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Decrease of P-glycoprotein activity in K562/ADR cells by MbetaCD and filipin and lack of effect induced by
Paiboon Reungpatthanaphong1, Carole Marbeuf-Gueye, Laurence Le Moyec
1Laboratoire de Physicochimie Biomoléculaire et Cellulaire (LPBC/CSSB UMR 7033), Université Paris Nord, 74 rue Marcel Cachin, 93017, Bobigny, France.
Abstract:
The effect of low-density membrane domains on function of the plasma membrane transporter P-glycoprotéine (P-gp), involved in multidrug resistance (MDR) phenotype, has been investigated in K562/ADR cells. To this end we reversibly altered the cholesterol content of K562/ADR cells by using methyl-beta-cyclodextrin as a cholesterol chelator and conversely we repleted them through incubation with cholesterol in culture medium. We also used the cholesterol-binding fluorochrome filipin and cholesterol oxidase. Our data show that either cholesterol depletion or complex formation with filipin resulted in a strong decrease of P-gp activity. However, when cells were incubated with cholesterol oxidase that are known to disrupt rafts, no modification of the P-gp activity was observed. In addition, using a free-detergent methodology to separate by ultracentrifugation, "light," "heavy," and "extra heavy" fractions we show that no P-gp is found in the "light" fraction where rafts are usually detected. Altogether, our data strongly suggest that, in this cell line, P-gp is not localized in rafts.
Insights
This study investigated how membrane domains affect P-glycoprotein (P-gp) activity in multidrug resistance. Results indicate P-gp is not located in membrane rafts, challenging previous assumptions.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- The plasma membrane transporter P-glycoprotein (P-gp) is crucial in the multidrug resistance (MDR) phenotype.
- Low-density membrane domains, or rafts, are implicated in regulating transporter function.
- Understanding P-gp localization within membrane domains is key to overcoming MDR.
Purpose of the Study:
- To investigate the effect of membrane cholesterol content on P-glycoprotein (P-gp) activity.
- To determine if P-gp is localized within low-density membrane domains (rafts) in K562/ADR cells.
Main Methods:
- Cholesterol levels in K562/ADR cells were manipulated using methyl-beta-cyclodextrin and cholesterol repletion.
- P-gp activity was assessed after cholesterol depletion, filipin complexation, and cholesterol oxidase treatment.
- Cellular fractions were analyzed using ultracentrifugation to determine P-gp localization.
Main Results:
- Cholesterol depletion and filipin treatment significantly reduced P-gp activity.
- Cholesterol oxidase, which disrupts rafts, did not alter P-gp activity.
- Ultracentrifugation analysis revealed P-gp is absent from the 'light' membrane fraction typically containing rafts.
Conclusions:
- P-glycoprotein activity is sensitive to changes in membrane cholesterol content.
- Contrary to expectations, P-gp is not localized in membrane rafts in the K562/ADR cell line.
- These findings suggest a non-raft localization for P-gp, impacting our understanding of MDR mechanisms.
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