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

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

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

TRANSPORT ASSAYS

Transepithelial transport of PhiP

Pavek P., et al: 2005; J Pharmacol Exp Ther, 312(1): 144-52.

ACCUMULATION EXPERIMENTS

Interactions between BCRP and antiepilepticsInteractions between BCRP and antiepileptics

Accumulation of BODIPY- prazosin in MEF3.8-BCRP cells

Cerveny L., et al., 2005

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

PLACENTAPLACENTA

BeWoBeWo

BeWo BeWo BeWo BeWo

in vitro model of human trophoblast – cell line BeWo

• 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

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

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

BCRP pumps potentially toxic xenobiotics from trophoblast cells

in feto-maternal direction

BeWoBeWo

BeWo BeWo BeWo BeWo

BCRP

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