Acid ceramidase as a therapeutic target in metastatic prostate cancer
2013; Elsevier BV; Volume: 54; Issue: 5 Linguagem: Inglês
10.1194/jlr.m032375
ISSN1539-7262
AutoresLuz Camacho, Óscar Meca‐Cortés, José Luı́s Abad, Simón García, Núria Rubio, Alba Díaz, Toni Celià-Terrassa, Francesca Cingolani, Raquel Bermudo, Pedro L. Fernández, Jerónimo Blanco, Antonio Delgado, Josefina Casas, Gemma Fabriàs, Timothy M. Thomson,
Tópico(s)Fibroblast Growth Factor Research
ResumoAcid ceramidase (AC) catalyzes the hydrolysis of ceramide into sphingosine, in turn a substrate of sphingosine kinases that catalyze its conversion into the mitogenic sphingosine-1-phosphate. AC is expressed at high levels in several tumor types and has been proposed as a cancer therapeutic target. Using a model derived from PC-3 prostate cancer cells, the highly tumorigenic, metastatic, and chemoresistant clone PC-3/Mc expressed higher levels of the AC ASAH1 than the nonmetastatic clone PC-3/S. Stable knockdown of ASAH1 in PC-3/Mc cells caused an accumulation of ceramides, inhibition of clonogenic potential, increased requirement for growth factors, and inhibition of tumorigenesis and lung metastases. We developed de novo ASAH1 inhibitors, which also caused a dose-dependent accumulation of ceramides in PC-3/Mc cells and inhibited their growth and clonogenicity. Finally, immunohistochemical analysis of primary prostate cancer samples showed that higher levels of ASAH1 were associated with more advanced stages of this neoplasia. These observations confirm ASAH1 as a therapeutic target in advanced and chemoresistant forms of prostate cancer and suggest that our new potent and specific AC inhibitors could act by counteracting critical growth properties of these highly aggressive tumor cells. Acid ceramidase (AC) catalyzes the hydrolysis of ceramide into sphingosine, in turn a substrate of sphingosine kinases that catalyze its conversion into the mitogenic sphingosine-1-phosphate. AC is expressed at high levels in several tumor types and has been proposed as a cancer therapeutic target. Using a model derived from PC-3 prostate cancer cells, the highly tumorigenic, metastatic, and chemoresistant clone PC-3/Mc expressed higher levels of the AC ASAH1 than the nonmetastatic clone PC-3/S. Stable knockdown of ASAH1 in PC-3/Mc cells caused an accumulation of ceramides, inhibition of clonogenic potential, increased requirement for growth factors, and inhibition of tumorigenesis and lung metastases. We developed de novo ASAH1 inhibitors, which also caused a dose-dependent accumulation of ceramides in PC-3/Mc cells and inhibited their growth and clonogenicity. Finally, immunohistochemical analysis of primary prostate cancer samples showed that higher levels of ASAH1 were associated with more advanced stages of this neoplasia. These observations confirm ASAH1 as a therapeutic target in advanced and chemoresistant forms of prostate cancer and suggest that our new potent and specific AC inhibitors could act by counteracting critical growth properties of these highly aggressive tumor cells. Cancer cells develop a lipogenic phenotype that supports the energy and membrane synthesis requirements associated with the enhanced proliferation and survival under stress inherent to malignant progression (1Hirsch H.A. Iliopoulos D. Joshi A. Zhang Y. Jaeger S.A. Bulyk M. Tsichlis P.N. Shirley Liu X. Struhl K. A transcriptional signature and common gene networks link cancer with lipid metabolism and diverse human diseases.Cancer Cell. 2010; 17: 348-361Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 2Menendez J.A. Lupu R. Fatty acid synthase and the lipogenic phenotype in cancer pathogenesis.Nat. Rev. Cancer. 2007; 7: 763-777Crossref PubMed Scopus (2024) Google Scholar). 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One pathway that many neoplastic cells activate to offset the toxic accumulation of palmitate is its peroxisome proliferator-activated receptor (PPAR)γ-dependent funneling to eventually form triglycerides, which can be further used as energy stores (4Kourtidis A. Srinivasaiah R. Carkner R.D. Brosnan M.J. Conklin D.S. Peroxisome proliferator-activated receptor-gamma protects ERBB2-positive breast cancer cells from palmitate toxicity.Breast Cancer Res. 2009; 11: R16Crossref PubMed Scopus (48) Google Scholar). A second pathway followed by palmitate is the condensation of palmitoyl CoA with L-serine, leading to the synthesis of ceramides (5Delgado A. Casas J. Llebaria A. Abad J.L. Fabrias G. Inhibitors of sphingolipid metabolism enzymes.Biochim. Biophys. Acta. 2006; 1758: 1957-1977Crossref PubMed Scopus (144) Google Scholar). The accumulation of ceramides also poses a problem for cell survival because of its proapoptotic consequences (6Kolesnick R.N. Kronke M. 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Acta. 2006; 1758: 1957-1977Crossref PubMed Scopus (144) Google Scholar), which is reduced to the sphingoid base sphinganine and acylated by ceramide synthase to generate dihydroceramide. This compound is oxidized to ceramide by introduction of a trans-4,5 double bond. This pathway can be stimulated by drugs and ionizing radiation and usually results in a prolonged ceramide accumulation (10Henry, B., Moller, C., Dimanche-Boitrel, M. T., Gulbins, E., Becker, K. A., . Targeting the ceramide system in cancer. Cancer Lett., Epub ahead of print. July 23, 2011; doi:10.1016/j.canlet.2011.07.010.Google Scholar). Once generated, ceramide may amass or be converted into a variety of metabolites. Phosphorylation by ceramide kinase (11Bornancin F. Ceramide kinase: the first decade.Cell. Signal. 2011; 23: 999-1008Crossref PubMed Scopus (62) Google Scholar) generates ceramide 1-phosphate, while deacylation by alkaline, neutral or acid ceramidases (the products of different genes) (12Gangoiti P. Camacho L. Arana L. Ouro A. Granado M.H. Brizuela L. Casas J. Fabrias G. Abad J.L. Delgado A. et al.Control of metabolism and signaling of simple bioactive sphingolipids: implications in disease.Prog. Lipid Res. 2010; 49: 316-334Crossref PubMed Scopus (119) Google Scholar) yields sphingosine, which may be phosphorylated by sphingosine kinase to S1P. Two distinct sphingosine kinases have been cloned. These two isoforms differ in temporal patterns of expression during development, are expressed in different tissues, and possess distinct kinetic properties (13Pitson S.M. Regulation of sphingosine kinase and sphingolipid signaling.Trends Biochem. Sci. 2011; 36: 97-107Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar), implying that they perform different cellular functions. Ceramide may also be converted back to SM by transfer of phosphorylcholine from phosphatidylcholine via SM synthases (14Holthuis J.C. Luberto C. Tales and mysteries of the enigmatic sphingomyelin synthase family.Adv. Exp. Med. Biol. 2010; 688: 72-85Crossref PubMed Scopus (36) Google Scholar). Alternatively, it can be glycosylated by glucosylceramide synthase to form glucosylceramide, which may be further modified by various enzymes in the Golgi apparatus to form complex glycosphingolipids (15Wennekes T. Berg R.J. Boot R.G. van der Marel G.A. Overkleeft H.S. Aerts J.M. Glycosphingolipids–nature, function, and pharmacological modulation.Angew. Chem. Int. Ed. Engl. 2009; 48: 8848-8869Crossref PubMed Scopus (237) Google Scholar). Many tumor types express high levels of acid ceramidase (AC). Specifically, the expression levels of AC in prostate cancer have been reported to be elevated relative to normal prostate tissue (16Norris J.S. Bielawska A. Day T. El-Zawahri A. ElOjeimy S. Hannun Y. Holman D. Hyer M. Landon C. Lowe S. et al.Combined therapeutic use of AdGFPFasL and small molecule inhibitors of ceramide metabolism in prostate and head and neck cancers: a status report.Cancer Gene Ther. 2006; 13: 1045-1051Crossref PubMed Scopus (53) Google Scholar, 17Seelan R.S. Qian C. Yokomizo A. Bostwick D.G. Smith D.I. Liu W. Human acid ceramidase is overexpressed but not mutated in prostate cancer.Genes Chromosomes Cancer. 2000; 29: 137-146Crossref PubMed Scopus (128) Google Scholar). Prostate cancer (PC) is the most prevalent neoplasia in men in industrialized nations (18Ferlay J. Parkin D.M. Steliarova-Foucher E. Estimates of cancer incidence and mortality in Europe in 2008.Eur. J. Cancer. 2010; 46: 765-781Abstract Full Text Full Text PDF PubMed Scopus (1887) Google Scholar). Although PC is frequently initially sensitive to hormonal deprivation therapies and follows indolent clinical courses, a significant proportion of cases eventually become resistant to such therapeutic approaches, accompanied with aggressive growth, establishment of metastasis, and tumors that are highly resistant to conventional chemotherapeutic regimes (19Pienta K.J. Bradley D. Mechanisms underlying the development of androgen-independent prostate cancer.Clin. Cancer Res. 2006; 12: 1665-1671Crossref PubMed Scopus (349) Google Scholar, 20Yap T.A. Zivi A. Omlin A. de Bono J.S. The changing therapeutic landscape of castration-resistant prostate cancer.Nat. Rev. Clin. Oncol. 2011; 8: 597-610Crossref PubMed Scopus (123) Google Scholar). Two major challenges in PC are to find predictive markers that identify those tumors most likely to follow a hormone-independent, aggressive clinical course as aids to decide early intervention and to identify molecular targets for improved therapies of castration-resistant cases that respond poorly to conventional chemotherapeutic regimes. Here, we provide new evidence to reinforce the notion that the acid ceramidase ASAH1 is a valid therapeutic target in advanced prostate cancer, and we characterize new potent and specific inhibitors of AC. PC-3/Mc and PC-3/S cells (21Celià-Terrassa T. Meca-Cortes O. Mateo F. de Paz A.M. Rubio N. Arnal-Estape A. Ell B.J. Bermudo R. Diaz A. Guerra-Rebollo M. et al.Epithelial-mesenchymal transition can suppress major attributes of human epithelial tumor-initiating cells.J. Clin. Invest. 2012; 122: 1849-1868Crossref PubMed Scopus (356) Google Scholar) were grown in RPMI1640 medium supplemented with 10% fetal bovine serum, nonessential amino acids, 2 mM glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from PAA, Ontario, Canada). Fibroblasts from a Farber patient (FD, wild-type) and FD transformed to stably overexpress AC (FD10X) were grown in a humidified 5% CO2 atmosphere at 37°C in DMEM medium supplemented as above. Twenty-four hours before transfection, cells were plated at a density of 2.5 × 105 cells in 35 mm diameter plates. Cells were then transfected with the specific constructs or empty vectors using lipofectamine 2000 (Invitrogen, Carlsbad, CA). Twenty-four hours after transfection, cells were either processed immediately or collected by trypsinization, washed twice with PBS, and centrifuged. Pellets were stored at −20°C until use. In transfections with neutral ceramidase (NC), the success of transfection was confirmed by activity assays with CerC12NBD in intact cells. Cells were collected by trypsinization, washed with PBS, resuspended in 0.25 M sucrose and lysed by ultrasonication in an ultrasonic bath. For the assay, 75 µl of reaction buffer (100 mM sodium acetate buffer, pH 4.5, for acid ceramidase activity), containing 40 µM RBM14C12 fluorogenic substrate (22Bedia C. Camacho L. Abad J.L. Fabrias G. Levade T. A simple fluorogenic method for determination of acid ceramidase activity and diagnosis of Farber disease.J. Lipid Res. 2010; 51: 3542-3547Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) (with or without test compounds), was mixed with 25 µl of the cell lysates (20 µg of total protein content) and incubated at 37°C for 3 h. For time dependence of inhibition, incubations were carried out for 0.5, 1, 2, and 3 h with different amounts of protein. The reaction was stopped by addition of methanol followed by NaIO4 (2.5 mg/ml in 200 mM glycine/NaOH buffer, pH 10.6). After 1 h at 37°C, 100 μl of 200 mM glycine/NaOH buffer were added and fluorescence detected at 355/460 nm excitation/emission wavelengths on a SpectraMax Microplate Reader (Molecular Devices, Sunnyvale, CA). To determine ceramidase activity in intact cells, 2 × 104 cells/well were seeded in 96-well plates (Nunc, Roskilde, Denmark). The following day, medium was replaced by 100 μl fresh medium containing 40 µM RBM14C12 fluorogenic substrate incubated for 3 h at 37°C in a 5% CO2 atmosphere, and the assay was continued as above. Papain activity was determined in 96-well plates by a modification of the reported procedure (23Filippova I.Yu Lysogorskaya E.N. Oksenoit E.S. Rudenskaya G.N. Stepanov V.M. L-Pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide–a chromogenic substrate for thiol proteinase assay.Anal. Biochem. 1984; 143: 293-297Crossref PubMed Scopus (99) Google Scholar). The reaction mixture contained 250 µl of 0.1 M phosphate buffer (pH 6.5) with 0.3 M KCl, 0.1 mM EDTA, and 3 mM DTT; 30 µl of substrate solution (L-pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide; 2.2 mM in DMSO, 0.22 mM final concentration); 20 µl of enzyme solution (30 µg/ml in reaction buffer); and 3 µl of inhibitor solution or vehicle. Chymostatin at 1 µM and 10 µM was used as positive control of inhibition of papain activity. The reaction was stopped by the addition of 20 µl of 1 N HCl, and the OD was measured at 410 nm. The liquid chromatography-mass spectrometry equipment consisted of a Waters Aquity UPLC system connected to a Waters LCT Premier orthogonal accelerated time of flight mass spectrometer (Waters, Millford, MA), operated in positive electrospray ionization mode. Full scan spectra from 50 to 1,500 Da were acquired, and individual spectra were summed to produce data points every 0.2 s. Mass accuracy and reproducibility were maintained by using an independent reference spray by the LockSpray interference. The analytical column was a 100 mm × 2.1 mm id, 1.7 µm C8 Acquity UPLC BEH (Waters). The two mobile phases were phase A: MeOH/H2O/HCOOH (74:25:1 v/v/v); phase B: MeOH/HCOOH (99/1 v/v), both also containing 5 mM ammonium formate. A linear gradient was programmed as follows: 0.0 min: 80% B; 3 min: 90% B; 6 min: 90% B; 15 min: 99% B; 18 min: 99% B; 20 min: 80% B, at 0.3 ml/min flow rate. The column was held at 30°C. Quantification was carried out using the extracted ion chromatogram of each compound, using 50 mDa windows. Linear dynamic range was determined by injecting standard mixtures, and positive identification of compounds was based on accurate mass measurement (< 5 ppm error) and LC retention time compared with that of a standard (± 2%). These analyses were carried out in an Alliance Waters 2695 HPLC system coupled to a Waters 2475 Multi λ fluorescence detector (Waters, Milford USA) equipped with an Atlantis T3 C18 (50 mm × 4.6 mm) column (Waters). The mobile phase was composed of a mixture of acetonitrile/H2O (80:20), and the flow rate was set at 1 ml/min. All solvents contained 0.1% trifluoroacetic acid. Fluorescent compounds were monitored at 420/483 nm excitation/emission wavelengths. Peak quantification was carried out using the Empower Pro 2.0 software (Waters). Cells were lysed (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton-X 100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, 1 mM PMSF, 1 mg/ml aprotinine, and leupeptine), proteins separated by SDS-polyacrylamide electrophoresis (Bio-Rad Laboratories, Hercules, CA) and transferred to nitrocellulose membrane blots (Bio-Rad). After blocking with 5% nonfat milk in PBS containing 0.1% Tween 20, membranes were incubated with primary antibody, washed, incubated with HRP-conjugated goat anti-rabbit IgG or anti-mouse IgG antibodies, and then washed again. Signals were detected by chemoluminescence (ECL Western Blotting Detection Kit, Amersham Biosciences, Barcelona, Spain). Actin signals were used as protein loading and transfer references. Total RNA was extracted with the RNeasy Kit (Qiagen, Venlo, Netherlands). Complementary DNAs were synthesized with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Real-time quantitative PCR assays were performed on a LightCycler 480 instrument (Roche Diagnostics, Mannheim, Germany) and analyzed with the LightCycler 480 Software release 1.5.0. The amplification levels of RN18S1 and HMBS were used as internal references to estimate the relative levels of specific transcripts, and relative quantification was determined by the ΔΔCp method. All determinations were done in triplicate. Cells were seeded in 6-well Corning plates (Corning, NY), detached with Trypsin/EDTA/1% BSA, washed twice, resuspended in PBS, and fixed at 4°C for at least 2 h by dropwise addition of 70% ethanol. Subsequently, cells were washed with PBS/50 mM EDTA/1% BSA and incubated with 1 mg/ml RNase A (Sigma) at 37°C for 1 h and 0.1 mg/ml propidium iodide (Sigma, Alcobendas, Madrid, Spain). DNA content was determined in a Cytomics FC500 instrument (Coulter, Hialeah, FL), and cell cycle distribution analyzed with Multicycle. All determinations were done in triplicate. For soft-agar colony formation assays, 0.5% agar in complete culture medium was placed at the bottom of 12-well plates, allowed to solidify, and overlayed with a suspension of 3 × 103 cells in 0.3% agar in complete medium. After solidification, wells were fed with complete medium twice a week. After three weeks, they were fixed with 0.5% glutaraldehyde, stained with 0.025% crystal violet, and visualized under a Leica magnifying glass (Wetzler, Germany) coupled to an Olympus digital camera (Olympus, Hamburg, Germany). Colonies ≥ 0.2 mm diameter were scored with the ImageJ software (National Institutes of Health, MD). Each experimental condition was performed in triplicate. For localized growth, 1 × 103 to 1 × 105 cells with stably integrated firefly luciferase were injected in a volume of 50 µl of RPMI 1640 (without FBS) intramuscularly in each hind limb of anesthetized six-week-old male NOD-SCID mice. Tumor growth was monitored by luminometry on an ORCA-2BT instrument (Hamamatsu Photonics, Hamamatsu, Japan), 5 min after intraperitoneal injection of luciferine (100 mg/kg in 150 µl of PBS). For lung colony formation, 5 × 105 cells in 150 µl RPMI1640 were injected through the dorsal caudal vein. Mice were imaged immediately after injection, and thereafter, tumor development was monitored by weekly imaging. For bioluminescence plots, photon flux was calculated relative to background values from luciferin-injected mice with no tumor cells and normalized to the value obtained immediately after xenografting. In lung colonization free survival analysis, lesions that had an increased photon flux value above day 0 were counted as events. Constructs based on the pLK0puro vector and bearing ASAH1-targeting shRNAs or control sequences were purchased from Sigma-Aldrich. The lentivirus packaging cell line HEK293T was cotransfected with these DNAs, together with pCMVdeltaR8.91 and pVSV-G (Clontech, Mountain View, CA) for 12 h using Fugene HD (Roche). Supernatants were collected for the following 48 h and filtered through 0.45 µm methylcellulose filters (Millipore, Billerica, MA). Lentiviral particles were concentrated by ultracentrifugation at 27,000 rpm for 90 min on 20% sucrose density gradients. Viral particles were resupended with medium and added to the cells together with 8 µg/ml polybrene (Sigma). Cells were infected for 24 h and allowed to recover in fresh medium for 24–48 h. Selection for cells with integrated sequences was carried out for three days in medium supplemented with 5 µg/ml puromycin (Biomol, Exeter, UK). MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma) was added to cultured cells at a final concentration of 0.5 mg/ml, incubated at 37°C for 3 h, and the resulting precipitates were solubilized with dimethyl sulfoxide. Absorbance was measured at 570 nm on a SpectraMax Microplate Reader (Molecular Devices, Sunnyvale, CA). A solution of 1-hydroxybenzotriazole (18 mg, 0.13 mmol), the corresponding carboxylic acid (0.1 mmol), and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (20 mg, 0.13 mmol) in CH2Cl2 (1 ml) was added to a mixture containing sphinganine (30 mg, 0.1 mmol), NEt3 (30 µl, 0.2 mmol) in THF or CH3CN (1 ml). The resulting solution was stirred for 1 h at room temperature and concentrated at reduced pressure. The residue was taken up in CH2Cl2 (2 ml), washed with saturated aqueous NaHCO3 solution (3 × 0.5 ml), and then the solvent was evaporated to give a crude mixture that was purified by flash chromatography on silica gel using a gradient of 0–5% CH2Cl2/MeOH to afford the pure amide in 70–85% yield. Spectroscopic data for the synthesized compounds: SABRAC: N-[(2S,3R)-1,3-dihydroxyoctadecan-2-yl]2-bromoacetamide. 1H-NMR (400 MHz, CDCl3): 7.40 (1H, NH), 4.20 (2H), 4.05 (1H), 3.85 (1H), 3.83 (1H), 3.81 (1H), 1.55 (2H), 1.25–1.30 (26H), 0.88 (t, 3H). 13C-NMR (101 MHz, CDCl3): 177.2, 74.2, 62.2, 54.0, 42.8, 34.7, 32.0, 29.8–29.6, 26.0, 22.8, 14.3. RBM1-12: N-[(2S,3R)-1,3-dihydroxyoctadecan-2-yl]2,2-dibromoacetamide. 1H-NMR (400 MHz, CD3OD): 6.23 (1H, NH), 3.80 (1H), 3.75 (2H), 3.65 (1H), 1.44 (2H), 1.25–1.30 (26H), 0.90 (3H). 13C-NMR (101 MHz, CD3OD): 166.9, 71.9, 61.7, 57.6, 37.9, 34.9, 33.1, 30.8–30.5, 26.6, 23.7, 14.5. RBM1-13: N-[(2S,3R)-1,3-dihydroxyoctadecan-2-yl]2-methylacrylamide. 1H-NMR (400 MHz, CDCl3): δ 6.75 (1H, NH), 5.72 (1H), 5.38 (1H), 4.05 (1H), 3.87 (1H), 3.81 (2H), 1.99 (3H), 1.42 (2H), 1.25–1.35 (26H), 0.88 (3H). 13C-NMR (101 MHz, CDCl3): δ 168.6, 139.7, 120.2, 74.2, 62.3, 53.7, 32.7, 31.8, 29.9–29.3, 25.9, 22.7, 14.1. RBM1-15: (E)-4-[(2S,3R)-N-1,3-dihydroxyoctadecan-2-ylamino]4-oxo-2-butenoic acid. 1H-NMR (400 MHz, CDCl3): δ 7.9 (1H, NH), 6.45 (1H), 6.35 (1H), 4.12 (1H), 3.88 (3H), 1.45 (2H), 1.25 (26H), 0.88 (3H). 13C-NMR (101 MHz, CDCl3): δ166.4, 166.2, 135.5, 132.5, 73.6, 61.2, 54.7, 34.5, 32.1, 29.8–29.5, 26.1, 22.8, 14.2. RBM1-16: N-[(2S,3R)-1,3-dihydroxyoctadecan-2-yl]3-methyl-2-butenamide. 1H-NMR (400 MHz, CDCl3): δ 6.26 (1H, NH), 5.64 (1H), 4.05 (1H), 3.85 (1H); 3.80 (1H), 2.17 (3H), 1.86 (3H), 1.54 (2H), 1.25 (26H), 0.88 (3H). 13C-NMR (101 MHz, CDCl3): δ 167.4, 151.2, 118.5, 74.3, 62.7, 53.9, 34.7, 32.0, 29.8–29.5, 27.3, 26.1, 22.8, 19.9, 14.2. RBM1-17: (2E,4E)-N-[(2S,3R)-1,3-dihydroxyoctadecan-2-yl]hexa-2,4-dienamide. 1H-NMR (400 MHz, CDCl3): δ 7.11 (dd, J = 14.8 Hz, J′=10 Hz, 1H), 6.90 (1H, NH), 6.05 (2H), 5.78 (d, J = 15.2 Hz, 1H), 3.95 (2H), 3.62 (1H), 1.78 (d, J = 6Hz, 3H), 1.45 (2H), 1.19 (26H), 0.82 (3H). 13C-NMR (101 MHz, CDCl3,CD3OD): δ 167.3, 141.7, 138.2, 129.7, 121.3, 73.3, 61.9, 54.5, 34.3, 31.9, 29.7–29.3, 26.0, 22.7, 18.5, 14.1. RBM1-18: (E)-N-[(2S,3R)-1,3-dihydroxyoctadecan-2-yl]but-2-enamide. 1H-NMR (400 MHz, CD3OD): δ 6.88 (1H), 6.02 (1H), 5.87 (d, J = 14.3, 1H), 3.85 (1H), 3.71 (2H), 3.63 (1H), 1.87 (d, J = 6.6, 3H), 1.52 (2H), 1.25 (26 H), 0.88 (3H). 13C-NMR (101 MHz, CD3OD): δ 168.5, 140.8, 126.2, 72.5, 62.3, 56.9, 35.0, 33.1, 30.9, 30.7–30.4, 26.9, 23.8, 18.0, 14.6. RBM1-19: N-[(2S,3R)-1,3-dihydroxyoctadecan-2-yl]acrylamide. 1H-NMR (400 MHz, CDCl3): δ 6.33 (d, J = 17.0, 1H), 6.17 (dd, J = 10.2, 16.9, 1H), 5.69 (d, J = 11.1, 1H), 4.16–3.99 (1H), 3.96–3.71 (3H), 1.68–1.46 (4H), 1.25 (24H), 0.88 (3H). 13C-NMR (101 MHz, CDCl3): δ 165.7, 131.0, 127.3, 74.5, 62.4, 54.0, 34.7, 32.1, 30.0, 29.7, 29.5, 26.3, 22.8, 14.2. The procurement of human tissues complied with Spanish legislation regarding informed consent, privacy, and all legal requirements after approval by the Hospital Clínic Institutional Ethics Committee. Sections (2 µm thick) were obtained for immunohistochemistry either from formalin-fixed and paraffin-embedded tissue blocks or from tissue microarrays (TMA) built with a Manual Tissue Arrayer 1 (Beecher Instruments, Sun Prairie, WI). A total of 33 samples, containing normal, prostate intraepithelial neoplasia, and carcinomatous glands were analyzed. Tissue sections were mounted on xylaned glass slides (Thermo Scientific, Braunschweig, Germany) and used for immunohistochemical staining using the Bond Polymer Refine Detection System (Leica Microsystems, Wetzlar, Germany). Samples were deparaffinized, antigen retrieval was performed at pH 6 for 20 min in citrate buffer, and primary antibody was incubated for 1 h at room temperature. Rabbit anti-ASAH1 (BD Transduction Laboratories, Franklin Lakes, NJ) was used at a dilution of 1/100. Staining was scored as the percentage of cells with clear positivity and the predominant staining intensity. Images were captured with an Olympus BX-51 microscope equipped with an Olympus DP70 camera. Significance was determined by the two-tailed unpaired t-test using the Graph Pad Prism 4.0 software. The PC-3 prostate cancer cell line was used to generate two distinct clonal populations. PC-3/S cells were isolated in vitro by single-cell cloning from luciferase-expressing PC-3 cells. A second single-cell progeny, hereafter designated PC-3/Mc, was isolated from luciferase-expressing PC-3/M cells, a PC-3 subline that had been selected in vivo for its high metastatic potential (21Celià-Terrassa T. Meca-Cortes O. Mateo F. de Paz A.M. Rubio N. Arnal-Estape A. Ell B.J. Bermudo R. Diaz A. Guerra-Rebollo M. et al.Epithelial-mesenchymal transition can suppress major attributes of human epithelial tumor-initiating cells.J. Clin. Invest. 2012; 122: 1849-1868Crossref PubMed Scopus (356) Google Scholar). Intramuscular grafting in NOD-SCID mice of 2.5 × 105 PC-3/Mc cells quickly produced large tumors (Fig. 1A) with the appearance of abdominal lymph node metastases by 19 days in 50% of mice (21Celià-Terrassa T. Meca-Cortes O. Mateo F. de Paz A.M. Rubio N. Arnal-Estape A. Ell B.J. Bermudo R. Diaz A. Guerra-Rebollo M. et al.Epithelial-mesenchymal transition can suppress major attributes of human epithelial tumor-initiating cells.J. Clin. Invest. 2012; 122: 1849-1868Crossref PubMed Scopus (356) Google Scholar). In vitro, PC-3/Mc cells grew much faster than PC-3/S cells (Fig. 1B). Moreover, PC-3/Mc cells were highly clonogenic, whereas PC-3/S cells showed limited anchorage-independent growth (Fig. 1C). To investigate the sphingolipid profiles of both cell lines, cells were seeded (0.25 × 106 cells/ml) and grown under standard conditions, and their sphingolipid composition determined after 48 h of culture. LC/MS analysis showed that total ceramide abundance in PC-3/S cells was 1.3-fold that of PC-3/Mc cells (Fig. 1D). Interestingly, this difference increased to 2.2 for the C14 and C16 N-acyl species (PC-3/S versus PC-3/Mc ratio: 2.2). Likewise, the cell content of SM and ceramide monohexosides (CMH, including both glucosylceramides and galactosylceramides) was 1.3–1.5 times higher in PC-3/S cells than in PC-3/Mc cells (Fig. 1E, F), and this difference was similar for all the differently N-acylated species. No significant differences between free bases and long-chain phosphates were detected between the two cell lines (not shown). Enzyme activity determination showed that the highly aggressive and metastatic PC-3/Mc cells displayed levels of AC activity that were 2.5 to 4 times higher than those of PC-3/S cells, determined either in cell lysates at acidic pH or in intact cells (Fig. 1G) using a fluorogenic assay (22Bedia C. Camacho L. Abad J.L. Fabrias G. Levade T. A simple fluorogenic method for determination of acid ceramidase activity and diagnosis of Farber disease.J. Lipid Res. 2010; 51: 3542-3547Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Several findings support that the bulk of t
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