Modulation of Cellular Cholesterol Alters P-Glycoprotein Activity in Multidrug-Resistant Cells

Abstract

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% Ϯ 43, p Ͻ 0.001; control ϭ 100%). Cholesterol repletion reduced intracellular substrate fluorescence (120% Ϯ 36, p Ͻ 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% Ϯ 10, p Ͻ 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.

Multidrug resistance protein 1 is not associated to detergent-resistant membranes

“…Inhibition of Pgp transport and ATPase activities by cholesterol-depleting agents like methyl-bcyclodextrin (MbCD) [5,6] supports this view. Moreover, cholesterol upregulates Pgp ATPase activity and drug transport in proteoliposomes and cell systems [5,7,8].…”

“…Recent works have suggested the association of Pgp to cholesterol and sphingolipid-rich membrane microdomains called detergent-resistant membranes (DRMs) [3,4]. Inhibition of Pgp transport and ATPase activities by cholesterol-depleting agents like methyl-bcyclodextrin (MbCD) [5,6] supports this view. Moreover, cholesterol upregulates Pgp ATPase activity and drug transport in proteoliposomes and cell systems [5,7,8].…”

The novel Akt inhibitor, perifosine, induces caspase-dependent apoptosis and downregulates P-glycoprotein expression in multidrug-resistant human T-acute leukemia cells by a JNK-dependent mechanism

“…However, it is established that P-gp exists in lipid rafts, 36 where it displays a more active state than when localized outside rafts. 37 Since perifosine specifically accumulates in lipid rafts, 38 it might be that this alkylphospholipid would somehow interfere with P-gp activity and/or localization in lipid rafts, ensuing in downregulation of the pump activity. Further experiments are clearly required to investigate this issue.…”

Lipid Regulation of the ABCB1 and ABCG2 Multidrug Transporters

“…Cholesterol depletion of the cell membranes has been reported to decrease both ABCB1 and ABCG2 function (Storch et al, 2007;Troost, Lindenmaier, Haefeli, & Weiss, 2004). For assessing a direct modulation of these transporters by membrane cholesterol, various functional assays should be combined, e.g., measurement of vanadate-sensitive, basal, or drug-stimulated ATPase activity, direct drug transport in isolated membranes, or combined ATPase and transport activity by isolated and reconstituted proteins (see Section 1).…”