In Vitro And In Vivo Antitumor Effects Of Fisetin On Bladder Cancer Cells

Objective:Bladder cancer is one of the most common malignancies and tumor recurrence is a major clinical concern. Unfortunately, current chemotherapeutics are insufficient. Therefore, there is an obvious urgent need for novel, effective and safe therapies to prevent both recurrence and progression. The present study was carried out to investigate the effect of fisetin on cell cycle arrest and the induction of apoptosis in human bladder cancer cell lines and the mechanism of intravesical activity in a rat bladder tumor model.Methods:The MTT assay and clonal growth assay was used to determine cell viability. To determine whether the decrease in cell viability after fisetin treatment was the result of induction of apoptosis in bladder cancer cells, Hoechst 33258 fluorescence staining was performed to detect the morphology of cells. Meanwhile, apoptosis of the cancer cell lines was assessed by flow cytometric analysis after propidium iodide staining and the apoptotic-associated proteins were measured by western blot. RT-PCR was used to detect the expression of Bax and Bcl-xL mRNA. In addition, this study established a rat bladder carcinoma animal model induced by MNU. We observed the pathology changes of bladder by the HE staining. The tumor cell proliferation and apoptosis in a rat bladder carcinogenesis model were evaluated by proliferating cell nuclear antigen and terminal deoxyribonucleotide transferase-mediated nick-end labeling method. time-dependent inhibition of bladder cancer cell proliferation. Our data demonstrated that treatment of fisetin (60-100?M; 48 h) was found to bring about the induction of apoptosis in bladder cancer cells, as evidenced by Hochest 33258 staining and sub-G1 peak of apoptotic markers detection in T24 and EJ cells exposed to fisetin. Our results also clearly showed that, in fisetin-treated T24 and EJ cells, apoptosis was accompanied by an increase in the percent of cells in G0/G1 and reduction in G2/M indicating the cell cycle arrest in G0/G1. G1-phase arrest was associated with a marked decrease in the protein expression of cyclin D1, cyclin E, CDK4 and CDK2 and the increase in the protein expression of p53 and p21. There was also induction of mitochondrial release of cytochrome c into cytosol. In addition, the study also found that exposure of human bladder cancer cells to fisetin could upregulated the expression of Bax and Bak as well as downregulated the expression of Bcl-2, Bcl-xL in a dose-dependent manner. Changes in expression of Bax and Bcl-2 molecules resulted in an increase in the bax/bcl-2 ratio. Our in vitro study reveals that 400?M fisetin has been completely lethal to the 2 cell lines after a 1h incubation period. Based on these results, the same dose of fisetin and 2h dwell time to more closely resemble clinical instillation has been selected for our in vivo experiments. In the 11 MNU group was evident in the bladder of 9 for an incidence rate of 81.8%. Of the 13 MNU-Fis group fisetin-treated rats 2 had tumor for an incidence rate of 15.4%. Fisetin treatment significantly reduced the number of PCNA-positive cells in tumors from the MNU-Fis group as compared to the MNU group. In the MNU-Fis group treatment with fisetin resulted in a significant increase in the number of TUNEL-positive cells. After instillation of fisetin, heart, liver, kidneys, lungs and spleen are safe without damages.Conclusion:In summary, our present study showed the anticancer efficacy of fisetin against bladder cancer cells in vitro and in vivo. Our data in vitro demonstrated that fisetin inhibit the growth of bladder cancer cells both through retardation of the cell cycle and activation of the cellular apoptotic response in the cancer cells. Moreover, we successfully constructed the model of bladder cancer of rat by infusing bladder with MNU and the process of development of bladder tumor induced by MNU was similar with the development of human bladder cancer disease. Based on the in vitro results, the 2h dwell time to more closely resemble clinical instillation has been selected for our in vivo experiments. Our data reveals fisetin can result in significant inhibition and apoptosis in the progression of bladder cancer in rat tumor model. Our in vitro and in vivo results suggest that fisetin, a naturally dietary flavonoid, should be developed as a chemopreventive and chemotherapeutic agent against bladder cancer.