The interaction of celecoxib with MDR transporters enhances the activity of mitomycin C in a bladder cancer cell line

Abstract

Background: 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.

Methods: UMUC-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.

Results: The 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.

Conclusions: The 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.

Figure 1

Protein expression levels of COX-2, P-gp, MRP1 and BCRP in UMUC3, UMUC-3-CX, 5637, 5637si-CX, TCCsup, and J82, as determined by western blot. Cell lysates were obtained from exponentially growing cells and subjected to immunoblotting with appropriate antibodies. Immunoblotting with an antibody to β-actin was used to ensure equal loading of proteins in each lane (bottom).

Figure 2

Secretion of prostagladin E2 in the culture medium of a selection of bladder cancer cell lines. PGE2 in cell supernatant was determined by enzyme immunoassay after treatment with or without known concentrations (0 - 1- 5–50 μM) of Celecoxib for 24 and 48 h.

Figure 3

Antiproliferative effect of celecoxib and MMC administered alone at increasing doses (0,1 - 0,5 - 1–5 - 10–30 - 50 μM) (A, B). Antiproliferative effect of MMC alone and in co-administration with known concentrations of celecoxib (C, D). All experiments conducted after 48 h of incubation in 5637 (A, C) and in 5637si-CX (B, D) cells.

Figure 4

Antiproliferative effect of celecoxib and MMC administered alone at increasing doses (0,1 - 0,5 - 1–5 - 10–30 - 50 μM) (A, B). Antiproliferative effect of MMC alone and in co-administration with known concentrations of celecoxib (C, D). All experiments conducted after 48 h of incubation in UMUC-3 (A, C) and in UMUC-3-CX (B, D) cells.

Figure 5

Flow cytometry analysis to study the time course of MMC intracellular accumulation and its modulation by celecoxib in UMUC-3 (A) and UMUC-3-CX (B). In both cases, cells were treated one day with MMC (1.69 μM) or celecoxib (14.2 μM) alone, or with both in co-administration (MMC 1.69 μM + celecoxib 14.2 μM). Celecoxib causes an increase in the intracellular concentration of MMC in UMUC-3-CX, but not in UMUC-3 cells.

Figure 6

Schematic view of the mechanism of calcein cellular retention in the absence (A) and in the presence (B) of an MDR pump substrate, such as celecoxib.