| - Purpose: Multidrug resistance is the major reason for chemotherapy treatment failure. This project proposes a multi-modal study on the molecular mechanisms by which sphingolipids modulate multidrug resistance, in concert with P-glycoprotein expression and function, by altering the physico-chemical properties of P-glycoprotein harboring membrane domains. - Approach: There is ample evidence that multidrug resistant tumor cells accumulate glucosylceramides, but the functional significance for drug resistance remains poorly understood. We have performed studies in (multidrug resistant) tumor cell lines, showing that up-regulation of ATP-binding cassette-transporters and glucosylceramide occur concomitantly, while both become enriched in detergent-resistant membrane domains. We will now investigate in a systematic manner how P-glycoprotein (ATPase/efflux) activity is affected by glucosylceramide (compared to other sphingolipids), focusing on the lipid's ability to modulate the physico-chemical properties of membranes, specifically raft membrane domain formation. For this we will use model systems ranging from intact cells to liposomes, including giant unilamellar vesicles, which allow direct visualisation of raft formation, the lateral organisation of (fluorescently tagged) P-glycoprotein and its partitioning into sphingolipid-containing domains by confocal microscopy. Reconstitution of P-glycoprotein into giant unilamellar vesicles will include (multidrug resistant) tumor cell-derived membrane domains, as well as their isolated lipid constituents or defined glucosylceramide species, based on results obtained from HPLC-MS/MS analysis of raft-associated sphingolipids. The final level involves the study of P-glycoprotein-glucosylceramide interactions in intact cells, employing (multidrug resistant) tumor cell lines with genetically or pharmacologically manipulated P-glycoprotein or glucosylceramide synthase expression/activity. - Relevance: We expect to obtain a detailed insight into the contribution of sphingoid base-specific sphingolipids (especially glucosylceramide) to the physico-chemical properties of membranes and in particular raft domains, which are important for the accommodation and function of ATP-binding-cassette transporters (P-glycoprotein). This knowledge can then be exploited as a novel means for reversal of multidrug resistance, based on genetic or pharmacological manipulation of sphingolipid metabolism. |
