Celecoxib and DFMO mechanisms of growth inhibition converge on G1 cell cycle arrest in transitional cell carcinoma of the bladder

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INTRODUCTION AND OBJECTIVE: Celecoxib and difluoromethylornithine (DFMO) are being evaluated as chemoprevention agents for bladder and other cancers. A combination prevention strategy that features synergy would permit lower dosing to minimize toxicity in long-term prevention studies. To investigate potential synergy in bladder cancer, we assessed celecoxib and DFMO effects alone and in combination on the growth of malignant urothelial cells. METHODS: RT4, UM-UC-3 and TCC 96-1 bladder cancer cell lines were treated with 0, 3.12, 6.25, 12.5, 25, or 50 μM celecoxib alone or combined with 0, 250, or 500 μM DFMO. Cell growth was quantified at 24-hour intervals using crystal violet elution. Flow cytometry with PI/APO-BrDU staining was utilized to evaluate cell cycle and apoptosis. Western blot analysis on cell lysates was performed for cyclooxygenase (COX)-2 and the cell cycle regulatory protein cyclin B1. RESULTS: Celecoxib alone at high doses (25-50 μM) significantly inhibited growth of all three cell lines as measured at 24, 48, and 72 hours. This growth inhibition was associated with G1 cell cycle arrest and apoptosis in a dose-dependent fashion for all three cell lines including UM-UC-3 which lacks COX-2 expression. DFMO also inhibited growth for all three cell lines with dose-dependent accumulation of cells in G1 cell cycle arrest in UM-UC-3 and TCC 96-1. Apoptosis was not observed with DFMO treatment. At non-inhibitory celecoxib doses (3.1 or 6.2 μM), up to 70% growth inhibition of all three cell lines was achieved by addition of 250 or 500 μM DFMO. Downregulation of cyclin B1 was identified with G1 cell cycle arrest for celecoxib but not DFMO. CONCLUSIONS: Celecoxib in combination with DFMO inhibits growth of bladder cancer cells to a greater extent than celecoxib alone. The mechanism of growth inhibition appears to converge on G1 cell cycle arrest for these agents via separate pathways. This finding indicates a synergistic effect for these agents and supports further investigation of their combined use in bladder cancer chemoprevention studies.