| Abstract:Bladder cancer (BC) is the most common malignant tumor of urinary system,about90%of which is comprised of urothelial cell carcinoma (UCC)(also known as transitional cell carcinoma, TCC).About70%-80%bladder cancer cases are Non-muscle-invasive bladder cancer (NMIBC).At present,all the patients with NMIBC are suggested to have surgical resection.But the recurrence rate is still above60%in3-5years after the surgery. So it is important to have intravesical chemotherapy drugs or immunomodulating agents,which can kill the epibiotic tumor cells and reduce the risk of relapses of the tumor.Because of NMIBC always are multiple malignant tumor and the tumor in Tis stage can hardly be diagnosed, about15%-61%of the patients presented to us with recurrent tumors after received intravesical therapy in the first year after operation and31%-78%in five years.Now the most common chemotherapy drugs are pirarubicin, mitomycin, pharmorubicin, doxorubicine, hydroxycamptothecine.All those drugs lead to side effect such as chemical cystitis, which is obvious at the time of the immediate postoperative intravesical. Bacillus Calmette-Guerin (BCG) is considered to be the most effective intravesical drug.But tumor recurrence in30%-45%of the patients received intravesical BCG therapy.Some of the patients have severe irritation symptoms of bladder, prostatitis, epididymitis, hepatitis. So its usage is greatly limited. Doctors among the world are making unremitting efforts to search new drug or new drug targets.In recent years,VR1(vanilloid receptor1) is rediscovered as a antineoplastic drug target. Vanilloid receptor1, a non-selective cation channel belonging to the transient receptor potential family of ion channels, is expressed in the spinal cord, brain and a wide-range of non-neuronal cells such as urothelium and some tumor tissue.Vanilloids (capsaicin and resiniferatoxin),as the VR1agonists can strongly activates VR1producing a large influx of calcium, resulting in calcium-induced inactivation and cytotoxicity lead to mitochondrial damage.Resiniferatoxin, a diterpine derived from the latex of the plant Euphorbia resinifera used by the ancient physicians of Roman times, was found to share structural similarity to capsaicin by containing a common vanilloid moiety essential for activity. In the anti-tumor field Hail investigate the inhibitory effect of resiniferatoxin on SCC cell line.Hartel investigate the inhibitory effect of resiniferatoxin on human pancreatic cancer line. What we interested in is if RTX has antineoplastic activity against the UCC cell line. Object:To investigate the inhibitory effect of resiniferatoxin on growth inhibition cell cycle and induction on apoptosis of human bladder carcinoma RT4cell.Methods:1.The inhibitory effect of RTX on human bladder carcinoma RT4cells was determined with varying concentration of RTX (50,100,200,500,1000,1500nmol/L) treatment for24,48and72hours by MTT assay taking0nmol/L RTX as a negtive control.2.Morphologic changes of RT4cells were studied with inverted phase contrast microscope with different concentration (0,50,100,200,500,1000nmol/L) of RTX at different time (Oh,8h,24h,48h)3.The cell cycle of RT4cells treated by RTX (0,500,1000nmol/L) for48hours were detected by flow cytometiy (FCM) with PI staining method.4.The extent of apoptosis of RT4cells treated by RTX (0,500,1000nmol/L) for48hours were detected by (FCM) with annexin V and PI staining method.Results:1. Using the human bladder carcinoma RT4cell line, we found that RTX treatment resulted in dose-dependent (50,100,200,500,1000,1500nmol/L) inhibition of cellular proliferation and cell viability. The inhibition rate of cellular proliferation with RTX treatment at concentrations of (50,100,200,500,1000,1500nmol/L) treatment after24hours ranged from3.48%to29.73%respectively. The inhibition rate of cellular proliferation with RTX treatment was significantly different compared with negative control group(P<0.01). The inhibition of cellular proliferation with RTX treatment at concentrations of (50,100,200,500,1000,1500nmol/L) after48and72hours ranged from8.49%to50.23%,13.54%to72.76%respectively in a time-dependent manner.2. In lower concentrations (50,100nmol/L) treatment of RTX for8hours, it shows a moderate apoptotic effect, while in higher concentrations1000nmol/L it showed more apoptotic cells observed with inverted phase contrast microscope.After24hours in higher concentrations(1000nmol/L), more typical apoptosis body was observed,while after48hours a large number of apoptotic cells was observed.3.As shown by FCM with PI staining method, RTX treatment (500,1000nmol/L) of the RT4cells resulted in significant G0/G1-phase cell arrest ranging from42.37to48.79%, and each concentration of RTX group was significantly different compared with negative control group35.02%(P<0.05).4. As shown by FCM with PI and Annexin V staining method, we found that RTX caused a dose-dependent increase in RT4cell apoptosis. It was observed that treatment of RT4cells with varying concentration of RTX (0,500,1000nmol/L) for48hours increased the number of early apoptotic cells from3.21%to5.42%, and total apoptotic cells from13.93%to25.29%in a dose-dependent manner, and significantly different compared with negative control group0.65%and2.99%respectively (P<0.05).Conclusion:1.Resiniferatoxin (RTX) can inhibit the proliferation of human bladder carcinoma RT4cells effectively in a dose and time-dependent manner in vitro.2. RTX can arrest the RT4cells in G0/G1phase at a dose-dependent manner.3. RTX induces human bladder carcinoma RT4cells apoptosis at a dose-dependent manner. Object:to investigate the mechnism of the inhibitory effect of resiniferatoxin on human bladder carcinoma RT4cell.Methods:1. After treated by RTX (0,500,1000nmol/L) for different time (Oh,8h,24h) the concentration of Ca2+in RT4cells is determined with fluoreacent labeling method.2.After treated by RTX (1000nmol/L) for different time (Oh,8h,24h,48h),detect three proteins of apoptosis related proteins caspase-8, caspase-3and evaluate cytochrome c release with the approsch of western-blot analysis.3.After treated by RTX (1000nmol/L) for different time (Oh,8h,24h,48h),detect three cell cycle regulatory protein p53, p21, CDK2with the approsch of western-blot analysis.Results:1. After a48hour treatment with RTX, the expressions of P53and P21were up-regulated in contrary to the expression of CDK2.3.RTX induced caspase-8activation at8h that increased at24-48h. Caspase-3activation occurred at12h and persisted until24h after treatment.8hours after treatment, a band of12kDa corresponding to cytochrome c was observed and declined at later time pointsConclusion:1. RTX can arrest the RT4cells in G0/G1phase by modulating the expression of P53, P21and CDK2.2.Treatment of cells with RTX induced apoptosis accompanied by activation of caspase-8,caspase-3.and increased mitochondrial cytochrome c release. |
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