Effect of the modulation of the membrane lipid composition on the localization and function of P-glycoprotein in MDR1-MDCK cells

In Vitro Cell Dev Biol Anim. 2005 Jul-Aug;41(7):207-16. doi: 10.1290/0502016.1.

Abstract

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.

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / metabolism*
  • Animals
  • Antineoplastic Agents / pharmacology
  • Caveolins / metabolism
  • Cell Line
  • Cell Membrane / chemistry*
  • Cell Membrane / drug effects
  • Cell Membrane / metabolism
  • Cell Survival
  • Cholesterol / metabolism
  • Dogs
  • Drug Resistance, Multiple
  • Fluorescent Dyes / metabolism
  • Kidney / cytology*
  • Membrane Lipids / chemistry*
  • Membrane Microdomains / chemistry
  • Membrane Microdomains / metabolism
  • Membrane Proteins / metabolism
  • Morpholines / pharmacology
  • Occludin
  • Rhodamine 123 / metabolism
  • beta-Cyclodextrins / pharmacology

Substances

  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • Antineoplastic Agents
  • Caveolins
  • Fluorescent Dyes
  • Membrane Lipids
  • Membrane Proteins
  • Morpholines
  • Occludin
  • beta-Cyclodextrins
  • methyl-beta-cyclodextrin
  • Rhodamine 123
  • RV 538
  • Cholesterol