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Characterization and application of a vinblastine-selected CACO-2 cell line for evaluation of p-glycoprotein
Dennis A Laska1, Jack O Houchins, Susan E Pratt
1Department of Toxicology, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA. dalpsu@lily.com
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.
Insights
We developed a new Caco-2 VinB cell line to study P-glycoprotein (Pgp) drug efflux in the intestine. This cell line enhances Pgp expression, aiding in the development of new drugs and understanding drug interactions.
Area of Science:
- Pharmacology
- Cell Biology
- Drug Metabolism
Background:
- Adenosine triphosphate-binding cassette (ABC) transporters, particularly P-glycoprotein (Pgp), are crucial in multidrug resistance and influence drug absorption and elimination.
- Investigating Pgp's role in intestinal xenobiotic efflux is vital for pharmaceutical development.
- Caco-2 cells are a widely used model for intestinal drug transport studies.
Purpose of the Study:
- To develop and characterize a novel Caco-2 cell line with enhanced P-glycoprotein (Pgp) expression for studying intestinal drug efflux.
- To establish a fluorescent-based assay for evaluating modulators of Pgp activity.
- To assess the morphological and functional changes in the selected cell line.
Main Methods:
- Caco-2 cells were propagated in the presence of vinblastine to select for cells with high Pgp expression.
- A fluorescent-based assay was developed to study Pgp activity and chemical modulators.
- Morphological analysis was performed using transmission, scanning, and confocal microscopy.
- Western blot analysis and confocal microscopy were used to confirm Pgp expression and localization.
- Transport assays were conducted to functionally demonstrate Pgp activity.
Main Results:
- The selected Caco-2 VinB cell line exhibited significantly increased Pgp expression compared to native Caco-2 cells.
- Pgp was localized to the apical surface of the Caco-2 VinB cells.
- The developed assay successfully demonstrated differential inhibition of Pgp activity by known inhibitors.
- Morphological evaluation showed polarized columnar epithelium in both cell lines, though Caco-2 VinB cells had reduced phenotypic diversity.
Conclusions:
- The Caco-2 VinB cell line is a valuable tool for studying intestinal Pgp-mediated xenobiotic efflux.
- This cell line facilitates the development of assays to identify Pgp modulators, aiding pharmaceutical drug development.
- The enhanced Pgp expression and functional activity make Caco-2 VinB cells suitable for predicting drug interactions and bioavailability.