Evaluation of a vincristine resistant Caco-2 cell line for use in a calcein AM extrusion screening assay for P-glycoprotein interaction

Eur J Pharm Sci. 2001 Jan;12(3):205-14. doi: 10.1016/s0928-0987(00)00117-2.


Aim: To develop a fast fluorometric screening assay based on vincristine resistant Caco-2 cells (Caco-2VCR) in order to elucidate potential P-glycoprotein (Pgp) interactions of compounds, and to characterise Caco-2VCR cells with regard to their expression of the efflux transporters Pgp, MRP1 and MRP2.

Methods: We applied the Caco-2VCR cells to a 96-well plate-based calcein AM extrusion assay. The Caco-2VCR cells were cultured as monolayers and incubated with calcein AM with/without addition of Pgp modulators. Fourteen known Pgp modulators were tested in the assay (chloropromazine, cyclosporin A, domperidone, digoxin, ivermectin, ketoconazole, loperamide, metoprolol, propranolol, progesterone, quinidine, quinine, verapamil and vincristine). For each compound an EC50 value was calculated. Protein and mRNA levels of the efflux transporters were analysed by Western blot and polymerase chain reaction techniques.

Results: All compounds with the exception of digoxin displayed increased calcein levels. Protein and mRNA analysis showed increased levels of Pgp after vincristine exposure, while expression of the efflux transporters MRP1 and MRP2 remained unchanged.

Conclusions: The calcein AM extrusion assay applied to Caco-2VCR cells can be a valuable tool as a screening assay for new compounds and their potential interaction with P-glycoprotein.

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / metabolism*
  • Algorithms
  • Antineoplastic Agents, Phytogenic / pharmacology*
  • Blotting, Western
  • Caco-2 Cells
  • Drug Evaluation, Preclinical
  • Drug Resistance, Neoplasm
  • Fluoresceins / metabolism*
  • Fluorescent Dyes / metabolism*
  • Glyceraldehyde-3-Phosphate Dehydrogenases / metabolism
  • Humans
  • Indicators and Reagents
  • Models, Biological
  • RNA, Messenger / biosynthesis
  • Reverse Transcriptase Polymerase Chain Reaction
  • Vincristine / pharmacology*


  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • Antineoplastic Agents, Phytogenic
  • Fluoresceins
  • Fluorescent Dyes
  • Indicators and Reagents
  • RNA, Messenger
  • calcein AM
  • Vincristine
  • Glyceraldehyde-3-Phosphate Dehydrogenases