Cytotoxicity of an Anti-cancer Lysophospholipid through Selective Modification of Lipid Raft Composition

Abstract

Edelfosine is a prototypical member of the alkylphosphocholine class of antitumor drugs. Saccharomyces cerevisiae was used to screen for genes that modulate edelfosine cytotoxicity and identified sterol and sphingolipid pathways as relevant regulators. Edelfosine addition to yeast resulted in the selective partitioning of the essential plasma membrane protein Pma1p out of lipid rafts. Microscopic analysis revealed that Pma1p moved from the plasma membrane to intracellular punctate regions and finally localized to the vacuole. Consistent with altered sterol and sphingolipid synthesis resulting in increased edelfosine sensitivity, mislocalization of Pma1p was preceded by the movement of sterols out of the plasma membrane. Cells with enfeebled endocytosis and vacuolar protease activities prevented edelfosine-mediated (i) mobilization of sterols, (ii) loss of Pma1p from lipid rafts, and (iii) cell death. The activities of proteins and signaling processes are meaningfully altered by changes in lipid raft biophysical properties. This study points to a novel mode of action for an anti-cancer drug through modification of plasma membrane lipid composition resulting in the displacement of an essential protein from lipid rafts. Edelfosine is a prototypical member of the alkylphosphocholine class of antitumor drugs. Saccharomyces cerevisiae was used to screen for genes that modulate edelfosine cytotoxicity and identified sterol and sphingolipid pathways as relevant regulators. Edelfosine addition to yeast resulted in the selective partitioning of the essential plasma membrane protein Pma1p out of lipid rafts. Microscopic analysis revealed that Pma1p moved from the plasma membrane to intracellular punctate regions and finally localized to the vacuole. Consistent with altered sterol and sphingolipid synthesis resulting in increased edelfosine sensitivity, mislocalization of Pma1p was preceded by the movement of sterols out of the plasma membrane. Cells with enfeebled endocytosis and vacuolar protease activities prevented edelfosine-mediated (i) mobilization of sterols, (ii) loss of Pma1p from lipid rafts, and (iii) cell death. The activities of proteins and signaling processes are meaningfully altered by changes in lipid raft biophysical properties. This study points to a novel mode of action for an anti-cancer drug through modification of plasma membrane lipid composition resulting in the displacement of an essential protein from lipid rafts. The synthetic lipid edelfosine (also known as 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine or ET-18-OCH3) is a prototypical member of the alkylphosphocholine class of cancer chemotherapeutic drugs. Alkylphosphocholines are effective drugs as they contain ether-linked fatty acids, as opposed to ester-linked fatty acids prevalent in endogenous phospholipids, and thus are much more resistant to cellular degradation by phospholipases (1Arthur G. Bittman R. Biochim. Biophys. Acta. 1998; 1390: 85-102Crossref PubMed Scopus (97) Google Scholar, 2Zaremberg V. McMaster C.R. J. Biol. Chem. 2002; 277: 39035-39044Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 3Gajate C. Mollinedo F. Curr. Drug Metab. 2002; 3: 491-525Crossref PubMed Scopus (155) Google Scholar). Because of their similarity in structure to phosphatidylcholine (PC), 5The abbreviations used are: PC, phosphatidylcholine; DRM, detergent-resistant membrane; Mes, 4-morpholineethanesulfonic acid. the main experimentations to determine a mechanism of action for edelfosine and other alkylphosphocholines have focused on PC metabolism. This course of action was supported by observations that only alkylphospholipids with choline head groups, but not head groups found on other phospholipids such as ethanolamine or serine, were effective antitumor agents (1Arthur G. Bittman R. Biochim. Biophys. Acta. 1998; 1390: 85-102Crossref PubMed Scopus (97) Google Scholar). Edelfosine and other choline-containing alkylphospholipids were found to