Evaluation of a vincristine resistant Caco-2 cell line for use in a calcein AM extrusion screening assay for P-glycoprotein interaction

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Abstract

Aim: To develop a fast fluorometric screening assay based on vincristine resistant Caco-2 cells (Caco-2VCR) in order to elucidate potential P-glycoprotein (Pgp) interactions of compounds, and to characterise Caco-2VCR cells with regard to their expression of the efflux transporters Pgp, MRP1 and MRP2. Methods: We applied the Caco-2VCR cells to a 96-well plate-based calcein AM extrusion assay. The Caco-2VCR cells were cultured as monolayers and incubated with calcein AM with/without addition of Pgp modulators. Fourteen known Pgp modulators were tested in the assay (chloropromazine, cyclosporin A, domperidone, digoxin, ivermectin, ketoconazole, loperamide, metoprolol, propranolol, progesterone, quinidine, quinine, verapamil and vincristine). For each compound an EC50 value was calculated. Protein and mRNA levels of the efflux transporters were analysed by Western blot and polymerase chain reaction techniques. Results: All compounds with the exception of digoxin displayed increased calcein levels. Protein and mRNA analysis showed increased levels of Pgp after vincristine exposure, while expression of the efflux transporters MRP1 and MRP2 remained unchanged. Conclusions: The calcein AM extrusion assay applied to Caco-2VCR cells can be a valuable tool as a screening assay for new compounds and their potential interaction with P-glycoprotein.

Introduction

The phenomenon of multidrug resistance (MDR) was first observed in tumour cell lines. These cell lines showed a capability to actively pump compounds out of the cells as a protective mechanism to limit distribution and toxicity of substrate drugs in cells. They did not only develop resistance to one cytotoxic compound, but also showed cross resistance to several other unrelated cytotoxic agents. This cross resistance was found later to be correlated to an efflux protein called P-glycoprotein, a Mr 170 kDa transmembrane protein (Endicott and Ling, 1989).

Thereafter it was found that some of these multidrug resistant tumour cell lines contained only low levels of P-glycoprotein (Pgp), and instead hyperexpressed another efflux protein called the multidrug resistance-associated protein, MRP, a Mr 190 kDa protein (Cole et al., 1992). Besides the MRP (MRP1) protein there are at least five MRP homologues expressed in humans, designated MRP2 to MRP6. Only MRP1 and MRP2 showed correlation between their expression and multidrug resistance (Kool et al., 1997).

P-Glycoprotein, MRP1 and MRP2 are all members of the ATP-binding cassette (ABC) superfamily of transporters. The enzyme MRP2 (cMOAT; canalicular multispecific organic anion transporter) is an isoform of MRP1 and with similar substrate specificity (Keppler et al., 1998).

The localisation of these efflux proteins are quite similar in human tissue.

P-Glycoprotein is found in kidney, adrenal, brain vessels, muscle, lung, pancreas, liver, intestine, placenta, testis and stomach, and in tumour cell lines. MRP1 is also found in these tissues except in the liver, where no or low levels are detected. MRP2 is found in the liver and intestine (Gatmaitan and Arias, 1993, Huai-Yun et al., 1998, Peng et al., 1999, Sagawara et al., 1997). On the cellular level MRP1 is expressed in the basolateral membrane, while Pgp and MRP2 are expressed mainly in the apical region in polarised cells.

Since it has been shown that Pgp can influence absorption of compounds (Schinkel et al., 1994, Schinkel et al., 1996, Sparreboom et al., 1997, Teraro et al., 1996), and thereby their distribution, especially in brain, it is of importance to screen new chemical entities (NCEs) for their Pgp affinity at an early phase of drug development. Recently, Döppenschmitt et al. (1998), have developed a receptor–ligand binding (RLB) screening assay for Pgp interaction. In this assay vinblastine treated Caco-2 cells and 3H-Verapamil were used as model cell line and radioligand, respectively.

The aim of this work was to establish a microtiter plate-based fluorometric assay as a fast screening tool for Pgp interaction of NCEs, and to characterise vincristine-resistant sublines of the adenocarcinoma Caco-2 cell line (Caco-2VCR) with regard to their Pgp, MRP1 and MRP2 expression. The Caco-2VCR cells were used as a model cell line and calcein AM (calcein acetoxymethyl ester) was applied as a probe for Pgp mediated transport.

Section snippets

Caco-2 cells

Wild type Caco-2 cell line (wtCaco-2) at passage 24 was a kind gift from AstraZeneca R&D (Mölndal, Sweden) and the cells were cultured in +DMEM (Dulbecco’s Modified Eagle Medium with glucose and l-glutamine) containing 10% foetal bovine serum (FBS), 100 IU/ml penicillin, 100 μg/ml streptomycin and 1% (v/v) 100x non essential amino acids (NEAs).

P-Glycoprotein over expressing cell lines Caco-2VCR25 and Caco-2VCR100

The vincristine sulfate resistant sublines were obtained from wtCaco-2 cells exposed to 10 nM vincristine sulfate at passage 41 for at least eight

Expression of MDR1/P-glycoprotein, MRP1 and MRP2

The analysis of mRNA and protein levels of the efflux proteins P-glycoprotein, MRP1 and MRP2 (Table 1 and Fig. 2.) showed that they all were present in wtCaco-2, Caco-2VCR25 and Caco-2VCR100 cell lines, but that only the expression of Pgp was increased in the Caco-2VCR cells. The expression levels of MRP1 and MRP2 remained unchanged after vincristine treatment.

In the vincristine resistant Caco-2 cells a two- and a fourfold increase of MDR1 mRNA expression were observed in Caco-2VCR25 and

Discussion

The importance of efflux proteins Pgp, MRP1 and MRP2 on intestinal drug absorption and permeation of the blood–brain barrier (BBB) of various compounds is still under debate. During the last 5 years, several reports have been published that show the importance of these proteins in drug distribution, especially in brain (Schinkel et al., 1994).

Schinkel et al. (1994) developed a mouse strain that was lacking the mdr1a gene (Pgp), which is normally abundant in the BBB in mice. They demonstrated

Acknowledgements

We thank Dr. Johan Gabrielsson (AstraZeneca R&D, Södertälje, Sweden) for his PK/PD assistance and Professor Per Artursson for valuable advice.

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