Studies On Inhibitory Effects And Its Mechanisms With Ursolic Acid In Bladder Cancer T24 Cells

Bladder cancer is the most common malignancy involving the urinary system, and surgery, chemotherapy, radiation therapy, biologic therapy are the main treatment options available, but side effects, like bladder irritation still exist, and there is also no evidence that adjuvant treatment is of long term benefit. The highly recurrent bladder cancer shows more malignant and invasive, and it directly links to high rates of mortality in cancer patients. So, it urgently requires new, innovative therapeutic strategies for the clinical treatment of bladder cancer.Ursolic acid is a pentacyclic triterpenes derived from nature plants, it has a wide spectrum of biological activities, such as anti- inflammation, anti- virus, enhencing immune function, hepatoprotection and so on. Recently ursolic acid is found to exhibit the acitivity against various malignancy.ObjectiveThe aim of the present study was to examine the potential antitumor activity of ursolic acid against bladder cancer T24 cells in vitro, such as anti- proliferative, proapoptotic potential of ursolic acid, its inhibition of invasiveness of tumor cells, then to discuss the mechanism of ursolic acid against bladder cancer T24 cells. And the antitumor activity of ursolic acid was also studied in tumor-bearing nude mice, This research hold promise for future clinical management the patient with bladder cancer.MethodsIn an independent set of experiments, The cells were exposed to different concentrations (0, 60,70,80μmol/L) of ursolic acid as well as curcumin (13μmol/L), the cytotoxic effects of ursolic acid and curcumin on cells were measured from 24 h to 72 h by dimethylthiazol- 2-yl- 2,5- diphenyltetrazolium bromide (MTT) assay, and morphological changes of cells was observed by inverted microscope. Regulation of the cell cycle and induction of apoptosis were evaluated using flow cytometry(FCM), migration of tumor cells was measured in a scratch wound assay and their invasiveness was measured with a transwell-invasion assay. The expression of cyclin D1, p21, Bcl-2, Bax, Caspase-3, MMP2 and TIMP2 was evaluated using RT-PCR and Western blotting. In the experiment in vivo, the average tumor size was calculated in tumor bearing mice treated with ursolic acid as well as curcumin, the apoptosis and migration related protein expression was also checked with Western blotting.All experiments were performed in triplicate and above along with proper control experiments. The results are expressed as mean±standard deviation. Multiple independent-samples tests were performed by one way ANOVA. P values less than 0.05 and 0.01 were considered statistically significant.Results1.In vitro,the results of MTT showed that ursolic acid kill bladder cancer T24 cells in a dosage and time dependent manner, light microscopy also revealed that most of the dead cells showed reduced cell volume, cytoplasmic membrane blebbing, vacuolization of the cytoplasm, and the cell lysis. And these were observed a similar pattern in the positive control experiments. flow cytometry was performed to confirm that most of cells were blocked in G1 phase after treated by ursolic acid and cell death was due to apoptosis. Ursolic acid down regulated expression of cyclinD1 and Bcl-2,up regulated expression of Bax and caspase-3 through PCR and western blotting.2. In vitro,Ursolic acid interfered with migration and invasiveness of T24 cells and down regulated expression of MMP-2, up regulated expression of TIMP-2.3.In vivo,experiments revealed that ursolic acid resulted in retardation of tumor growth and prolongation of mouse survival, the average tumor size decreased in tumor bearing mice with ursolic acid treatment. RT-PCR and Western blot proven that ursolic acid down regulated expression of Bcl-2 and MMP-2, activated Caspase-3 and up regulated expression of Bax, TIMP-2.Conclusion:In vitro ursolic acid blocks bladder cancer T24 cell cycle progression and inhibits cyclin D1 accumulation, it induces apoptosis of T24 cells through alteration of the Bcl-2 / Bax ratio and activation of Caspase-3, suggests that these important biological molecules interact at the level of the mitochondria to influence ursolic acid sensitivity. And ursolic acid also disrupts the equilibrium between MMP-2 and TIMP-2 and inhibits cellular migration and invasion. In vivo ursolic acid changed the Bcl-2 / Bax ratio and activated caspase-3, and down regulated MMP-2,resulted in inhibition of tumor growth.These properties of ursolic acid strongly suggest that it could be used as a cancer chemopreventive agent.