Abstract
P-glycoprotein expression was demonstrated in two human intestinal adenocarcinoma cell-lines (HCT-8, ileocaecal and T84, colonic) by immunoprecipitation of a 170-180 kDa protein with monoclonal antibody JSB-1. Both HCT-8 and T84 formed functional epithelial cell layers of high transepithelial electrical resistance (greater than 700 omega.cm2) when grown on permeable matrices. These epithelial layers demonstrated vectorial secretion (net vinblastine fluxes in the basal-to-apical direction of 0.135 and 0.452 pmol h-1 cm-2 in HCT-8 and T84 cell layers, respectively, from bathing solutions containing 10 nM vinblastine). These vectorial vinblastine secretions were sensitive to inhibition by verapamil. Passive transepithelial vinblastine permeation was limited by the presence of intercellular (tight) junctions, as demonstrated by the high transepithelial electrical resistance, and verapamil increased this passive vinblastine permeation concomitant with a reduction in the electrical resistance. Cellular vinblastine loading was significantly greater from the basal side, and this was also susceptible to inhibition by basal verapamil. The demonstration of vectorial transport of vinblastine in human intestinal colonic adenocarcinoma cell layers is direct evidence in favour of the hypothesis that the function of mdr1 in epithelial from the gastrointestinal tract is to promote detoxification by a process of epithelial secretion. This study also highlights that cellular vinblastine accumulation depends not only upon P-glycoprotein function, but also upon differential apparent membrane permeabilities and the presence of intercellular (tight) junctions that may restrict drug permeation and cellular accumulation to apical or basal membrane domains.
Highlights
Gastrointestinal Drug Delivery Research Centre and Department of Physiological Sciences, University of Newcastle upon Tyne, Medical School, Newcastle upon Tyne NE2 4HH, UK
With HCT-8 cells grown as monolayers on plastic plates, cellular uptake and loss of vinblastine sulphate (Figure 1) will represent the relative rates of influx and efflux mainly across the apical cell border
Verapamil increased the extent of cellular uptake of vinblastine by 38% at 5 h (Student's t-test, P
Summary
HCT-8 and T84 cells were obtained from the ATCC (Rockville, Maryland 20852 USA). HCT-8 cells were maintained in RPMI 1640 with 10% horse serum, 1 mM glutamine, 1 mM sodium pyruvate, 40 IU ml-' penicillin and 40 fig ml1I streptomycin. T84 cells were grown in a 1: 1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 with 5% newborn calf serum and 200 IU ml-' penicillin, 200 ,g ml-l streptomycin. It should be noted that the cell lines used in this present study have not been selected for drug resistance (Tompkins et al, 1974; Dharmsathaphorn et al, 1984). Confluent monolayers were subcultured every 5-7 (HCT-8) days or 14-16 days (T84), by treatment with 0.05% trypsin and 0.02% EDTA in Ca2+-and Mg2+-free phosphatebuffered saline (PBS). The cultures were incubated at 37°C in a humidified atmosphere of 5% C02/95% air
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