title: Characterization and application of a vinblastine-selected CACO-2 cell line for evaluation of p-glycoprotein.
abstract: The role of the adenosine triphosphate-binding cassette (ABC) superfamily of membrane transporters is well documented in tumor cell multidrug resistance. More recently, growing evidence of their influence on oral bioavailability, drug excretion rates, and drug-drug interaction potential at the intestinal level has stimulated much investigation. Our laboratory is interested in evaluating the apical (AP) ABC transporter P-glycoprotein (Pgp [mdr-1]) for its role in xenobiotic efflux at the intestinal level. We propagated Caco-2 cells in the presence of vinblastine (a cytotoxic, Pgp substrate) to promote transporter expression though selection. That is, the cell population expressing Pgp, or with the capacity to up-regulate Pgp expression, survived and expanded in the presence of vinblastine. We have used this selected cell line (Caco-2 VinB) to develop a fluorescent-based assay to study the chemical modulators of Pgp activity. Using the Caco-2 VinB cells, we have successfully demonstrated the differential potency of previously characterized Pgp inhibitors. In addition, we conducted a morphological evaluation of the two cell lines using transmission, scanning, and confocal microscopy. Both cell strains differentiated into highly functional, polarized columnar epithelium, although the vinblastine-selected cell line had lost the phenotypic diversity observed in native Caco-2 populations. Increased Pgp expression was noted in Caco-2 VinB cells compared with the native cell line on Western blot analysis, which was localized to the AP surface using confocal microscopy and functionally demonstrated using transport assays. We believe that the Caco2 VinB cell line is a versatile tool for application in pharmaceutical drug development.
authors.affiliation: Department of Toxicology, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA. dalpsu@lily.com
authors.forename: Dennis A
authors.initials: DA
authors.lastname: Laska
authors.forename: Jack O
authors.initials: JO
authors.lastname: Houchins
authors.forename: Susan E
authors.initials: SE
authors.lastname: Pratt
authors.forename: Jeffery
authors.initials: J
authors.lastname: Horn
authors.forename: Xialong
authors.initials: X
authors.lastname: Xia
authors.forename: Brenda R
authors.initials: BR
authors.lastname: Hanssen
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Williams
authors.forename: Anne H
authors.initials: AH
authors.lastname: Dantzig
authors.forename: Terry
authors.initials: T
authors.lastname: Lindstrom
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D015153:Blotting, Western; D018938:Caco-2 Cells; D002462:Cell Membrane / Q000187:drug effects / Q000648:ultrastructure; D002470:Cell Survival / Q000187:drug effects; D004077:Digoxin / Q000378:metabolism; D019008:Drug Resistance, Neoplasm; D005452:Fluoresceins / Q000378:metabolism; D005453:Fluorescence; D006801:Humans; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D014747:Vinblastine / Q000494:pharmacology*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D005452:Fluoresceins; D014747:Vinblastine; D004077:Digoxin; C007740:fluorexon