Short Communication INTERACTION OF PROGESTINS WITH THE HUMAN MULTIDRUG RESISTANCE-ASSOCIATED PROTEIN 2 (MRP2)

Progestins are widely used as oral contraceptives and hormone replacement therapy. Recently it has been demonstrated that many progestins are inhibitors of P-glycoprotein, possibly explaining gender differences in drug actions. In vitro evidence suggested that at least norgestimate might also inhibit other transporters like the multidrug resistance-associated protein 2 (MRP2). We therefore investigated whether norgestimate, desogestrel, medroxyprogesterone acetate, norethisterone, progesterone, cyproterone acetate, chlormadinone acetate, and levonorgestrel inhibit MRP2 in vitro using confocal laser scanning microscopy and 5-chloromethylfluorescein diacetate as a prodrug of the fluorescent 5-chloromethylfluorescein (CMF), which is actively transported by MRP2 as glutathione conjugate. Of the progestins tested, only norgestimate (50 M) and progesterone (100 M) significantly increased intracellular CMF fluorescence by 62% and 53%, respectively. In conclusion, the progestins norgestimate and progesterone significantly inhibit the transport activity of MRP2 in vitro. Multidrug resistance-associated proteins (MRPs) are a subfamily of the ATP-binding cassette transport protein family involved in drug resistance (Schinkel and Jonker, 2003). MRP2 is a key member of this group and was identified in the canalicular membrane of hepatocytes as a transporter for organic anions extruding a wide range of glutathione, glucuronate, and sulfate conjugates into the bile (Ishikawa 1993; Ito et al., 1997; Paulusma and Oude Elferink, 1997). It was also detected in renal brush-border membranes, the intestine (Schaub et al., 1997), placenta (St.-Pierre et al., 2000; Gerk and Vore, 2002), and brain (Török et al., 2003). In human carcinoma, MRP2 may confer resistance to chemotherapy (Norris et al., 1996; Nies et al., 2001; Young et al., 2001; Schinkel and Jonker, 2003), and it appears also to play a role in drug-drug interactions (Fromm et al., 2000; Bramow et al., 2001; Bode et al., 2002; Huisman et al., 2002; Giessmann et al., 2004). In recent years it has been suggested that gender could alter the activity of drug transporters like P-glycoprotein (P-gp) (Cummins et al., 2002). Hence in a previous study we have investigated whether P-gp is inhibited by progestins used in oral contraceptives and hormone replacement therapy. All progestins tested had P-gp inhibitor properties, with some of them being as potent as the well known P-gp inhibitor quinidine (Fröhlich et al., 2004). Interestingly, one of the tested progestins (norgestimate) revealed an inhibitory effect on the transport of calcein-acetoxymethylester (calcein-AM) not only in the P-gp-overexpressing cell line L-MDR1, but also in the parental cell line LLC-PK1, which only expresses low amounts of P-gp (Decorti et al., 2001; Weiss et al., 2003; Fröhlich et al., 2004), indicating that this effect was not selective. Because LLC-PK1 expresses MRP2 (Evers et al., 1996; Decorti et al., 2001) and MRP2 can transport calcein-AM (Evers et al., 2000), these data may suggest that norgestimate also inhibits MRP2. We thus aimed to characterize the MRP2inhibitory potencies of norgestimate and a series of frequently used progestins in vitro in the canine kidney cell line MDCK II/Par and its analog MDCK II/MRP2, which stably overexpresses human MRP2. The functional activity of MRP2 can be demonstrated with the cell tracker reagent 5-chloromethylfluorescein diacetate (CMFDA). The nonfluorescent CMFDA passively diffuses into the cells where cytosolic esterases cleave off its acetate residues, thereby releasing the fluorescent and membrane-impermeable product 5-chloromethylfluorescein (CMF), which can react, e.g., with glutathione, to form fluorescent conjugates. This methylfluorescein-sulfoglutathione complex (MF-SG) is then actively secreted by MRP2 (Bogman et al., 2003). CMF fluorescence was measured by confocal laser scanning microscopy. Materials and Methods Materials. Dulbecco’s modified Eagle’s medium was purchased from PAA Laboratories GmbH (Cölbe, Germany); fetal calf serum, medium supplements, antibiotics, glutamine, buffers, and cell culture medium M 199 were obtained from Invitrogen (Karlsruhe, Germany); and NaCl, MgSO4 and dimethyl sulfoxide were obtained from AppliChem (Darmstadt, Germany). Collagen-R was obtained from Serva (Heidelberg, Germany). A Cytotoxicity Detection Kit was obtained from Roche Applied Science (Mannheim, Germany). CMFDA was purchased from MoBiTec (Göttingen, Germany), norgestimate was a kind gift from Janssen-Cilag GmbH (Neuss, Germany), desogestrel was obtained from Grünenthal GmbH (Santiago, Chile), and medroxyprogesterone acetate, norethisterone, progesterone, cyproterone acetate, chlormadinone acetate, levonorgestrel, and pyruvate were obtained from Sigma-Aldrich (Taufkirchen, Germany), MK 571 was obtained from BIOMOL Research Laboratories (Plymouth Meeting, PA). Six-well plates were obtained from Biochrom AG (Berlin, Germany), 96-well microtiter plates and culturing bottles were obtained from NUNC GmbH & Co. KG (Wiesbaden, Germany), the coverslips for confocal laser scanning microscopy were obtained from H. Saur (Reutlingen, Germany), and the QuantiChrom Glutathione Assay Kit was obtained from BioAssay Systems (Hayward, CA). MDCK II/Par and MDCK II/MRP2 Cells. As an in vitro model for Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.105.005314. ABBREVIATIONS: MRP, human multidrug resistance-associated protein; CMF, 5-chloromethylfluorescein; CMFDA, 5-chloromethylfluorescein diacetate; MF-SG, methylfluorescein-sulfoglutathione; P-gp, P-glycoprotein; ANOVA, analysis of variance. 0090-9556/05/3311-1576–1579$20.00 DRUG METABOLISM AND DISPOSITION Vol. 33, No. 11 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 5314/3055581 DMD 33:1576–1579, 2005 Printed in U.S.A. 1576 at A PE T Jornals on A ril 2, 2017 dm d.aspurnals.org D ow nladed from human MRP2 we used MDCK II/MRP2 cells, a cell line generated by stable transfection of MRP2 cDNA into MDCK II cells (Evers et al., 1998). The cell line was kindly provided by Dr. Piet Borst (The Netherlands Cancer Institute, Amsterdam). MDCK II/Par cells served as a control. Both cell lines were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin sulfate. L-MDR1 and LLC-PK1 Cells. The porcine kidney epithelial cell line LLC-PK1 (available at the American Type Culture Collection, Manassas, VA) and the L-MDR1 cell line with overexpression of human P-gp (kindly provided by Dr. Alfred H. Schinkel, The Netherlands Cancer Institute, Amsterdam) were also tested for MRP2-activity. The cells were cultured under standard cell culture as described previously (Weiss et al., 2003). Confocal Laser Scanning Microscopy: CMFDA Accumulation Assay. Intracellular accumulation of the MRP2 substrate MF-SG in cells was analyzed with a DM IRE 2 TCS SP II confocal laser scanning microscope from Leica (Wetzlar, Germany) using an adapted protocol published by Bogman et al. (2003). Pilot experiments confirmed that a 15-min incubation with CMFDA is sufficient to achieve maximal loading with this compound. For excitation, a 488-nm argon laser line was used and a 500to 550-nm band-pass filter was used to detect emission. The objective used was a Leica HCX PL APO CS 63x with a numerical aperture of 1.2. Living cells (6 10) were seeded on poly-D-lysine and collagen-coated coverslips in a closed miniperfusion chamber (H. Saur) directly before the experiment and preincubated for 10 min with or without the test compound in darkness at 37°C in 1 ml of transport buffer consisting of Hanks’ balanced salt solution and 1 mM pyruvate for energy supply. Subsequently, CMFDA in a final concentration of 50 nM was added. After incubation for 15 min, three series of 20 sections in z-plane through the cells were acquired for each coverslip. The thickness of each optical section was about 0.15 to 0.2 m. The mean amplitude of fluorescence intensities between cells without (control) and those treated with the specific compound were compared. In each series, 30 regions of interest with an area of 350 m each were automatically drawn in the image. Fluorescence was quantified with the stack profile function in the quantify modus (Leica software), which calculates the statistical mean average. The experiments were performed at least in triplicate on different days. All progestins were tested in the highest soluble concentration (Fröhlich et al., 2004): norgestimate (50 M), desogestrel (30 M), medroxyprogesterone (20 M), norethisterone (20 M), progesterone (100 M), cyproterone acetate (20 M), chlormadinone acetate (50 M), and levonorgestrel (5 M). For compounds that revealed inhibitory activity on CMF transport by MRP2 (MK 571, progesterone, and norgestimate), the increase in intracellular fluorescence was also recorded over a period of 30 min. For the time kinetics, 5 10 cells/ml transport buffer were preincubated on a coverslip in a closed miniperfusion chamber with 50 nM CMFDA for 15 min to reach a steady state within the cells. Afterward, MK 571 (20 M), progesterone (100 M), or norgestimate (50 M) was added, and time series in xyzt-modus over a period of 30 min were recorded with one picture per minute. Time series were also recorded in LLC-PK1 and L-MDR1 cells with and without the MRP2-specific inhibitor MK 571. Quenching Test. The absence of potential errors in the quantification of CMF because of changes in its spectral behavior in the presence of other compounds was tested in a quenching assay by adding increasing concentrations of individual progestins to aliquots of the cell lysate after incubation with 50 nM CMFDA. Comparison of the fluorescence with control cell lysates without the respective progestin confirmed that none of the progestins showed any quenching effect on the fluorescence of CMF. Cytotoxicity Assay. None of the progestins exerted cytotoxic effects as evaluated with the Cytotoxicity Detection Kit (Roche Applied Science). Glutathione Assay. CMFDA requires conjugation with glutathione to generate the fluorescent MRP2 substrate MF-SG. To exclude that differences observed between MDCK II/Par and MDCK II/MRP2 cells can be attributed to differences in the glutathione level in the two cell lines and to ensure that norgestimate and progesterone do not alter glutathione levels, glutathione concentrations were measured in both cell lines before and after incubation with these progestins. Quantification of glutathione was measured with the QuantiChrom Glutathione Assay Kit (BioAssay Systems). In this assay, 5,5 dithiobis(2-nitrobenzoic acid) reacts with reduced glutathione to form a yellow product. The optical density, measured at 412 nm, is directly proportional to the glutathione concentration in the sample. The assay was conducted according to the manufacturer’s instruction with 5 10 cells/100 l. Statistical Analysis. p values were calculated by analysis of variance (ANOVA) with Dunnett’s multiple comparison test for post hoc pair-wise comparison of the results with the corresponding control (without inhibitor). All statistical analyses were performed with GraphPad InStat, version 3.05 (GraphPad Software Inc., San Diego, CA). A p value of 0.05 was considered significant.