cell culture models in the study of drug efflux transporters mgr. martina Čečková department of...
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Cell culture models in the Cell culture models in the study of study of
drug efflux transportersdrug efflux transporters
Mgr. Martina ČečkováMgr. Martina Čečková
Department of Pharmacology and ToxicologyDepartment of Pharmacology and ToxicologyCharles University in PragueCharles University in Prague
Faculty of Pharmacy in Hradec KrálovéFaculty of Pharmacy in Hradec Králové
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Cell culture modelsCell culture models
Primary cultures
Cell lines
• immortalized cells
• cancer cell lines
Genetically modified cell lines
Cell cultures as a model of biological barriers
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GENETICALLY MODIFIED CELLSGENETICALLY MODIFIED CELLS
MDCKII-parent
MDCKII-MDR1
MDCKII-BCRP
MDCKII-MRP2
MEF 3.8-parent
MEF 3.8-BCRP
• Transport assays
• Accumulation / efflux assays
• ATPase assays
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TRANSPORT ASSAYS
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Transepithelial transport of PhiP
Pavek P., et al: 2005; J Pharmacol Exp Ther, 312(1): 144-52.
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ACCUMULATION EXPERIMENTS
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Interactions between BCRP and antiepilepticsInteractions between BCRP and antiepileptics
Accumulation of BODIPY- prazosin in MEF3.8-BCRP cells
Cerveny L., et al., 2005
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CELL CULTURES AS A MODEL OF BIOLOGICAL CELL CULTURES AS A MODEL OF BIOLOGICAL BARRIERSBARRIERS
Intestine: Caco-2, HCT-8, HT-29, SW-480…
Kidney: HK-2Liver: Cultured hepatocytes HepG2
Blood-brain barrier: BCEC SV-HCEC, HBEC-51, BB19Blood-testis barrier: isolated Sertoli cellsPlacenta: BeWo, JEG3, JAR
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PLACENTAPLACENTA
BeWoBeWo
BeWo BeWo BeWo BeWo
in vitro model of human trophoblast – cell line BeWo
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• Number of transcripts per microgram of total RNA (Real time RT-PCR)
Transcript (104)
Human term placenta
BeWoMean ± S.D. Minimum-Maximum
BCRP 104.4 ± 16.2 77.6 - 128.1 626.7
MDR1 8.9 ± 2.5 6.3 - 13.3 N.D.
Expression of BCRP in BeWo cell line
Ceckova M., et al., Clin Exp Pharmacol Physiol, 2005
• Immunodetection of BCRP (Western blotting, BXP-21 antibody)
A - MDCKII
B - MDCKII – BCRP
C – BeWo
D – human term placenta
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Localization of BCRP in the BeWo cells
• Indirect immunofluorescence microscopy (BXP-21 antibody)
BCRP transporter is localized predominantly in the apical plasma membrane
Ceckova M., et al., Clin Exp Pharmacol Physiol, 2005
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Functional expression of BCRP in BeWo cellsFunctional expression of BCRP in BeWo cells
• Accumulation assay FACS analysisSubstrate: 20µM mitoxantrone
Inhibitors: GF120918, Ko143, PSC833
BCRP actively pumps mitoxantrone out of BeWo cells
Ceckova M., et al., Clin Exp Pharmacol Physiol, 2005
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BCRP pumps potentially toxic xenobiotics from trophoblast cells
in feto-maternal direction
BeWoBeWo
BeWo BeWo BeWo BeWo
BCRP
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Poděkování
• Department of Pharmacology and ToxicologyCharles University in Prague, Faculty of Pharmacy in Hradec Kralove
Doc. PharmDr. František Štaud, Ph.D.PharmDr. Petr Pávek, Ph.D.Mgr. Lukáš ČervenýMgr. Antonín LibraProf. MUDr. Zdeněk Fendrich, CSc.
• Department of Pathophysiology, Medical University of Vienna
Univ. Prof. DI Dr. Renate FuchsDr. Marianne Brabec
• Institute of Clinical Immunology and Alergology, Faculty Hospital, Hradec Kralove
PharmDr. Doris Vokurková