title: Fluorimetric Methods for Analysis of Permeability, Drug Transport Kinetics, and Inhibition of the ABCB1 Membrane Transporter.
abstract: The cell membrane P-glycoprotein (P-gp; MDR1, ABCB1) is an energy-dependent efflux pump that belongs to the ATP-binding cassette (ABC) family of transporters, and has been associated with drug resistance in eukaryotic cells. Multidrug resistance (MDR) is related to an increased expression and function of the ABCB1 (P-gp) efflux pump that often causes chemotherapeutic failure in cancer. Modulators of this efflux pump, such as the calcium channel blocker verapamil (VP) and cyclosporine A (CypA), can reverse the MDR phenotype but in vivo studies have revealed disappointing results due to adverse side effects. Currently available methods are unable to visualize and assess in a real-time basis the effectiveness of ABCB1 inhibitors on the uptake and efflux of ABCB1 substrates. However, predicting and testing ABCB1 modulation activity using living cells during drug development are crucial. The use of ABCB1-transfected mouse T-lymphoma cell line to study the uptake/efflux of fluorescent probes like ethidium bromide (EB), rhodamine 123 (Rh-123), and carbocyanine dye DiOC2, in the presence and absence of potential inhibitors, is currently used in our laboratories to evaluate the ability of a drug to inhibit ABCB1-mediated drug accumulation and efflux. Here we describe and compare three in vitro methods, which evaluate the permeability, transport kinetics of fluorescent substrates, and inhibition of the ABCB1 efflux pump by drugs of chemical synthesis or extracted from natural sources, using model cancer cell lines overexpressing this transporter, namely (1) real-time fluorimetry that assesses the accumulation of ethidium bromide, (2) flow cytometry, and (3) fluorescent microscopy using rhodamine 123 and DiOC2.
authors.affiliation: Grupo de Micobactérias, Unidade de Ensino e Investigação de Microbiologia Médica e Centro de Malária e Outras Doenças Tropicais, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisbon, Portugal.
authors.forename: Ana
authors.initials: A
authors.lastname: Armada
authors.affiliation: Centre for Toxicogenomics and Human Health, Genetics, Oncology and Human Toxicology, NOVA Medical School/Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Rua Câmara Pestana 6, Lisbon, 1150-008, Portugal.
authors.forename: Célia
authors.initials: C
authors.lastname: Martins
authors.affiliation: Department of Medical Microbiology and Immunobiology, Faculty of Medicine, University of Szeged, Szeged, Hungary.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Spengler
authors.affiliation: Department of Medical Microbiology and Immunobiology, Faculty of Medicine, University of Szeged, Szeged, Hungary.
authors.forename: Joseph
authors.initials: J
authors.lastname: Molnar
authors.affiliation: Department of Medical Microbiology and Immunobiology, Faculty of Medicine, University of Szeged, Szeged, Hungary.|Unidade de Medicina das Viagens, Centro de Malária e Outras Doenças Tropicais, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisbon, Portugal.
authors.forename: Leonard
authors.initials: L
authors.lastname: Amaral
authors.affiliation: Centre for Toxicogenomics and Human Health, Genetics, Oncology and Human Toxicology, NOVA Medical School/Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Rua Câmara Pestana 6, Lisbon, 1150-008, Portugal.
authors.forename: António Sebastião
authors.initials: AS
authors.lastname: Rodrigues
authors.affiliation: Grupo de Micobactérias, Unidade de Ensino e Investigação de Microbiologia Médica e Centro de Malária e Outras Doenças Tropicais, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Lisbon, Portugal. MViveiros@ihmt.unl.pt.
authors.forename: Miguel
authors.initials: M
authors.lastname: Viveiros
mesh_terms: D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D000818:Animals; D001688:Biological Products / Q000494:pharmacology; D001692:Biological Transport; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism*; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D007700:Kinetics; D051379:Mice; D008856:Microscopy, Fluorescence; D010539:Permeability
chemical_list: D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters; D001688:Biological Products; D005456:Fluorescent Dyes; C073119:TAP1 protein, human
keywords: ABCB1; Flow cytometry; MDR; MDR1; Real-time fluorimetry