Anti-tumor Effect And Underlying Mechanism Of Capsaicin On Bladder Cancer Cells

Bladder cancer(BCa)is one of the most common malignant tumors in urinary system.According to Burger's report in 2013,BCa is the 7th most common cancer in men and the 17th most common in women worldwide.BCa is more common in the developed countries and is the fourth and ninth most common cancer in men and women respectively,in the developed countries.Ferlay et al.reported in 2015 that BCa is the 9th most common cancer worldwide(both men and women)and is more common in developed countries.In Europe,the tumor specific mortality of BCa ranks eighth.In the United States,the morbidity of BCa in men ranks 4th and the mortality ranks 8th.In China,BCa is the most common urologic tumor.According to the China cancer statistics published in 2016,BCa is the 6th most common cancer in men and ranks after the top fifteen in women.Although the morbidity of BCa in China is lower than in the developed countries,an increasing trend has been showed in recent years.Non-muscle invasive bladder cancer(NMIBC)is usually treated by transurethral resection of bladder tumor.However,the postoperative recurrence rate of 5 years ups to 50%-70%.Furthermore,after one or more recurrences,about 10%-25%of the patients will progress to muscle invasive bladder cancer(MIBC).MIBC gradually infiltrates into the whole the bladder wall and spreads to the surrounding tissue.Moreover,MIBC often metastasize through lymphatic pathway.Radical cystectomy and urinary diversion are suggested for the treatment of MIBC.The wide surgical resection range and various postoperative complications seriously affect the patients'lives and quality of life.Furthermore,even received radical cystectomy,the BCa recurrence and distant metastasis rate remains approximately to 50%,and the five-year survival rate is only 50%-66%.Given current therapeutic approaches have a variety of adverse effects on patients,such as local recurrence,distant metastasis,low survival rate and high cost,it is urgent to explore more effective therapies for the treatment of BCa.Capsaicin(8-methyl-N-vanillyl-6-nonenamide)is the main pungent ingredient in Capsicum species plants and mainly consumed as food additives throughout the world.Capsaicin(CAP)is a highly selective agonist for the transient receptor potential vanilloid type 1(TRPV1).In addition to the prototypical function of Ca2+ channel,TRPV1 has been described to be correlated with BCa and also revealed as a target for drug development.Recently,CAP has been reported for its antioxidant,anti-inflammatory and anticancer activity.Moreover,CAP has been suggested a potential clinical significance in tumor therapy.ObjectiveWe detected the effects of CAP on the phenotype of 5637 and T24 bladder cancer cell lines in vitro.Combined with microarray analysis results of mRNA isolated from Bca and normal bladder tissues,the aim of this study was to explore the mechanism of CAP on Bca cells.We further established an immunodeficiency mouse model of subcutaneous xenograft to investigate the effect and mechanism of CAP in vivo.Methods1.MTT assay was used to analyze the effects of CAP on the proliferation in 5637 and T24 cells.2.Cell migration of 5637 and T24 cells was measured using transwell migration assay.3.Flow cytometry(FCM)analysis was performed to evaluate alterations of cell cycle after CAP treatment.4.FCM analysis was performed to evaluate alterations of apoptosis in 5637 and T24 cells after CAP treatment.5.Western blot analysis was taken to detect the alterations of proteins involved in epithelial mesenchymal transition and cell cycle regulation after CAP treatment.6.Cellular reactive oxygen species(ROS)production in 5637 and T24 cells after CAP treatment was detected by immunofluorescence staining and FCM.7.Western blot analysis was taken to detect alterations of proteins involved in ROS metabolism and Akt/FOXO3a pathway after CAP treatment.8.A subcutaneous xenograft model of NOD/SCID mouse model was established to observe the effect of CAP in vivo.9.Cell proliferation,ROS metabolic enzymes and FOXO3a expression were detected by immunofluorescence staining in xenograft tissues in vivo.Results1.CAP inhibited the proliferation of 5637 and T24 cells in a dose and time-dependent manner.2.CAP induced G0/G1 phase cell cycle arrest in 5637 cells and S phase cell cycle arrest in T24 cells.3.CAP could not induce apoptosis in 5637 or T24 cells.4.CAP induced an increase of ROS production in 5637 and T24 cells.5.CAP induced a strong upregulation of FOXO3a in 5637 and T24 cells.6.Injection of CAP suppressed xenograft tumor growth in vivo.7.CAP inhibited the proliferation of xenograft tumor cells and induced the increase of ROS production in vivo.8.CAP induced a strong increase of ROS metabolic enzymes and FOXO3a in xenograft tumor in vivo.Conclusions1.CAP can inhibit viability and growth of Bca cells in vitro and in vivo.2.CAP can increase ROS production and up-regulate FOXO3a in Bca cells in vitro and in vivo.3.CAP could induce FOXO3a up-regulation possibly via the increase of ROS and suppress BCa viability possibly via FOXO3a-mediated pathways.