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. 2013 May;54(5):1457-65.
doi: 10.1194/jlr.M036749. Epub 2013 Mar 7.

Sustained activation of sphingomyelin synthase by 2-hydroxyoleic acid induces sphingolipidosis in tumor cells

Maria Laura Martin  1 , Gerhard Liebisch, Stefan Lehneis, Gerd Schmitz, María Alonso-Sande, Joan Bestard-Escalas, Daniel H Lopez, José Manuel García-Verdugo, Mario Soriano-Navarro, Xavier Busquets, Pablo V Escribá, Gwendolyn Barceló-Coblijn
Affiliations

Sustained activation of sphingomyelin synthase by 2-hydroxyoleic acid induces sphingolipidosis in tumor cells

Maria Laura Martin et al. J Lipid Res. 2013 May.

Abstract

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.

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Figures

Fig. 1.
Fig. 1.
2OHOA treatment alters the sphingolipid composition in U118 cells. After U118 cells were exposed to 2OHOA for different times (200 µM, 0.5–72 h), the lipids were extracted and analyzed by mass spectrometry. (A) Total ceramide mass, (B) total HexCer mass, (C) total LacCer mass, (D) total dhSM mass, (E) Sph mass, and (F) dhSph mass. The values represent the mean ± SEM (n = 3–4). Asterisks (*) indicate significant effect of the treatment compared with controls: *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 2.
Fig. 2.
2OHOA modulates the profile of sphingolipid molecular species. After U118 cells were exposed to 2OHOA (200 µM, 72 h), lipids were extracted and analyzed by mass spectrometry. (A) Ceramide species mass; (B) HexCer species mass, (C) LacCer species mass, and (D) dhSM species mass. 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.
Fig. 3.
Fig. 3.
2OHOA induces de novo ceramide synthesis. U118 cells exposed to 2OHOA for 6 h (A) and 24 h (B) were pulse-labeled with [3H]palmitic acid for 5 min before the radioactive lipids were separated by TLC and quantified by liquid scintillation counting. HexCer includes GluCer+GalCer, [3H]SP and [3H]sphingolipids. Values represent the mean ± SEM (n = 3). Asterisks (*) indicate a significant effect of the treatment compared with controls: *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 4.
Fig. 4.
2OHOA 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 (21). 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.
Fig. 5.
Fig. 5.
Effect on ceramide mass of the SMS inhibition by D609 in U118 cells. Inhibition of SMS by D609 in the presence of 2OHOA induced a minor accumulation of ceramides. The values represent the mean ± SEM (n = 3) and the asterisks (*) indicate a significant effect of treatment compared with controls: *P < 0.05. In this case, and indicates a significant effect of the inhibition compared with the treatment (P < 0.05).
Fig. 6.
Fig. 6.
2OHOA treatment augments the number of lysosomes and induces the appearance of myelin-like bodies. (A) Representative micrographs of control and 2OHOA-treated cells (200 µM, 72 h) labeled with Lysosensor (see Experimental Procedures). Scale bar = 100 µM; (B-D) U118 cells were exposed to 2OHOA for 72 h, fixed and processed for electron microscopy. Numerous myelin-like bodies (arrows) are visible in the cell's cytoplasm (B, D). On occasion, these heterogeneous dense bodies form important aggregates, as shown in C. In addition to myelin-like bodies (arrows), lipid droplets (LD) were also observed and in the image shown, degeneration is also visible in the nucleus (*, D). N, nucleus; mit, mitochondrion.
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References

    1. Martínez J., Gutiérrez A., Casas J., Lladó V., López-Bellán A., Besalduch J., Dopazo A., Escribá P. V. 2005. The repression of E2F-1 is critical for the activity of Minerval against cancer. J. Pharmacol. Exp. Ther. 315: 466–474 - PubMed
    1. Terés 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. 2012. 2-Hydroxyoleate, a nontoxic membrane binding anticancer drug, induces glioma cell differentiation and autophagy. Proc. Natl. Acad. Sci. USA. 109: 8489–8494 - PMC - PubMed
    1. Lladó V., Gutiérrez A., Martínez J., Casas J., Terés S., Higuera M., Galmés A., Saus C., Besalduch J., Busquets X., et al. 2010. Minerval induces apoptosis in Jurkat and other cancer cells. J. Cell. Mol. Med. 14: 659–670 - PMC - PubMed
    1. Lladó V., Terés S., Higuera M., Alvarez R., Noguera-Salvà M. A., Halver J. E., Escribá P. V., Busquets X. 2009. Pivotal role of dihydrofolate reductase knockdown in the anticancer activity of 2-hydroxyoleic acid. Proc. Natl. Acad. Sci. USA. 106: 13754–13758 - PMC - PubMed
    1. Martínez J., Vögler O., Casas J., Barceló F., Alemany R., Prades J., Nagy T., Baamonde C., Kasprzyk P. G., Terés S., et al. 2005. Membrane structure modulation, protein kinase C alpha activation, and anticancer activity of minerval. Mol. Pharmacol. 67: 531–540 - PubMed

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