Sustained activation of sphingomyelin synthase by 2-hydroxyoleic acid induces sphingolipidosis in tumor cells
2013; Elsevier BV; Volume: 54; Issue: 5 Linguagem: Inglês
10.1194/jlr.m036749
ISSN1539-7262
AutoresM. Laura Martín, Gerhard Liebisch, Stefan Lehneis, Gerd Schmitz, María Alonso-Sande, Joan Bestard-Escalas, Daniel H. López, José Manuel García‐Verdugo, Mario Soriano‐Navarro, Xavier Busquets, Pablo V. Escribá, Gwendolyn Barceló‐Coblijn,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoThe mechanism of action of 2-hydroxyoleic acid (2OHOA), a potent antitumor drug, involves the rapid and specific activation of sphingomyelin synthase (SMS), leading to a 4-fold increase in SM mass in tumor cells. In the present study, we investigated the source of the ceramides required to sustain this dramatic increase in SM. Through radioactive and fluorescent labeling, we demonstrated that sphingolipid metabolism was altered by a 24 h exposure to 2OHOA, and we observed a consistent increase in the number of lysosomes and the presence of unidentified storage materials in treated cells. Mass spectroscopy revealed that different sphingolipid classes accumulated in human glioma U118 cells after exposure to 2OHOA, demonstrating a specific effect on C16-, C20-, and C22-containing sphingolipids. Based on these findings, we propose that the demand for ceramides required to sustain the SMS activation (ca. 200-fold higher than the basal level) profoundly modifies both sphingolipid and phospholipid metabolism. As the treatment is prolonged, tumor cells fail to adequately metabolize sphingolipids, leading to a situation resembling sphingolipidosis, whereby cell viability is compromised. The mechanism of action of 2-hydroxyoleic acid (2OHOA), a potent antitumor drug, involves the rapid and specific activation of sphingomyelin synthase (SMS), leading to a 4-fold increase in SM mass in tumor cells. In the present study, we investigated the source of the ceramides required to sustain this dramatic increase in SM. Through radioactive and fluorescent labeling, we demonstrated that sphingolipid metabolism was altered by a 24 h exposure to 2OHOA, and we observed a consistent increase in the number of lysosomes and the presence of unidentified storage materials in treated cells. Mass spectroscopy revealed that different sphingolipid classes accumulated in human glioma U118 cells after exposure to 2OHOA, demonstrating a specific effect on C16-, C20-, and C22-containing sphingolipids. Based on these findings, we propose that the demand for ceramides required to sustain the SMS activation (ca. 200-fold higher than the basal level) profoundly modifies both sphingolipid and phospholipid metabolism. As the treatment is prolonged, tumor cells fail to adequately metabolize sphingolipids, leading to a situation resembling sphingolipidosis, whereby cell viability is compromised. The mechanism of action of 2-hydroxyoleic acid (2OHOA), a potent antitumor drug, involves the specific and sequential induction of cell cycle arrest (1Martínez J. Gutiérrez A. Casas J. Lladó V. López-Bellán A. Besalduch J. Dopazo A. Escribá P.V. The repression of E2F-1 is critical for the activity of Minerval against cancer.J. Pharmacol. Exp. Ther. 2005; 315: 466-474Crossref PubMed Scopus (36) Google Scholar), cell differentiation (2Terés S. Lladó V. Higuera M. Barceló-Coblijn G. Martin M.L. Noguera-Salvà M.A. Marcilla-Etxenike A. García-Verdugo J.M. Soriano-Navarro M. Saus C. et al.2-Hydroxyoleate, a nontoxic membrane binding anticancer drug, induces glioma cell differentiation and autophagy.Proc. Natl. Acad. Sci. USA. 2012; 109: 8489-8494Crossref PubMed Scopus (82) Google Scholar), and cell death in human cancer cells (1Martínez J. Gutiérrez A. Casas J. Lladó V. López-Bellán A. Besalduch J. Dopazo A. Escribá P.V. The repression of E2F-1 is critical for the activity of Minerval against cancer.J. Pharmacol. Exp. Ther. 2005; 315: 466-474Crossref PubMed Scopus (36) Google Scholar, 2Terés S. Lladó V. Higuera M. Barceló-Coblijn G. Martin M.L. Noguera-Salvà M.A. Marcilla-Etxenike A. García-Verdugo J.M. Soriano-Navarro M. Saus C. et al.2-Hydroxyoleate, a nontoxic membrane binding anticancer drug, induces glioma cell differentiation and autophagy.Proc. Natl. Acad. Sci. USA. 2012; 109: 8489-8494Crossref PubMed Scopus (82) Google Scholar, 3Lladó V. Gutiérrez A. Martínez J. Casas J. Terés S. Higuera M. Galmés A. Saus C. Besalduch J. Busquets X. et al.Minerval induces apoptosis in Jurkat and other cancer cells.J. Cell. Mol. Med. 2010; 14: 659-670PubMed Google Scholar, 4Lladó V. Terés S. Higuera M. Alvarez R. Noguera-Salvà M.A. Halver J.E. Escribá P.V. Busquets X. Pivotal role of dihydrofolate reductase knockdown in the anticancer activity of 2-hydroxyoleic acid.Proc. Natl. Acad. Sci. USA. 2009; 106: 13754-13758Crossref PubMed Scopus (35) Google Scholar, 5Martínez J. Vögler O. Casas J. Barceló F. Alemany R. Prades J. Nagy T. Baamonde C. Kasprzyk P.G. Terés S. et al.Membrane structure modulation, protein kinase C alpha activation, and anticancer activity of minerval.Mol. Pharmacol. 2005; 67: 531-540Crossref PubMed Scopus (70) Google Scholar, 6Barceló-Coblijn G. Martin M.L. de Almeida R.F. Noguera-Salvà M.A. Marcilla-Etxenike A. Guardiola-Serrano F. Lüth A. Kleuser B. Halver J.E. Escribá P.V. Sphingomyelin and sphingomyelin synthase (SMS) in the malignant transformation of glioma cells and in 2-hydroxyoleic acid therapy.Proc. Natl. Acad. Sci. USA. 2011; 108: 19569-19574Crossref PubMed Scopus (124) Google Scholar). Based on its high efficacy and low toxicity compared with other existing chemotherapy agents, the European Medicines Agency has acknowledged the potential benefits of 2OHOA and has designated this molecule as an orphan drug for the treatment of glioma (7European Medicines Agency Committee for Orphan Medicinal Products. 2011 Public summary of opinion on orphan designation: 2-hydroxyoleic acid for the treatment of glioma. EMA/COMP/780686/2011. Available at: http://www.ema.europa.eu/docs/en_GB/document_library/Orphan_designation/2011/11/WC500117956.pdf. Accessed Feb 8, 2013Google Scholar). In the present study, we investigated the mechanism of action of 2OHOA to further clarify the features underlying its unique combination of high efficacy, low toxicity, and specificity. By activating sphingomyelin synthase (SMS) isozymes, 2OHOA increases SM mass in a rapid and highly specific manner (6Barceló-Coblijn G. Martin M.L. de Almeida R.F. Noguera-Salvà M.A. Marcilla-Etxenike A. Guardiola-Serrano F. Lüth A. Kleuser B. Halver J.E. Escribá P.V. Sphingomyelin and sphingomyelin synthase (SMS) in the malignant transformation of glioma cells and in 2-hydroxyoleic acid therapy.Proc. Natl. Acad. Sci. USA. 2011; 108: 19569-19574Crossref PubMed Scopus (124) Google Scholar). Indeed, the effect of 2OHOA on the cell cycle is partially reversed by SMS inhibition, suggesting that its activation is critical in its mechanism of action. SMS proteins catalyze the transfer of a phosphocholine moiety to the primary hydroxyl group of ceramide to form SM and 1,2-diacylglycerol (1,2-DAG) (8Huitema K. van den Dikkenberg J. Brouwers J.F. Holthuis J.C. Identification of a family of animal sphingomyelin synthases.EMBO J. 2004; 23: 33-44Crossref PubMed Scopus (469) Google Scholar). However, the source of the ceramides required to maintain the sustained synthesis of SM induced by 2OHOA has yet to be defined. The increase in sphingosine (Sph) mass following 2OHOA treatment previously described already suggests that the salvage pathway is activated (6Barceló-Coblijn G. Martin M.L. de Almeida R.F. Noguera-Salvà M.A. Marcilla-Etxenike A. Guardiola-Serrano F. Lüth A. Kleuser B. Halver J.E. Escribá P.V. Sphingomyelin and sphingomyelin synthase (SMS) in the malignant transformation of glioma cells and in 2-hydroxyoleic acid therapy.Proc. Natl. Acad. Sci. USA. 2011; 108: 19569-19574Crossref PubMed Scopus (124) Google Scholar, 9Kitatani K. Idkowiak-Baldys J. Hannun Y.A. The sphingolipid salvage pathway in ceramide metabolism and signaling.Cell. Signal. 2008; 20: 1010-1018Crossref PubMed Scopus (415) Google Scholar). However, ceramides can be also synthesized via two alternative mechanisms: the "de novo" pathway, through the condensation of L-serine and palmitic acid; and the SM cycle (10Kolesnick R. Golde D.W. The sphingomyelin pathway in tumor necrosis factor and interleukin-1 signaling.Cell. 1994; 77: 325-328Abstract Full Text PDF PubMed Scopus (915) Google Scholar). Accordingly, we examined the effect of 2OHOA on ceramide metabolism in U118 human brain cancer cells, showing that 2OHOA activated the pathway of de novo ceramide synthesis and the recycling pathway. Furthermore, exposure to 2OHOA induced a profound alteration in sphingolipid metabolism, resulting in the accumulation of SM, ceramide, and hexosylceramide (HexCer) species. This sphingolipid accumulation was associated with an increase in lysosomal number and the formation of unidentified intracellular structures that were probably composed of the sphingolipids accumulated. Based on these results, we propose that the rapid and sustained activation of SMS generates an unusual demand for ceramide, provoking a profound shift in sphingolipid metabolism in order to maximize ceramide production. When exposure to 2OHOA is prolonged (48–72 h), tumor cells fail to fulfill this high metabolic requirement, resulting in a situation that resembles sphingolipidosis, in which cell viability is compromised. At this point, tumor cells trigger the activation of different pathways, such as cell cycle arrest and differentiation or cell apoptosis, producing a decrease in tumor cell number. These results complement our previous findings and allow a temporal sequence of events to be established that accounts for the antitumor effects of 2OHOA. Human glioma cells (U118) and human lung adenocarcinoma cells (A549) were obtained from the American Type Culture Collection (Manassas, VA) and maintained as described previously (4Lladó V. Terés S. Higuera M. Alvarez R. Noguera-Salvà M.A. Halver J.E. Escribá P.V. Busquets X. Pivotal role of dihydrofolate reductase knockdown in the anticancer activity of 2-hydroxyoleic acid.Proc. Natl. Acad. Sci. USA. 2009; 106: 13754-13758Crossref PubMed Scopus (35) Google Scholar). The 2OHOA (99.7%) compound (Good Manufacturing Practice quality) was obtained from Avanti Polar Lipids. NBD-C6-ceramide (NBD-Cer) and NBD-C6-sphingomyelin (NBD-SM) were purchased from Invitrogen (Barcelona, Spain). NBD-glucosylceramide (NBD-GluCer) was purchased from Larodan (Malmö, Sweden), and NBD-phosphatidylethanolamine (NBD-PE) and NBD-phosphatidylcholine (NBD-PC) were obtained from Avanti Polar Lipids (AL). Spectroscopic-grade organic solvents for the lipid and probe solutions were purchased from Merck (Darmstadt, Germany). Lipid extraction and mass spectrometry-based targeted lipid analysis was performed as described previously (11Ecker J. Liebisch G. Englmaier M. Grandl M. Robenek H. Schmitz G. Induction of fatty acid synthesis is a key requirement for phagocytic differentiation of human monocytes.Proc. Natl. Acad. Sci. USA. 2010; 107: 7817-7822Crossref PubMed Scopus (163) Google Scholar, 12Leidl K. Liebisch G. Richter D. Schmitz G. Mass spectrometric analysis of lipid species of human circulating blood cells.Biochim. Biophys. Acta. 2008; 1781: 655-664Crossref PubMed Scopus (121) Google Scholar, 13Scherer M. Schmitz G. Liebisch G. High-throughput analysis of sphingosine 1-phosphate, sphinganine 1-phosphate, and lysophosphatidic acid in plasma samples by liquid chromatography-tandem mass spectrometry.Clin. Chem. 2009; 55: 1218-1222Crossref PubMed Scopus (124) Google Scholar, 14Scherer M. Leuthauser-Jaschinski K. Ecker J. Schmitz G. Liebisch G. A rapid and quantitative LC-MS/MS method to profile sphingolipids.J. Lipid Res. 2010; 51: 2001-2011Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 15Liebisch G. Drobnik W. Reil M. Trumbach B. Arnecke R. Olgemoller B. Roscher A. Schmitz G. Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS).J. Lipid Res. 1999; 40: 1539-1546Abstract Full Text Full Text PDF PubMed Google Scholar, 16Liebisch G. Lieser B. Rathenberg J. Drobnik W. Schmitz G. High-throughput quantification of phosphatidylcholine and sphingomyelin by electrospray ionization tandem mass spectrometry coupled with isotope correction algorithm.Biochim. Biophys. Acta. 2004; 1686: 108-117Crossref PubMed Scopus (247) Google Scholar), in the presence of isotopic labeled lipids or nonnaturally occurring lipid species (as internal standards). Briefly, cell pellets were lysed in 0.1% SDS and sonicated, and then lipids were extracted from aliquots corresponding to 100 µg total protein (BCA assay). Lipids were quantified by electrospray ionization tandem mass spectrometry (ESI-MS/MS) in positive ion mode. Samples were quantified by direct flow injection analysis using the analytical setup described by Liebisch et al. (16Liebisch G. Lieser B. Rathenberg J. Drobnik W. Schmitz G. High-throughput quantification of phosphatidylcholine and sphingomyelin by electrospray ionization tandem mass spectrometry coupled with isotope correction algorithm.Biochim. Biophys. Acta. 2004; 1686: 108-117Crossref PubMed Scopus (247) Google Scholar). Sphingosine-based ceramides (Cer) were analyzed using a fragment ion of m/z 264 (15Liebisch G. Drobnik W. Reil M. Trumbach B. Arnecke R. Olgemoller B. Roscher A. Schmitz G. Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS).J. Lipid Res. 1999; 40: 1539-1546Abstract Full Text Full Text PDF PubMed Google Scholar). For each lipid class two, nonnaturally occurring internal standards were added and quantification was achieved by calibration lines generated by addition of naturally occurring lipid species to the respective sample matrix. Liquid chromatography coupled to MS/MS (LC-MS/MS) was used to quantify HexCer, lactosylceramides (LacCer), sphingoid bases, and sphingosylphosphorylcholine (SPC) (14Scherer M. Leuthauser-Jaschinski K. Ecker J. Schmitz G. Liebisch G. A rapid and quantitative LC-MS/MS method to profile sphingolipids.J. Lipid Res. 2010; 51: 2001-2011Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) as well as lysophospholipids, sphingosine-1-phosphate, and lysophosphatidic acid (13Scherer M. Schmitz G. Liebisch G. High-throughput analysis of sphingosine 1-phosphate, sphinganine 1-phosphate, and lysophosphatidic acid in plasma samples by liquid chromatography-tandem mass spectrometry.Clin. Chem. 2009; 55: 1218-1222Crossref PubMed Scopus (124) Google Scholar). Deisotoping and data analysis for all lipid classes were performed by self-programmed Excel macros according to the principles described previously (14Scherer M. Leuthauser-Jaschinski K. Ecker J. Schmitz G. Liebisch G. A rapid and quantitative LC-MS/MS method to profile sphingolipids.J. Lipid Res. 2010; 51: 2001-2011Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 16Liebisch G. Lieser B. Rathenberg J. Drobnik W. Schmitz G. High-throughput quantification of phosphatidylcholine and sphingomyelin by electrospray ionization tandem mass spectrometry coupled with isotope correction algorithm.Biochim. Biophys. Acta. 2004; 1686: 108-117Crossref PubMed Scopus (247) Google Scholar). After extraction with n-hexane:2-propanol (3:2, by vol) (17Hara A. Radin N.S. Lipid extraction of tissues with a low-toxicity solvent.Anal. Biochem. 1978; 90: 420-426Crossref PubMed Scopus (2065) Google Scholar, 18Castagnet P.I. Golovko M.Y. Barceló-Coblijn G.C. Nussbaum R.L. Murphy E.J. Fatty acid incorporation is decreased in astrocytes cultured from alpha-synuclein gene-ablated mice.J. Neurochem. 2005; 94: 839-849Crossref PubMed Scopus (82) Google Scholar), individual phospholipid classes were separated by TLC, and the amount of protein was measured as described previously (6Barceló-Coblijn G. Martin M.L. de Almeida R.F. Noguera-Salvà M.A. Marcilla-Etxenike A. Guardiola-Serrano F. Lüth A. Kleuser B. Halver J.E. Escribá P.V. Sphingomyelin and sphingomyelin synthase (SMS) in the malignant transformation of glioma cells and in 2-hydroxyoleic acid therapy.Proc. Natl. Acad. Sci. USA. 2011; 108: 19569-19574Crossref PubMed Scopus (124) Google Scholar, 19Gellermann G.P. Appel T.R. Tannert A. Radestock A. Hortschansky P. Schroeckh V. Leisner C. Lutkepohl T. Shtrasburg S. Rocken C. et al.Raft lipids as common components of human extracellular amyloid fibrils.Proc. Natl. Acad. Sci. USA. 2005; 102: 6297-6302Crossref PubMed Scopus (181) Google Scholar, 20Marcheselli V.L. Scott B.L. Reddy S. Bazán N.G. Quantitative analysis of acyl group composition of brain phospholipids, neutral lipids, and free fatty acids..Neuromethods. 1989; 7: 83-110Google Scholar). Control and treated (200 µM, 24 h) U118 and A549 cells were incubated with NDB-C6-Cer, NDB-C6-GluCer, NDB-C6-SM, NDB-C6-PE, and NDB-C6-PC (3 μM) for 4 h prior to lipid extraction. After lipid extraction, NBD-C6-phospholipids were separated by HPTLC as described above, and the fluorescent lipids were visualized on a Bio-Rad Molecular Imager FX and quantified using Quantity One software (Bio-Rad). Control and treated (200 µM, 6 or 24 h) U118 cells were pulse labeled with [3H]palmitic acid (0.30 µCi/ml) for 5 min, and then total cell lipids were extracted and separated by TLC as described previously (21Kuerschner L. Ejsing C.S. Ekroos K. Shevchenko A. Anderson K.I. Thiele C. Polyene-lipids: a new tool to image lipids.Nat. Methods. 2005; 2: 39-45Crossref PubMed Scopus (150) Google Scholar). The plates were dried, and the side with the standards was sprayed with a solution of 8% (w/v) H3PO4 containing 10% (w/v) CuSO4 before they were dried and charred over a heater to develop the nonradioactive standard bands. The area corresponding to each lipid was scraped off, and the radioactivity was measured by liquid scintillation counting. The levels of [3H]ceramide produced were normalized to the cellular protein content. U118 cells were incubated with D609 for 16 h (200 μM) and 2OHOA (200 μM) was added 1 h after the addition of D609 (Tocris Bioscience, UK). After the incubation period, cell pellets were lysed in 0.1% SDS and sonicated. Lipids were extracted from aliquots corresponding to 100 µg total protein and analyzed by MS as previously described. U118 cells were plated at a density of 1.1·× 104 cell/cm2 on Chambered Coverglass (Lab-TekTM II, Thermo Fisher Scientific) as indicated above, in the presence or absence of 2OHOA (200 µM, 48 h). After treatment, the cells were incubated for 1 h with 1 µM LysoSensor Green DND-189 probe pH Indicator (pH 4.5–6; Invitrogen), with Hoechst (trihydrochloride trihidrate, 40 µg/ml; Invitrogen) added for the last 5 min. Stained samples were visualized on a Nikon Eclipse TE2000-S fluorescence microscope at 40× magnification. Cells were seeded at 1.1 × 104 cell/cm2 in 4-well Lab-Tek chamber slides (Nalge Nunc International, Naperville, IL) as indicated above, and they were maintained in the presence or absence of 2OHOA (200 µM) for 48 or 72 h. The cells were postfixed in 2% OsO4 for 1 h at room temperature and stained with 2% uranyl acetate (in 70% ethanol) in darkness for 2 h at 4°C. Finally, the cells were rinsed in sodium phosphate buffer (0.1 M, pH 7.2), dehydrated in ethanol, and infiltrated overnight with Araldite (Durcupan; Fluka, Buchs SG, Switzerland). Following polymerization, embedded cultures were detached from the chamber slide and glued to Araldite blocks. Serial semi-thin (1.5 µm) sections were cut with an Ultracut UC-6 (Leica, Heidelberg, Germany), mounted onto slides, and stained with 1% toluidine blue. Selected semi-thin sections were glued (Super Glue, Loctite) to araldite blocks and detached from the glass slide by repeated freezing (in liquid nitrogen) and thawing. Ultrathin (0.06–0.09 µm) ultracut sections were obtained and stained with lead citrate. Finally, photomicrographs were obtained by transmission electron microscopy (FEI Tecnai G2 Spirit Biotwin) using a digital camera (Morada, Soft Imaging System, Olympus). Statistical analyses were performed using GraphPad Prism 4.01 (GraphPad Software Inc., San Diego, CA). Unless otherwise indicated, data are expressed as the mean ± SEM of at least three independent experiments (n = 3). The statistical significance of the mean difference was determined using the Student t-test. Asterisks indicate a significant effect of treatment compared with controls: *P < 0.05; **P < 0.01; ***P < 0.001. Given the rapid effect of 2OHOA on SMS and the subsequent accumulation of SM (6Barceló-Coblijn G. Martin M.L. de Almeida R.F. Noguera-Salvà M.A. Marcilla-Etxenike A. Guardiola-Serrano F. Lüth A. Kleuser B. Halver J.E. Escribá P.V. Sphingomyelin and sphingomyelin synthase (SMS) in the malignant transformation of glioma cells and in 2-hydroxyoleic acid therapy.Proc. Natl. Acad. Sci. USA. 2011; 108: 19569-19574Crossref PubMed Scopus (124) Google Scholar), we used mass spectrometry to investigate the impact of 2OHOA (200 μM) on the rest of sphingolipids over a wide range of time points (0.5–72 h). Unexpectedly, no major changes in ceramide, HexCer, LacCer, Sph, dihydrosphingosine (dhSph), or dihydrosphingomyelin (dhSM) accumulation were detected following short exposures to 2OHOA (0.5–24 h; Fig. 1). The lack of changes does not exclude the possibility that the sphingolipid metabolism is not affected at that time. We addressed this point by using both radiolabeled and NBD-labeled substrates (see below). In addition, the lipidomic analysis showed that all these lipids, except LacCer, were increased after a 72 h exposure (Table 1 and Fig. 1). Thus, the mass of ceramide, HexCer, and dhSM increased 1.9-, 1.2- and 4.6-fold, respectively, and similarly, Sph and dhSph mass increased 1.7- and 4.4-fold, respectively, while that of LacCer decreased 28.6% in treated cells. Free dhSph is mostly generated by de novo sphingolipid biosynthesis, whereas free Sph (the product of hydrolysis of complex sphingolipids) appears to be derived exclusively from the turnover of complex sphingolipids (9Kitatani K. Idkowiak-Baldys J. Hannun Y.A. The sphingolipid salvage pathway in ceramide metabolism and signaling.Cell. Signal. 2008; 20: 1010-1018Crossref PubMed Scopus (415) Google Scholar). Hence, these results are the first evidence indicating that both de novo ceramide synthesis (via dhSph accumulation) and the salvage pathway (via Sph accumulation) activation occurred after exposure to 2OHOA.TABLE 1Effect of 2OHOA treatment on the mass of different sphingolipid classes after 72 h of treatmentSphingolipid Class (pmol/mg protein)ControlTreatedCeramides755.7 ± 236.61406 ± 187.1*Hexosylceramide367.1 ± 1.0456.5 ± 19.3*Lactosylceramide2124 ± 11.81525 ± 165.4*Sphingosine27.0 ± 3.450.8 ± 4.1**Sphinganine6.9 ± 0.930.4 ± 3.2**Dihydrosphingomyelin86.7 ± 21.96400.0 ± 54.9**After exposing U118 cells to 2OHOA for 72 h (200 µM), lipids were extracted and analyzed by mass spectrometry. The values represent the mean ± SEM (n = 3–4). Asterisks (*) indicate a significant effect of treatment compared with controls: *P < 0.05, **P < 0.01, and ***P < 0.001. Open table in a new tab After exposing U118 cells to 2OHOA for 72 h (200 µM), lipids were extracted and analyzed by mass spectrometry. The values represent the mean ± SEM (n = 3–4). Asterisks (*) indicate a significant effect of treatment compared with controls: *P < 0.05, **P < 0.01, and ***P < 0.001. MS analysis enabled the molecular species of each lipid class to be characterized in detail, which revealed a profound remodeling of the sphingolipid fatty acid composition following 2OHOA treatment (Fig. 2). The most consistent change among sphingolipids was the increase in C16- and C22-containing sphingolipid species, and after 72 h of treatment, the mass of C16-ceramide, C16-HexCer and C16-dhSM increased 2.2-, 3.0-, and 7.8-fold, respectively. Likewise, C22-ceramide, C22-HexCer and C22-LacCer increased 5.4-, 3.2-, and 2.2-fold, respectively. Interestingly, the mass of C24-sphingolipid species (24:0 and 24:1), which account for the 80% of total fatty acids, was either unaffected (in ceramides) or diminished (in HexCer and LacCer) following 2OHOA treatment. As a first approach to understand the specific effect on sphingolipid fatty acid composition, we investigated whether the treatment affected CerS mRNA expression levels (supplementary Fig. I). However, despite the large increase in C16- and C22-containing sphingolipids, no significant differences in CerS5 and CerS1 mRNA expression were observed in 2OHOA-treated cells and controls (200 μM, 72 h; supplementary Fig. IB). Based on the indirect evidence suggesting that both de novo synthesis and the salvage pathway are activated by 2OHOA, we further analyzed the effect of 2OHOA on these pathways in U118 cells. Activation of the de novo pathway was examined specifically by pulse-labeling with radioactive palmitate, the substrate of serine palmitoyltransferase (SPT) that is the first enzyme in this pathway (9Kitatani K. Idkowiak-Baldys J. Hannun Y.A. The sphingolipid salvage pathway in ceramide metabolism and signaling.Cell. Signal. 2008; 20: 1010-1018Crossref PubMed Scopus (415) Google Scholar). Cells were exposed to 2OHOA for 6 or 24 h and then pulse-labeled with [3H]palmitic acid for 5 min prior to lipid extraction. TLC analysis revealed a 7.8- and 5.6-fold increase in [3H]ceramide content following exposure to 2OHOA for 6 and 24 h, respectively (Fig. 3), confirming the activation of the de novo synthesis pathway. As expected, [3H]SM content increased after 2OHOA treatment (2.7-fold after 6 h and 3.4-fold after 24 h). Finally, the increases observed in the levels of [3H]LacCer and [3H]HexCer provided further evidence of a general modification of the sphingolipid metabolism. This was analyzed by incubating control and 2OHOA-treated (200 µM, 24 h) cells with the following NBD-C6 sphingolipid analogs (3 µM, 3 h): NBD-Cer, NBD-SM, and NBD-C6-GlcCer (Fig. 4A–D). Incubation with NBD-Cer or NBD-SM resulted in the accumulation of NBD-GlcCer (5.9- and 11.6-fold, respectively). Similarly, when cells were incubated with NBD-SM, we observed increased accumulation of NBD-Cer (2.7-fold) and NBD-GlcCer (3.0-fold). Finally, after exposure to NBD-GlcCer, the formation of NBD-Cer and NBD-SM increased 4.6- and 5.3-fold, respectively. Importantly, similar results were obtained in A549 cells (supplementary Fig. II). These results further confirm that both sphingolipid synthetic and degrading pathways are altered by exposing cells to 2OHOA for 24 h.Fig. 42OHOA alters the turnover of sphingolipids and phospholipids in U118 cells. (A) Representative TLC of lipid extracts from control and 2OHOA-treated cells (200 µM, 24 h) incubated with NBD-Cer (B), NBD-SM (C), or NBD-GlcCer (D: 3 µM, 3 h), from which lipids were extracted and analyzed by TLC. NBD-C6-SP, NBD-C6-sphingolipids. (E) Representative TLC of lipid extracts from control and 2OHOA-treated cells (200 μ, 24 h) incubated with NBD-C6-PE for 3 h, and from which lipids were extracted and analyzed by TLC. Separation was achieved using a two-solvent system (21Kuerschner L. Ejsing C.S. Ekroos K. Shevchenko A. Anderson K.I. Thiele C. Polyene-lipids: a new tool to image lipids.Nat. Methods. 2005; 2: 39-45Crossref PubMed Scopus (150) Google Scholar). The image contrast was saturated to better visualize the minor bands (image on right side of the panel indicated with an arrow). AU, arbitrary units. The values represent the mean ± SEM (n = 3). Asterisks (*) indicate a significant effect of treatment compared with controls: *P < 0.05, **P < 0.01, and ***P < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Collectively, our experiments using radioactive- and fluorescent-labeled lipids provide further evidence that de novo synthesis ([3H]palmitic acid labeling experiment), the salvage pathway (GluCer-NBD-labeling experiment) and the SM cycle (SM-NBD-labeling experiment) are activated after treatment with 2OHOA. Interestingly, the incubation of U118 cells with NBD-PE resulted in an unexpected increase in the formation of NBD-PC) (4-fold; Fig. 4E). The strong demand for PC as the second necessary substrate used by SMS along with ceramide may account for the activation of this pathway. In addition, this result may explain the significant decrease in PE mass previously described (6Barceló-Coblijn G. Martin M.L. de Almeida R.F. Noguera-Salvà M.A. Marcilla-Etxenike A. Guardiola-Serrano F. Lüth A. Kleuser B. Halver J.E. Escribá P.V. Sphingomyelin and sphingomyelin synthase (SMS) in the malignant transformation of glioma cells and in 2-hydroxyoleic acid therapy.Proc. Natl. Acad. Sci. USA. 2011; 108: 19569-19574Crossref PubMed Scopus (124) Google Scholar). Thus, the decrease in PE and PC masses (60 and 73 nmol/mg protein, respectively) correlated approximately with the increase in SM mass (151 nmol/mg protein; see Table 1 in Ref. 6Barceló-Coblijn G. Martin M.L. de Almeida R.F. Noguera-Salvà M.A. Marcilla-Etxenike A. Guardiola-Serrano F. Lüth A. Kleuser B. Halver J.E. Escribá P.V. Sphingomyelin and sphingomyelin synthase (SMS) in the malignant transformation of glioma cells and in 2-hydroxyoleic acid therapy.Proc. Natl. Acad. Sci. USA. 2011; 108: 19569-19574Crossref PubMed Scopus (124) Google Scholar). Taking into account the avid consumption of ceramides by SMS and that the amount present in the cell was approximately two orders of magnitude lower than the amount of SM, our hypothesis was that cells try to compensate this severe unbalance by activating ceramide-generating pathways. So far we demonstrated that the de novo, the salvage, and the SMase cycles are activated. To test that the driving force leading cells to activate them is the reestablishment of the chemical equilibrium rather than a direct effect of 2OHOA on any of the enzymes involved in those pathways, we inhibited SMS using D609 (potassium tricyclodecan-9-yl xanthate) (22González-Roura A. Casas J. Llebaria
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