Molecular expression and functional evidence of a drug efflux pump (BCRP) in human corneal epithelial cells.

PURPOSE: Breast Cancer Resistance Protein (BCRP) belongs to the family of efflux transporters involved in drug efflux leading to drug resistance. The objective of this study was to explore physical barriers for ocular drug absorption and to verify the presence and possible role of BCRP as a barrier for ocular drug resistance.

METHODS: Transfected human corneal epithelial cells (SV40-HCEC) were selected as an in vitro model for corneal epithelium with MDCKII-BCRP as positive control. [(3)H]-Mitoxantrone ([(3)H]-MTX), which is a proven substrate for organic anion transporter like BCRP, was selected as a model drug for functional expression studies. Fumetremorgin C (FTC), a known specific inhibitor for BCRP and GF120918, an inhibitor for BCRP and P-gp, were added to inhibit BCRP-mediated efflux. PGP-4008, a specific inhibitor of P-gp was used to delineate the contribution of P-gp. The mRNA extracted from cells was used for RT-PCR analysis and gene expression. Membrane fractions of SV40-HCEC and MDCKII-BCRP were used for immunoprecipitation followed by Western blot analysis.

RESULTS: Efflux was inhibited significantly in the presence of FTC and GF120918. Dose-dependent inhibition of efflux by BCRP was noticed in SV40-HCEC and MDCKII-BCRP in the presence of FTC and GF120918, and the efflux was ATP-dependent. The metabolic inhibitor, 2,4-DNP, significantly inhibited efflux. No pH-dependent efflux was noticed except at pH 5.5. RT-PCR analysis indicated a unique and distinct band at approximately 429 bp, corresponding to BCRP in SV40-HCEC and MDCKII-BCRP cells. Western Blot analysis indicated a specific band at approximately 70 kDa in the membrane fraction of SV40-HCEC and MDCKII-BCRP cells.

CONCLUSIONS: We have demonstrated the expression of BCRP in human corneal epithelial cells and, for the first time, demonstrated its functional activity leading to drug efflux. RT-PCR and Western blot analysis further confirmed this finding.