Breast Cancer Resistance Protein Protein Research Articles

Pharmacogenomics of drug resistance in Breast Cancer Resistance Protein (BCRP) and its mutated variants

AimDrugs in breast cancer treatment suffer resistance and drug efflux from ATP-binding cassette (ABC) efflux transporter protein. Drugs inhibiting BCRP suffer activity alteration due to sequence variants. It is imperative to investigate role of mutant variants using structure based aspects of drug binding. MethodIn present work, we included single nucleotide polymorphisms like F208S, S248P and F431L in BCRP structure and evaluated their role in alteration of drug binding affinities using computational approaches. Comparative modeling of BCRP 3D structure was achieved using various tools available followed by structure validation. Mutagenesis and its impact by SNPs was attained in 3D structure of BCRP. A set of selected and established BCRP inhibitors were further docked into binding site to record the drug resistance in mutant variants. ResultsNucleotide binding (NB) domain (258 AA) and transmembrane (TM) domain (291 AA) of BCRP were modeled separately and assembled together to generate a single structure. Ramachandran Plot confirmed quality of modeled structures along with main chain and side chain parameters. Mutagenesis included three main variants (F208S, S248P and F431L) using Triton program. Molecular docking results showed inhibitor CID_25223199 binding effectively to wild and F431L mutant structure of BCRP while inhibitors CID_25223002 to F208S and CID_119373 to S248P. ConclusionDistortion in spatial arrangement of amino acids in BCRP protein due to mutations led to low efficacy in drug response with respect to wild isoform. Results of present work demand to probe pharmacogenomic aspects in drug development efforts for breast cancer.

High Doses of Ursodeoxycholic Acid Up-Regulate the Expression of Placental Breast Cancer Resistance Protein in Patients Affected by Intrahepatic Cholestasis of Pregnancy

BackgroundUrsodeoxycholic acid (UDCA) administration in intrahepatic cholestasis of pregnancy (ICP) induces bile acids (BA) efflux from the foetal compartment, but the molecular basis of this transplacental transport is only partially defined.AimTo determine if placental breast cancer resistance protein (BCRP), able to transport BA, is regulated by UDCA in ICP.Methods32 pregnant women with ICP (14 untreated, 34.9±5.17 years; 18 treated with UDCA - 25 mg/Kg/day, 32.7±4.62 years,) and 12 healthy controls (33.4±3.32 years) agreed to participate in the study. Placentas were obtained at delivery and processed for membrane extraction. BCRP protein expression was evaluated by immunoblotting techniques and chemiluminescence quantified with a luminograph measuring emitted photons; mRNA expression with real time PCR. Statistical differences between groups were evaluated by ANOVA with Dunn’s Multiple Comparison test.ResultsBCRP was expressed only on the apical membrane of the syncytiotrophoblast. A significant difference was observed among the three groups both for mRNA (ANOVA, p = 0.0074) and protein (ANOVA, p<0.0001) expression. BCRP expression was similar in controls and in the untreated ICP group. UDCA induced a significant increase in placental BCRP mRNA and protein expression compared to controls (350.7±106.3 vs 100±18.68% of controls, p<0.05 and 397.8±56.02 vs 100±11.44% of controls, p<0.001, respectively) and untreated ICP (90.29±17.59% of controls, p<0.05 and 155.0±13.87%, p<0.01).ConclusionOur results confirm that BCRP is expressed only on the apical membrane of the syncytiotrophoblast and show that ICP treatment with high dose UDCA significantly upregulates placental BCRP expression favouring BA efflux from the foetal compartment.

Radioligands targeting P‐glycoprotein and other drug efflux proteins at the blood–brain barrier

Brain penetration of radiopharmaceuticals or therapeutic drugs may be restricted by adenosine triphosphate-binding cassette (ABC) transporters, such as P-glycoprotein (Pgp), breast cancer resistance protein (BCRP), or the multidrug resistance-associated proteins. These transporters are expressed in the luminal membrane of brain capillary endothelial cells forming the blood-brain barrier (BBB), where they actively efflux a wide range of chemically unrelated compounds from the brain back into the blood. Most efforts to visualize ABC transporters at the BBB with positron emission tomography have concentrated on Pgp. Pgp imaging probes can be classified as radiolabeled substrates or inhibitors. The radiolabeled substrates (R)-[(11) C]verapamil and [(11) C]-N-desmethyl-loperamide have been successfully used to assess Pgp function at the BBB of animals and humans. Radiolabeled Pgp inhibitors, such as [(11) C]tariquidar, [(11) C]elacridar, or [(11) C]laniquidar, were developed to measure Pgp expression levels at the BBB, which has so far remained unsuccessful as these probes were unexpectedly recognized at tracer concentrations by Pgp and BCRP as substrates resulting in low brain uptake. Studies on positron emission tomography tracers for other ABC transporters than Pgp (BCRP and multidrug resistance-associated proteins) are still in their infancy. It is hoped that the experience gained with the imaging of Pgp will be successfully translated to the development of radiotracers to visualize other ABC transporters.

Differential regulation of drug transporter expression by all-trans retinoic acid in hepatoma HepaRG cells and human hepatocytes

All-trans retinoic acid (atRA) is the active form of vitamin A, known to activate retinoid receptors, especially the heterodimer retinoid X receptor (RXR):retinoic acid receptor (RAR) that otherwise may play a role in regulation of some drug transporters. The present study was designed to characterize the nature of human hepatic transporters that may be targeted by atRA and the heterodimer RXR:RAR. Exposure of human hepatoma HepaRG cells and primary human hepatocytes to 5μM atRA down-regulated mRNA levels of various sinusoidal solute carrier (SLC) influx transporters, including organic anion transporting polypeptide (OATP) 2B1, OATP1B1, organic cation transporter (OCT) 1 and organic anion transporter (OAT) 2, and induced those of the canalicular breast cancer resistance protein (BCRP). The retinoid concomitantly reduced protein expression of OATP2B1 and OATP1B1 and activity of OATPs and OCT1 and induced BCRP protein expression in HepaRG cells. Some transporters such as OATP1B3 and the bile salt export pump (BSEP) were however down-regulated by atRA in primary human hepatocytes, but induced in HepaRG cells, thus pointing out discrepancies between these two liver cell models in terms of detoxifying protein regulation. atRA-mediated repressions of OATP2B1, OATP1B1, OAT2 and OCT1 mRNA expression were finally shown to be counteracted by knocking-down expression of RARα and RXRα through siRNA transfection in HepaRG cells. atRA thus differentially regulated human hepatic drug transporters, mainly in a RXR:RAR-dependent manner, therefore establishing retinoids and retinoid receptors as modulators of liver drug transporter expression.

Progesterone Negatively Regulates BCRP in Progesterone Receptor-Positive Human Breast Cancer Cells

Background/Aims: Breast cancer resistance protein (BCRP) plays a crucial role in multidrug resistance (MDR). Previous studies have shown that steroid hormones, like progesterone (PROG), regulate BCRP expression. The presence of a progesterone response element (PRE) in the BCRP promoter, suggests that PROG may regulate transcription of BCRP. Methods: To investigate the role of PROG in the regulation of BCRP expression, two constructs encoding full-length BCRP driven by either an endogenous PRE promoter or a constitutive CMV promoter, were transfected into T47D cells that express the progesterone receptor (PR) or into PR-negative MDA-MB-231 cells. Results: After treatment with PROG, qPCR and Western blotting analyses indicated that BCRP mRNA and BCRP protein levels were significantly reduced in a dose-dependent manner in PR-positive cells, but PROG had no significant effect on BCRP levels in the PR-negative cells. The effect observed in PR-positive cells was reversed by co-treatment with RU-486, a specific PROG inhibitor. Cytometric analysis confirmed that BCRP-mediated drug efflux was inhibited and chemosensitivity to mitoxantrone was markedly increased by PROG treatment. Conclusion: These results suggest that PROG reverses BCRP-mediated MDR by down-regulating BCRP expression in breast cancer cells by affecting transcription from the PRE-containing BCRP promoter. Our studies suggest that breast cancer patients with BCRP-mediated MDR may be successfully treated with PROG.

Susceptibility of juvenile and adult blood–brain barrier to endothelin-1: regulation of P-glycoprotein and breast cancer resistance protein expression and transport activity

BackgroundP-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) play a critical role in keeping neurotoxic substances from entering the brain. We and others have previously reported an impact of inflammation on the regulation of adult blood–brain barrier (BBB) efflux transporters. However, studies in children have not been done. From the pediatric clinical perspective, it is important to understand how the central nervous system (CNS) and BBB drug efflux transporters differ in childhood from those of adults under normal and inflammatory conditions. Therefore, we examined and compared the regulation of P-gp and BCRP expression and transport activity in young and adult BBB and investigated the molecular mechanisms underlying inflammatory responses.MethodsRats at postnatal day (P) P21 and P84, corresponding to the juvenile and adult stages of human brain maturation, respectively, were treated with endothelin-1 (ET-1) given by the intracerebroventricular (icv) route. Twenty-four hours later, we measured P-gp and BCRP protein expression in isolated brain capillary by immunoblotting as well as by transport activity in vivo by measuring the unbound drug partitioning coefficient of the brain (Kp,uu,brain) of known efflux transporter substrates administered intravenously. Glial activation was measured by immunohistochemistry. The release of cytokines/chemokines (interleukins-1α, 1-β (IL-1β), -6 (IL-6), -10 (IL-10), monocyte chemoattractant protein (MCP-1/CCL2), fractalkine and tissue inhibitor of metalloproteinases-1 (TIMP-1)) were simultaneously measured in brain and serum samples using the Agilent Technology cytokine microarray.ResultsWe found that juvenile and adult BBBs exhibited similar P-gp and BCRP transport activities in the normal physiological conditions. However, long-term exposure of the juvenile brain to low-dose of ET-1 did not change BBB P-gp transport activity but tended to decrease BCRP transport activity in the juvenile brain, while a significant increase of the activity of both transporters was evidenced at the BBB in the adult brain. Moreover, juvenile and adult brain showed differences in their expression profiles of cytokines and chemokines mediated by ET-1.ConclusionsBBB transporter activity during neuroinflammation differs between the juvenile and adult brains. These findings emphasize the importance of considering differential P-gp and BCRP transport regulation mechanisms between adult and juvenile BBB in the context of neuroinflammation.

Oleic acid decreases BCRP mediated efflux of mitoxantrone in Caco-2 cell monolayers

Breast cancer resistance protein (BCRP) efflux restricts intestinal absorption of substances like heterocyclic amines, mycotoxins and certain human and veterinary drugs. Fat rich meals seem to increase absorption of drugs which are BCRP substrates or inhibitors. We therefore hypothesize that absorption of toxicants normally effluxed by BCRP are increased by fatty acids in food. Transport across and accumulation of 3H-Mitoxantrone (MXR) in Caco-2 cell monolayers were measured after 60min exposure to emulsions of 3H-MXR (1μM) and oleic acid (0.5–5mM). In addition, BCRP gene expression (RT-PCR) and the amount of BCRP protein (Western blot) were measured in oleic acid exposed Caco-2 cells. Oleic acid increased transport of MXR in a concentration dependent manner and 2mM oleic acid or higher increased accumulation of MXR in cells, without any signs of cytotoxicity. Gene expression of BCRP was increased after exposure to oleic acid for 6h, but the amount of BCRP protein was not increased. In conclusion, oleic acid clearly induced BCRP gene expression and reduced BCRP mediated efflux, although the amount of BCRP in cells was not affected. Consequently, effects of fatty acids on BCRP mediated efflux are important to consider in risk assessment of toxicants in food.

Intestinal Expression of Mouse Abcg2/Breast Cancer Resistance Protein (BCRP) Gene Is under Control of Circadian Clock-activating Transcription Factor-4 Pathway

ABCG2, encoding breast cancer resistance protein (BCRP), is a member of the ATP-binding cassette transporter family and is often associated with cancer chemotherapeutic resistance. BCRP is also expressed in a variety of normal cells and acts as a xenobiotic efflux transporter. Because intestinal BCRP limits systemic exposure to xenobiotics, alterations in the function and expression of this transporter could account for part of the variation in oral drug absorption. In this study, we show that ATF4, a molecular component of the circadian clock, induces circadian expression of the Abcg2 gene in mouse small intestine. Three types of leader exons (termed exons 1A, 1B, and 1C) are identified in the 5'-untranslated region of mouse Abcg2 transcripts. The exon 1B-containing Abcg2 transcript was the only isoform detected in mouse small intestine, and its mRNA levels oscillated in a circadian time-dependent manner. ATF4 bound time-dependently to the cAMP response element within the exon 1B promoter region of the Abcg2 gene, thereby causing the oscillation of BCRP protein abundance and its efflux pump function. The circadian clock-ATF4 pathway appears to enhance the function of BCRP during a specific time window and to modulate intestinal drug absorption. Our findings suggest a mechanism underlying circadian change in xenobiotic detoxification.

Expression of ABC Efflux Transporters in Placenta from Women with Insulin-Managed Diabetes

Drug efflux transporters in the placenta can significantly influence the materno-fetal transfer of a diverse array of drugs and other xenobiotics. To determine if clinically important drug efflux transporter expression is altered in pregnancies complicated by gestational diabetes mellitus (GDM-I) or type 1 diabetes mellitus (T1DM-I), we compared the expression of multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 2 (MRP2) and the breast cancer resistance protein (BCRP) via western blotting and quantitative real-time polymerase chain reaction in samples obtained from insulin-managed diabetic pregnancies to healthy term-matched controls. At the level of mRNA, we found significantly increased expression of MDR1 in the GDM-I group compared to both the T1DM-I (p<0.01) and control groups (p<0.05). Significant changes in the placental protein expression of MDR1, MRP2, and BCRP were not detected (p>0.05). Interestingly, there was a significant, positive correlation observed between plasma hemoglobin A1c levels (a retrospective marker of glycemic control) and both BCRP protein expression (r = 0.45, p<0.05) and BCRP mRNA expression (r = 0.58, p<0.01) in the insulin-managed DM groups. Collectively, the data suggest that the expression of placental efflux transporters is not altered in pregnancies complicated by diabetes when hyperglycemia is managed; however, given the relationship between BCRP expression and plasma hemoglobin A1c levels it is plausible that their expression could change in poorly managed diabetes.

Selective increase of two ABC drug efflux transporters at the blood–spinal cord barrier suggests induced pharmacoresistance in ALS

ATP-binding cassette (ABC) drug efflux transporters in the CNS are predominantly localized to the luminal surface of endothelial cells in capillaries to impede CNS accumulation of xenobiotics. Inflammatory mediators and cellular stressors regulate their activity. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of upper and lower motor neurons characterized by extensive neuroinflammation. Here we tested the hypothesis that disease-driven changes in ABC transporter expression and function occur in ALS. Given the multitude of ABC transporters with their widespread substrate recognition, we began by examining expression levels of several ABC transporters. We found a selective increase in only two transporters: P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) both at mRNA and protein levels, in the SOD1-G93A mouse model of ALS, specifically in disease-affected CNS regions. Detailed analysis revealed a similar disease-driven increase in P-gp and BCRP levels in spinal cord microvessels, indicating that their altered expression occurs at the blood spinal cord barrier. Transport activity of P-gp and BCRP increased with disease progression in spinal cord and cerebral cortex capillaries. Finally, P-gp and BCRP protein expression also increased in spinal cords of ALS patients. Preclinical drug trials in the mouse model of ALS have failed to decisively slow or arrest disease progression; pharmacoresistance imparted by ABC transporters is one possible explanation for these failures. Our observations have large implications for ALS therapeutics in humans and suggest that the obstacle provided by these transporters to drug treatments must be overcome to develop effective ALS pharmacotherapies.

Progesterone receptor downregulates breast cancer resistance protein expression via binding to the progesterone response element in breast cancer

Breast cancer resistance protein (BCRP) plays a major role in multidrug resistance (MDR). Sequence analysis reveals there is a novel progesterone response element (PRE) in the BCRP promoter, suggesting progesterone receptor (PR) may have a function in the regulation of BCRP expression. We examined the expressions of BCRP, PR, estrogen receptor α (ERα), androgen receptor (AR) and Her-2 in 95 breast cancer samples by immunohistochemistry. Then, to identify the role of PR in the regulation of BCRP expression, two constructs encoding full length BCRP cDNA driven by putative PRE promoter or constitutive CMV promoter were transfected into MCF-7 and MDA-MB-231, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) and western blotting were used to detect the expression of BCRP. Further electrophoretic mobility shift assay (EMSA) was used to verify the nuclear protein-DNA specific binding. We innovatively found that the expression of BCRP negatively related with that of PR and ERα in breast cancer by immunohistochemistry. While at a cellular level, after being treated by progesterone and 17β-estradiol, BCRP mRNA and protein levels were significantly reduced in a concentration-dependent manner in MCF-7/P-BCRP cells with PR bound to the identified PRE in BCRP promoter. Our results demonstrated that active PR inactived BCRP expression via progesterone-PR complexes binding to PRE in BCRP promoter in breast cancer cells.

Breast cancer resistance protein (BCRP/ABCG2) localises to the nucleus in glioblastoma multiforme cells

The breast cancer resistance protein (BCRP), an ATP binding cassette (ABC) efflux transporter, plays a role in multiple drug resistance (MDR). Previous studies of the subcellular location of the ABC transporter P-glycoprotein indicated that this protein is expressed in nuclear membranes. This study examines the nuclear distribution of BCRP in seven human-derived glioblastoma (GBM) and astrocytoma cell lines.BCRP expression was observed in the nuclear extracts of 6/7 cell lines. Using the GBM LN229 cell line as a model, nuclear BCRP protein was detected by immunoblotting and confocal laser microscopy. Importantly, nuclear BCRP staining was found in a subpopulation of tumour cells in a human brain GBM biopsy.Mitoxantrone cytotoxicity in the LN229 cell line was determined with and without the BCRP inhibitor fumitremorgin C (FTC) and after downregulation of BCRP with small interfering RNA (siRNA). FTC inhibition of BCRP increased mitoxantrone cytotoxicity with a ~7-fold reduction in the IC50 and this effect was further potentiated in the siRNA -treated cells.In conclusion, BCRP is expressed in the nuclear extracts of select GBM and astrocytoma cell lines and in a human GBM tumour biopsy. Its presence in the nucleus of cancer cells suggests new role for BCRP in MDR.

Breast cancer resistance protein BCRP (ABCG2)-mediated transepithelial nitrofurantoin secretion and its regulation in human intestinal epithelial (Caco-2) layers

In order to determine the capacity and regulation of the breast cancer resistance protein (BCRP)-mediated transport in intact human intestinal epithelial monolayers (Caco-2) in which multiple ABC transporters are expressed, nitrofurantoin has been used as a selective transported substrate. Nitrofurantoin transepithelial secretion was confirmed in both human BCRP and mouse bcrp-transfected MDCKII epithelia, whereas no net transepithelial secretion was observed in native or human MDR1-MDCKII epithelia. Furthermore, nitrofurantoin transepithelial secretion by BCRP-MDCKII monolayers was inhibited by Ko143 (10 μM), but not verapamil (100 μM). In Caco-2 cells grown upon permeable supports, nitrofurantoin displayed a dose-dependent transepithelial secretion with an apparent Km = 69.41 ± 22.3 μM and Vmax = 14.03 ± 2.27 nmol/(cm 2.h). Net nitrofurantoin transepithelial secretion by Caco-2 epithelia was inhibited 92% by 10 μM Ko143. Regulation of expression and function of BCRP in Caco-2 epithelial monolayers was determined after 72-h pre-exposure of the monolayers to a number of potential inducing agents. Quantitative real-time PCR and Western blotting were used to correlate induction of BCRP transcript and protein levels with transport activity. 72-h pre-treatment with β-napthoflavone and rosiglitazone up-regulates BCRP mRNA and protein expression and transport of nitrofurantoin. Ko143-sensitive transepithelial secretion of the bi-substrate (MDR1/BCRP) prazosin was also increased in the presence of rosiglitazone. We conclude that nitrofurantoin may be used to unambiguously measure BCRP-mediated fluxes in Caco-2 epithelial layers. Since dynamic regulation of BCRP expression and function is retained, the Caco-2 cell-line is useful as a screen for drug–drug and drug–diet interactions mediated by BCRP.

P-glycoprotein and breast cancer resistance protein in acute myeloid leukaemia cells treated with the Aurora-B Kinase Inhibitor barasertib-hQPA

BackgroundAurora kinases play an essential role in orchestrating chromosome alignment, segregation and cytokinesis during mitotic progression, with both aurora-A and B frequently over-expressed in a variety of human malignancies. Over-expression of the ABC drug transporter proteins P-glycoprotein (Pgp) and Breast cancer resistance protein (BCRP) is a major obstacle for chemotherapy in many tumour types with Pgp conferring particularly poor prognosis in acute myeloid leukaemia (AML). Barasertib-hQPA is a highly selective inhibitor of aurora-B kinase that has shown tumouricidal activity against a range tumour cell lines including those of leukaemic AML origin.MethodsEffect of barasertib-hQPA on the pHH3 biomarker and cell viability was measured in a panel of leukaemic cell lines and 37 primary AML samples by flow cytometry. Pgp status was determined by flow cytometry and BCRP status by flow cytometry and real-time PCR.ResultsIn this study we report the creation of the cell line OCI-AML3DNR, which over-expresses Pgp but not BCRP or multidrug resistance-associated protein (MRP), through prolonged treatment of OCI-AML3 cells with daunorubicin. We demonstrate that Pgp (OCI-AML3DNR and KG-1a) and BCRP (OCI-AML6.2) expressing AML cell lines are less sensitive to barasertib-hQPA induced pHH3 inhibition and subsequent loss of viability compared to transporter negative cell lines. We also show that barasertib-hQPA resistance in these cell lines can be reversed using known Pgp and BCRP inhibitors. We report that barasertib-hQPA is not an inhibitor of Pgp or BCRP, but by using 14[C]-barasertib-hQPA that it is effluxed by these transporters. Using phosphoHistone H3 (pHH3) as a biomarker of barasertib-hQPA responsiveness in primary AML blasts we determined that Pgp and BCRP positive primary samples were less sensitive to barasertib-hQPA induced pHH3 inhibition (p = <0.001) than samples without these transporters. However, we demonstrate that IC50 inhibition of pHH3 by barasertib-hQPA was achieved in 94.6% of these samples after 1 hour drug treatment, in contrast to the resistance of the cell lines.ConclusionWe conclude that Pgp and BCRP status and pHH3 down-regulation in patients treated with barasertib should be monitored in order to establish whether transporter-mediated efflux is sufficient to adversely impact on the efficacy of the agent.

Posttranslational negative regulation of glycosylated and non-glycosylated BCRP expression by Derlin-1

Human breast cancer resistance protein (BCRP)/MXR/ABCG2 is a well-recognized ABC half-transporter that is highly expressed at the apical membrane of many normal tissues and cancer cells. BCRP facilitates disposition of endogenous and exogenous harmful xenobiotics to protect cells/tissues from xenobiotic-induced toxicity. Despite the enormous impact of BCRP in the physiological and pathophysiological regulation of the transport of a wide variety of substrates, little is known about the factors that regulate posttranslational expression of BCRP. Here, we identified Derlin-1, a member of a family of proteins that bears homology to yeast Der1p, as a posttranslational regulator of BCRP expression. Overexpression of Derlin-1 suppressed ER to Golgi transport of wild-type (WT) BCRP that is known to be efficiently trafficked to the plasma membrane. On the other hand, protein expression of N596Q variant of BCRP, N-linked glycosylation-deficient mutant that preferentially undergoes ubiquitin-mediated ER-associated degradation (ERAD), was strongly suppressed by the overexpression of Derlin-1, whereas knockdown of Derlin-1 stabilized N596Q protein, suggesting a negative regulatory role of Derlin-1 for N596Q protein expression. Notably, knockdown of Derlin-1 also stabilized the expression of tunicamycin-induced deglycosylated WT BCRP protein, implying the importance of glycosylation state for the recognition of BCRP by Derlin-1. Thus, our data demonstrate that Derlin-1 is a negative regulator for both glycosylated and non-glycosylated BCRP expression and provide a novel posttranslational regulatory mechanism of BCRP by Derlin-1.

BCRP at the Blood−Brain Barrier: Genomic Regulation by 17β-Estradiol

At the blood-brain barrier (BBB), the ABC transporter breast cancer resistance protein (BCRP) actively extrudes a variety of therapeutic drugs, including cytostatics, and diminishes their pharmacological efficacy in the brain. Consequently, new strategies to circumvent BCRP-mediated multidrug resistance in the CNS are required. One major approach to increase brain drug levels is to manipulate signaling mechanisms that control transporter expression and function. In the present study, we investigated the long-term effect of 17β-estradiol on BCRP in an ex vivo model of isolated rat brain capillaries. BCRP function and protein expression were decreased after 6 h of incubation with nanomolar concentrations of 17β-estradiol in capillaries from male and female rats. Concomitantly, levels of BCRP mRNA were also reduced by 17β-estradiol suggesting that the transporter is down-regulated via a genomic pathway. Additionally, we identified the presence of both estrogen receptor (ER) subtypes α and β at the rat BBB. Experiments using selective ER agonists and antagonists revealed that ER subtype β is responsible for the hormone-induced reduction of BCRP function and protein expression. These findings were confirmed by the use of ERKO mice. Blocking the proteasome-dependent degradation by lactacystin reversed the 17β-estradiol-mediated decrease of BCRP supposing that transcriptional down-regulation of the efflux transporter is paralleled by protein degradation. This study demonstrates that 17β-estradiol induces the down-regulation of BCRP on transcriptional and translational levels via the activation of ERβ in rat brain capillaries after 6 h. These results could help to improve brain targeting of BCRP substrates in the treatment of CNS diseases such as brain tumors and also contribute to an enlarged understanding of BCRP-drug interactions at a chronic intake of phytoestrogens and oral contraceptives.

ABC efflux transporters in brain vasculature of Alzheimer's subjects

Multidrug efflux transporters of the ATP-Binding cassette (ABC) family, P-glycoprotein (Pgp), multidrug-resistance associated protein 4 (MRP4) and breast cancer resistance protein (BCRP), located on endothelial cells lining brain vasculature play important roles in limiting movement of substances into and enhancing their efflux from the brain. Signals from the surrounding brain normally maintain such barrier function but these may become altered in CNS pathologies such as Alzheimer's disease (AD). Previous studies have reported decreases in the glucose transporter, Glut-1, in brain vasculature of AD patients. The present study investigates the status of the multidrug efflux transporters. Sections of frozen brain from hippocampal region obtained from male AD and age-matched non-demented cases were examined for amyloid plaques and Dkk-1 expression and subjected to dual fluorescence immunochemical staining using antibodies against Pgp, BCRP or MRP4 and von Willebrand factor. Protein expression of each transporter was assessed using confocal microscopy, quantifying peak fluorescence values of cross sectional profiles across brain microvessels. Results in brain microvessels revealed expression of Pgp protein to be significantly lower in hippocampal vessels of patients with AD compared to normal individuals whereas that of MRP4 or BCRP protein was not. By contrast, analysis of the sections at protein level via Western blotting or at transcript level by qRT-PCR did not reveal significantly lower expression for either Pgp or BCRP. Such analysis did however reveal higher than normal expression in the AD brains of MRP4, probably due to gliosis, MRP4 being present also in glial cells.

Estrogen Receptor β Signaling through Phosphatase and Tensin Homolog/Phosphoinositide 3-Kinase/Akt/Glycogen Synthase Kinase 3 Down-Regulates Blood-Brain Barrier Breast Cancer Resistance Protein

Breast cancer resistance protein (BCRP) is an ATP-driven efflux pump at the blood-brain barrier that limits central nervous system pharmacotherapy. Our previous studies showed rapid loss of BCRP transport activity in rat brain capillaries exposed to low concentrations of 17-beta-estradiol (E2); this occurred without acute change in BCRP protein expression. Here, we describe a pathway through which sustained, extended exposure to E2 signals down-regulation of BCRP at the blood-brain barrier. Six-hour exposure of isolated rat and mouse brain capillaries to E2 reduced BCRP transport activity and BCRP monomer and dimer expression. Experiments with brain capillaries from estrogen receptor (ER)alpha and ERbeta knockout mice and with ER agonists and antagonists showed that E2 signaled through ERbeta to down-regulate BCRP expression. In rat brain capillaries, E2 increased unphosphorylated, active phosphatase and tensin homolog (PTEN); decreased phosphorylated, active Akt; and increased phosphorylated, active glycogen synthase kinase (GSK)3. Consistent with this, inhibition of phosphoinositide 3-kinase (PI3K) or Akt decreased BCRP activity and protein expression, and inhibition of PTEN or GSK3 reversed the E2 effect on BCRP. Lactacystin, a proteasome inhibitor, abolished E2-mediated BCRP down-regulation, suggesting internalization followed by transporter degradation. Dosing mice with E2 reduced BCRP activity in brain capillaries within 1 h; this reduction persisted for 24 h. BCRP protein expression in brain capillaries was unchanged 1 h after E2 dosing but was substantially reduced 6 and 24 h after dosing. Thus, E2 signals through ERbeta, PTEN/PI3K/Akt/GSK3 to stimulate proteasomal degradation of BCRP. These in vitro and in vivo findings imply that E2-mediated down-regulation of blood-brain barrier BCRP has the potential to increase brain uptake of chemotherapeutics that are BCRP substrates.

17-β-Estradiol: A Powerful Modulator of Blood–Brain Barrier BCRP Activity

The ATP-driven efflux transporter, breast cancer resistance protein (BCRP), handles many therapeutic drugs, including chemotherapeutics, limiting their ability to cross the blood-brain barrier. This study provides new insight into rapid, nongenomic regulation of BCRP transport activity at the blood-brain barrier. Using isolated brain capillaries from rats and mice as an ex vivo blood-brain barrier model, we show that BCRP protein is highly expressed in brain capillary membranes and functionally active in intact capillaries. We show that nanomolar concentrations of 17-β-estradiol (E2) rapidly reduced BCRP transport activity in the brain capillaries. This E2-mediated effect occurred within minutes and did not involve transcription, translation, or proteasomal degradation, indicating a nongenomic mechanism. Removing E2 after 1 h fully reversed the loss of BCRP activity. Experiments using agonists and antagonists for estrogen receptor (ER)α and ERβ and brain capillaries from ERα and ERβ knockout mice demonstrated that E2 could signal through either receptor to reduce BCRP transport function. We speculate that this nongenomic E2-signaling pathway could potentially be used for targeting BCRP at the blood-brain barrier, in brain tumors, and in brain tumor stem cells to improve chemotherapy of the central nervous system.

The synthesis and characterization of cellular membrane affinity chromatography columns for the study of human multidrug resistant proteins MRP1, MRP2 and human breast cancer resistant protein BCRP using membranes obtained from Spodoptera frugiperda (Sf9) insect cells

CMAC (cellular membrane affinity chromatography columns) have been developed for the study of the human multidrug transporters MRP1, MRP2 and the breast cancer resistance protein (BCRP). The columns were constructed using the immobilized artificial membrane (IAM) stationary phase and cellular membrane fragments obtained from Spodoptera frugiperda (Sf9) cells that had been stably transfected with human Mrp1, Mrp2 or Bcrp cDNA, using a baculovirus expression system. The resulting CMAC(Sf9(MRP1)), CMAC(Sf9(MRP2)) and CMAC(Sf9(BCRP)) columns and a control column produced using membrane fragments from non-transfected Sf9 cells, CMAC(Sf9), were characterized using frontal affinity chromatography using [(3)H]-etoposide as the marker ligand and etoposide, benzbromarone and MK571 as the displacers on the CMAC(Sf9(MRP1)) column, etoposide and furosemide on the CMAC(Sf9(MRP2)) column and etoposide and fumitremorgin C on the CMAC(Sf9(BCPR)) column. The binding affinities (K(i) values) obtained from the chromatographic studies were consistent with the data obtained using non-chromatographic techniques and the results indicate that the immobilized MRP1, MRP2 and BCRP transporters retained their ability to selectively bind known ligands. (S)-verapamil displaced [(3)H]-etoposide on the CMAC(Sf9(MRP1)) column to a greater extent than (R)-verapamil and the relative IC(50) values of the enantiomers were calculated using the changes in the retention times of the marker. The observed enantioselectivity and calculated IC(50) values were consistent with previously reported data. The results indicated that the CMAC(Sf9(MRP1)), CMAC(Sf9(MRP2)) and CMAC(Sf9(BCRP)) columns can be used for the study of binding to the MRP1, MRP2 and BCRP transporters and that membranes from the Sf9 cell line can be used to prepare CMAC columns. This is the first example of the use of membranes from a non-mammalian cell line in an affinity chromatographic system.