Cell culture models in the study of drug efflux transporters

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Cell culture models in the study of drug efflux
transporters

• Mgr. Martina Cecková
• Department of Pharmacology and
  ToxicologyCharles University in PragueFaculty
  of Pharmacy in Hradec Králové

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Cell 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 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 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
BARRIERS
Intestine Caco-2, HCT-8, HT-29,
SW-480 Kidney HK-2 Liver Cultured
hepatocytes HepG2 Blood-brain barrier
BCEC SV-HCEC, HBEC-51, BB19 Blood-testis
barrier isolated Sertoli cells Placenta
BeWo, JEG3, JAR
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PLACENTA
in vitro model of human trophoblast cell line
BeWo
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Expression of BCRP in BeWo cell line

• Number of transcripts per microgram of total RNA
  (Real time RT-PCR)

Transcript (104) Human term placenta Human term placenta BeWo
Transcript (104) Mean S.D. Minimum-Maximum BeWo
BCRP 104.4 16.2 77.6 - 128.1 626.7
MDR1 8.9 2.5 6.3 - 13.3 N.D.

• Immunodetection of BCRP (Western blotting,
  BXP-21 antibody)

Ceckova M., et al., Clin Exp Pharmacol Physiol,
2005
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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 cells

• Accumulation assay ? FACS analysis
• Substrate 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

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Podekování

• Department of Pharmacology and Toxicology
• Charles University in Prague, Faculty of
  Pharmacy in Hradec Kralove
• Doc. PharmDr. František Štaud, Ph.D.
• PharmDr. Petr Pávek, Ph.D.
• Mgr. Lukáš Cervený
• Mgr. Antonín Libra
• Prof. MUDr. Zdenek Fendrich, CSc.
• Department of Pathophysiology, Medical University
  of Vienna
• Univ. Prof. DI Dr. Renate Fuchs
• Dr. Marianne Brabec
• Institute of Clinical Immunology and Alergology,
  Faculty Hospital, Hradec Kralove
• PharmDr. Doris Vokurková