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Free keywords:
SPECTROMETRY-BASED QUANTIFICATION; MULTIENZYME DIGESTION FASP; CANCER
RESISTANCE PROTEIN; MDCK CELLS; P-GLYCOPROTEIN; CACO-2 CELLS; DRUG
TRANSPORTERS; 2 STRAINS; ABSOLUTE QUANTIFICATION; MULTIDRUG-RESISTANCEResearch & Experimental Medicine; Pharmacology & Pharmacy; Label-freeproteomics; total protein analysis (TPA); MDCK; epithelial
proteome; ADME protein abundances;
Abstract:
Madin-Darbycanine kidney (MDCK) cells are widely used tostudy epithelial cell functionality. Their low endogenous drug transporterprotein levels make them an amenable system to investigate transepithelialpermeation and drug transporter protein activity after their transfection.MDCK cells display diverse phenotypic traits, and as such, laboratory-to-laboratoryvariability in drug permeability assessments is observed. Consequently,in vitro-in vivo extrapolation (IVIVE) approaches using permeabilityand/or transporter activity data require calibration. A comprehensiveproteomic quantification of 11 filter-grown parental or mock-transfectedMDCK monolayers from 8 different pharmaceutical laboratories usingthe total protein approach (TPA) is provided. The TPA enables estimationsof key morphometric parameters such as monolayer cellularity and volume.Overall, metabolic liability to xenobiotics is likely to be limitedfor MDCK cells due to the low expression of required enzymes. SLC16A1 (MCT1) was the highest abundant SLC transporterlinked to xenobiotic activity, while ABCC4 (MRP4)was the highest abundant ABC transporter. Our data supports existingfindings that claudin-2 levels may be linked to tight junction modulation,thus impacting trans-epithelial resistance. This unique database providesdata on more than 8000 protein copy numbers and concentrations, thusallowing an in-depth appraisal of the control monolayers used in eachlaboratory.
