Structure and function of the human breast cancer resistance protein (BCRP/ABCG2).
ABSTRACT: 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.
Project description:Since cloning of the ATP-binding cassette (ABC) family member breast cancer resistance protein (BCRP/ABCG2) and its characterization as a multidrug resistance efflux transporter in 1998, BCRP has been the subject of more than two thousand scholarly articles. In normal tissues, BCRP functions as a defense mechanism against toxins and xenobiotics, with expression in the gut, bile canaliculi, placenta, blood-testis and blood-brain barriers facilitating excretion and limiting absorption of potentially toxic substrate molecules, including many cancer chemotherapeutic drugs. BCRP also plays a key role in heme and folate homeostasis, which may help normal cells survive under conditions of hypoxia. BCRP expression appears to be a characteristic of certain normal tissue stem cells termed "side population cells," which are identified on flow cytometric analysis by their ability to exclude Hoechst 33342, a BCRP substrate fluorescent dye. Hence, BCRP expression may contribute to the natural resistance and longevity of these normal stem cells. Malignant tissues can exploit the properties of BCRP to survive hypoxia and to evade exposure to chemotherapeutic drugs. Evidence is mounting that many cancers display subpopulations of stem cells that are responsible for tumor self-renewal. Such stem cells frequently manifest the "side population" phenotype characterized by expression of BCRP and other ABC transporters. Along with other factors, these transporters may contribute to the inherent resistance of these neoplasms and their failure to be cured.
Project description:Breast cancer resistant protein (BCRP, ABCG2) is an ATP-binding cassette (ABC) transporter, which together with two other ABC efflux drug pumps, namely P-glycoprotein (P-gp, ABCB1) and multidrug resistance-related protein 1 (MRP1, ABCC1) is the most important multidrug resistance protein foun d in eukaryotic cells including cells in the testis. However, unlike P-gp and MRP1, which are components of the Sertoli cell blood-testis barrier (BTB), BCRP is not expressed at the BTB in rodents and human testes. Instead, BCRP is expressed by peritubular myoid cells and endothelial cells of the lymphatic vessel in the tunica propria, residing outside the BTB. As such, the testis is equipped with two levels of defense against xenobiotics or drugs, preventing these harmful substances from entering the adluminal compartment to perturb meiosis and post-meiotic spermatid development: one at the level of the BTB conferred by P-gp and MRP1 and one at the tunica propria conferred by BCRP. The presence of drug transporters at the tunica propria as well as at the Sertoli cell BTB thus poses significant obstacles in developing non-hormonal contraceptives if these drugs (e.g., adjudin) exert their effects in germ cells behind the BTB, such as in the adluminal (apical) compartment of the seminiferous epithelium. Herein, we summarize recent findings pertinent to adjudin, a non-hormonal male contraceptive, and molecular interactions of adjudin with BCRP so that this information can be helpful to devise delivery strategies to evade BCRP in the tunica propria to improve its bioavailability in the testis.
Project description:1. The umbilical cord is a direct conduit to the fetus hence transporters could have roles in partitioning substances between the maternal-placental-fetal units. Here we determined the expression and localization of the ATP-Binding Cassette (ABC) transporters BCRP (ABCG2), P-gp (ABCB1) and MRP1 (ABCC1) in human umbilical cords. 2.?The mRNA for BCRP and MRP1 was detected in 25/25 samples, but P-gp was detected in only 5/25. ABC transporter mRNA expression relative to 18S was 25.6?±?0.3, 26.5?±?0.6 and 22.2?±?0.2 cycles for BCRP, MRP1 and P-gp respectively. 3.?Using a subset of 10 umbilical cords, BCRP protein was present in all samples (immunoblot) with positive correlation between mRNA and proteins (p?=?0.07, r?=?0.62) and between immunoblotting and immunohistochemistry (IHC) (p?=?0.03, r?=?0.67). P-gp protein was observed in 4/10 samples by both immunoblot and IHC, with no correlation between mRNA and protein (p?=?0.45, r?=?0.55) or immunoblotting and IHC (p?=?0.2, r?=?0.72), likely due to small sample size. MRP1 protein was not observed. 4. Localization of BCRP and P-gp proteins was to Wharton's jelly with no specific staining in arterial or venous endothelia. 5. Understanding ABC transporter expression in the umbilical cord may be useful for determining fetal exposures to xenobiotics if functional properties can be defined.
Project description:The breast cancer resistance protein (BCRP, ABCG2) is an efflux transporter that removes xenobiotics that cross the placenta back to the maternal circulation, thereby limiting exposure of the fetus to drugs and chemicals. Currently, variability of BCRP expression within the placenta is not known. Ten placentas were collected from healthy women undergoing elective Cesarean sections at term. Villous samples were dissected in defined regions (medial, intermediate, and peripheral) and BCRP mRNA and protein were quantified. There were no regional differences in mRNA expression of housekeeping genes (GAPDH, RPL13a, PRL, 18S). GAPDH had the lowest correlation with BCRP Ct values and was used for BCRP mRNA normalization. No differences in placental BCRP mRNA and protein were observed among the sample sites (<20% variability). Sampling site does not affect the expression of BCRP, supporting the utility of single site sampling protocols to assess the interindividual regulation of this transporter in human placentas.
Project description:The breast cancer resistance protein (BCRP) is an ABC transporter playing a crucial role in the pharmacokinetics of drugs. The early identification of substrates and inhibitors of this efflux transporter can help to prevent or foresee drug-drug interactions. In this work, we built a ligand-based in silico classification model to predict the inhibitory potential of drugs toward BCRP. The model was applied as a virtual screening technique to identify potential inhibitors among the small-molecules subset of DrugBank. Ten compounds were selected and tested for their capacity to inhibit mitoxantrone efflux in BCRP-expressing PLB985 cells. Results identified cisapride (IC50 = 0.4 µM) and roflumilast (IC50 = 0.9 µM) as two new BCRP inhibitors. The in silico strategy proved useful to prefilter potential drug-drug interaction perpetrators among a database of small molecules and can reduce the amount of compounds to test.
Project description: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.
Project description:ATP-binding cassette (ABC) transporters, such as breast cancer resistance protein (BCRP), are key players in resistance to multiple anti-cancer drugs, leading to cancer treatment failure and cancer-related death. Currently, there are no clinically approved drugs for reversal of cancer drug resistance caused by ABC transporters. This study investigated if a novel drug candidate, SCO-201, could inhibit BCRP and reverse BCRP-mediated drug resistance. We applied in vitro cell viability assays in SN-38 (7-Ethyl-10-hydroxycamptothecin)-resistant colon cancer cells and in non-cancer cells with ectopic expression of BCRP. SCO-201 reversed resistance to SN-38 (active metabolite of irinotecan) in both model systems. Dye efflux assays, bidirectional transport assays, and ATPase assays demonstrated that SCO-201 inhibits BCRP. In silico interaction analyses supported the ATPase assay data and suggest that SCO-201 competes with SN-38 for the BCRP drug-binding site. To analyze for inhibition of other transporters or cytochrome P450 (CYP) enzymes, we performed enzyme and transporter assays by in vitro drug metabolism and pharmacokinetics studies, which demonstrated that SCO-201 selectively inhibited BCRP and neither inhibited nor induced CYPs. We conclude that SCO-201 is a specific, potent, and potentially non-toxic drug candidate for the reversal of BCRP-mediated resistance in cancer cells.
Project description:Cannabidiol (CBD) is currently being investigated as a novel therapeutic for the treatment of CNS disorders like schizophrenia and epilepsy. ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) mediate pharmacoresistance in these disorders. P-gp and Bcrp are expressed at the blood brain barrier (BBB) and reduce the brain uptake of substrate drugs including various antipsychotics and anticonvulsants. It is therefore important to assess whether CBD is prone to treatment resistance mediated by P-gp and Bcrp. Moreover, it has become common practice in the drug development of CNS agents to screen against ABC transporters to help isolate lead compounds with optimal pharmacokinetic properties. The current study aimed to assess whether P-gp and Bcrp impacts the brain transport of CBD by comparing CBD tissue concentrations in wild-type (WT) mice versus mice devoid of ABC transporter genes. P-gp knockout (Abcb1a/b (-?-)), Bcrp knockout (Abcg2 (-?-)), combined P-gp/Bcrp knockout (Abcb1a/b (-?-) Abcg2 (-?-)) and WT mice were injected with CBD, before brain and plasma samples were collected at various time-points. CBD results were compared with the positive control risperidone and 9-hydroxy risperidone, antipsychotic drugs that are established ABC transporter substrates. Brain and plasma concentrations of CBD were not greater in P-gp, Bcrp or P-gp/Bcrp knockout mice than WT mice. In comparison, the brain/plasma concentration ratios of risperidone and 9-hydroxy risperidone were profoundly higher in P-gp knockout mice than WT mice. These results suggest that CBD is not a substrate of P-gp or Bcrp and may be free from the complication of reduced brain uptake by these transporters. Such findings provide favorable evidence for the therapeutic development of CBD in the treatment of various CNS disorders.
Project description: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 IC?? 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.
Project description:Breast cancer resistance protein (BCRP) protects tissues by actively transporting xenobiotics and their metabolites out of the cells. BCRP is expressed in the apical membrane of normal intestinal and colonic epithelium. The BCRP substrates include a number of structurally unrelated compounds, such as drugs, pesticides, carcinogens and endogenous compounds. Although the functional and common BCRP alleles, 34G>A and 421C>A, are shown to vary by ethnicity, their potential mechanism has not been adequately described with regards to affecting the susceptibility to colorectal cancer. The present study aimed to evaluate the effects of the BCRP variants on the susceptibility to colorectal cancer and to predict the individual responses to xenobiotics transferred by BCRP. BCRP 421C>A was significantly associated with the colorectal cancer risk (odds ratio, 16.12; P=0.005). These findings are the first results of BCRP allele distributions in the Turkish population and provide an understanding of the correlation between therapeutic approaches and etiology of colorectal cancer.