Abstract
BackgroundAn in vitro model was developed to understand if celecoxib could synergize with Mitomycin C (MMC), commonly used for the prevention of non-muscle invasive bladder cancer recurrence, and eventually elucidate if the mechanism of interaction involves multi drug resistance (MDR) transporters.MethodsUMUC-3, a non COX-2 expressing bladder cancer cell line, and UMUC-3-CX, a COX-2 overexpressing transfectant, as well as 5637, a COX-2 overexpressing cell line, and 5637si-CX, a non COX-2 expressing silenced 5637 cell line, were used in the present study. The expression of COX-2 and MDR pumps (P-gp, MDR-1 and BCRP) was explored through western blot. The anti-proliferative effect of celecoxib and MMC was studied with MTT test. Three biological permeability assays (Drug Transport Experiment, Substrate Transporter Inhibition, and ATP cell depletion) were combined to study the interaction between MDR transporters and celecoxib. Finally, the ability of celecoxib to restore MMC cell accumulation was investigated.ResultsThe anti-proliferative effect of celecoxib and MMC were investigated alone and in co-administration, in UMUC-3, UMUC-3-CX, 5637 and 5637si-CX cells. When administered alone, the effect of MMC was 8-fold greater in UMUC-3. However, co-administration of 1 μM, 5 μM, and 10 μM celecoxib and MMC caused a 2,3-fold cytotoxicity increase in UMUC-3-CX cell only. MMC cytotoxicity was not affected by celecoxib co-administration either in 5637, or in 5637si-CX cells. As a result of all finding from the permeability experiments, celecoxib was classified as P-gp unambiguous substrate: celecoxib is transported by MDR pumps and interferes with the efflux of MMC. Importantly, among all transporters, BCRP was only overexpressed in UMUC-3-CX cells, but not in 5637 and 5637si-CX.ConclusionsThe UMUC-3-CX cell line resembles a more aggressive phenotype with a lower response to MMC compared to the wt counterpart. However, the administration of celecoxib in combination to MMC causes a significant and dose dependent gain of the anti-proliferative activity. This finding may be the result of a direct interaction between celecoxib and MDR transporters. Indeed, BCRP is overexpressed in UMUC-3-CX, but not in UMUC-3, 5637, and 5637si-CX, in which celecoxib is ineffective.
Highlights
An in vitro model was developed to understand if celecoxib could synergize with Mitomycin C (MMC), commonly used for the prevention of non-muscle invasive bladder cancer recurrence, and eventually elucidate if the mechanism of interaction involves multi drug resistance (MDR) transporters
The UMUC-3-CX cell line resembles a more aggressive phenotype with a lower response to MMC compared to the wt counterpart
Expression of COX-2 and ATP-Binding Cassette (ABC) trasporters in bladder cancer cells COX-2 and ABC transporters expression was studied via western blot in several bladder cancer cell lines (Figure 1)
Summary
An in vitro model was developed to understand if celecoxib could synergize with Mitomycin C (MMC), commonly used for the prevention of non-muscle invasive bladder cancer recurrence, and eventually elucidate if the mechanism of interaction involves multi drug resistance (MDR) transporters. Early recurrence is common after MMC treatment and may recognize several causes, such as disease phenotype [1], the presence of residual tumor following a TUR [4], as well as downstaging [5] Another possible cause may be the failure of cancer treatment due to resistance to structurally unrelated chemotherapeutic agents, defined Multi Drug Resistance (MDR) [6,7]. MDR is usually associated with a decreased intracellular concentration of cytostatic drugs and with the overexpression of ATP-Binding Cassette (ABC) transporters, such as P-glycoprotein (P-gp), Breast Cancer Resistance Proteins (BCRP) and Mutidrug Resistance associated Proteins (MRPs) [8,9,10] These transporters cause the efflux of drugs and xenobiotics out from the cells [11]. Their overexpression has been observed in urothelial cancer cells [12] and may be useful for selecting patients with bladder cancer to be candidates for neoadjuvant chemotherapy [13]
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