Contribution of cholesterol and phospholipids to inhibitory effect of dimethyl-beta-cyclodextrin on efflux function of P-glycoprotein and multidrug resistance-associated protein 2 in vinblastine-resistant Caco-2 cell monolayers

• Published in: Pharmaceutical research Vol. 21; no. 4; pp. 625 - 634
• Main Authors: Arima, Hidetoshi, Yunomae, Kiyokazu, Morikawa, Tadatoshi, Hirayama, Fumitoshi, Uekama, Kaneto
• Format: Journal Article
• Language: English
• Published: United States 01.04.2004
• Subjects: ATP Binding Cassette Transporter, Subfamily B, Member 1 - antagonists & inhibitors; ATP Binding Cassette Transporter, Subfamily B, Member 1 - metabolism; beta-Cyclodextrins - pharmacology; Caco-2 Cells; Cholesterol - metabolism; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm - drug effects; Drug Resistance, Neoplasm - physiology; Humans; Membrane Transport Modulators; Membrane Transport Proteins - antagonists & inhibitors; Multidrug Resistance-Associated Proteins - antagonists & inhibitors; Phospholipids - metabolism; Vinblastine - pharmacology
• ISSN: 0724-8741; 1573-904X
• DOI: 10.1023/B:PHAM.0000022409.27896.d4

The purpose of this study is to reveal the contribution of membrane components to the inhibitory effect of 2,6-di-O-methyl-beta-cyclodextrin (DM-beta-CyD) on P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) function in vinblastine-resistant Caco-2 (Caco-2R) cell monolayers. The transport of rhodamine-123 and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was studied in Caco-2R cell monolayers. P-gp and MRP2 residing in the monolayers and releasing in cell supernatants were detected by Western blotting. The mRNA levels of MDR1 and MRP2 were detected by reverse transcription-polymerase chain reaction (RT-PCR) method. Cholesterol, phospholipids, and proteins were mainly determined by each assay kit. Of various beta-cyclodextrin derivatives (beta-CyDs), DM-beta-CyD most significantly impaired the efflux function of P-gp and MRP2 without changing cell viability and membrane integrity. The treatment with CyDs did not change the mRNA levels of MDR1 and MRP2. DM-beta-CyD lowered cholesterol content and P-gp level in caveolar membranes. In addition, DM-beta-CyD released not only cholesterol and phospholipids but also proteins including P-gp and MRP2 from apical membranes of the monolayers. DM-beta-CyD may impair P-gp and MRP2 function in Caco-2R cell monolayers, probably, at least in part, through the release of these transporters from the apical membranes of monolayers, and the exertion of the inhibitory effect of DM-beta-CyD may require the extraction of not only cholesterol but also phospholipids from the monolayers.