hours)manner.2.The cell viability with resveratrol treatment at concentrations of 0-250μM after 12 hours ranged from 49.8%-97.0%,whereas after 24 and 48 hours ranged from 27.7%-98.0%and 20.6%-95.7%respectively.3.Compared with the vehicle-treated controls,the resveratrol treatment resulted in an appreciable arrest of T24 cells in G1 phase of cell cycle.It was observed that the G1 phase population of the untreated cells was 47.6%and the percentage of cells in G1 phase was significantly increased(74.7%,78.2%,78.3%and 73.5% cells at 50,100,150,and 200μM concentrations of resveratrol,respectively)after 24 hours of the treatment.4.Using immunoblot analysis,we found that resveratrol treatment resulted in a significant dose-dependent activation of WAF-1/p21.Also,the results revealed that resveratrol treatment of T24 cells leads to a dose-dependent decrease in protein expression of cyclin D1 and CDK4.Conclusions Our data suggested resveratrol treatment resulted in a significant doseand time-dependent Inhibition in the growth of T24 Cells.Our study also suggests that resveratrol causes cell cycle arrest in G1 phase from the modulation of WAF-1/p21,cyclin D1 and CDK 4.Part 2 Resveratrol induced apoptosis of human bladder cancer T24 cellsObjective The present study was undertaken to examine the effects of resveratrol on apoptosis of human bladder cancer T24 cells and to identify the altered signaling pathway(s)underlying the response to resveratrol exposure.Methods We evaluated the effects of resveratrol on apoptosis of T24 cell by annexin V-FITC and flow cytometry.The extent of apoptosis was quantified by flow-cytometric analysis of resveratrol-treated cells labeled with PI and annexin V. Alterations in signaling events were determined in Western blot analysis probing for phosphorylated MAPK(ERK1/2 and p38)and Akt proteins,indicative of activation. The role of caspase-3,caspase-9,PARP,Bcl-2 family proteins in apoptosis was also analyzed by Western blotting.Results 1.Using the T24 human bladder cancer cell line,we found that resveratrol treatment resulted in induction of apoptosis in dose-dependent manner(0-200μM).2.After the treatment for 24 hours,it was observed that treatment of T24 cells with 0-100μM of resveratrol increased the number of early apoptotic cells from 4%to 14.1%,which decreased from 14.1%to 10.0%further after 100μM resveratrol treatment.The number of late apoptotic cells increased from 2.6%to 36.7%.The total percent of apoptotic cells was directly related to resveratrol concentration increasing from 6.6%to 47.5%3.Western blot analysis indicated that treatment of T24 cells with resveratrol resulted in a dose-dependent activation of caspase-3,caspase-9 and PARP proteins 24 hours after resveratrol treatment.4.After 24 hours treatment ofresveratrol,the phosphorylation of Akt and ERK1/2 was decreased,while the phosphorylation of p38 was increased and the results suggested that the levels of both phosphorylated Akt,ERK1/2 and phosphorylated p38 are dose-dependent in response to resveratrol.However,the total Akt,ERK1/2 and p38 expression were not affected in T24 cells.5.Resveratrol treatment of T24 cell lines resulted in a decrease in antiapoptotic Bcl-2 and Bcl-xL protein levels,whereas a concomitant increase in proapoptotic Bax proteins.The levels of these proteins are dose-dependent in response to resveratrol.6.The ratio of Bax/Bcl-2 was significantly increased in a dose-dependent manner with resveratrol treatment.Conclusions Our data suggested that treatment of resveratrol on T24 cells could leads to modulation of Akt and MAPK(ERK1/2 and p38)pathways that,in turn, results in modulations in Bcl-2 family proteins,such as Bcl-2,Bax and Bcl-xL.Then, caspases are activated in such a way that the apoptosis of T24 cells is initiated. Based on these studies,we suggest that resveratrol could be developed as an agent for the management of bladder cancer. Part 3 Effects of resveratrol on invasion and metastasis of human bladder cancer T24 cellsObjective The present study was undertaken to examine the effects of resveratrol on invasion and metastasis of human bladder cancer T24 cells and to identify the possible mechanisms.Methods To evaluate the antimetastatic activity of resveratrol,we first assessed the inhibitive effect of resveratrol 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 effect of resveratrol on the migration ability and invasion ability of T24 cells.The role of MMP-9 protein levels was analyzed by Western blotting.Results1.Usingthe T24 human bladder cancer cell line,we found that resveratrol treatment resulted in dose-dependent(0-200μM)inhibition of the adhesion activities.2.The cellular migration ability was evidently inhibited in dose-dependent(0-50μM)and time-dependent(0-24 hours)manner by resveratrol.3.Similarly,the results of the in vitro invasion assay also displayed that resveratrol was able to inhibit invasion ability of T24 cells in dose-dependent manner(0-50μM).4.Western blot analysis indicated that treatment of T24 cells with resveratrol resulted in a dose-dependent(0-50μM)decrease in MMP-9 protein levels.Conclusions resveratrol 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 resveratrol was shown to may be attributed to decreases of the expression of MMP-9.Thus,clinical application of resveratrol may contribute to the potential benefit for suppression of bladder cancer invasion and metastasis. Part 4 Inhibition effects of resveratrol on bladder cancer xenografts in vivoObjective Evaluate the effects of resveratrol on bladder cancer xenografts in vivo and investigate the underlying mechanism.Methods T24 cells were injected subcutaneously on the right flank of Male BALB/c-nude mice.At a tumor size of approximately 30 mm~3 the mice were divided into three groups.Group A received no treatment.Groups B and C were treated with intraperitoneal injection of propylene glycol(vehicle,0.1 ml),30 mg/kg of resveratrol (in 0.1 ml ofpropylene glycol),once daily for 4 weeks,respectively.CD34 expression of xenografts was evaluated by immunohistochemistry.VEGF and FGF-2 mRNA level were analyzed by RT-PCR.Results1.The tumor growth in the resveratrol group was significantly slower than that in the vehicle and control groups.In the first 10 days,tumor volume was not different among three groups.In contrast,tumor volume in group A and group B were significant larger than group C from days 12 to 28.2.By immunohistochemistry,CD34 expression of xenografts was significantly reduced in the resveratrol group.3.VEGF and FGF-2 mRNA level was decreased significantly in the resveratrol group analyzed by RT-PCR.Conclusions In the nude mice xenograft model,resveratrol were capable of inhibiting tumor growth.The inhibition effects of resveratrol may be attributed to decreases of the expression of CD34,VEGF and FGF-2.