Apoptosis of bladder cancer cells induced by short-term and low-dose Mitomycin-C: Potential molecular mechanism and clinical implication

Adjuvant chemotherapies have been used to prevent recurrence of bladder transitional cell carcinoma (TCC), but their efficacies are variable due to the side effects of anti-cancer agents and the drug resistant property of the target cells. To provide experimental evidence for improving clinical management of TCCs, two human TCC cell lines, EJ and BIU, were treated for 1 or 2 h by 50, 100, 150 and 200 µg/ml, respectively and their growth and death patterns were elucidated in 12-h intervals. The status of Fas, FasL and caspase-3 in the two cell lines were analyzed with immunocytochemical staining and Western blot hybridization, and their potential link to MMC-induced cell death was investigated by treating the cells with anti-Fas antibody (150 ng/ml) and by incubating the cells with an inhibitor of caspase-3 related proteases Ac-DEVO-CHO (250 µM) 1 h before 100 µg/ml MMC treatment. The results demonstrated that the lower dose (100 µg/ml) and short-term (1 h) MMC treatment could induce sufficient apoptosis in EJ and BIU populations within 48 or 60 h. Constitutive soluble and membrane Fas and FasL were found in both cell lines, and caspase-3 could be upregulated after MMC treatment. Anti-Fas antibody could commit the target cells to die of apoptosis, while Ac-DEVD-CHO inhibited MMC-induced apoptosis. Our data thus suggest that MMC-induced apoptosis in EJ and BIU cells is mediated by Fas and upregulation and activation of caspase-3 is an essential element for the apoptotic process. Reduced dose and short-term MMC strategy would be of practical value either in determining the apoptotic susceptibility of individual TCC cases or in the clinical instillation of urothelial cancers.