(R)- and (S)-Verapamil Differentially Modulate the Multidrug-resistant Protein MRP1

The multidrug-resistant protein MRP1 (involved in the cancer cell multidrug resistance phenotype) has been found to be modulated by racemic verapamil (through stimulation of glutathione transport), inducing apoptosis of human MRP1 cDNA-transfected baby hamster kidney 21 (BHK-21) cells and not of control BHK-21 cells. In this study, we show that the two enantiomers of verapamil have different effects on MRP1 activity. Only the S-isomer (not the R-isomer) potently induced the death of MRP1-transfected BHK-21 cells. The decrease in cellular glutathione content induced by the S-isomer, which was not observed with the R-isomer, was stronger than that induced by the racemic mixture, indicating that the R-isomer antagonized the S-isomer effect. Both enantiomers altered leukotriene C4 and calcein transport by MRP1. Thus, the R-isomer behaved as an inhibitor, which was confirmed by its ability to revert the multidrug resistance phenotype toward vincristine. Molecular studies on purified MRP1 using fluorescence spectroscopy showed that both enantiomers bound to MRP1 with high affinity, with the binding being prevented by glutathione. Furthermore, conformational changes induced by the two enantiomers (monitored by sodium iodide accessibility of MRP1 tryptophan residues) were quite different, correlating with their distinct effects. (S)-Verapamil induces the death of potentially resistant tumor cells, whereas (R)-verapamil sensitizes MRP1-overexpressing cells to chemotherapeutics. These results might be of great potential interest in the design of new compounds able to modulate MRP1 in chemotherapy. The multidrug-resistant protein MRP1 (involved in the cancer cell multidrug resistance phenotype) has been found to be modulated by racemic verapamil (through stimulation of glutathione transport), inducing apoptosis of human MRP1 cDNA-transfected baby hamster kidney 21 (BHK-21) cells and not of control BHK-21 cells. In this study, we show that the two enantiomers of verapamil have different effects on MRP1 activity. Only the S-isomer (not the R-isomer) potently induced the death of MRP1-transfected BHK-21 cells. The decrease in cellular glutathione content induced by the S-isomer, which was not observed with the R-isomer, was stronger than that induced by the racemic mixture, indicating that the R-isomer antagonized the S-isomer effect. Both enantiomers altered leukotriene C4 and calcein transport by MRP1. Thus, the R-isomer behaved as an inhibitor, which was confirmed by its ability to revert the multidrug resistance phenotype toward vincristine. Molecular studies on purified MRP1 using fluorescence spectroscopy showed that both enantiomers bound to MRP1 with high affinity, with the binding being prevented by glutathione. Furthermore, conformational changes induced by the two enantiomers (monitored by sodium iodide accessibility of MRP1 tryptophan residues) were quite different, correlating with their distinct effects. (S)-Verapamil induces the death of potentially resistant tumor cells, whereas (R)-verapamil sensitizes MRP1-overexpressing cells to chemotherapeutics. These results might be of great potential interest in the design of new compounds able to modulate MRP1 in chemotherapy. Resistance of tumors to multiple structurally unrelated anticancer drugs is one of the major obstacles to successful cancer chemotherapy. Failure to achieve complete and long-lasting responses is a common clinical problem that limits the curative potential of anticancer drugs in clinical oncology. Human multidrug resistance (MDR) 2The abbreviations used are: MDR, multidrug resistance; ABC, ATP-binding cassette; LTC4, leukotriene C4; BHK-21, baby hamster kidney 21; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; AM, acetoxymethyl ester; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PBS, phosphate-buffered saline.2The abbreviations used are: MDR, multidrug resistance; ABC, ATP-binding cassette; LTC4, leukotriene C4; BHK-21, baby hamster kidney 21; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; AM, acetoxymethyl ester; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PBS, phosphate-buffered saline. is frequently associated with the overexpression of three transporters belonging to the ATP-binding cassette (ABC) protein superfamily, P-glycoprotein (ABCB1), the multidrug-resistance protein MRP1 (ABCC1), and the breast cancer resistance protein (ABCG2) (1Glavinas H. Kim P. Cserepes J. Sarkadi B. Curr. Drug Deliv. 2004; 1: 27-42Crossref PubMed Scopus (449) Google Scholar), which can actively extrude anticancer drugs from the cell at the expense of ATP hydrolysis. MRP1 transports organic anions, many of which are conjugated to GSH, sulfate, or glucuronate; physiological substrates of MRP1 include GSH and leukotriene C4 (LTC4) (2Deeley R.G. Kim S.P. FEBS Lett. 2006; 580: 1103-1111Crossref PubMed Scopus (230) Google Scholar). Cytotoxic drugs in clinical use against cancer such as vincristine, vinblastine, and daunorubicin are cotransported with GSH (2Deeley R.G. Kim S.P. FEBS Lett. 2006; 580: 1103-1111Crossref PubMed Scopus (230) Google Scholar). Although a number of modulators have been found (3Boumendjel A. Kim H. Trompier D. Perrotton T. Di Pietro A. Med. Res. Rev. 2005; 25: 453-472Crossref PubMed Scopus (146) Google Scholar), the mechanism of inhibition is poorly understood. In many cases, a competitive inhibition toward substrate was described, as for the leukotriene antagonist MK571, which is considered a reference inhibitor for MRP1 (4Gekeler V. Kim W. Sanders K.H. Ulrich W.R. Beck J. Biochem. Biophys. Res. Commun. 1995; 208: 345-352Crossref PubMed Scopus (372) Google Scholar). Interesting but complex results have been obtained with two classes of modulators: flavonoids and verapamil. Dietary flavonoids such as apigenin have been shown to interact with MRP1 (5Leslie E.M. Kim Q. Oleschuk C.J. Deeley R.G. Cole S.P. Mol. Pharmacol. 2001; 59: 1171-1180Crossref PubMed Scopus (223) Google Scholar), and quercetin induces reversion of drug resistance (6Leslie E.M. Kim R.G. Cole S.P. Drug Metab. Dispos. 2003; 31: 11-15Crossref PubMed Scopus (113) Google Scholar). Another flavonoid, dehydrosilybin, efficiently chemosensitizes cell growth to vincristine (7Trompier D. Kim H. Chang X.-B. Maitrejean M. Barron D. Riordan J.R. Di Pietro A. CMLS Cell. Mol. Life Sci. 2003; 60: 2164-2177Crossref PubMed Scopus (66) Google Scholar). Some bioflavonoids have been shown to stimulate glutathione transport by MRP1 (6Leslie E.M. Kim R.G. Cole S.P. Drug Metab. Dispos. 2003; 31: 11-15Crossref PubMed Scopus (113) Google Scholar). Verapamil, which is a P-glycoprotein inhibitor (8Naito M. Kim T. Cancer Res. 1989; 49: 1452-1455PubMed Google Scholar), has been reported to be ineffective in restoring drug sensitivity of MRP1-overexpressing cells (9Cole S.P. Kim K.E. Fraser K. Loe D.W. Grant C.E. Wilson G.M. Deeley R.G. Cancer Res. 1994; 54: 5902-5910PubMed Google Scholar, 10Cullen K.V. Kim R.A. Davey M.W. Biochem. Pharmacol. 2001; 62: 417-424Crossref PubMed Scopus (46) Google Scholar, 11Davey R.A. Kim T.J. Davey M.W. Belov L. Harvie R.M. Hancox D. Wheeler H. Leuk. Res. 1995; 19: 275-282Crossref PubMed Scopus (64) Google Scholar). However, although verapamil alone poorly inhibits LTC4 transport by MRP1, the addition of GSH strongly enhances the inhibition (10Cullen K.V. Kim R.A. Davey M.W. Biochem. Pharmacol. 2001; 62: 417-424Crossref PubMed Scopus (46) Google Scholar, 12Loe D.W. Kim R.G. Cole S.P. J. Pharmacol. Exp. Ther. 2000; 293: 530-538PubMed Google Scholar, 13Trompier D. Kim X.-B. Barattin R. du Moulinet D'Hardemare A. Di Pietro A. Baubichon-Cortay H. Cancer Res. 2004; 64: 4950-4956Crossref PubMed Scopus (107) Google Scholar). Verapamil and its analogs as well as bioflavonoids stimulate glutathione transport by MRP1 (6Leslie E.M. Kim R.G. Cole S.P. Drug Metab. Dispos. 2003; 31: 11-15Crossref PubMed Scopus (113) Google Scholar, 12Loe D.W. Kim R.G. Cole S.P. J. Pharmacol. Exp. Ther. 2000; 293: 530-538PubMed Google Scholar, 14Loe D.W. Kim C.J. Deeley R.G. Cole S.P. Biochem. Biophys. Res. Commun. 2000; 275: 795-803Crossref PubMed Scopus (49) Google Scholar) but are not transported themselves by MRP1. A new mechanism of modulation of the MDR phenotype induced by MRP1 recently emerged (13Trompier D. Kim X.-B. Barattin R. du Moulinet D'Hardemare A. Di Pietro A. Baubichon-Cortay H. Cancer Res. 2004; 64: 4950-4956Crossref PubMed Scopus (107) Google Scholar) wherein the modulator, i.e. verapamil, triggers apoptosis of resistant cells through stimulation of MRP1-mediated glutathione efflux. This new concept could lead to finding new inducers of GSH depletion as suggested (6Leslie E.M. Kim R.G. Cole S.P. Drug Metab. Dispos. 2003; 31: 11-15Crossref PubMed Scopus (113) Google Scholar, 15Kachadourian R. Kim B.J. Free Radic. Biol. Med. 2006; 41: 65-76Crossref PubMed Scopus (108) Google Scholar). Verapamil being a chiral molecule, Loe et al. (14Loe D.W. Kim C.J. Deeley R.G. Cole S.P. Biochem. Biophys. Res. Commun. 2000; 275: 795-803Crossref PubMed Scopus (49) Google Scholar) showed that the enantiomers of various verapamil analogs display differential abilities to stimulate GSH transport. Therefore, in this work, we investigated the individual effects of the two R- and S-enantiomers of verapamil on MRP1-overexpressing baby hamster kidney 21 (BHK-21) cells. Surprisingly, we found that, whereas the S-isomer was responsible for the death of MRP1-overexpressing cells through GSH depletion, in contrast, the R-isomer sensitized the cell growth to vincristine by inhibiting drug transport. In addition, we demonstrate that (R)-verapamil antagonized the (S)-verapamil-induced GSH efflux and that the two isomers directly bound to purified MRP1. The two isomers induced specific conformational changes in MRP1, likely correlating with their different effects on MRP1 activity. Materials—Reduced GSH, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5,5′-dithiobis(2-nitrobenzoic acid), β-NADPH, calcein acetoxymethyl ester (AM), nucleotides, MgCl2, dithiothreitol, verapamil enantiomers, penicillin/streptomycin, mammalian protease inhibitor mixture, asolectin, and fetal bovine serum were obtained from Sigma (Saint Quentin Fallavier, France). Triton X-100 was from Merck (Darmstadt, Germany); CHAPS was from Euromedex (Souffelweyersheim, France); and methotrexate was from Rhône-Poulenc (Montrouge, France). ATP and glutathione reductase (EC 1.8.1.7) was from Roche Applied Science (Meylan, France). Bradford reagent was from Pierce (Perbio Science France SA). Cell Lines—BHK-21 cells were stably transfected with wild-type MRP1 as described previously (16Chang X.-B. Kim Y.-X. Riordan J.R. J. Biol. Chem. 1997; 272: 30962-30968Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). No MRP1 expression was detected in control BHK-21 cells by Western blot analysis (16Chang X.-B. Kim Y.-X. Riordan J.R. J. Biol. Chem. 1997; 272: 30962-30968Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Cells were grown at 37 °C and 5% CO2 in culture medium containing 1% (v/v) penicillin/streptomycin, 5% (v/v) fetal bovine serum, and 200 μm methotrexate for transfected cells. Cell Proliferation as Determined by MTT Assay—The MTT colorimetric assay was used to assess the sensitivity of control and MRP1-transfected BHK-21 (MRP1-BHK-21) cells to verapamil enantiomers. Briefly, growth inhibition (IC50) assays were performed by plating cells at a density of 1.0 × 104 cells/well in 96-well plates. Cells were cultured for 17 h before the addition of verapamil enantiomers diluted in complete culture medium in the presence of 0.5% (v/v) Me2SO. The cells were then incubated for 72 h at 37 °C and 5% CO2. Surviving cells were quantified by the MTT colorimetric assay as described previously (17Mosmann T. J. Immunol. Methods. 1983; 65: 55-63Crossref PubMed Scopus (46262) Google Scholar). The IC50 values were derived from a typical MTT assay. were performed in and on different were from of a typical the of verapamil enantiomers on the transport of calcein by MRP1, cells were using by at × and in culture medium with μm was to × cells in of culture medium with to cell containing μm or verapamil enantiomers. The cells were incubated at 37 °C in the for and then and in of phosphate-buffered containing bovine serum fluorescence was using an of a cellular glutathione content was the as described Biochem. PubMed Scopus Google Scholar) and PubMed Scopus Google Scholar). Cells were in 96-well at a density of 1.0 × cells/well and cultured for 17 h before the addition of verapamil enantiomers, with the not 0.5% various cells were with 200 of and then with of with (v/v) Triton The was and two of and were to new for GSH and protein GSH was performed by of containing μm μm 5,5′-dithiobis(2-nitrobenzoic acid), and GSH The was then in a and the was at for The was for and the GSH was by with glutathione was performed by the Bradford assay to the The glutathione were as of and the results were from The was on different LTC4 LTC4 transport was by a Kim D. J. Biochem. 1994; PubMed Scopus Google Scholar, D.W. Kim Deeley R.G. Cole S.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) as described Kim L. Riordan J.R. Chang J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar) in the or presence of GSH, racemic or at and were performed in in the presence of or ATP at The of LTC4 bound to the in the presence of was considered as the and from the in the presence of MRP1 was performed to Chang et al. (16Chang X.-B. Kim Y.-X. Riordan J.R. J. Biol. Chem. 1997; 272: 30962-30968Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). BHK-21 cells MRP1 with a were with sodium for 17 h to MRP1 Cells were then with PBS, with and by at × The was in containing and and with a protease inhibitor were by cells with a France). were by at × for The was then at × for to the The was in A CHAPS, (v/v) and to a protein of The were for at °C and the was at × for The was then with with and for h at The was with of A containing CHAPS and and then with of A containing CHAPS, (v/v) and The protein was with of A containing and (v/v) The was in CHAPS, (v/v) and (v/v) was then The protein was by the Bradford assay. MRP1 was used than h and was to (v/v) on a and with Assay—The of purified MRP1 was using a colorimetric assay Kim Biophys. PubMed Scopus Google Scholar). Briefly, of purified MRP1 was incubated at 37 °C for h in of assay containing dithiothreitol, MgCl2, and ATP in the presence or of GSH or μm verapamil enantiomers. were performed in and for the of the of MRP1 by Verapamil were with a at °C using a and were for to and effects were on were performed using μm purified MRP1 in of with an of and an of The studies were performed and of were performed with the of to of tryptophan accessibility to sodium iodide from to were the as used for the of The was with and of the MRP1 fluorescence was were using the and are the fluorescence in the and presence of the is the and is the The accessibility was obtained from the is the and and is the accessibility the were performed three of Verapamil in verapamil was shown previously to cells by apoptosis through glutathione by MRP1 (13Trompier D. Kim X.-B. Barattin R. du Moulinet D'Hardemare A. Di Pietro A. Baubichon-Cortay H. Cancer Res. 2004; 64: 4950-4956Crossref PubMed Scopus (107) Google Scholar). In this study, we investigated the individual effects of by with the racemic The cellular effects were on both control and cells In with results (13Trompier D. Kim X.-B. Barattin R. du Moulinet D'Hardemare A. Di Pietro A. Baubichon-Cortay H. Cancer Res. 2004; 64: 4950-4956Crossref PubMed Scopus (107) Google Scholar), racemic verapamil was for cells at which were ineffective in control BHK-21 cells. The R- and S-enantiomers were (R)-verapamil not for the two of whereas a than that observed with the racemic at the of Verapamil on the GSH the of GSH the addition of racemic verapamil or its enantiomers to the cell an and decrease in the GSH content in the presence of racemic verapamil, as reported previously (13Trompier D. Kim X.-B. Barattin R. du Moulinet D'Hardemare A. Di Pietro A. Baubichon-Cortay H. Cancer Res. 2004; 64: 4950-4956Crossref PubMed Scopus (107) Google Scholar). the on the GSH whereas the induced a decrease in the GSH in with the shown in In addition, μm a stronger on the GSH content with μm racemic verapamil, which μm that the might the modulation of MRP1 activity. this we investigated the of the on the of the GSH decrease induced by the analysis showed an inhibition in the with by and of MRP1-mediated inhibition that both enantiomers were able to interact with MRP1. investigated their effects on the transport of MRP1 by using as a The control BHK-21 cells of calcein of verapamil was or not In contrast, both S-enantiomers induced an in calcein in MRP1-overexpressing cells, to the inhibitor that the can efficiently the of MRP1. of LTC4 by and in the of racemic LTC4 transport in a in the presence of GSH alone the transport of in with Loe et al. D.W. Kim R.G. Cole S.P. J. Pharmacol. Exp. Ther. 2000; 293: 530-538PubMed Google In with the results obtained with the racemic mixture, individual could not efficiently LTC4 transport in the of GSH the addition of GSH, (R)-verapamil LTC4 whereas strongly with the racemic These results are with derived from and on calcein of Resistance by (R)-verapamil MRP1 transport but showed for BHK-21 or cells we that (R)-verapamil might cells to anticancer this the IC50 values of vincristine for BHK-21 and cells were in the or presence of or μm (R)-verapamil on the IC50 values of vincristine for control BHK-21 cells, whereas values for cells from to in the presence of μm and in the presence of μm μm to complete reversion of the resistance to of the MDR phenotype by (R)-verapamil in cells BHK-21 and cells were with of vincristine in the or presence of or μm 72 h of the cells were by the MTT assay. IC50 values were in using for μm μm μm in a new Molecular of the Verapamil the of the two enantiomers with the we purified the MRP1 protein by a from Chang et al. (16Chang X.-B. Kim Y.-X. Riordan J.R. J. Biol. Chem. 1997; 272: 30962-30968Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) in the presence of that the purified MRP1 protein was detected on with The purified MRP1 protein was confirmed on a Western blot with the of MRP1 tryptophan fluorescence was to the of the or the indicating that both enantiomers bound to MRP1. These results suggested that the inhibition of MRP1 transport by the enantiomers in from a with the The values for the R- and were and indicating that the two isomers bound to purified MRP1. The addition of GSH prevented the and from efficiently for the S-isomer than for the R-isomer The binding of GSH could not be of the of on tryptophan Furthermore, the of MRP1 tryptophan accessibility to sodium iodide in the presence of the and was different in the of the tryptophan to be to sodium iodide as shown by a with In the presence of or the were indicating that the tryptophan were not to sodium iodide and as from the In both cases, the binding of the isomers to distinct conformational changes in the the obtained in the presence of was whereas was in the presence of This likely with the differential effects on MRP1 as described was by the of MRP1. The was by or (R)-verapamil GSH MRP1 in with the GSH stimulation reported by et al. Kim W. J. FEBS Lett. 2000; PubMed Scopus Google Scholar) and et al. L. Kim X.-B. Y.-X. A. Riordan J.R. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The S-isomer the GSH whereas the R-isomer effect. In this work, we have new of verapamil enantiomers, one of which two different effects on MRP1. These new the mechanism of this and could be of great interest in reversion of multidrug Although verapamil is a well inhibitor of the MDR phenotype (8Naito M. Kim T. Cancer Res. 1989; 49: 1452-1455PubMed Google Scholar), the results are for MRP1. although Cole et al. (9Cole S.P. Kim K.E. Fraser K. Loe D.W. Grant C.E. Wilson G.M. Deeley R.G. Cancer Res. 1994; 54: 5902-5910PubMed Google Scholar) not reversion of Davey et al. R.A. Kim T.J. Davey M.W. Belov L. Harvie R.M. Hancox D. Wheeler H. Leuk. Res. 1995; 19: 275-282Crossref PubMed Scopus (64) Google Scholar) and et al. (10Cullen K.V. Kim R.A. Davey M.W. Biochem. Pharmacol. 2001; 62: 417-424Crossref PubMed Scopus (46) Google Scholar) described a to However, verapamil has been to stimulate GSH transport (10Cullen K.V. Kim R.A. Davey M.W. Biochem. Pharmacol. 2001; 62: 417-424Crossref PubMed Scopus (46) Google Scholar, 12Loe D.W. Kim R.G. Cole S.P. J. Pharmacol. Exp. Ther. 2000; 293: 530-538PubMed Google Scholar, K.V. Kim R.A. Davey M.W. Biochem. Pharmacol. PubMed Scopus Google Scholar) and to strongly LTC4 transport by MRP1 in the presence of GSH D.W. Kim R.G. Cole S.P. J. Pharmacol. Exp. Ther. 2000; 293: 530-538PubMed Google Scholar, 13Trompier D. Kim X.-B. Barattin R. du Moulinet D'Hardemare A. Di Pietro A. Baubichon-Cortay H. Cancer Res. 2004; 64: 4950-4956Crossref PubMed Scopus (107) Google Scholar). This the complex mechanism of verapamil as suggested previously Kim R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The inhibition of calcein transport observed with cells with results an in induced by verapamil in MRP1-overexpressing cells (10Cullen K.V. Kim R.A. Davey M.W. Biochem. Pharmacol. 2001; 62: 417-424Crossref PubMed Scopus (46) Google Scholar). new could with cell racemic verapamil stimulation of GSH transport by MRP1 to apoptosis of MRP1-overexpressing cells (13Trompier D. Kim X.-B. Barattin R. du Moulinet D'Hardemare A. Di Pietro A. Baubichon-Cortay H. Cancer Res. 2004; 64: 4950-4956Crossref PubMed Scopus (107) Google Scholar). This cell death was shown to be induced by the In the racemic mixture, the the specific inhibition of the of the GSH decrease showed a inhibition the two The transport as efficiently as and an of MRP1-transfected cell growth to vincristine at the used in this (S)-Verapamil to calcein strongly with the R-isomer, by MRP1 from substrate transport to the of GSH transport. The S-isomer LTC4 transport and strongly with the R-isomer this is the that two enantiomers of an MDR phenotype have been described to display different effects. for is in the effects of verapamil on transport Kim J. Sci. Full Text PDF PubMed Scopus Google Scholar) or of compounds on drug efflux from MDR cells D. Kim J. Res. Google Scholar), whereas but not inhibits transport Kim J. Sci. Full Text PDF PubMed Scopus Google Scholar). In contrast, for MRP1, we the of chiral verapamil, i.e. the of the S-isomer and the of the are both potentially for MDR phenotype the to potentially resistant tumor cells (13Trompier D. Kim X.-B. Barattin R. du Moulinet D'Hardemare A. Di Pietro A. Baubichon-Cortay H. Cancer Res. 2004; 64: 4950-4956Crossref PubMed Scopus (107) Google Scholar) and the to the sensitivity of tumor cells to chemotherapy. the enantiomers to the MRP1 protein with high and the bound verapamil is not transported by MRP1 D.W. Kim R.G. Cole S.P. J. Pharmacol. Exp. Ther. 2000; 293: 530-538PubMed Google Scholar), we that of have a long-lasting the of high in However, the two isomers to be used in not in a mixture, we have shown that showed previously that of verapamil to a in the of racemic verapamil in the for control cells (13Trompier D. Kim X.-B. Barattin R. du Moulinet D'Hardemare A. Di Pietro A. Baubichon-Cortay H. Cancer Res. 2004; 64: 4950-4956Crossref PubMed Scopus (107) Google Scholar). Furthermore, has been on the of the molecule, to be for Kim P. R. A. A. D. J. Med. Chem. PubMed Scopus Google Scholar). Therefore, the of the R- and S-enantiomers of verapamil is in to decrease the of the S-isomer, to their effects. verapamil which the MDR phenotype induced by were found by et al. Kim A. Biochem. Pharmacol. 1995; PubMed Scopus Google Scholar). of be performed on to of the Although GSH has been shown to the binding of the R- and we of verapamil enantiomers on the for GSH GSH binding be directly by tryptophan fluorescence However, et al. J. Kim R. J. P. 2003; PubMed Scopus Google Scholar) showed that a GSH is in the presence of verapamil. or GSH alone the of purified MRP1 that binding of individual to MRP1 induced a conformational in the GSH the to the as GSH alone In contrast, GSH the of GSH In the presence of stimulation of was whereas GSH transport. can that the of not with activity. results that the S-isomer, but not the R-isomer, could with the GSH directly or through induced conformational This could have an on the GSH stimulation of activity. of this at the to the transport mechanism of MRP1. showed previously that cellular GSH efflux to apoptosis of MRP1-overexpressing BHK-21 cells (13Trompier D. Kim X.-B. Barattin R. du Moulinet D'Hardemare A. Di Pietro A. Baubichon-Cortay H. Cancer Res. 2004; 64: 4950-4956Crossref PubMed Scopus (107) Google Scholar). A was described in and cells Kim J. Biochem. Pharmacol. PubMed Scopus Google Scholar), and et al. Kim R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that glutathione apoptosis multidrug-resistant However, studies reported a GSH but cell death was not observed (10Cullen K.V. Kim R.A. Davey M.W. Biochem. Pharmacol. 2001; 62: 417-424Crossref PubMed Scopus (46) Google Scholar, 12Loe D.W. Kim R.G. Cole S.P. J. Pharmacol. Exp. Ther. 2000; 293: 530-538PubMed Google Scholar, K.V. Kim R.A. Davey M.W. Biochem. Pharmacol. PubMed Scopus Google Scholar). The for verapamil is results could be of great potential interest in The verapamil enantiomers different effects on MRP1 activity. (S)-Verapamil as a by of MRP1-mediated GSH to the death of potentially resistant tumor cells. In contrast, (R)-verapamil as an inhibitor to MRP1-mediated in of MRP1-overexpressing cells to anticancer In we have to the two enantiomers to the of verapamil on cells MRP1. The of such a major in the of different GSH transport and drug resistance Furthermore, new to be in obtained using verapamil or its for and for the