Human BCRP (Arg482) membranes (5 mg/ml) and a control membrane preparation for ABC transporters were purchased from Gentest; Corning Incorporated (Corning, NY, USA). Anti-BCRP mouse monoclonal antibody (clone BXP21; cat. no. MC-236) and horseradish peroxidase-labeled goat anti-mouse secondary antibody (cat. no. 5220-0341) were purchased from Kamiya Biomedical Company (Tukwila, WA, USA) and SeraCare Life Sciences (Milford, MA, USA), respectively. β-Actin mouse monoclonal antibody (cat. no. AF0003) was purchased from Beyotime Institute of Biotechnology (Shanghai, China). PAL, Ko143, sulfasalazine, and dimethyl sulfoxide were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Fetal bovine serum (FBS), trypsin, Dulbecco's modified Eagle's medium (DMEM), Ham's F12 nutrient (F12) medium, PBS and TRIzol reagent were obtained from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Microplates (with 6 and 96 wells) and cell culture flasks were obtained from Corning Costar; Merck KGaA. Methanol of high-performance liquid chromatography (HPLC) grade was obtained from Merck KGaA and the other solvents used were of analytical grade. The lentiviral vector encoding the BCRP gene was purchased from Shanghai GeneChem Co., Ltd. (Shanghai, China).

The porcine renal endothelial cell line LLC-PK1 and LLC-PK1/BCRP cells were provided by Professor Zeng Su from the College of Pharmaceutical Sciences, Zhejiang University (Hangzhou, China). LLC-PK1/BCRP cells are transgenic cells that were established by using liposome as vehicles to import human BCRP genes into LLC-PK1 cells (18). Both types of cell were cultured in F12 medium supplemented with 20% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in an atmosphere of 5% CO₂ and 95% relative humidity. Cells were supplemented with fresh media every 2–3 days. All of the cultured cells used in subsequent experiments were at passages 5–25.

In addition, 293 cells were seeded in 6-well plates at a density of 1×10⁴ cells/well and were cultured in DMEM supplemented with 20% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in an atmosphere of 5% CO₂ and 95% relative humidity. Cells were exposed to lentiviral vector encoding the BCRP gene with a green fluorescence protein (GFP)-tag at a multiplicity of infection of 50 in the presence of 8 µg/ml polybrene (Sigma-Aldrich; Merck KGaA) added to enhance the transduction efficiency. The transfection medium was replaced with DMEM after 24 h. Cells were observed under a fluorescence microscope at 4 days following transfection, at which time >90% of the cells were found to be positive for green fluorescence protein-tag fluorescence. Both 293 cells and BCRP-transfected 293 (293/BCRP) cells were then cultured in DMEM supplemented with 20% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in an atmosphere of 5% CO₂ and 95% relative humidity. Cells were supplemented with fresh media every 2–3 days. All of the cultured cells used in the subsequent experiments were at passages 2–5.

RT-qPCR was used to detect gene expression levels in 293 cells and lentivirus-BCRP-transduced 293 cells at 10 and 20 days following transduction. Total RNA was isolated from cells using TRIzol reagent (Life Technologies; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RNA samples (0.5 µg) were used for RT using the Prime Script™ 174 RT reagent kit (Takara, Biotechnology, Dalian, China) according to the manufacturer's instructions. SYBR Green Realtime PCR Master mix (Qiagen, Inc., Valencia, CA, USA) was used to detect the expression level of ABC subfamily G member 2 (ABCG2), with a 20 µl reaction mixture containing 2 µl synthesized cDNA, 10 µl SYBR Premix Ex Taq, 0.2 µl each primer and 7.6 µl ddH₂O. The cycling conditions were as follows: Initial denaturation at 95°C for 30 sec followed by 36 cycles of 95°C for 5 sec and 60°C for 30 sec. The primers used for human ABCG2 were forward, 5′-GTTGTGATGGGCACTCTGAC-3′ and reverse), 5′-CCCTGTTAATCCGTTCGTTT-3′; and for β-actin forward, 5′-CTCTTCCAGCCTTCCTTCCT-3′ and reverse), 5′-AGCACTGTGTTGGCGTACAG-3′ were used for human β-actin (GenBank accession no. NM 01101). Each sample was analyzed in triplicate. Endogenous β-actin mRNA was used to determine the relative quantitative expression using the 2^−ΔΔCq method (19).

293 and lentivirus-BCRP-transduced 293 cells were cultured in 6-well plates for harvesting the cells and the cell monolayer was lysed at 4°C for 30 min in radioimmunoprecipitation assay buffer with protease inhibitors (Sigma-Aldrich; Merck KGaA) and the amount of protein was determined by bicinchoninic acid assay. Proteins (60 µg) were separated on 12% SDS-PAGE gels and transferred onto a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). The membrane was blocked in Dulbecco's PBS containing 0.1% Tween-20 and 5% non-fat dry milk (Bio-Rad Laboratories, Inc., Hercules, CA, USA) for 2 h at room temperature. The PVDF membranes were then washed three times for 10 min with tris-buffered saline with Tween-20 (TBST) buffer and incubated overnight at room temperature with a mouse monoclonal antibody (1:1,000) against human BCRP and β-actin. Following extensive washing, the membranes were further incubated with secondary antibody (1:5,000) for 1 h at room temperature. The membranes were then washed three times with TBST and immunoreactivity was visualized using an enhanced chemiluminescence kit and the membranes were exposed to photographic film (Kodak, Rochester, NY, USA). Protein levels were expressed as the ratio of the band intensities of BCRP to the endogenous control β-actin. Each sample was analyzed in triplicate.

Intracellular accumulation experiments were conducted with 293 and 293/BCRP cells. Cells were cultured in 6-well plates at a density of 2×10⁵ cells/well for 2 days and on the day of the experiment the culture medium was replaced with DMEM with, containing PAL at varying concentrations (0.1, 1, 10, 25 and 50 µM) with or without BCRP inhibitor Ko143 (5 µM). Following incubation for 2 h in a humidified atmosphere of 5% CO₂ at 37°C, the cells were washed three times with PBS and then were lysed by repeated freezing and thawing. The lysed cells were centrifuged at 16,770 × g at 4°C for 5 min and 150 µl liquid supernatant was collected for PAL detection by high performance liquid chromatography/mass spectrometry (HPLC/MS), whereas 20-µl samples were taken for bicinchoninic acid assays to determine the protein concentration. Data are presented as the mean ± standard deviation (n=3) following normalization to the total protein concentration in each well.

For the bidirectional transport experiments, LLC-PK1 and LLC-PK1/BCRP cells were seeded into Transwell inserts in 6-well plates at a density of 2×10⁵ cells/well. The cells were then cultured in F12 medium supplemented with 20% FBS, at 37°C for 7 days, at which time the integrity of the monolayer was evaluated by measuring the transepithelial electrical resistance (TEER) as described previously (20) across the monolayer (EVOM2; World Precision Instruments, Inc., Sarasota, FL, USA). Monolayers with TEER values exceeding 200 Ω/cm² were chosen for use in the transport experiments.

On the day of transport experiments, the culture medium was replaced with F12 medium containing PAL at varying concentrations (0.1, 1, 10, 25 and 50 µM) on the upper or lower side and with blank Hank's balanced salt solution (HBSS; Thermo Fisher Scientific, Inc.) on the other side. Cells were incubated at 37°C. Six sequential samples (0.2 ml) were collected at different times (0, 0.5, 1, 2, 3 and 4 h) from both sides of the cell monolayer. The same volume of testing solution or blank HBSS was immediately added to replace the samples obtained. Transport experiments were also performed in the presence of the BCRP inhibitor (5 µM Ko143) loaded on the same side of the monolayers with PAL.

The permeability of PAL was estimated by calculating the apparent permeability coefficient (Papp) as follows: Papp=dQ/dtxAxC₀, where C₀ is the concentration of PAL added to the upper or lower side, A is the surface area of the monolayer (4.67 cm² in the present study) and dQ/dt is the rate at which the drug(s) appeared on the opposite side.

The efflux ratio was calculated as: Efflux ratio=Papp_(BL→AP)/Papp_(AP→BL), where apical (AP) and basal (BL) are the two sides of the compartment, with the indicated transport direction. Data are presented as the mean ± standard deviation (n=3).

PAL concentrations following cellular accumulation and bidirectional transport experiments were determined using HPLC/MS. An 80-µl aliquot obtained from the transport investigation and 20 µl internal standard moclobemide (100 ng/ml; Sigma-Aldrich; Merck KGaA) in the mobile phase were mixed on a high-speed vortex for 30 sec. Methyl tert-butyl ether (400 µl) was added to the mixture and vortexed for 5 min. Following centrifugation at 22,160 × g for 10 min at 4°C, the supernatant (300 µl) was transferred to an 1.5-ml Eppendorf tube and solvent was evaporated to dryness for 40 min at 37°C using a vacuum drying system (RVT4104-230; Thermo Fisher Scientific, Inc.). The dry residue was reconstituted in 80 µl mobile phase and mixed on a vortex for 5 min. The mixture was then centrifuged at 22,160 × g for 5 min at 4°C, and a 5-µl aliquot of the supernatant was injected into an HPLC-MS system for analysis. The calibration standards ranged from 2–5,000 ng/ml.

PAL concentration was measured using an HPLC system (2690; Waters Corporation, Milford, MA, USA) and a mass spectrometer (Micromass; Waters Corporation) equipped with an electrospray ionization (ESI) ion source. Separation was performed on an Ultimate XB-C18 column (3 µm; 2.1× 100 mm; Welch Materials, Inc., Shanghai, China). The mobile phase was water (25 mM ammonium acetate, 0.06% ammonium) and acetonitrile (68:32) at a flow rate of 0.25 ml/min. The total run time was 3 min and the column and autosampler were maintained at 40 and 25°C, respectively.

The compounds were ionized in the positive ESI ion source mode of the mass spectrometer. The detection conditions were a capillary voltage of 3.00 kV, a cone voltage of 22.00 V, an extractor voltage of 2.00 V, a source temperature of 120°C, a desolvation temperature of 350°C and cone and desolvation gas flows of 50 and 500 l/h, respectively. The selected ion recording mode was used for quantification, with m/z=427.5 for PAL and m/z=269.5 for moclobemide.

The in vitro BCRP affinity for PAL was investigated as described by Boulton et al (21) with residue R482/BCRP membranes analyzed for both basal and drug-stimulated ATP hydrolysis by colorimetric detection of inorganic phosphate release. The reaction was conducted in 96-well plates, and initiated by adding 20 µl Mg-ATP (12 mM) solution (heated to 37°C) and 20 µl (heated to 37°C for 5 min) BCRP membrane (2 mg/ml) to the reaction system (20 µl), which contained 100 µM sodium orthovanadate and PAL or sulfasalazine (a substrate for ABCG2) at varying concentrations (0.1, 1, 10, 25, 50 and 100 µM). All of the reagents were made up in Tris and 2-(N-morpholino) ethanesulfonic acid buffer (pH 6.8). Following 60 min of incubation at 37°C, the reaction was terminated by the addition of 30 ml ice-cold 10% sodium dodecylsulfate plus 0.1% antifoam A. The phosphate concentration (Pi) was assayed by adding 200 µl 35 mM ammonium molybdate in 15 mM zinc acetate: 10% ascorbic acid (pH 5.0, freshly prepared) at a ratio of 1:4 to each well and the mixture was incubated for 20 min at 37°C. The liberation of Pi was detected by the ultraviolet absorption of the Pi-molybdate complex at 620 nm by a spectrophotometer and the Pi concentration was calculated from the standard curve. Each sample was analyzed in triplicate. Data were plotted as drug concentration vs. Pi release and analyzed by least-squares non-linear regression fits using the Michaelis-Menten equation. The maximal reaction velocity (V_max) and the substrate concentration at 50% of V_max (K_m) were determined using GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA, USA).

Statistical analyses were performed with SPSS software (version 17.0; SPSS, Inc., Chicago, IL, USA). The effect of BCRP on PAL accumulation in 293 and 293/BCRP cells was analyzed with one-way analysis of variance, using Fisher's least significant difference test for post hoc comparisons. P<0.05 was considered to indicate a statistically significant difference.

The results from the RT-qPCR and western blotting assays indicated high expression levels of BCRP mRNA and protein in 293 cells transfected with the BCRP gene, whereas no notable BCRP was detected in the 293 cells (Fig. 1).

The cellular accumulation of PAL in both 293 and 293/BCRP cells increased markedly with the drug concentration. However, the rate of increase was markedly lower in 293/BCRP cells than in 293 cells (Fig. 2), indicating that the cellular bioavailability of PAL was attenuated by BCRP. Conversely, combination treatment with 5 µM Ko143 significantly increased the PAL accumulation in 293/BCRP cells but had no influence on its accumulation in 293 cells. These results suggest that PAL can be effluxed by BCRP.

Table I lists the permeability parameters of the transcellular transport of PAL. The transport of PAL was asymmetric, with the transport rates being distinctly lower in the AP→BL direction than in the BL→AP direction. Transfection of the BCRP gene further reduced the transport rates in the AP→BL direction, while it increased the rates in the BL→AP direction. The net efflux ratios of PAL in were >1.7 for concentrations from 0.1 to 25 µM, which indicated that PAL may be a substrate of BCRP. The net efflux ratio of PAL decreased as the concentration increased to 50 µM, for which the net efflux ratio was 1.22, suggesting that the BCRP in PAL transport had saturated.

The addition of the BCRP inhibitor Ko143 (5 µM) increased transport rates in the AP→BL direction but decreased those in the BL→AP direction were observed in LLC-PK1/BCRP cells and the net ratios of PAL were markedly decreased. These results further support that PAL is a substrate of BCRP.

It is known that ABC drug transporters utilize the energy of ATP hydrolysis to transport drugs outside cells (11,21). As presented in Fig. 3, both sulfasalazine and PAL stimulated vanadate-sensitive BCRP ATPase activity in a concentration-dependent manner, with the degree of stimulation being less for PAL than for sulfasalazine at all concentrations. The concentrations of sulfasalazine and PAL required for half-maximal stimulation (i.e., EC₅₀ values) of the vanadate-sensitive ATPase activity were 25.09±4.96 and 31.68±9.60 µM, respectively, and the V_max values were 108.5 and 98.01 nM/min; the V_max/K_m values of the two compounds were therefore 4.32×10⁻³ and 3.09×10⁻³ min⁻¹.