Epithelial secretion of vinblastine by human intestinal adenocarcinoma cell (HCT-8 and T84) layers expressing P-glycoprotein

Abstract

Summary P-glycoprotein expression was demonstrated in two human intestinal adenocarcinoma cell-lines 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 (>700 Q 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-' 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 mdrl in epithelia 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.

Volume‐activated Cl− secretion and transepithelial vinblastine secretion mediated by P‐glycoprotein are not correlated in cultured human T84 intestinal epithelial layers

“…P-glycoprotein is expressed in the apical poles of intestinal enterocytes [6] and the existence of polarised secretion of vinblastine by intact T~ monolayers is consistent with its iocalisation within the apical membrane [4].…”

“…Measurements of transepithelial solute flux were made essentially as described by Hunter et al [4]. Functional epithelial layers in filter cups were washed with 2×3 ml serum.free medium and placed into fresh 6-well plates containing 3 ml serum-free med;xm (basal solution), a further 3 ml serum-free medium was then pipetted into the upper chamber (apical solution) of the filter cup.…”

“…beeco's modified Eagles Medium and Hams's F12 w':th 5% new-born calf serum, 200 IU.ml -~ of penicillin, 200/~g.ml -~ streptomycin. T~ cells were grown as epithelial layers upon Anocell 25 mm culture inserts coated with rat-tail collagen using high-density seeding, as previously described [4,14]. Cultures were grown for typically 2 weeks, in 6-well plates at 37°C, 5% CO., witla media replacement ever~ 3 days.…”

“…Epithelial monolayers of T~ human colonic adenocarcinoma cells express both CFTR [3] and MDR1 [4] and this is associated with cAMP-stimulated transepithelial CI-secretion [2,5] and transepithelial vinblastine secretion [4], respectively. P-glycoprotein is expressed in the apical poles of intestinal enterocytes [6] and the existence of polarised secretion of vinblastine by intact T~ monolayers is consistent with its iocalisation within the apical membrane [4].…”

Cholangiocarcinoma: current knowledge and future perspectives consensus statement from the European Network for the Study of Cholangiocarcinoma (ENS-CCA)

“…Export pumps of the ATP-binding cassette (ABC) superfamily of proteins are key elements determining the intracellular concentration of drugs (MOC-1b). Multidrug resistance protein 1, highly expressed in healthy cholangiocytes 280 , is able to export a large variety of anticancer drugs (for example, doxorubicin, etoposide, paclitaxel and vinblastine) 281,282 , hence reducing their efficacy in CCA 283,284 . Members of the ABCC family, MRP1, MRP3, MRP4 and MRP5, might also be involved in CCA chemoresistance 279,285,286 .…”