The Effects And Mechanism Of Diallyl Trisulfide (DATS) On Human Bladder Carcinoma T24 Cells

Part 1 Effects of DATS on proliferation of human bladder carcinoma T24 cellsObjective To investigate the effects of diallyl trisulfide(DATS)on growth inhibition, cell cycle and corresponding molecular mechanism of T24 cells.Methods The cytotoxic effect of DATS on human bladder cancer T24 cells was determined with varying concentration of DATS(10,20,40,and 80μmol/L)treatment for 24,48,and 72 hours by MTT assay.The antiproliferative or anticarcinogenic effect of DATS 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 DATS on human bladder cancer T24 cells was determined with varying concentration of DATS treatment for 24 hours by flow cytometry.The cell cycle modulating protein Cdc25C was determined by immunoblotting.Results1.Using the T24 human bladder cancer cell line,we found that DATS treatment resulted in dose-dependent(2.5/100μmol/L)and time-dependent(24～72 hours) inhibition of cellular proliferation and cell viability.2.Reduction in cell viability with DATS treatment at concentrations of 5～100μmol/L after 24 hours ranged from 13%to 81%,whereas after 48 and 72 hours ranged from 25%to 87%,31%to 96%,respectively.3.There was a drastic decrease in the ability of the T24 cells to form colonies with increasing doses of DATS(0.5～20μmol/L).DATS at dosages of 20μmol/L completely inhibited the proliferation of cells with no colonies formed by the end of 7 days.4.As shown by flow cytometry,DATS treatment(10,20,40,and 80μmol/L for 24h) of the T24 cells resulted in significant G2/M phase cell cycle arrest.5.As shown by immunoblot analysis,DATS treatment(10,20,40,and 80μmol/L for 24 h)of the T24 cells resulted in significant dose-dependent downmodulation of the protein expression of Cdc25C.Conclusions Our data suggested DATS treatment resulted in a significant dose- and time-dependent inhibition in the growth and colonogenic survival of T24 Cells.Our study also suggests that DATS causes a decrease in the protein levels of the Cdc25C, thereby causing a G2/M-phase arrest of the cell cycle.Part 2 Effects of DATS on apoptosis of human bladder carcinoma T24 cellsObjective To investigate the effects of DATS on apoptosis of human bladder carcinoma T24 cells and to identify the altered signaling pathway underlying the response to EGCG exposure.Methods We determined whether DATS -mediated loss of T-24 cell viability was the result of the induction of apoptosis.The induction of apoptosis by DATS (10～80μmol/L)was measured by acridine orange/ethidium bromide assays,and the proapoptotic effect of DATS 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 DATS-treated cells labeled with PI and annexin V. Alterations in signaling events were determined in Western blot analysis probing for Akt proteins,indicative of activation.The role of caspase 3,PARP-1,Bcl-2 family in apoptosis was analyzed by Western blotting.Results1.Using the T24 human bladder cancer cell line,we found that DATS treatment resulted in induction of apoptosis in dose-dependent manner(10～80μmol/L).2.Phase-contrast photomicrographs were taken 24 h after DATS 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 annexinⅤmethod,we found that DATS caused a dosage dependent increase in T24 cell apoptosis.It was observed that treatment of T24 cells with 10 and 80μmol/L of DATS for 24 hours increased the number of early apoptotic cells(LR)from 4.8%to 12.2%respectively,in a dose-dependent manner compared to 0.1%in untreated control cells.The number of late apoptotic cells(UR)increased from 1.7%to 7.8%compared with 0.2%in non-DATS treated cells.4.Western blot analysis indicated that treatment of T24 cells with DATS resulted in a dose-dependent activation of caspase-3 and PARP proteins 24 hours after DATS treatment.5.DATS 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.DATS treatment of T24 cell lines resulted in a decrease in antiapoptotic Bcl-2 and a concomitant increase in proapoptotic Bax proteins.7.The ratio of Bax/Bcl-2 was significantly increased in a dose-dependent manner with DATS treatment.8.DATS caused an increase in Bad and a decrease in Bcl-xL protein levels.Conclusions Our data suggested that DATS causes an inhibition of phosphatidylinositol 3'-kinase/Akt activation that,in turn,results in modulations in Bcl-2 family proteins in such a way that the apoptosis of T24 cells is promoted.Based on these studies,we suggest that DATS could be developed as an agent for the management of bladder cancer.Part 3 Effects of DATS on invasion and metastasis of human bladder carcinoma T24 cellsObjective The present study was undertaken to examine the effects of DATS on invasion and metastasis of human bladder carcinoma T24 cells and to identify the possible mechanisms.Methods To evaluate the antimetastatic activity of DATS,we first assessed the inhibitive effect of DATS on the adhesion of T24 cells to fibronectin by MTT assay. Wound healing assay and the in vitro invasion assay were used to investigate the antimetastatic activities of DATS against T24 cells.The effect of DATS on the gene expression of MMPs was examined by treating human bladder carcinoma T24 cells with DATS(10～80μmol/L).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 DATS treatment resulted in dose-dependent(10～80μmol/L)inhibition of the adhesion activities.2.The cellular motility was evidently inhibited in dose-dependent(10-80μmol/L) and time-dependent(6～24 hours)manner by DATS.3.Similarly,the results of the in vitro invasion assay also displayed that DATS was able to inhibit invasion ability of T24 cells dose-dependently.4.RT-PCR analysis indicated that treatment of T24 cells with DATS resulted in a dose-dependent decrease in MMP-9 mRNA levels.5.Western blot analysis indicated that treatment of T24 cells with DATS resulted in a dose-dependent decrease in MMP-9 protein levels.Conclusions DATS 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 ceils by DATS was shown to may be attributed to decreases of the expression of MMP-9.Thus,clinical application of DATS may contribute to the potential benefit for suppression of bladder cancer invasion and metastasis.