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

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 inhibit PC synthesis and this correlated with inhibition of cell growth in various cancer cell lines (4Boggs K.P. Rock C.O. Jackowski S. J. Biol. Chem. 1995; 270: 7757-7764Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 5Boggs K.P. Rock C.O. Jackowski S. J. Biol. Chem. 1995; 270: 11612-11618Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 6Van Der Luit A.H. Budde M. Verheij M. Van Blitterswijk W.J. Biochem. J. 2003; 374: 747-753Crossref PubMed Scopus (50) Google Scholar, 7Baburina I. Jackowski S. J. Biol. Chem. 1998; 273: 2169-2173Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Further metabolic labeling demonstrated that alkylphosphocholine drugs can inhibit the synthesis of PC-derived sphingomyelin, and this correlated with increased ceramide mass. Inhibition of de novo ceramide synthesis by the addition of the ceramide synthase inhibitor fumonisin B1 decreased ceramide levels and this was associated with increased resistance to alkylphosphocholines (8Wieder T. Orfanos C.E. Geilen C.C. J. Biol. Chem. 1998; 273: 11025-11031Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). As ceramide is a lipid second messenger whose accumulation can result in cytostasis or apoptosis (9Kolesnick R. J. Clin. Investig. 2002; 110: 3-8Crossref PubMed Scopus (382) Google Scholar, 10Cremesti A.E. Goni F.M. Kolesnick R. FEBS Lett. 2002; 531: 47-53Crossref PubMed Scopus (296) Google Scholar), an increase in cellular ceramide-mediated signaling was an alternate hypothesis proposed for alkylphosphocholine-mediated cell death (8Wieder T. Orfanos C.E. Geilen C.C. J. Biol. Chem. 1998; 273: 11025-11031Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). To gain further insight into the mechanism of action of alkylphosphocholine-mediated cytotoxicity we performed a genetic screen in Saccharomyces cerevisiae to isolate genes that increased edelfosine susceptibility. The genetic screen provided evidence that ceramide structure and sphingolipid metabolism modulate edelfosine sensitivity and implicate lipid rafts as a site of action for edelfosine. Edelfosine addition resulted in the movement of plasma membrane sterols into the cell, suggesting that edelfosine-mediated cytotoxicity is through modification of the biophysical structure of lipid rafts. Analysis of lipid raft protein composition revealed that edelfosine was responsible for the selective partitioning of the plasma membrane lipid raft protein Pma1p from plasma membrane rafts to the vacuole. Yeast defective in endocytosis and vacuolar protease activity prevented edelfosine-mediated sterol movement, Pma1p loss from lipid rafts, and cellular cytotoxicity. Our results indicate a novel mode of action for an antitumor drug through drug insertion into lipid rafts and selective displacement of an essential protein from lipid rafts. These results are consistent with an emerging phenomenon whereby the activity of biological signal processes that assemble on lipid raft scaffolds (9Kolesnick R. J. Clin. Investig. 2002; 110: 3-8Crossref PubMed Scopus (382) Google Scholar, 10Cremesti A.E. Goni F.M. Kolesnick R. FEBS Lett. 2002; 531: 47-53Crossref PubMed Scopus (296) Google Scholar) are meaningfully altered by changes in raft biophysical properties. Plasmids and Yeast Strains—Yeast strain W303-1a (MATa ura3-1 his3-11,15 leu2-3,112 trp1-1 ade2-1 can1-100) was used for the genetic screen. Other strains used were RH1800 (MATa leu2 his4 ura3-52 bar1), RH3809 (MATa lcb1-100 leu2 his4 ura3 bar1), RH448 (MATa his4 lys2 leu2 ura3 bar1), RH732 (MATa his4 lys2 leu2 ura3 bar1 pep4::URA3), RH2765 (MATa his4 leu2 ura3 lys2 bar1 sla2-3 (end4)), RH2763 (MATa his4 leu2 ura3 bar1 pep4::URA3 sla2-1 (end4)), SEY6210 (MATa ura3-52 leu2-3,112 his3-100 trp1-901 lys2-801 suc2-9), SEY6210-PMA1-dsRFP (MATa ura3-52 leu2-3,112 his3-100 trp1-901 lys2-801 suc2-9 PMA1::tdimer2 (12)::kanMX4), and BY4741 (MATa his3Δ0 leu2Δ0 met15Δ0 ura3Δ0). Multicopy PMA1 plasmid pCM10.4 was kindly provided by Dr. Raffael Schaffrath, Martin-Luther University, Germany (48Zink S. Mehlgarten C. Kitamoto H.K. Nagase J. Jablonowski D. Dickson R.C. Stark M.J. Schaffrath R. Eukaryot. Cell. 2005; 4: 879-889Crossref PubMed Scopus (22) Google Scholar). The AST1 gene ± 500 bp was amplified with specific primers (AST1-forward, 5′-GACTTAGCAACGCAAGCAGAAAAGAAGGTTTGC-3′, and AST1-reverse, 5′-GCCATATCTTCTCGAGACTCTGAATTCATTTTTGCTGG-3′) using BY4741 genomic DNA as template. The PCR product was cloned into the pCR2.1-TOPO vector (Invitrogen) and then subcloned into pRS426 (HindIII and NotI sites). Yeast were grown at 30 °C unless otherwise indicated. Yeast Genetic Screens—Yeast cells were transformed with a genomic and were to with or edelfosine of that we and PubMed Scopus Google Scholar) have that the of on cell growth in can by cell Plasmids were from yeast cells on the that to the yeast strain that not on edelfosine. The were transformed into yeast to that edelfosine sensitivity was of plasmid using a Plasmids from that increased sensitivity to the drug were and their DNA of only increased sensitivity to the Plasmids from were and their DNA was plasmid only gene identified as the other plasmid The specific gene responsible for the is Yeast lipid protein strains were for edelfosine sensitivity in synthetic using of BY4741 (MATa his3Δ0 leu2Δ0 met15Δ0 and and and These strains were from and detergent-resistant membrane was as M. S. S. PubMed Scopus Google Scholar) with were with or the edelfosine for the and in the edelfosine a on cell and a in protein Cells as were in and and at Cells were in of protease inhibitor and by with for 30 with on The was of cells and by at 500 for was and of protein in 500 of was with for 30 on The was to by of with of in with and then with of The were at for in a and of were from the of the The the and the and was identified and as 30 was for at °C in and and by by or were to and were with to of de and of or and with by using The of edelfosine on and was in were as J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) of the cell were used for the Yeast cells were grown to and an of cells to was or in the of edelfosine for the Cells were then with and with and and the cell was at Cells were in of and and protease inhibitor and with the of in the of at cells were by a 500 for at and the resulting cell was for at °C in an at to cellular The was in Mes, and was to a of at were further with in a of the were with of in at the of an and with a from to in at at at at and at were in an at for at °C in a and then were from the of the and of was to and of edelfosine in lipid rafts, cells were with for the and to lipid raft as were for for the of Pma1p as V. McMaster C.R. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar) and using a with a were using a and using or was used for and Cells a genomic PMA1 gene to J. M. Biol. Cell. 2003; PubMed Scopus Google Scholar) were using an with a of were using and were used for and labeling of was as a in of cells or cells with edelfosine in for at 30 °C were and in ± edelfosine for Cells were using the of a with a of was as a in was to cells at a of Cells were then by and in the of addition T. T. F. Cell. Biol. PubMed Scopus Google Scholar). To of the cells for of used in cells were in of as 2002; PubMed Scopus Google Scholar). cells were grown to and with the edelfosine for 30 at 30 by a with to with the cells were by and with and were and as V. McMaster C.R. J. Biol. Chem. 2002; 277: 39035-39044Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). was used for drug and lipid raft cells were with for the and with to cell drug was in the cell by as F. C. R. M. Google Scholar). was by of the of cells at 30 °C in and were and in and at 30 °C for Cells were then with protein labeling for and with addition of and at a of of cells of were at and to in of was as with of the with were by or analysis were with at °C for and to a of and with cells Pma1p was from the using a and protein were by and with a using analysis by the protein was in with in the using a and identified by of in were on a and analysis was performed using a with a were identified from in the mode by the using the and sterols were as S. R. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar) using the from and a of cells at 30 °C in were with edelfosine. and were and lipid was performed as sterols were to and of lipid in were performed in was as by and J. Biol. Chem. Full Text PDF PubMed Google Scholar), and protein using the J. Biol. Chem. Full Text PDF PubMed Google Scholar). of protein from was performed using a The of PC in in the of edelfosine on yeast cells was or a for with various of edelfosine was with of edelfosine that edelfosine was to yeast cells with yeast cells to edelfosine at to for cells I. Jackowski S. J. Biol. Chem. 1998; 273: 2169-2173Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, Luit A.H. Budde M. Verheij M. Blitterswijk W.J. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). edelfosine we that yeast cells were for to of drug by a in to at and and was in cells that edelfosine the of the for PC hypothesis for the of edelfosine is through inhibition of PC the addition of edelfosine we a in the of choline into PC was found in the PC product consistent with edelfosine PC synthesis in the inhibition of PC synthesis with a accumulation of of a at the Yeast cells a in their sensitivity to edelfosine in to cells yeast cells are to a for PC synthesis of the of a that for the synthesis of PC from McMaster C.R. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). As we that edelfosine-mediated was in yeast a is that edelfosine is other gene to yeast and Edelfosine in a mode of action for edelfosine we a yeast genomic as an to genes whose increased resulted in sensitivity to edelfosine. plasmid that resulted in increased sensitivity to edelfosine was and found to contain the gene Yeast is for of the fatty of yeast ceramide D. T. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). To determine edelfosine sensitivity was in ceramide or in sphingolipid synthesis edelfosine we edelfosine sensitivity of yeast a defective The gene an essential that the in sphingolipid The lcb1-100 is defective in at such that the of sphingolipid synthesis at only that of at this S. C. J. PubMed Scopus Google Scholar). that the lcb1-100 strain was to edelfosine at °C the of a that the biophysical of D. T. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Bittman R. G. D. C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. V. M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), or the of sphingolipid resulted in increased edelfosine To in membrane synthesis in edelfosine we the edelfosine sensitivity of a of yeast strains of known lipid that strains defective in or the fatty responsible for the fatty composition of as as cells a sterol in the of were by the and As of genes in sphingolipid and synthesis increased edelfosine sensitivity this a specific for lipid as opposed to in lipid synthesis in as of cellular sensitivity to edelfosine. Edelfosine and sterols are to at the plasma membrane into specific to as lipid rafts C. G. S. 2003; 4: PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). cells edelfosine was found to the death into lipid rafts resulting in the of and cell death C. F. Mollinedo F. J. 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Cell. 2003; PubMed Scopus Google Scholar) that Pma1p was the protein from lipid rafts. this yeast strain the Pma1p was by a protein of that was from lipid rafts as by with Pma1p protein and of the not As was in cells the endogenous Pma1p was with we were to Pma1p levels through and protein by or Further analysis of the by as Analysis of the of the by revealed a in Pma1p to and of a in lipid the of Pma1p in not for the in Pma1p levels in that of was only in cells in Pma1p S. PubMed Scopus Google Scholar), and in with was to Pma1p in yeast cells defective in vacuolar protease activity not The protein was used as a with to protein as as to course was performed to edelfosine resulted in the loss of Pma1p from lipid rafts. a loss of Pma1p from with a ± ± in Pma1p in and a ± ± in Pma1p a to edelfosine The loss of Pma1p from was by using The of Pma1p from to edelfosine addition was using a second for that loss from was not of a specific but is a consistent and of lipid raft M. S. S. PubMed Scopus Google Scholar, M. C. R. Biol. Cell. 2002; PubMed Scopus (97) Google Scholar, Biochim. Biophys. Acta. PubMed Scopus Google Scholar), revealed edelfosine not with in the as was for but of in was of edelfosine and of cell by that Pma1p as as levels of the protein not the course of edelfosine Pma1p associated with lipid rafts synthesis in the and is to plasma membrane rafts of de novo Pma1p The loss of Pma1p from rafts displacement of plasma membrane Pma1p from rafts or an to assemble Pma1p into lipid rafts. To determine edelfosine the of Pma1p to with lipid rafts, cells were with for and for the in the or of edelfosine. were and Pma1p was by of the associated with the we to and by Pma1p by Edelfosine not the of Pma1p to with lipid raft Pma1p from protein was and we not in the of edelfosine that edelfosine not the of To the of edelfosine on Pma1p that raft cells were for that to of Pma1p with lipid rafts M. Biol. Cell. PubMed Scopus Google Scholar) to the addition of edelfosine resulted in the loss of of Pma1p from and that edelfosine Pma1p lipid raft and not into lipid rafts. and partitioning Pma1p was in the plasma membrane of and edelfosine resulted in movement of Pma1p and to intracellular punctate regions of edelfosine addition in and and with Pma1p with the yeast vacuole. This in is consistent with observations that Pma1p was found in activity S. PubMed Scopus Google Scholar) in cells but not in cells and and not of Pma1p from lipid and from the plasma membrane is to have on intracellular and membrane have used the intracellular to the of edelfosine in yeast cells for 30 a of is not but a of Pma1p from is a that in the of yeast cells PubMed Scopus Google Scholar). with the changes for edelfosine intracellular as by accumulation of in the of cells with vacuolar accumulation in cells These results that intracellular is an of edelfosine The of Pma1p to the of edelfosine was by to to edelfosine sensitivity of yeast by Pma1p cellular edelfosine sensitivity of cells Pma1p was of PMA1 from a plasmid edelfosine sensitivity of yeast cells of whose product is to Pma1p with lipid rafts, edelfosine of genes from not further cells from edelfosine we that loss of Pma1p from plasma membrane lipid rafts was a of edelfosine then of Pma1p plasma membrane lipid rafts resistance to edelfosine. revealed that Pma1p in lipid rafts in cells with decreased that Pma1p degradation endocytosis from the cell or in cells that Pma1p degradation vacuolar activity S. PubMed Scopus Google Scholar, M. C. R. Biol. Cell. 2002; PubMed Scopus (97) Google Scholar, T. M. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). was used for as a in endocytosis from the and of the of defective endocytosis and loss of of to Pma1p in lipid rafts edelfosine To the endocytosis cells were for edelfosine sensitivity at a for the Cells only the defective not or were at as as cells to edelfosine cells the defective and the gene were resistant to edelfosine. of cells to edelfosine was not to of drug cells at a The are known to defective in edelfosine and were used as a J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). analysis of the of Pma1p in of cells with cells with and revealed that Pma1p was in edelfosine addition in cells with and This that the edelfosine-mediated of Pma1p out of in cells was not of on to cell and as Pma1p in in cells with and and cells the of edelfosine as to further was a for inhibition of PC synthesis in edelfosine we the of PC synthesis from that provided edelfosine resistance in cells with the defective and with loss of of PC synthesis was by edelfosine at the to an cells not consistent with that inhibition of PC synthesis not to edelfosine in the results indicate a from the plasma membrane and increased edelfosine of at the Pma1p from and Edelfosine cells edelfosine was to into lipid rafts Luit A.H. Budde M. Verheij M. Blitterswijk W.J. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). performed using edelfosine and results were with of edelfosine in of addition to and to not edelfosine was rafts by using to as to a specific for yeast Biochim. Biophys. Acta. PubMed Scopus Google Scholar, J. J. PubMed Scopus (147) Google Scholar). As can biological we the of cells to to as this was a that to not cellular lipid in yeast T. T. F. Cell. Biol. PubMed Scopus Google Scholar). was in the plasma membrane of and yeast cells and movement of out of the plasma membrane and into the cell was of edelfosine addition and increased movement out of the plasma membrane was 30 to edelfosine addition and was The for to decreased as the of edelfosine increased as increased of to edelfosine. Edelfosine was by a increase in in cells to the increase in was a in Edelfosine for not the of in cells not that the in changes in the of sterols The cells with decreased endocytosis of defective not in with an gene not Pma1p from lipid rafts and were resistant to edelfosine and sterol was resulting in edelfosine that in cells edelfosine not result in loss of sterol from the plasma membrane in that associated with the plasma membrane in cells of the of edelfosine As edelfosine was in and cells we can that the increase in in cells was not of increased membrane by edelfosine but sterol this study we for of lipid and protein lipid rafts and in have a novel mechanism of antitumor drug that the prototypical member of the alkylphosphocholine class of anti-cancer resulted in a of from the plasma membrane into the cell resulting in the selective loss of an essential from plasma membrane lipid rafts. This is consistent with that that a of Pma1p defective in was from lipid rafts and then S. PubMed Scopus Google Scholar). of decreased endocytosis and loss of vacuolar activity prevented the in from the plasma Pma1p lipid rafts, and prevented edelfosine cytotoxicity. As edelfosine in the and vacuolar cells was to cells we are that edelfosine resistance is to cells sphingolipid metabolism is with sterol metabolism and a sphingolipid levels and cell is J. V. M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, 4: Scopus Google Scholar, M. S. S. T. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (50) Google Scholar, J. A.H. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). that and are for intracellular of proteins M. S. S. PubMed Scopus Google Scholar, S. R. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). Our of and lcb1-100 strains to edelfosine to from edelfosine on defective lipid rafts. was of Pma1p in and and decreased of Pma1p with lipid rafts in lcb1-100 cells M. S. S. 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FEBS Lett. 2002; 531: 47-53Crossref PubMed Scopus (296) Google Scholar) and points to a novel mode of action for an anti-cancer drug the modification of plasma membrane lipid composition resulting in selective of essential proteins with lipid raft The are consistent with an emerging phenomenon whereby biophysical in membrane composition growth protein that a specific lipid raft for and for their of for the yeast genomic and and Raffael Schaffrath for yeast strains and The of and as as the of the and the Yeast are

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