Evaluation of a vincristine resistant Caco-2 cell line for use in a calcein AM extrusion screening assay for P-glycoprotein interaction
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.
References (33)
- et al.
Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction
Anal. Biochem.
(1987) - et al.
Transport properties of the multidrug resistance-associated protein (MRP) in human tumour cells
FEBS Lett.
(1996) - et al.
Expression of multidrug resistance-associated protein (MRP) in brain microvessel endothelial cells
Biochem. Biophys. Res. Commun.
(1998) - et al.
Induction of cmrp/cmoat gene expression by cisplatin, 2-acetylaminofluorene, or cycloheximide in hepatocytes
Hepatology
(1997) - et al.
ATP-dependent transport of glutathione S-conjugates by the multidrug resistance protein MRP1 and its apical isoform MRP2
Chemico-Biol. Int.
(1998) - et al.
Microfluorometric evaluation of calcein acetoxymethyl ester as a probe for P-glycoprotein-mediated resistance: effects of cyclosporin A and its nonimmunosuppresive analogue SDZ PSC 833
Exp. Cell Res.
(1994) Photoaffinity labels for characterizing drug interaction sites of P-glycoprotein
- et al.
Disruption of the mouse mdr1a p-glycoprotein gene leads to a deficiency in blood–brain barrier and to increased sensitivity to drugs
Cell
(1994) - et al.
Experimental modulation of MRP (multidrug resistance-associated protein)-mediated resistance
Eur. J. Cancer
(1996) - et al.
Expression of the multidrug resistance-associated protein (MRP) gene correlates with amplification and over-expression of the N-myc oncogene in childhood neuroblastoma
Cancer Res.
(1994)
Transport and epithelial secretion of the cardiac glycoside, digoxin, by human intestinal epithelial (Caco-2) cells
Br. J. Pharmacol.
Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line
Science.
Handling of doxorubicin by the LLC-PK1 kidney epithelial cell line
J. Pharmacol. Exp. Ther.
Characterization of binding properties to human P-glycoprotein: development of a [3H]verapamil radioligand-binding assay
J. Pharmacol. Exp. Ther.
Radioligand-binding assay employing P-glycoprotein-overexpressing cells: testing drug affinities to the secretory intestinal multidrug transporter
Pharm. Res.
The biochemistry of P-glycoprotein-mediated multidrug resistance
Annu. Rev. Biochem.
Cited by (98)
Role of P-glycoprotein in deoxynivalenol-mediated in vitro toxicity
2018, Toxicology LettersMultiparametric luminescent cell viability assay in toxicology models: A critical evaluation
2016, Journal of Pharmacological and Toxicological MethodsCitation Excerpt :The fluorescent signal derived from FDB showed good correlation with Jurkat cell number (R2 = 0.959, FDB: 1.6 μM, 1 × 104–1 × 106 cells/mL, n = 42). It is well known that fluorescence polarization technique gives information on the motional freedom of fluorescent molecules (Cholujová et al., 2008; Eneroth et al., 2001). Due to the low background fluorescence of non-hydrolyzed dyes, in the case of cell suspensions, there is no need to lyse the cells and although the formed fluorescein easily leaks out from the cells this fact does not falsify the results.
Haemonchus contortus P-glycoprotein-2: In situ localisation and characterisation of macrocyclic lactone transport
2015, International Journal for ParasitologyA novel concentration dependent amino acid ion pair strategy to mediate drug permeation using indomethacin as a model insoluble drug
2014, European Journal of Pharmaceutical SciencesCitation Excerpt :After differentiation, the cells express many enzymes such as alkaline phosphatase, sucrase isomaltase and aminopeptidase which are characteristic for enterocyte brush border microvilli. Caco-2 cells also express a lot of transporter protein and therefore they have been used successively to evaluate carrier mediated uptake of B-lactam antibiotics (Inui et al., 1988), amino acids (Thwaites et al., 1995), amino acids analogue (Hu and Borchardt, 1990) and efflux transport (Eneroth et al., 2001). Indomethacin (TLC ⩾ 99%), l-arginine (non-animal source), l-lysine, 1-octanol (ACS spectrophotometric grade ⩾99%) and ninhydrin reagent (2% solution) were purchased from Sigma Aldrich, UK.