Effect of the modulation of the membrane lipid composition on the localization and function of P-glycoprotein in MDR1-MDCK cells
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Multidrug resistance (MDR) is a major obstacle in cancer therapy. It results from different mechanisms; among them is P-glycoprotein (P-gp)-mediated drug efflux out of cells. The mechanism of action remains elusive. The membrane lipid surrounding of P-gp, especially cholesterol, has been postulated to play an important role. To determine the effect of cholesterol depletion on P-gp, Madin Darby canine kidney (MDCK) cells, transfected with the mdr1 gene (MDR1-MDCK cells), were treated with methyl-β-cyclodextrin (MβCD). The localization and function of P-gp were analyzed using confocal laser scanning microscopy. Treatment with 100 mM MβCD did not affect viability but altered the structural appearance of the cells and abolished efflux of rhodamine 123, a P-gp substrate. The MβCD treatment released P-gp from intact cells into the supernatant and reduced the amount of P-gp in total membrane preparations. The P-gp was shifted from the raft fractions (1% Triton X-100, 4° C) to higher density fractions in MβCD-treated cells. The amount of cholesterol was significantly decreased in the raft fractions. Treatment of cells with 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, a glucosylceramide synthase inhibitor, also led to a shift of P-gp to higher density fractions. These results show that removal of cholesterol modulates the membrane lipid composition, changes the localization of P-gp, and results in loss of P-gp function.
Key wordsP-glycoprotein cholesterol glycosphingolipids rafts
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- Arima, H.; Yunomae, K.; Morikawa, T.; Hirayama, F.; Uekama, K. Contribution of cholesterol and phospholipids to inhibitory effect of dimethyl-beta-cyclodextrin on efflux function of P-glycoprotein and multidrug resistance-associated protein 2 in vinblastine-resistant Caco-2 cell monolayers. Pharm. Res. 21:625–634; 2004.PubMedCrossRefGoogle Scholar
- Hangumaran, S.; Hoesli, D. C. Effects of cholesterol depletion by cyclodextrin on the sphingolipid microdomains of the plasma membrane, Biochem. J. 335(Pt. 2):433–440; 1998.Google Scholar
- Kramer, S. D.; Hurley, J. A.; Abbott, N. J.; Begley, D. J. Lipids in blood-brain barrier models in vitro I: thin-layer chromatography and high-performance liquid chromatography for the analysis of lipid classes and loing-chain polyunsaturated fatty acids. In Vitro Cell. Dev. Biol. 38A:557–565; 2002.CrossRefGoogle Scholar
- Lavie, Y.; Cao, H.; Volner, A.; Lucci, A.; Han, T. Y.; Geffen, V.; Giuliano, A. E.; Cabot, M. C. Agents that reverse multidrug resistance, tamoxifen, verapamil, and cyclosporin A, block glycosphingolipid metabolism by inhibiting ceramide glycosylation in human cancer cells. J. Biol. Chem. 272:1682–1687; 1997.PubMedCrossRefGoogle Scholar
- Nagafuku, M.; Kabayama, K.; Oka, D., et al. Reduction of glycosphingolipid levels in lipid rafts affects the expression state and function of glycosylphosphatidylinositol-anchored proteins but does not impair signal transduction via the T cell receptor. J. Biol. Chem. 278:51920–51927; 2003.PubMedCrossRefGoogle Scholar
- Rothen-Rutishauser, B.; Kramer, S. D.; Braun, A.; Gunthert, M.; Wunderli-Allenspach, H. MDCK cell cultures as an epithelial in vitro model: cytoskeleton and tight junctions as indicators for the definition of age-related stages by confocal microscopy. Pharm. Res. 15:964–971; 1998.PubMedCrossRefGoogle Scholar
- van de Loosdrecht, A. A.; Beelen, R. H.; Ossenkoppele, G. J.; Broekhoven, M. G.; Langenhuijsen M. M. A tetrazolium-based colorimetric MTT assay to quantitate human monocyte mediated cytotoxicity against leukemic cells from cell lines and patients with acute myeloid leukemia. J. Immunol. Methods 174:311–320; 1994.PubMedCrossRefGoogle Scholar
- von Tresckow, B.; Kallen, K. J.; von Strandmann, E. P.; Borchmann, P.; Lange H.; Engert, A.; Hansen, H. P. Depletion of cellular cholesterol and lipid rafts increases shedding of CD30. J. Immunol. 172:4324–4331; 2004.Google Scholar
- Zhou, M.; Diwu, Z.; Panchuk-Voloshina, N.; Haugland, R. P. A stable non-fluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: applications in detecting the activity of phagocyte NADPH oxidase and other oxidases. Anal. Biochem. 253:162–168; 1997.PubMedCrossRefGoogle Scholar