Abstract. The human breast cancer resistance protein (BCRP, gene symbol ABCG2) is an ATP-binding cassette (ABC) efflux transporter. It was so named because it was initially cloned from a multidrugresistant breast cancer cell line where it was found to confer resistance to chemotherapeutic agents such as mitoxantrone and topotecan. Since its discovery in 1998, the substrates of BCRP have been rapidly expanding to include not only therapeutic agents but also physiological substances such as estrone-3-sulfate, 17β-estradiol 17-(β-D-glucuronide) and uric acid. Likewise, at least hundreds of BCRP inhibitors have been identified. Among normal human tissues, BCRP is highly expressed on the apical membranes of the placental syncytiotrophoblasts, the intestinal epithelium, the liver hepatocytes, the endothelial cells of brain microvessels, and the renal proximal tubular cells, contributing to the absorption, distribution, and elimination of drugs and endogenous compounds as well as tissue protection against xenobiotic exposure. As a result, BCRP has now been recognized by the FDA to be one of the key drug transporters involved in clinically relevant drug disposition. We published a highly-accessed review article on BCRP in 2005, and much progress has been made since then. In this review, we provide an update of current knowledge on basic biochemistry and pharmacological functions of BCRP as well as its relevance to drug resistance and drug disposition.

The 72-kDa breast cancer resistance protein (BCRP) is the second member of the subfamily G of the human ATP binding cassette (ABC) transporter superfamily and thus also designated as ABCG2. Unlike P-glycoprotein and MRP1, which are arranged in 2 repeated halves, BCRP is a half-transporter consisting of only 1 nucleotide binding domain followed by 1 membrane-spanning domain. Current experimental evidence suggests that BCRP may function as a homodimer or homotetramer. Overexpression of BCRP is associated with high levels of resistance to a variety of anticancer agents, including anthracyclines, mitoxantrone, and the camptothecins, by enhancing drug efflux. BCRP expression has been detected in a large number of hematological malignancies and solid tumors, indicating that this transporter may play an important role in clinical drug resistance of cancers. In addition to its role to confer resistance against chemotherapeutic agents, BCRP actively transports structurally diverse organic molecules, conjugated or unconjugated, such as estrone-3-sulfate, 17beta-estradiol 17-(beta-D-glucuronide), and methotrexate. BCRP is highly expressed in the placental syncytiotrophoblasts, in the apical membrane of the epithelium in the small intestine, in the liver canalicular membrane, and at the luminal surface of the endothelial cells of human brain microvessels. This strategic and substantial tissue localization indicates that BCRP also plays an important role in absorption, distribution, and elimination of drugs that are BCRP substrates. This review summarizes current knowledge of BCRP and its relevance to multidrug resistance and drug disposition.

The 190-kDa phosphoglycoprotein multidrug resistance protein 1 (MRP1) (ABCC1) confers resistance to a broad spectrum of anticancer drugs and also actively transports certain xenobiotics with reduced glutathione (GSH) (cotransport) as well as conjugated organic anions such as leukotriene C(4) (LTC(4)). In the present study, we have investigated a series of bioflavonoids for their ability to influence different aspects of MRP1 function. Most flavonoids inhibited MRP1-mediated LTC(4) transport in membrane vesicles and inhibition by several flavonoids was enhanced by GSH. Five of the flavonoids were competitive inhibitors of LTC(4) transport (K(i), 2.4-21 microM) in the following rank order of potency: kaempferol > apigenin (+ GSH) > quercetin > myricetin > naringenin (+ GSH). These flavonoids were less effective inhibitors of 17beta-estradiol 17beta-(D-glucuronide) transport. Moreover, their rank order of inhibitory potency for this substrate differed from that for LTC(4) transport inhibition but correlated with their relative lipophilicity. Several flavonoids, especially naringenin and apigenin, markedly stimulated GSH transport by MRP1, suggesting they may be cotransported with this tripeptide. Quercetin inhibited the ATPase activity of purified reconstituted MRP1 but stimulated vanadate-induced trapping of 8-azido-alpha-[(32)P]ADP by MRP1. In contrast, kaempferol and naringenin stimulated both MRP1 ATPase activity and trapping of ADP. In intact MRP1-overexpressing cells, quercetin reduced vincristine resistance from 8.9- to 2.2-fold, whereas kaempferol and naringenin had no effect. We conclude that dietary flavonoids may modulate the organic anion and GSH transport, ATPase, and/or drug resistance-conferring properties of MRP1. However, the activity profile of the flavonoids tested differed from one another, suggesting that at least some of these compounds may interact with different sites on the MRP1 molecule.

The human breast cancer resistance protein (BCRP/ABCG2) is the second member of the G subfamily of the large ATP-binding cassette (ABC) transporter superfamily. BCRP was initially discovered in multidrug resistant breast cancer cell lines where it confers resistance to chemotherapeutic agents such as mitoxantrone, topotecan and methotrexate by extruding these compounds out of the cell. BCRP is capable of transporting non-chemotherapy drugs and xenobiotiocs as well, including nitrofurantoin, prazosin, glyburide, and 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine. BCRP is frequently detected at high levels in stem cells, likely providing xenobiotic protection. BCRP is also highly expressed in normal human tissues including the small intestine, liver, brain endothelium, and placenta. Therefore, BCRP has been increasingly recognized for its important role in the absorption, elimination, and tissue distribution of drugs and xenobiotics. At present, little is known about the transport mechanism of BCRP, particularly how it recognizes and transports a large number of structurally and chemically unrelated drugs and xenobiotics. Here, we review current knowledge of structure and function of this medically important ABC efflux drug transporter.

Breast cancer resistance protein (BCRP) is a recently discovered ATP-binding cassette drug transporter. Hence, the full spectrum of therapeutic agents that interact with BCRP remains to be elucidated. Because human immunodeficiency virus protease inhibitors (HPIs) are well known P-glycoprotein (P-gp) substrates, and there is an overlap in substrate specificity between P-gp and BCRP, this study was performed to investigate whether HPIs are substrates and/or inhibitors of BCRP. First, the effect of HPIs on BCRP efflux activity in human embryonic kidney (HEK) cells stably expressing wild-type BCRP (482R) and its two mutants (482T and 482G) was studied by measuring intracellular mitoxantrone fluorescence using flow cytometry. We found that ritonavir, saquinavir, and nelfinavir were effective inhibitors of wild-type BCRP (482R) with IC 50 values of 19.5 Ϯ 0.8 M, 19.5 Ϯ 7.6 M, and 12.5 Ϯ 4.1 M, respectively. Ritonavir, saquinavir, and nelfinavir inhibited 482T and 482G with IC 50 values that were approximately 2 times greater than that for 482R. Indinavir and amprenavir had no significant inhibition on BCRP activity. Direct efflux of radiolabeled HPIs in HEK cells was measured to determine whether the HPIs are substrates of BCRP. None of the HPIs were found to be transported by BCRP. Together, ritonavir, saquinavir, nelfinavir, indinavir, and amprenavir are not substrates for BCRP. However, ritonavir, saquinavir, and nelfinavir are effective inhibitors of the transporter. These results suggest that BCRP may play an important role in drug-drug interactions involving coadministration of the HPIs with drugs that are substrates of the transporter.

The breast cancer resistance protein (BCRP) is abundant in the placenta and protects the fetus by limiting placental drug penetration. We hypothesize that pregnancy-specific hormones regulate BCRP expression. Hence, we examined the effects of progesterone (P 4) and 17␤-estradiol (E2) on BCRP expression in the human placental BeWo cells. P 4 and E2 significantly increased and decreased BCRP protein and mRNA, respectively. Likewise, treatment with P 4 and E2 increased and decreased, respectively, fumitremorgin C-inhibitable mitoxantrone efflux activity of BeWo cells. Reduction in BCRP expression by E 2 was abrogated by the estrogen receptor (ER) antagonist ICI-182,780. However, the progesterone receptor (PR) antagonist RU-486 had no effect on P 4-mediated induction of BCRP. P4 together with E2 further increased BCRP protein and mRNA compared with P 4 treatment alone. This combined effect on BCRP expression was abolished by RU-486, ICI-182,780, or both. Further analysis revealed that E 2 significantly decreased ER␤ mRNA and strongly induced PRB mRNA in a dose-dependent manner but had no effect on PR A and ER␣. P4 alone had no significant effect on mRNA of ER␣, ER␤, PRA, and PR B. E2 in combination with P4 increased PRB mRNA, but the level of induction was significantly reduced compared with E 2 treatment alone. Taken together, these results indicate that E 2 by itself likely downregulates BCRP expression through an ER, possibly ER␤. P 4 alone upregulates BCRP expression via a mechanism other than PR. P 4 in combination with E2 further increases BCRP expression, presumably via a nonclassical PR-and/or E 2-mediated synthesis of PRB. hormonal regulation; ATP-binding cassette transporter; pregnancy THE BREAST CANCER RESISTANCE PROTEIN (BCRP) is the second member (gene symbol ABCG2) of the subfamily G of the large ATP-binding cassette (ABC) transporter superfamily (1, 9, 25). BCRP is highly expressed in many normal tissues, including the epithelium of the small intestine and the liver canalicular membrane (22). Therefore, in addition to conferring resistance in cancer cells to chemotherapeutic agents such as mitoxantrone (MX), topotecan, and methotrexate (8,9,25,36), BCRP has been shown to mediate apically-directed drug transport and play a significant role in absorption, distribution, and elimination of BCRP substrates (4,19,21,32,35). Of interest is that BCRP is also abundantly expressed in the apical membrane of placental syncytiotrophoblasts (22). Whereas the precise physiological role of BCRP in the placenta is still unclear, existing data suggest that BCRP may protect the fetus against toxic substances/drugs and metabolites by extruding them across the placental barrier. For example, Bcrp1, the murine homolog of BCRP, has been shown to significantly alter fetal distribution of topotecan, a BCRP substrate. The fetus/plasma ratio of topotecan was increased twofold in pregnant mice treated with the BCRP inhibitor GF-120918 compared with the vehicle-treatment control (19).Distribution of drugs that are BCRP substrate...

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-binding cassette (ABC) polytopic membrane transporter of considerable clinical importance that confers multidrug resistance on tumor cells by reducing drug accumulation by active efflux. MRP1 is also an efficient transporter of conjugated organic anions. Like other ABC proteins, including the drug resistance conferring 170-kDa P-glycoprotein (ABCB1), the 190-kDa MRP1 has a core structure consisting of two membrane-spanning domains (MSDs), each followed by a nucleotide binding domain (NBD). However, unlike P-glycoprotein and most other ABC superfamily members, MRP1 contains a third MSD with five predicted transmembrane segments with an extracytosolic NH 2 terminus. Moreover, the two nucleotide-binding domains of MRP1 are considerably more divergent than those of P-glycoprotein. In the present study, the first structural details of MRP1 purified from drug-resistant lung cancer cells have been obtained by electron microscopy of negatively stained single particles and two-dimensional crystals formed after reconstitution of purified protein with lipids. The crystals display p2 symmetry with a single dimer of MRP1 in the unit cell. The overall dimensions of the MRP1 monomer are ϳ80 ؋ 100 Å. The MRP1 monomer shows some pseudo-2-fold symmetry in projection, and in some orientations of the detergent-solubilized particles, displays a stain filled depression (putative pore) appearing toward the center of the molecule, presumably to enable transport of substrates. These data represent the first structural information of this transporter to ϳ22-Å resolution and provide direct structural evidence for a dimeric association of the transporter in a reconstituted lipid bilayer.The 190-kDa multidrug resistance protein MRP1 1 (ABCC1) is a polytopic membrane transport protein that belongs to the ATP-binding cassette (ABC) superfamily and has been detected in many different drug-resistant cell lines and tumor tissues since it was first cloned in 1992 (1-6). When overexpressed in tumor cells, MRP1 confers multidrug resistance by reducing intracellular drug concentrations in an ATP-dependent manner (6 -8). In this respect, MRP1 is similar to another ABC transporter, the well characterized 170-kDa P-glycoprotein (Pgp) (ABCB1) (9, 10). ABC proteins play important physiological and protective functions in bacteria, yeast, plants, and mammals and are capable of transporting a wide variety of molecules across biological membranes. Known substrates for ABC transporters include ions, phospholipids, steroids, polysaccharides, amino acids, peptides, and in the case of several MRPrelated proteins, anionic conjugated endo-and xenobiotics (6, 10 -12). In addition to MRP1 and P-gp, other examples of clinically important human ABC proteins include the cystic fibrosis transmembrane conductance regulator CFTR (13), and the sulfonylurea receptor (SUR), which is part of an ATPsensitive potassium channel involved in insulin secretion (14).The amino acid sequence of MRP1 predicts that it contains a cor...

The ATP-binding cassette transporter protein, multidrug resistance protein MRP1, was purified from doxorubicin-selected H69AR lung tumor cells which express high levels of this protein. A purification procedure comprised of a differential two-step solubilization of MRP1 from plasma membranes with 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate followed by immunoaffinity chromatography using the MRP1-specific monoclonal antibody QCRL-1 was developed. Approximately 300 microgram of MRP1 was obtained from 6 mg of plasma membranes at 80-90% purity, as indicated by silver staining of protein gels. After reconstitution of purified MRP1 into proteoliposomes, kinetic analyses indicated that its K(m) for ATP hydrolysis was 104+/-22 microM with maximal activity of 5-10 nmol min(-1) mg(-1) MRP1. MRP1 ATPase activity was further characterized with various inhibitors and exhibited an inhibition profile that distinguishes it from P-glycoprotein and other ATPases. The ATPase activity of reconstituted MRP1 was stimulated by the conjugated organic anion substrates leukotriene C(4) (LTC(4)) and 17beta-estradiol 17-(beta-D-glucuronide) with 50% maximal stimulation achieved at concentrations of 150 nM and 1.6 microM, respectively. MRP1 ATPase was also stimulated by glutathione disulfide but not by reduced glutathione or unconjugated chemotherapeutic agents. This purification and reconstitution procedure is the first to be described in which the ATPase activity of the reconstituted MRP1 retains kinetic characteristics with respect to ATP-dependence and substrate stimulation that are very similar to those deduced from transport studies using MRP1-enriched plasma membrane vesicles.