Drug resistance restricts the efficacy of short term low dose mitomycin-C treatment in UMUC-3 bladder cancer cells

OBJECTIVE: Mitomycin-c (MMC) is the most used intravesical adjuvant agent in non-muscle invasive bladder cancer to prevent recurrence. However, a consensus on about appropriate dosage and treatment schedule of MMC is lacking. We, therefore, aimed to evaluate the most appropriate MMC dosage using an in vitro model of high-grade human bladder cancer. METHODS: UMUC-3 cells, a model for high-grade bladder cancer, were exposed to MMC in different time courses to assess its toxicological effects. XTT cell proliferation kit was used to evaluate the effect of MMC on the proliferation of UMUC-3 cell line. Gene expression analysis for the MDR1, BCL2 and ANXA5 genes was performed by Real-time PCR and flow cytometry analysis were conducted to evaluate the cell death mechanism and acquired resistance after MMC exposure. An ANXA5 kit was used to detect apoptotic cells, and 7-AAD was used to detect necrotic cells. RESULTS: Cell proliferation was prevented to a large extent (IC50, 0.175-0.081 mg/mL) and cytotoxic effects were observed after 5 µg/mL and 10 µg/mL MMC administrations for 1 and 2-h, after the 4th and 2nd dose cycles, respectively. Moreover, cell death was observed at 5 µg/mL and 10 µg/mL MMC applications for 1-h and 2-h by the sixth and second week, respectively. Flow cytometry exhibits increased subpopulation of drug-extruding UMUC-3 cells after a single dose of MMC for 1-h. MMC did not increase the number of apoptotic or necrotic cells; yet, MDR1 (multiple drug resistance) and ANXA5 (apoptotic) expression levels were increased and BCL2 (anti-apoptotic) expression was decreased. Limitations: In-vitro nature of the study and working with only one cell culture are inherit limitations of this project. CONCLUSION: A single dose of MMC administration for 1 or 2-h results in drug-resistance. If maintenance treatment is administered for one hour, it should be continued throughout a 6-week period.