The selective COX-2 inhibitor celecoxib modulates sphingolipid synthesis
2008; Elsevier BV; Volume: 50; Issue: 1 Linguagem: Inglês
10.1194/jlr.m800122-jlr200
ISSN1539-7262
AutoresSusanne Schiffmann, Jessica Sandner, Ronald Schmidt, Kerstin Birod, Ivonne Wobst, Helmut Schmidt, Carlo Angioni, Gerd Geißlinger, Sabine Grösch,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoSphingolipids such as ceramides (Cers) play important roles in cell proliferation, apoptosis, and cell cycle regulation. An increased Cer level is linked to the cytotoxic effects of several chemotherapeutics. Various selective cyclooxygenase-2 (COX-2) inhibitors induce anti-proliferative effects in tumor cells. We addressed the possible interaction of the selective COX-2 inhibitors, coxibs, with the sphingolipid pathway as an explanation of their anti-proliferative effects. Sphingolipids were measured using liquid chromatography tandem mass spectrometry. Treatment of various cancer cell lines with celecoxib significantly increased sphinganine, C16:0-, C24:0-, C24:1-dihydroceramide (dhCer) and led to a depletion of C24:0-, C24:1-Cer in a time- and concentration-dependent manner, whereas other coxibs had no effect. Using 13C,15N-labeled l-serine, we demonstrated that the augmented dhCers after celecoxib treatment originate from de novo synthesis. Celecoxib inhibited the dihydroceramide desaturase (DEGS) in vivo with an IC50 of 78.9 ± 1.5 μM and increased total Cer level about 2-fold, indicating an activation of sphingolipid biosynthesis. Interestingly, inhibition of the sphingolipid biosynthesis by specific inhibitors of l-serine palmitoyltransferase diminished the anti-proliferative potency of celecoxib. In conclusion, induction of de novo synthesis of sphingolipids and inhibition of DEGS contribute to the anti-proliferative effects of celecoxib. Sphingolipids such as ceramides (Cers) play important roles in cell proliferation, apoptosis, and cell cycle regulation. An increased Cer level is linked to the cytotoxic effects of several chemotherapeutics. Various selective cyclooxygenase-2 (COX-2) inhibitors induce anti-proliferative effects in tumor cells. We addressed the possible interaction of the selective COX-2 inhibitors, coxibs, with the sphingolipid pathway as an explanation of their anti-proliferative effects. Sphingolipids were measured using liquid chromatography tandem mass spectrometry. Treatment of various cancer cell lines with celecoxib significantly increased sphinganine, C16:0-, C24:0-, C24:1-dihydroceramide (dhCer) and led to a depletion of C24:0-, C24:1-Cer in a time- and concentration-dependent manner, whereas other coxibs had no effect. Using 13C,15N-labeled l-serine, we demonstrated that the augmented dhCers after celecoxib treatment originate from de novo synthesis. Celecoxib inhibited the dihydroceramide desaturase (DEGS) in vivo with an IC50 of 78.9 ± 1.5 μM and increased total Cer level about 2-fold, indicating an activation of sphingolipid biosynthesis. Interestingly, inhibition of the sphingolipid biosynthesis by specific inhibitors of l-serine palmitoyltransferase diminished the anti-proliferative potency of celecoxib. In conclusion, induction of de novo synthesis of sphingolipids and inhibition of DEGS contribute to the anti-proliferative effects of celecoxib. Sphingolipids constitute an essential component of the eucaryotic plasma membrane and are also utilized as an important second messenger in a variety of cellular events, including cell senescence, cellular differentiation, apoptosis, and proliferation (1Pettus B.J Chalfant C.E. Hannun Y.A. Ceramide in apoptosis: an overview and current perspectives..Biochim. Biophys. Acta. 2002; 1585: 114-125Crossref PubMed Scopus (678) Google Scholar, 2Ruvolo P.P Intracellular signal transduction pathways activated by ceramide and its metabolites..Pharmacol. Res. 2003; 47: 383-392Crossref PubMed Scopus (300) Google Scholar, 3Hannun Y.A Obeid L.M. Principles of bioactive lipid signalling: lessons from sphingolipids..Nat. Rev. Mol. Cell Biol. 2008; 9: 139-150Crossref PubMed Scopus (2484) Google Scholar). Endogenous sphingolipid levels can be controlled by activation of sphingomyelinases (SMases) and de novo synthesis as well as by specific degradation mechanisms (e.g., ceramidases, lyases). The sphingolipid biosynthesis commences in the endoplasmic reticulum with the condensation of palmitoyl-CoA and l-serine by l-serine palmitoyltransferase (l-SPT). The intermediate 3-ketosphinganine is rapidly converted into sphinganine (dhSph) by 3-ketosphinganine reductase. Acyl-CoA thioesters of variable chain lengths are then attached to dhSph by chain-length-specific (dihydro)ceramide synthases (CerSs) (4Pewzner-Jung Y. Ben-Dor S. Futerman A.H. When do Lasses (longevity assurance genes) become CerS (ceramide synthases)? Insights into the regulation of ceramide synthesis..J. Biol. Chem. 2006; 281: 25001-25005Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). Dihydroceramide desaturase (DEGS) introduces a 4, 5-trans double bond in dihydroceramides (dhCers), resulting in formation of the final product, Cer. Via sphingosine (Sph), Cer is metabolized by the enzymes ceramidase and sphingosine kinase to sphingosine-1-phosphate (Sph1P). In the Golgi apparatus, the polar group phosphatidylcholine is attached to Cer by sphingomyeline synthase to produce sphingomyeline. SMase catalyzes the reverse reaction. Glucosylceramide synthase (GCS) transfers activated glucose to Cer to form glucosylceramide, which is the principle component of most glycosphingolipids (Fig. 1). Cer, Sph, and dhSph all have been shown to activate a number of enzymes involved in stress signaling cascades, including both protein kinases and protein phosphatases that suppress growth and survival pathways upstream of apoptotic events (2Ruvolo P.P Intracellular signal transduction pathways activated by ceramide and its metabolites..Pharmacol. Res. 2003; 47: 383-392Crossref PubMed Scopus (300) Google Scholar, 5Schmelz E.M Roberts P.C. Kustin E.M. Lemonnier L.A. Sullards M.C. Dillehay D.L. Merrill Jr., A.H. 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Mechanisms involved in exogenous C2- and C6-ceramide-induced cancer cell toxicity..Biochem. Pharmacol. 2003; 65: 1633-1642Crossref PubMed Scopus (52) Google Scholar, 20Zhu J. Huang J.W. Tseng P.H. Yang Y.T. Fowble J. Shiau C.W. Shaw Y.J. Kulp S.K. Chen C.S. From the cyclooxygenase-2 inhibitor celecoxib to a novel class of 3-phosphoinositide-dependent protein kinase-1 inhibitors..Cancer Res. 2004; 64: 4309-4318Crossref PubMed Scopus (240) Google Scholar, 21Kulp S.K Yang Y.T. Hung C.C. Chen K.F. Lai J.P. Tseng P.H. Fowble J.W. Ward P.J. Chen C.S. 3-Phosphoinositide-dependent protein kinase-1/Akt signaling represents a major cyclooxygenase-2-independent target for celecoxib in prostate cancer cells..Cancer Res. 2004; 64: 1444-1451Crossref PubMed Scopus (225) Google Scholar), which are also targets of sphingolipids. Celecoxib, therefore, may mediate its cytotoxic effects via modulation of sphingolipid metabolism. In addition, altered sphingolipid levels mediate the cytotoxic effects of several chemotherapeutics, such as etoposide (22Perry D.K Carton J. Shah A.K. Meredith F. Uhlinger D.J. Hannun Y.A. Serine palmitoyltransferase regulates de novo ceramide generation during etoposide-induced apoptosis..J. Biol. Chem. 2000; 275: 9078-9084Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 23Lin C.F Chen C.L. Chang W.T. Jan M.S. Hsu L.J. Wu R.H. Tang M.J. Chang W.C. Lin Y.S. Sequential caspase-2 and caspase-8 activation upstream of mitochondria during ceramide and etoposide-induced apoptosis..J. Biol. Chem. 2004; 279: 40755-40761Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), paclitaxel (24Charles A.G Han T.Y. Liu Y.Y. Hansen N. Giuliano A.E. Cabot M.C. Taxol-induced ceramide generation and apoptosis in human breast cancer cells..Cancer Chemother. Pharmacol. 2001; 47: 444-450Crossref PubMed Scopus (125) Google Scholar), and N-(4-hydroxyphenyl) retinamide (25Wang H. Maurer B.J. Reynolds C.P. Cabot M.C. N-(4-hydroxyphenyl)retinamide elevates ceramide in neuroblastoma cell lines by coordinate activation of serine palmitoyltransferase and ceramide synthase..Cancer Res. 2001; 61: 5102-5105PubMed Google Scholar). Therefore, we hypothesize that the sphingolipid pathway may be an important target for the anti-proliferative effects of coxibs. The human cancer cell lines HCA-7, MCF-7, MDA-MB231, and HT-29 were purchased from European Collection of Cell Cultures (ECC; Salisbury, UK), and HEK293 and HCT-116 carcinoma cells were ordered from Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany). HEK293, MCF-7, and MDA-MB231 cells were cultured in DMEM without sodium pyruvate, containing glucose (4,500 mg/l) and GlutaMAX™. HCA-7 cells were cultured in DMEM containing sodium pyruvate, pyridoxine, and 8 mM l-glutamine. HCT-116 cells were incubated in McCoy's 5A medium, and HT-29 cells were cultured in RPMI medium containing GlutaMAX™ and 50 mM HEPES. All media contained 100 U/ml penicillin G and 100 μg/ml streptomycine, 10% fetal calf serum (FCS) for culturing, and 7.5% FCS for treatment. Cells were cultured at 37°C in an atmosphere containing 5% CO2. 13C3,15N-labeled l-serine, myriocin, desipramine, l-cycloserine, glutathione, para-nitrophenylphosphate, alkaline phosphatase, cholesterol, palmitic acid, and 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol were purchased from Sigma-Aldrich (Schnelldorf, Germany). Fumonisin B1 (FB1) was purchased from Alexis Biochemicals (Lausen, Switzerland). C8-cyclopropenylceramide (C8-CPPC) was purchased from Matreya LLC (Pleasant Gap, PA). The sphingolipids were purchased either from Avanti Polar Lipids (Alabaster, AL) or Matreya LLC. Celecoxib, valdecoxib, etoricoxib, methylcelecoxib, and rofecoxib were synthesized by WITEGA Laboratorien Berlin-Adlershof GmbH. Lumiracoxib was provided by Novartis (Basel, Switzerland). The identity and purity of all coxibs were determined using [1H]NMR spectroscopy and HPLC as described previously (26Brautigam L. Vetter G. Tegeder I. Heinkele G. Geisslinger G. Determination of celecoxib in human plasma and rat microdialysis samples by liquid chromatography tandem mass spectrometry..J. Chromatogr. B Biomed. Sci. Appl. 2001; 761: 203-212Crossref PubMed Scopus (47) Google Scholar) and were >99%. For the quantification of sphingolipid amounts, cells were seeded at a density of 0.5 × 106 and incubated for 24 h. Cells subsequently were treated with various coxib concentrations (0–200 μM) over various periods of time. The cells were washed with 1 ml PBS, treated with 0.5 ml trypsin, and collected in 0.5 ml PBS. Cells were counted in a Neubauer chamber and centrifuged at 745 g and 4°C for 5 min. The PBS supernatant was removed, and the cells were stored at −80°C. Lipids were extracted with 200 μl methanol after the addition of the internal standards (C17:0-Cer, C17:0-Sph, C17:0-Sph1P). The suspension was incubated at 25°C with shaking at 1,400 rpm for 30 min and then centrifuged for 30 min at 25°C and 20,500 g. The supernatants were collected, and the extraction step was repeated. The combined organic phases were dried under a stream of nitrogen and resolved in methanol for quantification. After liquid-liquid extraction, amounts of C16:0-Cer, C24:1-Cer, C24:0-Cer, C16:0-dhCer, C24:1-dhCer, C24:0-dhCer, dhSph, Sph, sphinganine-1-phosphate (dhSph1P), and Sph1P, labeled analogs and the internal standards were determined by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Chromatographic separation was accomplished under gradient conditions using a Luna C18 column (150 mm × 2 mm ID, 5 μm particle size, and 10 nm pore size; Phenomenex, Aschaffenburg, Germany). The HPLC mobile phases consisted of water-formic acid (100:0.1, v/v) (A) and acetonitrile-tetrahydrofuran-formic acid (50:50:0.1, v/v/v) (B). A gradient program was used for the HPLC separation at a flow rate of 0.3 ml/min. The initial buffer composition 60% (A) / 40% (B) was hold for 0.6 min, then linearly changed to 0% (A) / 100% (B) in 4.4 min and hold for 5 min, then linearly changed to 60% (A) / 40% (B) in 0.5 min and hold for further 5.5 min. Ten microliters of each sample was injected, and the total run time was 16 min. MS/MS analyses were performed on an API 4000 triple quadrupole mass spectrometer with a Turbo V source (Applied Biosystems; Darmstadt, Germany). Precursor-to-product ion transitions of m/z 408→264 for C8:0-Cer, of m/z 538 (541)→264 (267) for C16:0-Cer (C16:0-Cer*), of m/z 648 (651)→264 (267) for C24:1-Cer (C24:1-Cer*), of m/z 651 (654)→264 (267) for C24:0-Cer (C24:0-Cer*), of m/z 540 (543)→284 (287) for C16:0-dhCer (C16:0-dhCer*), of m/z 651 (654)→284 (287) for C24:1-dhCer (C24:1-dhCer*), of m/z 653 (657)→284 (287) for C24:0-dhCer (C24:0-dhCer*), of m/z 300 (303)→282 (285) for Sph (Sph*), of m/z 302 (305)→284 (287) for dhSph (dhSph*), of m/z 380 (383)→264 (267) for Sph1P (Sph1P*), of m/z 382 (385)→266 (269) for dhSph1P (dhSph1P*), of m/z 300 (303)→270 (273) for 3-ketosphinganine (3-ketosphinganine*), of m/z 552→534 for C17:0-Cer, of m/z 286→268 for C17:0-Sph and of m/z 366→250 for C17:0-Sph1P were used for multiple reaction monitoring with a dwell time of 15 msec. Concentrations of the calibration standards, quality controls, and unknowns were evaluated by Analyst software 1.4.2 (Applied Biosystems). A representative chromatogram of C16:0-Cer, C24:1-Cer, C24:0-Cer, C16:0-dhCer, C24:1-dhCer, C24:0-dhCer, and dhSph is displayed in Supplementary Figure I. Suppression effects were assessed by extraction of cell samples (n = 6), which were reconstructed with 10 ng/ml internal standard in methanol. The mean peak areas of the samples reconstructed with internal standard were compared with the mean peak area of the internal standard in methanol. An ion suppression effect was observed between 15% and 25% for the internal standard. Linearity of the calibration curve was proven for 3-ketosphinganine, C24:0-Cer, C24:1-Cer from 1.5 to 300 ng/ml, for C16:0-Cer, C16:0-dhCer, C24:0-dhCer, C24:1-dhCer from 2 to 1,000 ng/ml, dhSph, Sph, Sph1P from 0.4 to 400 ng/ml. The coefficient of correlation for all measured sequences was at least 0.99. Variations in accuracy and intraday and interday precision [n = 2 for each concentration in five various cell lines (HCT-116, HeLa, HCA-7, MCF-7, and HEK293)] were less than 15% over the range of calibration. The WST-1 assay (Roche Diagnostic GmbH; Mannheim, Germany) was used to determine the viability and proliferation rate of the cells after treatment with coxibs and the various inhibitors of sphingolipid biosynthesis. The cells were seeded at a density of 2–5 × 104cells/well in 100 μl of culture medium in 96-well microplates and incubated for 24 h at 37°C under an atmosphere containing 5% CO2. Medium was removed, and the cells were treated with increasing concentrations of celecoxib for 20 h in the presence or the absence of myriocin (150 nM) and l-cycloserine (500 μM) (inhibitors of L-SPT). The inhibitors were preincubated for 90 min. After 20 h of incubation, 10 μl of WST-1 reagent was added to each well, and the cells were incubated for 90 min. The absorbance of the dye was measured at 450 nm against a reference wavelength of 620 nm using a 96-well spectrophotometric plate reader (SPECTRAFluor Plus, Tecan; Crailsheim, Germany). Induction of apoptosis after celecoxib treatment was measured by the APOPercentage apoptosis assay (Biocolor; Dublin, Ireland). This assay is dye-based and takes advantage of the phosphatidylserine transmembrane movement that takes place during the onset of apoptosis. When this occurs, the dye is able to penetrate the cell membrane and accumulates inside the cell. HCT-116 cells/well (15 × 104) were seeded in 100 μl of 0.4% gelatin and 200 μl of medium in 96-well plates. HCT-116 cells were either treated with 80 μM celecoxib or coincubated with 80 μM celecoxib and 150 nM myriocin for 6 h. The dye was added 30 min before analysis. For quantification of apoptosis, analytical digital photomicroscopy was used according to the manufacturer's manual. For the L-SPT and CerS assay, the microsomal fraction of HCT-116 cells was isolated. The reaction mixture for the L-SPT assay contained 100 μg of the microsomal fraction, 1 mM l-serine, 150 μM palmitoyl-CoA, 50 μM pyridoxal phosphate, 100 mM HEPES, 2.5 mM EDTA, and 5 mM DTT (pH 7.4). The assay mixture was preincubated for 10 min at 37°C. The reaction was initiated by the simultaneous addition of pyridoxal phosphate and palmitoyl-CoA, and the mixture was incubated for different time points (5, 10, 15, 20 min). For the CerS assay, the reaction mixture consisted of 50 μg of the microsomal fraction, 30 μM dhSph, 40 μM palmitoyl-CoA in 20 mM HEPES, and 2 mM MgCl2 (pH 7.4). After 10 min of preincubation at 37°C, the reaction was initiated by the addition of palmitoyl-CoA, and the mixture was incubated for different time points (5, 10, 15, 30 min). Both reactions were terminated by the addition of the extraction solvent chloroform-methanol (7:1). The extraction step was repeated, and the organic solution was separated and dried under a stream of nitrogen. The products, 3-ketosphinganine (L-SPT assay) and C16:0-dhCer (CerS assay) were measured using LC-MS/MS. The specific activity was calculated based on the values of different time points. Proliferation and sphingolipid data are presented as mean ± SEM. The IC50 value was calculated using a nonlinear regression analysis and a sigmoidal Emax model. SPSS 9.01 software was used for statistical analyses. The sphingolipid levels and the cell viability rates were analyzed using an independent t-test, and the confidence interval was set at 95%. We first investigated whether coxibs have an influence on the sphingolipid pathway in the human colon carcinoma cell line HCT-116 after a 2 h treatment with various coxibs (rofecoxib, etoricoxib, valdecoxib, lumiracoxib, methylcelecoxib, and celecoxib). Subsequently, the cells were harvested and the sphingolipids isolated and determined by LC-MS/MS (27Schmidt H. Schmidt R. Geisslinger G. LC-MS/MS-analysis of sphingosine-1-phosphate and related compounds in plasma samples..Prostaglandins Other Lipid Mediat. 2006; 81: 162-170Crossref PubMed Scopus (53) Google Scholar). As shown in Fig. 2A, celecoxib, and its close structural derivative methylcelecoxib, significantly increased C16:0-, C24:1- and C24:0-dhCers and dhSph. Under the same conditions, celecoxib and methylcelecoxib decreased C24:1- and C24:0-ceramides. In contrast, the levels of C14:0-, C16:0-, C18:1-, C18:0-, and C20:0-ceramides, sphingosine, Sph1P, and dhSph1P were not influenced by coxib treatment (data only shown for celecoxib, Supplement 2). Because of this result, we focused our efforts on the sphingolipids (C16:0-, C24:1-, C24:0-dhCer, C24:1-, C24:0-ceramide, and dhSph) that are affected by celecoxib and methylcelecoxib. Other COX-2 inhibitors, such as etoricoxib (up to 200 μM), valdecoxib (up to 200 μM), rofecoxib (up to 100 μM), and lumiracoxib (up to 200 μM), showed no influence on any of the tested sphingolipids even at very high concentrations, indicating that the effect of celecoxib and methylcelecoxib is substance specific and occurs independently of COX-2 inhibition. Celecoxib-modulated changes in sphingolipid levels were concentration dependent and detectable after a 2 h treatment of HCT-116 cells with 20 μM celecoxib (Fig. 2B). The effect is more pronounced, however, with 80 μM celecoxib, the concentration used in further experiments. To demonstrate that the effects of celecoxib on the sphingolipid levels are not cell type-specific, we measured sphingolipid levels in different human cancer cell lines after treatment with 80 μM celecoxib for 2 h. Figure 3 shows that in human colon cancer cells (HCT-116, COX-2 depeleted/COX-1 expressing), in human cervix carcinoma cells (HeLa, COX-2 inducible/COX-1 expressing), and in human lung cancer cells (A549, COX-2 expressing/COX-1 expression very low), celecoxib treatment increased dhCer and dhSph levels and decreased C24:1- and C24:0-Cer levels. Other human colon cancer cells (HCA-7, HT-29), human breast cancer cells (MDA-MB231, MCF-7), and human embryonic kidney cells (HEK293) were tested and showed similar changes in the sphingolipid levels after celecoxib treatment (data not shown). These data clearly demonstrate that celecoxib-induced alterations in sphingolipid levels are a general COX-2-independent phenomenon that occurs in cell lines of various origins. Sphinganine and dhCers are nondurable intermediates of the sphingolipid pathway (Fig. 1). To investigate whether the increase of dhCer levels after celecoxib treatment originates from de novo sphingolipid biosynthesis or from the salvage pathway, an incorporation experiment with 13C3,15N-labeled l-serine was performed. The addition of 13C3,15N-labeled l-serine, a precursor of the sphingolipid de novo synthesis, leads to the production of labeled sphingolipids. This new labeling method enables us to discriminate by tandem mass spectrometry between sphingolipids generated by de novo synthesis and those generated by the salvage pathway. HCT-116 cells were treated with either 400 μM 13C,15N-labeled l-serine alone or with a combination of 400 μM 13C,15N-labeled l-serine and 80 μM celecoxib for times up to 6 h. In HCT-116 cells treated with 13C,15N-labeled l-serine, only labeled Cers were detected (Fig. 4; yellow/red bars). DhCers and dhSph were always low, probably owing to their immediate metabolism to Cer. In HCT-116 cells treated with 13C,15N-labeled l-serine and celecoxib, labeled dhCers and dhSph significantly increased up to 6 h post treatment in a time-dependent manner, whereas the levels of C24:1- and C24:0-Cer significantly decreased (Fig. 4; white/blue bars). The strong accumulation of labeled dhCers and dhSph indicates that de novo sphingolipid biosynthesis is affected after celecoxib treatment and, additionally, that a significant decrease of C24:0- and C24:1-Cer is caused by an inhibition of the DEGS by celecoxib. To confirm that celecoxib activates de novo sphingolipid biosynthesis, HCT-116 cells were treated with the l-SPT inhibitors myriocin (150 nm) or l-cycloserine (500 μM) (both concentrations do not compromise cell viability) and celecoxib (80 μM) for 2 h. Both inhibitors prevented the celecoxib-induced upregulation of dhCers (Supplement 3). Additionally, we compared the total amount of Cer (dhCer and Cer) in HCT-116 cells after treatment of cells with 80 μM celecoxib, 1 μM C8-CPPC (inhibitor of DEGS), 50 μM FB1 (inhibitor of CerS) and a DMSO control for 2 h (Fig. 5A). The inhibitors C8-CPPC and FB1 reduced Cer levels and increased dhCer or dhSph levels (data not shown) but mediated no alteration in the total Cer levels. In contrast, celecoxib significantly increased the total Cer level 2-fold. These data suggest that celecoxib activates de novo sphingolipid biosynthesis. However, in vitro (microsomal fractions of HCT-116 cells) activity assays of l-SPT and the dhCer synthase, the two pacemaker enzymes of the sphingolipid pathway, showed no increase in enzyme activity in the presence of celecoxib (Supplement 4), indicating that a cellular context is necessary for celecoxib-induced sphingolipid biosynthesis. The concomitant increase of dhCers (C16:0-, C24:1-, and C24:0-dhCer) and decrease of Cers (C24:1- and C24:0-Cer) after celecoxib treatment suggest inhibition of the DEGS. To test this hypothesis, we performed an in vivo activity assay using the nonphysiological C8:0-dhCer as a substrate for the DEGS and measured the product C8:0-Cer by LC-MS/MS. HCT-116 cells were incubated for 2 h with 10 μM C8:0-dhCer and coincubated with increasing concentrations of celecoxib (0–120 μM) (Fig. 5B). The IC50 value for DEGS inhibition was 78.9 ± 1.5 μM with a maximum inhibition of 75% at 120 μM (higher celecoxib concentrations induce cell death already after 2 h). This IC50 value fits with the observation that the levels of C24:1- and C24:0-Cer significantly decrease only at celecoxib concentrations ≥60 μM (Fig. 2B). The interaction of celecoxib with the acid or neutral SMase was also examined. These two enzymes were specifically inhibited with desipramine (inhibitor of the acid SMase) or glutathione (inhibitor of the neutral SMase) and coincubated the cells with celecoxib (80 μM). Both inhibitors had no influence on celecoxib-induced sphingolipid alterations (data not shown). To determine whether the elevated dhCer levels contributed to the apoptotic potency of celecoxib, cells were coincubated with myriocin, an inhibitor of the l-SPT. HCT-116 cells were either treated with 80 μM celecoxib alone or cotreated with 150 nM myriocin (a concentration that does not induce apoptosis) for 6 h, and the rate of apoptosis was determined using the APOPercentage assay. Cotreatment of cells with myriocin and celecoxib prevented upregulation of dhCers and dhSph after celecoxib treatment (Supplement 3) and reduced significantly celecoxib-induced apoptosis (Fig. 6A). To address the question of whether the increased dhCer levels reduce cell viability, HCT-116 cells were treated with 80 μM celecoxib alone or cotreated with either 150 nM myriocin or with 500 μM l-cycloserine (inhibitor of the l-SPT) and assayed for cell viability using the WST-1 protocol. Cotreatment (20 h) of cells with increasing concentrations of celecoxib and myriocin (Fig. 6B) or l-cycloserine (Fig. 6C) (both substances inhibit the celecoxib-induced upregulation of dhCers; Supplement 3) had protective ef
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