Modulation of cellular cholesterol alters P-glycoprotein activity in multidrug-resistant cells.
The drug transporter P-glycoprotein (ABCB1) plays an important role in drug distribution and elimination, and when overexpressed it may confer multidrug resistance (MDR). P-glycoprotein is localized in the plasma membrane, especially within rafts and caveolae, characterized as detergent-resistant membranes (DRMs). This study investigated the effect of cholesterol depletion and repletion as well as saturation on subcellular localization and function of P-glycoprotein to determine the effect of DRM localization on P-glycoprotein-mediated drug efflux. In L-MDR1 overexpressing human P-glycoprotein, cholesterol depletion removed P-glycoprotein from the raft membranes into non-DRM fractions, whereas repletion fully reconstituted raft localization. P-glycoprotein function was assessed by realtime monitoring with confocal laser scanning microscopy using BODIPY-verapamil as substrate. Cholesterol depletion reduced P-glycoprotein function in L-MDR1 cells resulting in intracellular substrate accumulation (159% +/- 43, p < 0.001; control = 100%). Cholesterol repletion reduced intracellular substrate fluorescence (120% +/- 36, p < 0.001) and restored the transporter activity. Addition of surplus cholesterol (saturation) even enhanced drug efflux in L-MDR1 cells, leading to reduced intracellular accumulation of BODIPY-verapamil (69% +/- 10, p < 0.001). Transport of BODIPY-verapamil in cells not expressing human P-glycoprotein (LLC-PK1) was not susceptible to cholesterol alterations. These results demonstrate that cholesterol alterations influence P-glycoprotein localization and function, which might contribute to the large interindividual variability of P-glycoprotein activity known from in vivo studies.
Department of Internal Medicine VI, Clinical Pharmacology and Pharmacoepidemiology, University of Heidelberg, Im Neuenheimer Feld 410, D-69120 Heidelberg, Germany.
Joachim
J
Troost
Heike
H
Lindenmaier
Walter Emil
WE
Haefeli
Johanna
J
Weiss
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D001896:Boron Compounds / Q000493:pharmacokinetics; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism*; D019008:Drug Resistance, Neoplasm / Q000502:physiology*; D006801:Humans; D018374:LLC-PK1 Cells; D013552:Swine; D014700:Verapamil / Q000493:pharmacokinetics
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001896:Boron Compounds; D051242:Caveolin 1; D022461:Caveolins; D002784:Cholesterol; D014700:Verapamil