Human Breast Cancer Resistance Research Articles

Acyclovir is a substrate for the human breast cancer resistance protein (BCRP/ABCG2): implications for renal tubular transport and acyclovir-induced nephrotoxicity

The human breast cancer resistance protein (BCRP/ABCG2) is widely expressed in human tissues, including the kidney. In mice, Bcrp1 (murine BCRP ortholog) mediates the transport of acyclovir into breast milk. It is plausible that acyclovir is also a substrate for the human BCRP. The objective of the study was to determine whether acyclovir is a substrate for human BCRP. Transfected human embryonic kidney (HEK293) cells (containing the wild-type ABCG2 gene) were exposed to [8-(14)C]acyclovir (1µmol/L) in the presence or absence of the BCRP inhibitor fumitremorgin C (FTC). Intracellular acyclovir accumulation was assessed using a liquid scintillation counter. Coexposure to FTC resulted in a significant (5-fold) increase in the intracellular accumulation of acyclovir. The results suggest that acyclovir is a substrate for human BCRP. The study is the first to provide direct evidence for the role of human BCRP in acyclovir transport and its potential significance with respect to renal tubular transport of acyclovir and the direct renal tubular insult induced by the drug.

Double-Transduced MDCKII Cells To Study Human P-Glycoprotein (ABCB1) and Breast Cancer Resistance Protein (ABCG2) Interplay in Drug Transport across the Blood−Brain Barrier

P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) combination knockout mice display disproportionately increased brain penetration of shared substrates, including topotecan and several tyrosine kinase inhibitors, compared to mice deficient for only one transporter. To better study the interplay of both transporters also in vitro, we generated a transduced polarized MDCKII cell line stably coexpressing substantial levels of human ABCB1 and ABCG2 (MDCKII-ABCB1/ABCG2). Next, we measured concentration-dependent transepithelial transport of topotecan, sorafenib and sunitinib. By blocking either one or both of the transporters simultaneously, using specific inhibitors, we aimed to mimic the ABCB1-ABCG2 interplay at the blood-brain barrier in wild-type, single or combination knockout mice. ABCB1 and ABCG2 contributed to similar extents to topotecan transport, which was only partly saturable. For sorafenib transport, ABCG2 was the major determinant at low concentrations. However, saturation of ABCG2-mediated transport occurred at higher sorafenib concentrations, where ABCB1 was still fully active. Furthermore, sunitinib was transported equally by ABCB1 and ABCG2 at low concentrations, but ABCG2-mediated transport became saturated at lower concentrations than ABCB1-mediated transport. The relative impact of these transporters can thus be affected by the applied drug concentrations. A comparison of the in vitro observed (inverse) transport ratios and cellular accumulation of the drugs at low concentrations with in vivo brain penetration data from corresponding Abcb1a/1b⁻/⁻, Abcg2⁻/⁻ and Abcb1a/1b;Abcg2⁻/⁻ mouse strains revealed very similar qualitative patterns for each of the tested drugs. MDCKII-ABCB1/ABCG2 cells thus present a useful in vitro model to study the interplay of ABCB1 and ABCG2.

Transport of Selected PET Radiotracers by Human P-Glycoprotein (ABCB1) and Breast Cancer Resistance Protein (ABCG2): An In Vitro Screening

Radiolabeled compounds used for brain imaging with PET must readily cross the blood-brain barrier (BBB) to reach their target. Efflux transporters at the BBB-P-glycoprotein (P-gp) and the breast cancer resistance protein (BCRP)-could limit their uptake by the brain. We developed and validated an in vitro model using MDCKII cells transfected with human multidrug resistance (MDR1) or BCRP genes and assessed the transport of selected PET ligands by the concentration equilibrium technique. The tested compounds included befloxatone, (R,S)-CGP-12177, clorgyline, R-(-)-deprenyl, diprenorphine, DPA-714, fallypride, flumazenil, 2-fluoro-A-85380, LBT-999, loperamide, p-MPPF, PE2I, Pittsburgh compound B (PIB), (R,S)-PK11195, raclopride, R-(+)-verapamil, and WAY-100635. The assays were performed using the nonradioactive form of each compound (ultraviolet high-performance liquid chromatography analysis) and, when available, the (18)F-labeled analogs (γ-counting). Befloxatone appeared to be transported solely by BCRP. Loperamide, verapamil, and diprenorphine were the only P-gp substrates. Other ligands were transported by neither P-gp nor BCRP. The present method can readily be used to screen new-compound transport by P-gp or BCRP, even before any radiolabeling. Compounds that were previously thought to be transported by P-gp in rodents, such as p-MPPF, WAY-100635, and flumazenil, cannot be considered substrates of human P-gp. The impact of BCRP and P-gp at the BBB on the transport of befloxatone and diprenorphine in vivo remains to be evaluated with PET.

Breast cancer resistance protein BCRP/ABCG2 regulatory microRNAs (hsa-miR-328, -519c and -520h) and their differential expression in stem-like ABCG2+ cancer cells

Recent studies have shown that a number of microRNAs (miRNA or miR) may regulate human breast cancer resistance protein (BCRP/ABCG2), an important efflux transporter responsible for cellular drug disposition, whereas their effects on ABCG2 protein expression are not compared. In this study, we first identified a new proximal miRNA response element (MRE) for hsa-miR-519c within ABCG2 3′-untranslated region (3′UTR) through computational analyses. This miR-519c MRE site was confirmed using dual luciferase reporter assay and site-directed mutagenesis. Immunoblot analyses indicated that ABCG2 protein expression was significantly down-regulated in MCF-7/MX100 cells after transfection with hsa-miR-328- or -519c expression plasmids, and was markedly up-regulated in MCF-7 cells after transfection with miR-328 or -519c antagomir. However, ABCG2 protein expression was unchanged in MCF-7/MX100 cells after transfection with hsa-miR-520h expression plasmids, which was associated with undetectable miR-520h expression. Furthermore, ABCG2 mRNA degradation was accelerated dramatically in cells transfected with miR-519c expression plasmid, suggesting the involvement of mRNA degradation mechanism. Intervention of miR-328 or -519c signaling led to significant change in intracellular mitoxantrone accumulation, as determined by flow cytometry analyses. In addition, we separated RB143 human retinoblastoma cells into stem-like (ABCG2+) and non-stem-like (ABCG2−) populations through immunomagnetic selection, and found that miR-328, -519c and -520h levels were 9-, 15- and 3-fold lower in the ABCG2+ cells, respectively. Our data suggest that miR-519c and -328 have greater impact on ABCG2 expression than miR-520h in MCF-7 human breast cancer cells, and the presence of proximal miR-519c MRE explains the action of miR-519c on shortened ABCG2 3′UTR.

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.

Structure and Function of the Human Breast Cancer Resistance Protein (BCRP/ABCG2)

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.

Transmembrane helices 1 and 6 of the human breast cancer resistance protein (BCRP/ABCG2): identification of polar residues important for drug transport

The human breast cancer resistance protein (BCRP/ABCG2) mediates efflux of drugs and xenobiotics. In this study, we investigated the role of polar residues within or near the predicted transmembrane α-helices 1 and 6 of BCRP in drug transport. We substituted Asn(387), Gln(398), Asn(629), and Thr(642) with Ala, Thr(402) with Ala and Arg, and Tyr(645) with Phe, and the mutants were stably expressed in human embryonic kidney-293 or Flp-In-293 cells. Immunoblotting and confocal microscopy analysis revealed that all of the mutants were well expressed and predominantly targeted to the plasma membrane. While T402A and T402R showed a significant global reduction in the efflux of mitoxantrone, Hoechst 33342, and BODIPY-prazosin, N629A exhibited significantly increased efflux activities for all of the substrates. N387A and Q398A displayed significantly impaired efflux for mitoxantrone and Hoechst 33342, but not for BODIPY-prazosin. In contrast, T642A and Y645F showed a moderate reduction in Hoechst 33342 efflux only. Drug resistance profiles of human embryonic kidney-293 cells expressing the mutants generally correlated with the efflux data. Furthermore, N629A was associated with a marked increase, and N387A and T402A with a significant reduction, in BCRP ATPase activity. Mutations of some of the polar residues may cause conformational changes, as manifested by the altered binding of the 5D3 antibody to BCRP in the presence of prazosin. The inward-facing homology model of BCRP indicated that Thr(402) within transmembrane 1 may be important for helical interactions, and Asn(629) may be involved in BCRP-substrate interaction. In conclusion, we have demonstrated the functional importance of some of these polar residues in BCRP activity.

Role of Basic Residues within or near the Predicted Transmembrane Helix 2 of the Human Breast Cancer Resistance Protein in Drug Transport

The human breast cancer resistance protein (BCRP/ABCG2) mediates efflux of drugs and xenobiotics out of cells. In this study, we investigated the role of five basic residues within or near transmembrane (TM) 2 of BCRP in transport activity. Lys(452), Lys(453), His(457), Arg(465), and Lys(473) were replaced with Ala or Asp. K452A, K453D, H457A, R465A, and K473A were stably expressed in human embryonic kidney (HEK) cells, and their plasma membrane expression and transport activities were examined. All of the mutants were expressed predominantly on the plasma membrane of HEK cells. After normalization to BCRP levels, the activities of K452A and H457A in effluxing mitoxantrone, boron-dipyrromethene-prazosin, and Hoechst33342 were increased approximately 2- to 6-fold compared with those of wild-type BCRP, whereas the activities of K453D and R465A were decreased by 40 to 60%. Likewise, K452A and H457A conferred increased resistance to mitoxantrone and 7-ethyl-10-hydroxy-camptothecin (SN-38), and K453D and R465A exhibited lower resistance. The transport activities and drug-resistance profiles of K473A were not changed. These mutations also differentially affected BCRP ATPase activities with a 2- to 4-fold increase in V(max)/K(m) for K452A and H457A and a 40 to 70% decrease for K453D and R465A. These mutations may induce conformational changes as manifested by the altered binding of the 5D3 antibody to BCRP in the presence of prazosin and altered trypsin digestion. Molecular modeling and docking calculations indicated that His(457) and Arg(465) might be directly involved in substrate binding. In conclusion, we have identified several basic residues within or near TM2 that may be important for interaction of substrates with BCRP.

The Human Breast Cancer Resistance Protein (BCRP/ABCG2) Shows Conformational Changes with Mitoxantrone

BCRP/ABCG2 mediates efflux of drugs and xenobiotics. BCRP was expressed in Pichia pastoris, purified to > 90% homogeneity, and subjected to two-dimensional (2D) crystallization. The 2D crystals showed a p12(1) symmetry and projection maps were determined at 5 A resolution by cryo-electron microscopy. Two crystal forms with and without mitoxantrone were observed with unit cell dimensions of a = 55.4 A, b = 81.4 A, gamma = 89.8 degrees , and a = 57.3 A, b = 88.0 A, gamma = 89.7 degrees , respectively. The projection map without mitoxantrone revealed an asymmetric structure with ring-shaped density features probably corresponding to a bundle of transmembrane alpha helices, and appeared more open and less symmetric than the map with mitroxantrone. The open and closed inward-facing forms of BCRP were generated by homology modeling, representing the substrate-free and substrate-bound conformations in the absence of nucleotide, respectively. These models are consistent with the experimentally observed conformational change upon substrate binding.

Warburg effect in chemosensitivity: Targeting lactate dehydrogenase-A re-sensitizes Taxol-resistant cancer cells to Taxol

BackgroundTaxol is one of the most effective chemotherapeutic agents for the treatment of patients with breast cancer. Despite impressive clinical responses initially, the majority of patients eventually develop resistance to Taxol. Lactate dehydrogenase-A (LDH-A) is one of the predominant isoforms of LDH expressed in breast tissue, which controls the conversion of pyruvate to lactate and plays an important role in glucose metabolism. In this study we investigated the role of LDH-A in mediating Taxol resistance in human breast cancer cells.ResultsTaxol-resistant subclones, derived from the cancer cell line MDA-MB-435, sustained continuous growth in high concentrations of Taxol while the Taxol-sensitive cells could not. The increased expression and activity of LDH-A were detected in Taxol-resistant cells when compared with their parental cells. The downregulation of LDH-A by siRNA significantly increased the sensitivity of Taxol-resistant cells to Taxol. A higher sensitivity to the specific LDH inhibitor, oxamate, was found in the Taxol-resistant cells. Furthermore, treating cells with the combination of Taxol and oxamate showed a synergistical inhibitory effect on Taxol-resistant breast cancer cells by promoting apoptosis in these cells.ConclusionLDH-A plays an important role in Taxol resistance and inhibition of LDH-A re-sensitizes Taxol-resistant cells to Taxol. This supports that Warburg effect is a property of Taxol resistant cancer cells and may play an important role in the development of Taxol resistance. To our knowledge, this is the first report showing that the increased expression of LDH-A plays an important role in Taxol resistance of human breast cancer cells. This study provides valuable information for the future development and use of targeted therapies, such as oxamate, for the treatment of patients with Taxol-resistant breast cancer.

Interaction of drugs of abuse and maintenance treatments with human P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2)

Drug interaction with P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) may influence its tissue disposition including blood-brain barrier transport and result in potent drug-drug interactions. The limited data obtained using in-vitro models indicate that methadone, buprenorphine, and cannabinoids may interact with human P-gp; but almost nothing is known about drugs of abuse and BCRP. We used in vitro P-gp and BCRP inhibition flow cytometric assays with hMDR1- and hBCRP-transfected HEK293 cells to test 14 compounds or metabolites frequently involved in addiction, including buprenorphine, norbuprenorphine, methadone, ibogaine, cocaine, cocaethylene, amphetamine, N-methyl-3,4-methylenedioxyamphetamine, 3,4-methylenedioxyamphetamine, nicotine, ketamine, Delta9-tetrahydrocannabinol (THC), naloxone, and morphine. Drugs that in vitro inhibited P-gp or BCRP were tested in hMDR1- and hBCRP-MDCKII bidirectional transport studies. Human P-gp was significantly inhibited in a concentration-dependent manner by norbuprenorphine>buprenorphine>methadone>ibogaine and THC. Similarly, BCRP was inhibited by buprenorphine>norbuprenorphine>ibogaine and THC. None of the other tested compounds inhibited either transporter, even at high concentration (100 microm). Norbuprenorphine (transport efflux ratio approoximately 11) and methadone (transport efflux ratio approoximately 1.9) transport was P-gp-mediated; however, with no significant stereo-selectivity regarding methadone enantiomers. BCRP did not transport any of the tested compounds. However, the clinical significance of the interaction of norbuprenorphine with P-gp remains to be evaluated.

Culture Period-Dependent Change of Function and Expression of ATP-Binding Cassette Transporters in Caco-2 Cells

The objective of this study was to determine an appropriate culture period to assess whether a compound of interest is transported by efflux transporters such as human multidrug resistance 1 (hMDR1), human multidrug resistance-associated protein 2 (hMRP2), and human breast cancer resistance protein (hBCRP) in Caco-2 cells. Caco-2 cells were cultured on a Transwell for 1 to 6 weeks. The expression of these transporters in the mRNA and protein levels was examined using a real-time polymerase chain reaction and Western blotting, respectively. Transcellular transport activities using digoxin, ochratoxin A, olmesartan, and estrone-3-sulfate were also examined. Except for digoxin, the permeability coefficient (P(app)) ratio of the three compounds at 2 weeks was the highest in the periods tested. The P(app) ratio of digoxin at 2 weeks was higher than that at 3 weeks. The temporal expression profile of each transporter in the mRNA level was similar to that in the protein level, and the functions of hMRP2 and hBCRP were roughly correlated with the expression in the mRNA and protein levels, but that of hMDR1 was not. These data suggest that among all the culture periods evaluated a 2-week culture is the best culture period for transport studies to identify whether a compound is a substrate for hMDR1, hMRP2, and hBCRP.

Connective Tissue Growth Factor Confers Drug Resistance in Breast Cancer through Concomitant Up-regulation of Bcl-xL and cIAP1

Connective tissue growth factor (CTGF) expression is elevated in advanced breast cancer and promotes metastasis. Chemotherapy response is only transient in most metastatic diseases. In the present study, we examined whether CTGF expression could confer drug resistance in human breast cancer. In breast cancer patients who received neoadjuvant chemotherapy, CTGF expression was inversely associated with chemotherapy response. Overexpression of CTGF in MCF7 cells (MCF7/CTGF) enhanced clonogenic ability, cell viability, and resistance to apoptosis on exposure to doxorubicin and paclitaxel. Reducing the CTGF level in MDA-MB-231 (MDA231) cells by antisense CTGF cDNA (MDA231/AS cells) mitigated this drug resistance capacity. CTGF overexpression resulted in resistance to doxorubicin- and paclitaxel-induced apoptosis by up-regulation of Bcl-xL and cellular inhibitor of apoptosis protein 1 (cIAP1). Knockdown of Bcl-xL or cIAP1 with specific small interfering RNAs abolished the CTGF-mediated resistance to apoptosis induced by the chemotherapeutic agents in MCF7/CTGF cells. Inhibition of extracellular signal-regulated kinase (ERK)-1/2 effectively reversed the resistance to apoptosis as well as the up-regulation of Bcl-xL and cIAP1 in MCF7/CTGF cells. A neutralizing antibody against integrin alpha(v)beta(3) significantly attenuated CTGF-mediated ERK1/2 activation and up-regulation of Bcl-xL and cIAP1, indicating that the integrin alpha(v)beta(3)/ERK1/2 signaling pathway is essential for CTGF functions. The Bcl-xL level also correlated with the CTGF level in breast cancer patients. We also found that a COOH-terminal domain peptide from CTGF could exert activities similar to full-length CTGF, in activation of ERK1/2, up-regulation of Bcl-xL/cIAP1, and resistance to apoptosis. We conclude that CTGF expression could confer resistance to chemotherapeutic agents through augmenting a survival pathway through ERK1/2-dependent Bcl-xL/cIAP1 up-regulation.

Membrane Topology of the Human Breast Cancer Resistance Protein (BCRP/ABCG2) Determined by Epitope Insertion and Immunofluorescence

The human breast cancer resistance protein (BCRP/ABCG2) mediates efflux of drugs and organic anions across the plasma membrane. Hydropathy analysis suggests that BCRP consists of a nucleotide-binding domain (residues approximately 1-395) and a membrane-spanning domain (MSD) (residues approximately 396-655); however, its exact topology structure remains unknown. In this study, we determined the topology structure of BCRP by inserting hemagglutinin (HA) tags in its predicted hydrophilic regions of the MSD. HA-tagged BCRP mutants were expressed in HEK cells and tested for their ability to efflux mitoxantrone and BODIPY-prazosin. Polarity of the inserted tags with respect to the plasma membrane was determined by immunofluorescence. All of the mutants were expressed at levels comparable to wild-type BCRP as revealed by immunoblotting with specific antibodies against BCRP and the HA tag. Insertions at residues 423, 454, 462, 499, 529, 532, and 651 produced functional mutants, whereas insertions at residues 560, 594, and 623 resulted in mutants with significantly reduced activity and insertions at residues 387, 420, 474, and 502 completely abrogated the activity. HA tags inserted at residues 387, 474, 529, 532, 560, and 651 were localized intracellularly, whereas those inserted at residues 420, 423, 454, 499, 502, 594, and 623 revealed an extracellular location. Residue 462 was localized in a transmembrane (TM) segment. These results provide the first direct experimental evidence in support of a 6-TM model for BCRP with the amino and carboxyl termini of the MSD located intracellularly. These data may have important implications for understanding the transport mechanism of BCRP.

Proteomic analysis of tumor necrosis factor-α resistant human breast cancer cells reveals a MEK5/Erk5-mediated epithelial-mesenchymal transition phenotype

IntroductionDespite intensive study of the mechanisms of chemotherapeutic drug resistance in human breast cancer, few reports have systematically investigated the mechanisms that underlie resistance to the chemotherapy-sensitizing agent tumor necrosis factor (TNF)-α. Additionally, the relationship between TNF-α resistance mediated by MEK5/Erk5 signaling and epithelial-mesenchymal transition (EMT), a process associated with promotion of invasion, metastasis, and recurrence in breast cancer, has not previously been investigated.MethodsTo compare differences in the proteome of the TNF-α resistant MCF-7 breast cancer cell line MCF-7-MEK5 (in which TNF-α resistance is mediated by MEK5/Erk5 signaling) and its parental TNF-a sensitive MCF-7 cell line MCF-7-VEC, two-dimensional gel electrophoresis and high performance capillary liquid chromatography coupled with tandem mass spectrometry approaches were used. Differential protein expression was verified at the transcriptional level using RT-PCR assays. An EMT phenotype was confirmed using immunofluorescence staining and gene expression analyses. A short hairpin RNA strategy targeting Erk5 was utilized to investigate the requirement for the MEK/Erk5 pathway in EMT.ResultsProteomic analyses and PCR assays were used to identify and confirm differential expression of proteins. In MCF-7-MEK5 versus MCF-7-VEC cells, vimentin (VIM), glutathione-S-transferase P (GSTP1), and creatine kinase B-type (CKB) were upregulated, and keratin 8 (KRT8), keratin 19 (KRT19) and glutathione-S-transferase Mu 3 (GSTM3) were downregulated. Morphology and immunofluorescence staining for E-cadherin and vimentin revealed an EMT phenotype in the MCF-7-MEK5 cells. Furthermore, EMT regulatory genes SNAI2 (slug), ZEB1 (δ-EF1), and N-cadherin (CDH2) were upregulated, whereas E-cadherin (CDH1) was downregulated in MCF-7-MEK5 cells versus MCF-7-VEC cells. RNA interference targeting of Erk5 reversed MEK5-mediated EMT gene expression.ConclusionsThis study demonstrates that MEK5 over-expression promotes a TNF-α resistance phenotype associated with distinct proteomic changes (upregulation of VIM/vim, GSTP1/gstp1, and CKB/ckb; and downregulation of KRT8/krt8, KRT19/krt19, and GSTM3/gstm3). We further demonstrate that MEK5-mediated progression to an EMT phenotype is dependent upon intact Erk5 and associated with upregulation of SNAI2 and ZEB1 expression.

Acid Sphingomyelinase Overexpression Enhances the Antineoplastic Effects of Irradiation In Vitro and In Vivo

Exposure of cells or animals to stress frequently induces acid sphingomyelinase (ASM)-mediated ceramide production that leads to cell death. Consistent with this, overexpression of ASM in subcutaneous B16-F10 mouse melanomas, in combination with irradiation, resulted in tumors that were up to 12-fold smaller than irradiated control melanomas. Similarly, when irradiated melanomas were pretreated with a single, peritumoral injection of recombinant ASM (rhASM), the tumors were up to threefold smaller. The in vivo effect of ASM was likely due to enhanced cell death of the tumor cells themselves, as well as the surrounding microvascular endothelial cells. In vitro, rhASM had little or no effect on the growth of tumor cells, even in combination with irradiation. However, when the culture media was acidified to mimic the acidic microenvironment of solid tumors, rhASM-mediated cell death was markedly enhanced when combined with irradiation. Microscopic analysis suggested that this was associated with an increase in autophagy. rhASM has been produced for the treatment of the lysosomal storage disorder, type B Niemann-Pick disease, and is currently being evaluated in a phase-1 clinical trial. Based on the data presented in this article, we propose that further investigation of this protein and gene as antineoplastic agents also is warranted.

Interactions of azole antifungal agents with the human breast cancer resistance protein (BCRP)

Breast cancer resistance protein (BCRP) is an efflux transporter that plays an important role in drug disposition. The goal of this study was to investigate the interactions of azole antifungal agents, ketoconazole, itraconazole, fluconazole, and voriconazole, with BCRP. First, the effect of the azoles on BCRP efflux activity in BCRP-overexpressing HEK cells was determined by measuring intracellular pheophorbide A (PhA) fluorescence using flow cytometry. We found that keotoconazole and itraconazole significantly inhibited BCRP-mediated efflux of PhA at low microM concentrations. However, fluconazole only mildly inhibited and voriconazole did not inhibit BCRP efflux activity at concentrations up to 100 microM. The IC(50) value of ketoconazole for inhibition of BCRP-mediated PhA efflux was 15.3 +/- 6.5 microM. Ketoconazole and itraconazole also effectively reversed BCRP-mediated resistance of HEK cells to topotecan. When direct efflux of [(3)H]ketoconazole was measured in BCRP-overexpressing HEK cells, we found that [(3)H]ketoconazole was not transported by BCRP. Consistent with this finding, BCRP did not confer resistance to ketoconazole and itraconazole in HEK cells. Taken together, ketoconazole and itraconazole are BCRP inhibitors, but fluconazole and voriconazole are not. These results suggest that BCRP could play a significant role in the pharmacokinetic interactions of ketoconazole or itraconazole with BCRP substrate drugs.

A Functional Steroid-Binding Element in an ATP-Binding Cassette Multidrug Transporter

The human breast cancer resistance protein is an ATP-binding cassette (ABC) multidrug transporter that affects the bioavailability of chemotherapeutic drugs and can confer drug resistance on cancer cells. It is the second member of the ABCG subfamily, other members of which are associated with human steroid disorders such as hypercholesterolemia, sitosterolemia, and atherosclerosis. The molecular bases of protein-steroid interactions in ABC transporters are unknown. Here, we identify a steroid-binding element in the membrane domain of ABCG2 with a similarity to steroid hormone/nuclear receptors. The element facilitates steroid hormone binding and mediates modulation of ABCG2 activity. The identification of this element might provide an opportunity for the development of new therapeutic ligands for ABCG2.

Localization of the Human Breast Cancer Resistance Protein (BCRP/ABCG2) in Lipid Rafts/Caveolae and Modulation of Its Activity by Cholesterol in Vitro

Breast cancer resistance protein (BCRP/ABCG2) is an active efflux pump that belongs to the ATP-binding cassette (ABC) transporter family. It is located in various tissues involved in drug absorption, distribution, and elimination and plays an important role in multidrug resistance. For P-glycoprotein, another member of the ABC transporter family, it is well established that it is at least partly located in cholesterol and sphingolipid-enriched domains of the plasma membrane called "lipid rafts" and that the composition of the membrane lipids may modulate its efflux activity. This study addressed the compartmentalization of BCRP in the plasma membrane and the influence of membrane cholesterol on the efflux activity of BCRP. As a cell model, we used the canine kidney epithelial cell line MDCKII-BCRP transfected with the cDNA encoding human BCRP and the corresponding parental cell line MDCKII. Cholesterol depletion with methyl-beta-cyclodextrin (MbetaCD) provoked a 40% decrease in BCRP activity (p < 0.01) assessed with flow cytometry (pheophorbide A efflux assay). Cholesterol repletion with MbetaCD/cholesterol-inclusion complexes restored BCRP function, and cholesterol saturation of native cells did not further enhance BCRP activity. Coimmunoprecipitation experiments indicated a physical interaction between BCRP and caveolin-1, and Western blot analysis after density gradient ultracentrifugation demonstrated that BCRP is located in detergent-resistant membranes that also contain caveolin-1. In conclusion, our results demonstrate for the first time that BCRP is located in membrane rafts and that cholesterol has impact on its efflux activity.

Abcg2/Bcrp1 Mediates the Polarized Transport of Antiretroviral Nucleosides Abacavir and Zidovudine

The bioavailability and targeted distribution of abacavir (ABC) and zidovudine (AZT) to viral reservoirs may be influenced by efflux transporters. The purpose of this study was to characterize the interaction of these nucleoside reverse transcriptase inhibitors with the Abcg2/Bcrp1 transporter, the murine homolog of human breast cancer resistance protein (BCRP), using a Bcrp1-transfected Madin-Darby canine kidney II cell model. Intracellular accumulation of ABC and AZT was significantly reduced by approximately 90% and approximately 70%, respectively, in Bcrp1-transfected cells compared with the wild-type cells. Both ABC and AZT showed significantly increased basolateral-to-apical (B-to-A) and decreased apical-to-basolateral (A-to-B) transport in Bcrp1 cells compared with wild-type directional flux. The efflux ratio (ratio of B-to-A to A-to-B) in Bcrp1-transfected cells was 22 for ABC and 11 for AZT. N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918) inhibited this difference in accumulation between the two cell variants with an EC(50) of 1.32 +/- 0.3 microM for ABC and 0.31 +/- 0.1 microM for AZT. Potent and highly cooperative inhibition by Ko143 (3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)-propionic acid tert-butyl ester) was observed with an EC(50) of 121 +/- 5 nM for ABC and 19.2 +/- 1.5 nM for AZT (Hill coefficient approximately 3-6). Probenecid, an organic anion inhibitor known to influence AZT biodistribution, had no effect on cellular accumulation in the Bcrp1 model. These studies characterize the Bcrp1-mediated transport of ABC and AZT and show that prototypical BCRP inhibitors GF120918 and Ko143 can inhibit the Bcrp1-mediated transport of these important antiretroviral compounds. The functional expression of BCRP at critical barriers, such as the intestinal enterocytes, brain capillary endothelium, and target lymphocytes, could influence the bioavailability and targeted delivery of these drugs to sanctuary sites.