Summary
The role of the adenosine triphosphate-binding cassette (ABC) superfamily of membrane transporters is well documented in tumor cell multidrug resistance. More recently, growing evidence of their influence on oral bioavailability, drug excretion rates, and drug-drug interaction potential at the intestinal level has stimulated much investigation. Our laboratory is interested in evaluating the apical (AP) ABC transporter P-glycoprotein (Pgp [mdr-1]) for its role in xenobiotic efflux at the intestinal level. We propagated Caco-2 cells in the presence of vinblastine (a cytotoxic, Pgp substrate) to promote transporter expression though selection. That is, the cell population expressing Pgp, or with the capacity to upregulate Pgp expression, survived and expanded in the presence of vinblastine. We have used this selected cell line (Caco-2 VinB) to develop a fluorescent-based assay to study the chemical modulators of Pgp activity. Using the Caco-2 VinB cells, we have successfully demonstrated the differential potency of previously characterized Pgp inhibitors. In addition, we conducted a morphological evaluation of the two cell lines using transmission, scanning, and confocal microscopy. Both cell strains differentiated into highly functional, polarized columnar epithelium, although the vinblastine-selected cell line had lost the phenotypic diversity observed in native Caco-2 populations. Increased Pgp expression was noted in Caco-2 VinB cells compared with the native cell line on Western blot analysis, which was localized to the AP surface using confocal microscopy and functionally demonstrated using transport assays. We believe that the Caco2 VinB cell line is a versatile tool for application in pharmaceutical drug development.
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Alderman, C.; Allcroft, P. Digoxin-itraconazole interaction: possible mechanisms. Ann. Pharmacother. 31:438–440;1998.
Anderle, P.; Neiderer, E.; Rubas, W.; Hilgendorf, C.; Spahn-Langguth, H.; Wunderli-Allenspach, H.; Merkle, H.; Langguth, P. P-glycoprotein (Pgp) mediated efflux in Caco-2 monolayers: the influence of culturing conditions and drug exposure on Pgp expression levels. J. Pharm. Sci. 87(6):757–762; 1998.
Arthursson, P.; Karlsson, J. Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells. Biochem. Biophys. Res. Commun. 175:880–885; 1991.
Cavet, M.; West, M.; Simons, N. Transport and epithelial secretion of the cardiac glycoside digoxin by human intestinal epithelial (Caco-2) cells. Br. J. Pharmacol. 118:1389–1396; 1996.
Cole, P.; Sparks, K.; Fraser, K.; Loe, D.; Grant, C.; Wilso, G.; Deeley, R. Pharmacological characterization of multi-drug resistant MRP-transfected human tumor cells. Cancer Res. 54:5902–5910; 1994.
Cui, Y.; König, J.; Buchholz, U.; Spring, H.; Leier, I.; keppler, D. Drug resistance and ATP-dependant conjugate transport mediated by the apial multi-drug resistance protein, MRP2, permanently expressed in human and canine cells. Mol. Pharmacol. 55:929–937; 1999.
Dantzig, A. H.; Bergin, L. Uptake of the cephalosporin, cephalexin, by a dipeptide transport carrier in the human intestinal cell line, Caco-2. Biochem. Biophys. Acta 1027:211–217; 1990.
Doppenschnitt, S.; Spahn-Langguth, H.; Regardh, C.; Langguth, P. Role of P-glucoprotein-mediated secretion in absorptive drug permeability: an approach using passive membrane permeability and affinity to P-glycoprotein. J. Pharm. Sci. 88:1067–1072; 1999.
Endicott, J.; Ling, V. The biochemistry of P-glycoprotein-mediated multi-drug resistance. Annu. Rev. Biochem. 58:137–171; 1989.
Eneroth, A.; Äström, E.; Hoogstraate, J.; Schrenk, D.; Conrad, S.; Kauffmann, H.; Gjellan, K. Evaluation of a vincristine resistant Caco-2 cell line for use in a calcein-AM extrusion screening assay for P-glycoprotein interactions. Eur J. Pharm. Sci. 12:205–214; 2001.
Essodaïgui, M.; Broxtermann, H.; Garnier-Suillerot, A. Kinetic analysis of calcein and calcein-acetoxymethylester efflux mediated by the multi-drug resistance protein and P-glycoprotein. Biochemistry 37:2243–2250; 1998.
Feller, N.; Broxterman, H.; Wahrer, D.; Pinedo, H. ATP-dependent efflux of calcein by the multi-drug resistance protein (MRP): no inhibition by intracellular glutathione depletion. FEBS Lett. 368:385–388; 1995.
Fogh, J. Submission of Caco-2 cell line to the American Type Culture Collection (ATCC). J. Natl. Cancer Inst. (Bethesda) 58:221–226; 1977.
Ford, J. M.; Hait, W. N. Pharmacology of drugs that alter multi-drug resistance in cancer. Pharmacol. Rev. 42:155–199; 1990.
Fujita, T.; Yamada, H.; Fukuzumi, M.; Nishimaki, A.; Yamamoto, A.; Muranishi, S. Calcein is excreted from intestinal mucosal cell membrane by the active transport system. Life Sci. 60:307–313; 1997.
Hidalgo, I.; Raub, T.; Borchardt, R. Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal permeability. Gastroenterology 96:736–749; 1989.
Hollo, Z.; Homolya, L.; Davis, C. Calcein accumulation as a fluorometric functional assay of the multi-drug transporter. Biochem. Biophys. Acta 1991:384–388; 1994.
Homolya, L.; Hollo, A.; Germann, U.; Pastn, I.; Gottsman, M. Fluorescent cellular indicators are extruded by the multi-drug resistance protein. J. Biol. Chem. 268:21493–21496; 1993.
Hoskins, J.; DiHerdt, S.; Moore, R.; Bumol, T. The development and characterization of Vinca alkaloid-resistant Caco-2 human colorectal cell lines expressing mdr-1. Int. J. Cancer 53:680–688; 1993.
Hunter, J.; Jepson, M.; Tsuruo, T.; Simmons, N.; Hirst, B. Functional expression of P-glycoprotein in apical membranes of human intestinal Caco-2 cells. J. Biol. Chem. 268:14991–14997; 1993.
Keppler, D.; Cui, Y.; König, J.; Leier, I.; Nies, A. Export pumps for anionic conjugates encoded by MRP genes. Adv. Enzyme Regul. 39:237–246; 1999.
Klein, I.; Sarkadi, B.; Varadi, A. An inventory of the human ABC proteins. Biochem. Biophys. Acta 1461:237–262; 1999.
Lentz, K.; Polli, J.; Wring, S.; Humphreys, J.; Polli, J. Influence of passive permeability on apparent P-glycoprotein kinetics. Pharm. Res. 17:1456–1460; 2000.
Liminga, G.; Nygren, P.; Larson, R. Microfluorometric evaluation of calcein acetoxymethyl ester as a probe for P-glycoprotein-mediated resistance: effects of cyclosporin A and its nonimmunosuppressive analogues. Exp. Cell Res. 212:291–296; 1994.
Loe, D.; Almquist, K.; Deeley, R.; Cole, S. Multi-drug resistance protein (MRP)-mediated transport of leukotriene C4 and chemotherapeutic agents in membrane vesicles. Demonstration of glutathione-dependent vincristine transport. J. Biol. Chem. 271:9675–9682: 1996.
Mandagere, A.; Thompson, T.; Hwang, K. Graphical model for estimating oral bioavailability of drugs in humans and other species from their Caco-2 permeability and in vitro liver enzyme metabolic stability rates. J. Med. Chem. 45:304–311; 2002.
Oprea, T.; Gottfries, J. Toward minimalistic modeling of oral drug absorption. J. Mol. Graph. Model. 17:261–274; 1999.
Pinto, M.; Robine-Leon, S.; Appay, M. D., et al. Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture. Biol. Chem. 47:323–330; 1983.
Roelofsen, H.; Vos, T.; Schippers, I.; Kuipers, F.; Koning, H.; Moshage, H.; Jansen, P.; Muller, M. Increased levels of human multi-drug resistance protein in lateral membranes of proliferating hepatocyte-derived cells. Gastroenterology 112:511–521; 1997.
Safa, A. Photoaffinity labels for characterizing drug interaction sites of P-glycoprotein. In: Ambedkar, S.; Gottesman, M., ed. Methods in enzymology. ABC transporters: biochemical, cellular, and molecular aspects. Vol. 292. London: Academic Press; 1998:289–307.
Shapiro, A.; Fox, K.; Lam, P.; Ling, V. Stimulation of P-glycoprotein-mediated drug transport by prazosin and progesterone: evidence for a third drug-binding site. Eur. J. Biochem. 259:841–850; 1999.
Tanaka, Y.; Taki, Y.; Sakane, T.; Nadai, F.; Sezaki, H.; Yamashits, S. Characterization of drug transport through tight-junctional pathway in Caco-2 monolayers: comparison with isolated rat jejunal fluid of normal subjects. Pharm. Res. 12:523–528; 1995.
Tiberghein, F.; Loor, F. Ranking of P-glycoprotein substrate and inhibitors by a calcein-AM fluorometry screening assay. Anticancer Drugs 7:568–578; 1996.
Vachon, P.; Beaulieu, J. Transtent mosaic patterns of morphological and functional differentiation in the Caco-2 cell line. Gastroenterology 103:414–423; 1992.
Van Aubel, R.; Koenderink, J.; Peters, J.; Van Os, C.; Russel, F. Mechanisms and interaction of vinblastine and reduced glutathione transport in membrane vesicles by the rabbit multi-drug resistance protein MRP2 expressed in insect cells. Mol. Pharmacol. 56:714–719; 1999.
Volm, M. Multi-drug resistance and its resersal. Anticancer Res. 18:2905–2918; 1998.
Volpe, D.; Faustino, P.; Hussain, A. Towards standardization of an in vitro method of drug absorption. Pharmacopeial Forum 27(4):2916–2922; 2001.
Wacher, V.; Silverman, J.; Zhang, Y.; Benet, L. Role of P-glycoprotein and cytochrome P-450 3A in limiting oral absorption of peptides and peptidomimetics. J. Pharm. Sci. 87:1322–1330; 1998.
Wacher, V.; Wu, C.; Benet, L. Overlapping substrate specificities and tissue distribution of cytochrome P450 3A and P-glycoprotein: implications for drug delivery and activity in cancer chemotherapy. Mol. Carcinog. 13:129–134; 1995.
Yu, D. The contribution of P-glycoprotein to pharmacokinetic drug-drug interactions. J. Clin. Pharmacol. 39:1203–1211; 1999.
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Laska, D.A., Houchins, J.O., Pratt, S.E. et al. Characterization and application of a vinblastine-selected Caco-2 cell line for evaluation of P-glycoprotein. In Vitro Cell.Dev.Biol.-Animal 38, 401–410 (2002). https://doi.org/10.1290/1071-2690(2002)038<0401:CAAOAV>2.0.CO;2
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DOI: https://doi.org/10.1290/1071-2690(2002)038<0401:CAAOAV>2.0.CO;2