A fluorescence-based in vitro assay for drug interactions with breast cancer resistance protein (BCRP, ABCG2)

Eur J Pharm Biopharm. 2009 Aug;72(3):605-13. doi: 10.1016/j.ejpb.2009.01.010.


Purpose: To establish a fluorescence-based assay for drug interactions with the ABC-export-protein BCRP (ABCG2).

Methods: BCRP expression was verified by immunostaining and Western blots in intact porcine brain capillaries, isolated endothelial cells (PBCECs) and in MDCKII-cells over-expressing human wild-type BCRP (MDCKII-hBCRP). Transport of fluorescent mitoxantrone across cells was determined to assess a preferred transport direction. Sensitivity of cultured cells versus mitoxantrone in the absence and in the presence of transport modulators was examined at increasing concentrations of the cytostatic using the AlamarBlue assay. In addition, cells were incubated with mitoxantrone in the absence and presence of increasing concentrations of different compounds with the potential to interact with BCRP. Intracellular fluorescence accumulation was measured using a flow cytometer.

Results: Isolated capillaries as well as 7-day old PBCECs showed expression of BCRP. Cell sensitivity to mitoxantrone significantly increased in the presence of the BCRP inhibitors KO143 and GF120918. Transport of mitoxantrone across PBCEC monolayers was directed with P(app) (apical to basolateral) 5.6 x 10(-6) cm s(-1) and with P(app) (basolateral to apical) 2.8 x 10(-5) cm s(-1). FACS analysis revealed a different extent of fluorescence accumulation dependent on the kind and concentration of BCRP modulating compounds.

Conclusions: The mitoxantrone-based assay can be used as a rapid FACS screening system to assess drug interactions with BCRP at the blood-brain barrier and therefore represents a useful tool in drug profiling.

Publication types

  • Comparative Study

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily G, Member 2
  • ATP-Binding Cassette Transporters / analysis
  • ATP-Binding Cassette Transporters / metabolism*
  • Animals
  • Antineoplastic Agents / pharmacokinetics
  • Boron Compounds
  • Cell Line
  • Cell Separation / methods*
  • Cell Survival / drug effects
  • Cell Survival / physiology
  • Cells, Cultured
  • Dogs
  • Drug Interactions / physiology
  • Drug Resistance, Multiple / physiology*
  • Female
  • Flow Cytometry / methods*
  • Fluorescent Dyes / metabolism*
  • Humans
  • Neoplasm Proteins / analysis
  • Neoplasm Proteins / metabolism*
  • Rats
  • Swine


  • 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
  • ABCG2 protein, human
  • ATP Binding Cassette Transporter, Subfamily G, Member 2
  • ATP-Binding Cassette Transporters
  • Antineoplastic Agents
  • Boron Compounds
  • Fluorescent Dyes
  • Neoplasm Proteins