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Papers In Press, published online ahead of print January 1, 2009
J. Lipid Res., doi:10.1194/jlr.M800122-JLR200

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Journal of Lipid Research, Vol. 50, 32-40, January 2009
Copyright © 2009 by American Society for Biochemistry and Molecular Biology

The selective COX-2 inhibitor celecoxib modulates sphingolipid synthesis

Susanne Schiffmann¹, Jessica Sandner, Ronald Schmidt, Kerstin Birod, Ivonne Wobst, Helmut Schmidt, Carlo Angioni, Gerd Geisslinger and Sabine Grösch

Institut für Klinische Pharmakologie, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, 60590 Frankfurt/Main, Germany

The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of five figures.

Published, JLR Papers in Press, 18 August 2008.

This work was supported by the Deutsche Forschungsgemeinschaft Forschergruppe FOG 784/TP5 (GR2011/2-1).

¹ To whom correspondence should be addressed. e-mail: susanne.schiffmann{at}med.uni-frankfurt.de

	ABSTRACT

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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, C_16:0-, C_24:0-, C_24:1-dihydroceramide (dhCer) and led to a depletion of C_24:0-, C_24:1-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.

Supplementary key words cancer • cyclooxygenase-2 • apoptosis • dihydroceramide desaturase • ceramide • dihydroceramide

Abbreviations: Cer, ceramide; CerS, (dihydro)ceramide synthase; C8-CPPC, C8-cyclopropenylceramide; COX-2, cyclooxygenase-2; DEGS, dihydroceramide desaturase; dhCer, dihydroceramide; dhSph, sphinganine; dhSph1P, sphinganine-1-phosphate; FB1, fumonisin B1; FCS, fetal calf serum; GCS, glucosylceramide synthase; HPLC, high-performance liquid chromatography; LC-MS/MS, liquid chromatography tandem mass spectrometry; SMase, sphingomyelinase; Sph, sphingosine; Sph1P, sphingosine-1-phosphate; Sph1PP, sphingosine-1-phosphate phosphatase; L-SPT, L-serine palmitoyltransferase

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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 (1–3). 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) (4). 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 ).

View larger version (12K): [in this window] [in a new window]   Fig. 1. Scheme of the sphingolipid pathway. L-SPT, L-serine palmitoyltransferase; KSR, 3-ketosphinganine reductase, CerS, (dihydro)ceramide synthase, DEGS, dihydroceramide desaturase, SMS, sphingomyeline synthase, SMase, sphingomyelinase, GCS, glucosylceramide synthase, SK, sphingosine kinase, Sph1PP, sphingosine 1-phosphate phosphatase, Sph1P, sphingosine 1-phosphate.

Coxibs have been shown to induce apoptosis and cell cycle arrest in various types of cancer cells, including colon, breast, lung, and cervical cancers (9–12). In addition to inhibition of cyclooxygenase-2 (COX-2), which is a key enzyme in the conversion of arachidonic acid to prostaglandins, COX-2-independent mechanisms are known to contribute to the anti-proliferative effects of coxibs (13). Treatment of clinical trial patients with celecoxib or rofecoxib produced pronounced reduction of the occurrence of colorectal adenomas relative to placebo (14–17).

The molecular mechanisms through which celecoxib or other coxibs exert their cytotoxic effects are not completely understood. Interestingly, celecoxib influences the β-catenin (5, 18) and caspase signaling pathways (19–21), 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 (22, 23), paclitaxel (24), and N-(4-hydroxyphenyl) retinamide (25). Therefore, we hypothesize that the sphingolipid pathway may be an important target for the anti-proliferative effects of coxibs.

	MATERIALS AND METHODS

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Cells and reagents
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^TM. 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^TM 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 (26) and were >99%.

Determination of sphingolipid amounts in cells
For the quantification of sphingolipid amounts, cells were seeded at a density of 0.5 x 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 (C_17:0-Cer, C_17:0-Sph, C_17:0-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 C_16:0-Cer, C_24:1-Cer, C_24:0-Cer, C_16:0-dhCer, C_24:1-dhCer, C_24:0-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 x 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 408264 for C_8:0-Cer, of m/z 538 (541)264 (267) for C_16:0-Cer (C_16:0-Cer*), of m/z 648 (651)264 (267) for C_24:1-Cer (C_24:1-Cer*), of m/z 651 (654)264 (267) for C_24:0-Cer (C_24:0-Cer*), of m/z 540 (543)284 (287) for C_16:0-dhCer (C_16:0-dhCer*), of m/z 651 (654)284 (287) for C_24:1-dhCer (C_24:1-dhCer*), of m/z 653 (657)284 (287) for C_24:0-dhCer (C_24:0-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 552534 for C_17:0-Cer, of m/z 286268 for C_17:0-Sph and of m/z 366250 for C_17:0-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 C_16:0-Cer, C_24:1-Cer, C_24:0-Cer, C_16:0-dhCer, C_24:1-dhCer, C_24:0-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, C_24:0-Cer, C_24:1-Cer from 1.5 to 300 ng/ml, for C_16:0-Cer, C_16:0-dhCer, C_24:0-dhCer, C_24:1-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.

In vitro cell viability assay
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 x 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).

Apoptosis assay
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 x 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.

Activity assay for L-SPT and CerS
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 C_16:0-dhCer (CerS assay) were measured using LC-MS/MS. The specific activity was calculated based on the values of different time points.

Statistics
Proliferation and sphingolipid data are presented as mean ± SEM. The IC₅₀ value was calculated using a nonlinear regression analysis and a sigmoidal E_max 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%.

	RESULTS

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Concentration-dependent alteration of the sphingolipid levels in HCT-116 cells after treatment with coxibs
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 (27). As shown in Fig. 2A , celecoxib, and its close structural derivative methylcelecoxib, significantly increased C_16:0-, C_24:1- and C_24:0-dhCers and dhSph. Under the same conditions, celecoxib and methylcelecoxib decreased C_24:1- and C_24:0-ceramides. In contrast, the levels of C_14:0-, C_16:0-, C_18:1-, C_18:0-, and C_20:0-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 (C_16:0-, C_24:1-, C_24:0-dhCer, C_24:1-, C_24:0-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.

View larger version (26K): [in this window] [in a new window]   Fig. 2. The effects of coxibs on the sphingolipid level. HCT-116 cells were treated for 2 h either with various coxibs [rofecoxib (100 µM), etoricoxib (200 µM), valdecoxib (200 µM), lumiracoxib (200 µM), methylcelecoxib (80 µM), celecoxib (80 µM)] or DMSO as control (A) or with increasing concentrations of celecoxib as indicated (B). The sphingolipid amounts determined by liquid chromatography tandem mass spectrometry (LC-MS/MS) were related to the number of treated cells. Data are mean ± SEM of one of three comparable independent experiments, each performed in duplicate. * P < 0.05, significant difference between coxib-treated and control cells. Cer, ceramide; dhCer, dihydroceramide.

View larger version (29K): [in this window] [in a new window]   Fig. 3. Comparison of celecoxib-induced alterations of sphingolipid levels in various human cancer cells. Human cervix carcinoma cells HeLa (yellow), lung cancer cells A549 (red), and colon cancer cells HCT-116 (blue) were treated with either DMSO (control) or 80 µM celecoxib (striped bars) for 2 h. The sphingolipid amounts determined by LC-MS/MS were related to the number of treated cells. Data are mean ± SEM of one of three comparable independent experiments, each performed in duplicate. * P < 0.05, significant difference between celecoxib-treated and control cells.

View larger version (17K): [in this window] [in a new window]   Fig. 4. A 13C,15N-labeled L-serine incorporation experiment. HCT-116 cells were treated either with 400 µM 13C,15N-labeled L-serine (yellow/red bars) as control or cotreated with 400 µM 13C,15N-labeled L-serine and 80 µM celecoxib (white/blue bars) up to 6 h. The labeled sphingolipids are marked with an asterisk. The labeled sphingolipid amounts determined by LC-MS/MS were related to the number of treated cells. Data are mean ± SEM of three independent experiments, each performed in duplicate. * P < 0.01, significant difference in case of dhCers/sphinganine (dhSph) between celecoxib-treated cells at different time points and celecoxib-treated cells for 5 min and in case of Cers between celecoxib-treated and control cells at the indicated time points.

View larger version (12K): [in this window] [in a new window]   Fig. 5. A: The effect of celecoxib, C8-cyclopropenylceramide (C8-CPPC), and fumonisin B1 (FB1) on the total ceramide level. HCT-116 cells were treated with either DMSO (white bars), 80 µM celecoxib (black bars), 50 µM FB1 (light-gray bars), or 5 µM C8-CPPC (gray bars) for 2 h. The sphingolipid amounts determined by LC-MS/MS were related to the sphingolipid amounts of DMSO-treated cells. Data are mean ± SEM of three independent experiments, each performed in duplicate. * P < 0.05, significant difference between celecoxib- or C8-CPPC-treated cells versus untreated cells. B: In vivo DEGS activity in the presence of celecoxib. HCT-116 cells were cotreated with 10 µM C8:0-dhCer and increasing concentrations of celecoxib for 2 h. The amount of C8:0-Cer was determined by LC-MS/MS. To calculate the IC50 value for DEGS inhibition, the nonlinear regression analysis and a sigmoid Emax model were used. Experiments were done in triplicate; data are mean ± SEM.

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).

The Cer biosynthesis inhibitors myriocin and L-cycloserine protect cells from celecoxib-induced anti-proliferative effects
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 ).

View larger version (11K): [in this window] [in a new window]   Fig. 6. Effect of myriocin and L-cycloserine on celecoxib-induced reduction of cell viability and apoptosis. A: HCT-116 cells were either incubated with 80 µM celecoxib or cotreated with 80 µM celecoxib and 150 nM myriocin for 6 h. The rate of apoptosis was analyzed using the APOPercentage kit as described. * P < 0.01, significant difference between celecoxib-treated cells versus celecoxib/myriocin-treated cells. B, C: HCT-116 cells were treated with increasing concentrations of celecoxib (black squares), as indicated, for 20 h or preincubated with either 150 nM myriocin (white squares) (B) or 500 µM L-cycloserine (white squares) (C) for 90 min and then treated with increasing concentrations of celecoxib in the presence of 150 nM myriocin or 500 µM L-cycloserine for 20 h. The cell viability was determined using WST-1 proliferation assay. The proliferation rate of untreated cells or cells treated only with myriocin or L-cycloserine was used as an index for 100% cell viability. The relative viability rate was then expressed as the ratio of the cell viability of treated versus untreated cells. Data are mean ± SEM of three comparable independent experiments, each performed in triplicate. * P < 0.05, significant difference between celecoxib and celecoxib/myriocin- or celecoxib/L-cycloserine-treated cells.

	DISCUSSION

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Sphingolipids are important signaling molecules that regulate proliferation as well as apoptotic processes (28, 29). Because disruption of the balance between proliferating and anti-proliferating sphingolipids has dramatic consequences for the cell, and because certain coxibs mediate anti-proliferative effects through COX-2-independent pathways, we investigated the interaction of coxibs with the sphingolipid pathway. In this study, we showed that celecoxib and methylcelecoxib are the only coxibs that lead to an accumulation of dhCers in cancer cells through activation of de novo sphingolipid biosynthesis and inhibition of DEGS. All other coxibs tested had no effect on sphingolipid levels. The increase of dhCers after celecoxib treatment occurs already after 5 min, indicating modulation of nontranscriptional processes. Interestingly, accumulation of dhCers depends on the presence of celecoxib, because removing celecoxib from the cell culture medium returns the dhCer concentrations (data not shown) to their basal levels. Furthermore, in vitro activity assays of the L-SPT and the dhCerS (Supplement 4) show no activation of either enzyme in the presence of celecoxib, indicating that a whole cell is necessary for celecoxib-induced de novo sphingolipid biosynthesis. Activation/inhibition of an enzyme may depend on post-translational modifications as well as cofactors and substrate availability. It is not clear which of these is responsible for the observed elevation of sphingolipid biosynthesis. Activation of SMase by accumulation of arachidonic acid due to inhibition of COX-2, as proposed by Chan et al. (30) for sulindac sulfide, cannot explain celecoxib-induced upregulation of dhCers because we show 1) that dhCers are produced via de novo synthesis and that SMases are not involved and 2) that other coxibs, which inhibit COX-2 even more potently than celecoxib, do not alter sphingolipid levels.

A decrease of C_24:0- and C_24:1-Cer levels after celecoxib treatment indicates that celecoxib inhibits DEGS. By performing an in vivo dhCer desaturase assay with nonphysiological C_8:0-dhCer as substrate, we could demonstrate that celecoxib inhibits DEGS in vivo with an IC₅₀ of 78.9 ± 1.5 µM. Dihydroceramide desaturase 1 (DEGS1; with delta-4 desaturase activity) plays the predominant role in human cells, whereas DEGS2 (with an additional hydroxylase activity), which is less characterized, appears to play a minor role (31, 32). DEGS1 prefers short-chain dhCer substrates (33, 34). Incomplete inhibition of these enzymes, possibly by indirect mechanisms, could be mediated by changes in the membrane fluidity, owing to celecoxib (unpublished observations). Planar substances like celecoxib (35) and unsaturated lipids, like the upregulated dhCers, induce membrane rigidity. Membrane stiffening is proposed to affect the conformation and function of integral membrane proteins (36). DEGS is a protein complex consisting of a flavin-containing cytochrome b5 reductase, a heme-containing b5, and a non-heme-containing desaturase (33), whose quaternary structure would be sensitive to membrane consistency. Johnson et al. (37) observed an inhibition of the membrane protein Ca²⁺-ATPase by higher concentrations of celecoxib (IC₅₀ = 35 µM), which was connected to membrane effects of celecoxib by Gamerdinger, Clement, and Behl (35), indicating that celecoxib can influence enzyme activities by increasing membrane rigidity.

The use of the Cer biosynthesis inhibitors myriocin and L-cycloserine suggests that changes in dhCer levels contribute to the apoptosis-inducing and anti-proliferative activity of celecoxib. Moreover, the specific inhibitor C8-CPPC (5 µM) reduced the cell viability of HCT-116 cells to 15% (Supplement 5). In the presence of myriocin, the reduction is attenuated to only 28%. In contrast, exogenously added dhCers induce no apoptosis or cell cycle arrest. A recent report demonstrated that the location of the synthesized Cer is essential for the mode of action (3). Additionally, in vivo studies support the notion that endogenously produced dhCers act like Cers. Munoz-Olaya et al. (38) showed that Jurkat A3 cells treated with XM462 (inhibitor of DEGS) exhibit increased dhCer levels and reduced cell viability. Additionally, Jiang et al. (39) could also demonstrate for the chemotherapeutic -tocopherole, which blocks the DEGS, that an accumulation of endogenously produced C_16:0-dhCer induces apoptosis in lung cancer cells (A549). Moreover, Tserng and Griffin (40) elucidated that natural C_16:0-Cer exogenously added to human leukemia cells induces apoptosis via accumulation of dhCers. Deregulation of dhCer and Cer levels in fibroblasts, caused by the loss of the CLN9 gene, has dramatic consequences for cell growth, inasmuch as these cells show rapid growth on the one hand but increased apoptosis on the other hand (41). Kraveka et al. (32) demonstrated that fenretinide increases the concentration of dhCer through inhibition of DEGS, which was associated with an upregulated dhCer level, activation of protein phosphatase PP1, and a subsequent dephosphorylation of retinoblastoma protein (RB), a set of events that resulted in cell cycle arrest. Furthermore, fenretinide induces in human prostate cancer cells also an increase of C_16:0-dhCer, C_24:1-dhCer, and C_24:0-dhCer and a slight decrease of C_24:1-Cer and C_24:0-Cer. The authors linked this increase of dhCers to the induction of autophagy (42). Together, the body of evidence strongly indicates that dhCers could be relevant mediators of cytotoxic effects. In addition to raised dhCer levels, it is also possible that the changes in Cer levels contribute to the induction of apoptosis after celecoxib treatment. Liu et al. (43) recently proposed that the balance between C_16:0- and C_24:0-Cer plays an important role in inducing apoptosis. Interestingly, the pro-apoptotic C_16:0-Cer remains unchanged after celecoxib treatment, whereas the anti-apoptotic C_24:0-Cer decreases; the balance is therefore tipped toward apoptosis. In the case of celecoxib, the modulation of sphingolipid levels appears to be a unique response, because it was observed in various cancer cell lines, and it seems to be COX-2 independent, because it occurs in COX-2-overexpressing and COX-2-depleted cell lines (Fig. 3). This tenet was further confirmed by the fact that other selective COX-2 inhibitors show no effect on sphingolipid levels (Fig. 2A).

In conclusion, we demonstrated for the first time that treatment of cells with celecoxib activates de novo sphingolipid synthesis and inhibits DEGS, which leads to a rapid cellular accumulation of C_16:0-, C_24:0-, and C_24:1dhCers and depletion of long-chain C_24:0- and C_24:1-Cers. Furthermore, the increase of dhCer levels appears to be relevant for the cytotoxic effect of celecoxib.

Submitted on March 6, 2008
Revised on July 3, 2008 and in re-revised form August 4, 2008.

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