Effect of the modulation of the membrane lipid composition on the localization and function of P-glycoprotein in MDR1-MDCK cells.
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-beta-cyclodextrin (MbetaCD). The localization and function of P-gp were analyzed using confocal laser scanning microscopy. Treatment with 100 mM MbetaCD did not affect viability but altered the structural appearance of the cells and abolished efflux of rhodamine 123, a P-gp substrate. The MbetaCD 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 degrees C) to higher density fractions in MbetaCD-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.
Kamau
Sarah W
SW
Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. kamau@vetbio.unizh.ch
Krämer
Stefanie D
SD
Günthert
Maja
M
Wunderli-Allenspach
Heidi
H
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D022461:Caveolins / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival; D002784:Cholesterol / Q000378:metabolism; D004285:Dogs; D018432:Drug Resistance, Multiple; D005456:Fluorescent Dyes / Q000378:metabolism; D007668:Kidney / Q000166:cytology*; D008563:Membrane Lipids / Q000737:chemistry*; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D062793:Occludin; D020112:Rhodamine 123 / Q000378:metabolism; D047392:beta-Cyclodextrins / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D022461:Caveolins; D005456:Fluorescent Dyes; D008563:Membrane Lipids; D008565:Membrane Proteins; D009025:Morpholines; D062793:Occludin; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D020112:Rhodamine 123; C033110:RV 538; D002784:Cholesterol