Affiliations of authors: Northern Institute for Cancer Research, University of Newcastle upon Tyne, Newcastle upon Tyne, U.K. (PEL, ADJP, AGH, CPFR); Department of Biology, University of Rome, Rome, Italy (FDS, BF, MP); Istituto Nazionale Malattie InfettiveIstituto di Ricovero e Cura a Carattere Scientifico Lazzaro Spallanzani, Rome (MC, MP); The Regina Elena National Cancer Institute, Rome (RPD)
Correspondence to: Chris P. F. Redfern, PhD, Northern Institute for Cancer Research, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, U.K. (e-mail: chris.redfern{at}ncl.ac.uk)
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ABSTRACT |
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INTRODUCTION |
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In neuroblastoma cells, fenretinide-induced apoptosis involves mitochondria and is caspase-dependent (10). Retinoic acid receptor (RAR) antagonists and antioxidants inhibit fenretinide-induced apoptosis, suggesting that signaling pathways involving RARs and reactive oxygen species (ROS) are both required for fenretinide-induced apoptosis (10). However, the ability of fenretinide to induce ROS may be the key to its apoptosis-inducing properties. Recent studies (11,12) have shown that, in two neuroblastoma cell lines, oxidative stress induced by fenretinide is mediated by 12-lipoxygenase activity. Sustained induction of the growth and DNA damage (GADD)-inducible transcription factor GADD153 (11) is a consequence of increased ROS (11), and subsequent induction of the proapoptotic protein Bak (13) accompanies mitochondrial cytochrome c release and caspase-3-dependent apoptosis (10).
In addition to its effects on ROS, fenretinide has been reported to increase intracellular levels of the lipid secondary messenger ceramide in tumor cells (5,1416). At high concentration, fenretinide appears to increase ceramide levels in some human neuroblastomas, prostate cancers, leukemias, and endothelial cells through a de novo synthesis pathway by activating the rate-limiting enzyme serine palmitoyltransferase (1417). Paradoxically, fumonisin B1, an inhibitor of ceramide synthase, does not inhibit fenretinide-induced apoptosis of neuroblastoma cells (14). High concentrations of fenretinide can induce necrosis as well as apoptosis (5); therefore, it is possible that more subtle apoptotic effects are mediated by mechanisms that increase ceramide levels by means other than de novo synthesis. One such mechanism is membrane sphingomyelin hydrolysis by sphingomyelinases (18,19) (Fig. 1). Both neutral and acidic sphingomyelinases, the two major classes of this enzyme, are important in ceramide generation and apoptosis (2024).
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METHODS |
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The human neuroblastoma cell lines SH-SY5Y (MYCN not amplified) (25) and HTLA230 (MYCN amplified) (12) were grown in complete culture medium consisting of a 1 : 1 mixture of Dulbeccos Modified Eagle Medium and Hams F12 (Life Technologies, Paisley, U.K.) supplemented with 10% fetal bovine serum (Life Technologies) and grown in a humidified atmosphere of 5% CO2 in air.
For all experiments, cells were seeded into tissue culture flasks and allowed to attach for 24 hours (HTLA230 cells) or 48 hours (SH-SY5Y cells) before treatment. The seeding density varied according to the type of experiment; for flow cytometry and RNA interference experiments to measure apoptosis and ROS generation, 1 x 106 cells were seeded into 25-cm2 flasks (Costar, High Wycombe, U.K.) in 8 mL of culture medium. For immunofluorescence experiments, 2 x 106 cells were seeded into 25-cm2 flasks in 8 mL of culture medium.
Ethanol was the diluent for fenretinide (Janssen-Cilag, Basserdorf, Switzerland) and for the disialogangliosides Neu5Ac8Neu5Ac
3Gal
4GlcCer (GD3) and GalNAc
4(Neu5Ac
8Neu5Ac
3)Gal
4GlcCer (GD2) (both from Calbiochem, San Diego, CA). Control cells were treated with a volume of ethanol (less than 0.1% of the final culture volume) equivalent to that used for fenretinide, GD3, or GD2. Fumonisin B1, a specific inhibitor of ceramide synthase (final concentration = 20 µM; Sigma Chemical, Poole, U.K.), was diluted in ethanol. Desipramine hydrochloride, a broad-spectrum inhibitor of sphingomyelinase (final concentration = 5 µM; Sigma), neutral sphingomyelinase spiroepoxide inhibitor (final concentration = 1 µM; Alexis Biochemicals, San Diego, CA), and 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP, an inhibitor of glucosylceramide synthase; final concentration = 1030 µM; Sigma) were diluted in dimethyl sulfoxide. For experiments involving inhibitors, cells were treated with the inhibitor or control diluent for 1 hour and then treated with fenretinide for 24 hours. Unless stated otherwise, the concentration of fenretinide used was 3 µM. For experiments involving baicalein (final concentration = 1 µM, a specific inhibitor of 12-lipoxygenase at this concentration, diluted in ethanol), cells were treated for 2 hours and then treated with fenretinide, GD3, or GD2, as previously described (11).
Flow Cytometry
Flow cytometry was used to evaluate apoptosis, ROS generation, and endogenous GD3 and GD2 levels. Apoptosis was evaluated by staining cells with propidium iodide (100 µg/mL final concentration), as previously described (10). ROS generation was evaluated by staining cells with 10 µM 5- (and 6-)chloromethyl-2',7'-dihydrodichlorofluorescein diacetate (CMH2DCFA) for 20 minutes at 37 °C in the dark, as previously described (10,11). Endogenous GD3 and GD2 were detected by immunofluorescence flow cytometry; cells were detached with trypsin, washed with phosphate-buffered saline (PBS), and fixed in 4% paraformaldehyde in PBS for 10 minutes at room temperature. The cells were permeabilized in a buffer containing 0.5% Triton X-100 for 2 minutes at room temperature and stained with 200 µL of a mouse monoclonal anti-GD3 antibody (Calbiochem, diluted 1 : 100) or with a mouse anti-GD2 antibody (mouse immunoglobulin G2 [IgG2a], diluted to 1 µg/mL; a kind gift from Dr. Holger N. Lode, Charite Universitatsmedizin, Berlin, Germany) for 1 hour at room temperature. Primary antibodies were diluted in PBS containing 5% bovine serum albumin, and were detected with 10 µg of a secondary goat anti-mouse IgG antibody conjugated with fluorescein isothiocyanate (Molecular Probes, Leiden, The Netherlands, for detection of GD3; Sigma for detection of GD2) diluted in PBS containing 5% bovine serum albumin in a final volume of 200 µL. For each sample, 20 000 events were acquired for flow cytometry, as previously described (11). Control samples stained with secondary antibody alone were included in each experiment.
RNA Interference of Acidic Sphingomyelinase and GD3 Synthase
The Ambion Silencer small interfering RNA (siRNA) construction kit (product 1620, Ambion Europe, Huntingdon, U.K.) was used to generate double-stranded small interfering RNAs (ds-siRNAs) according to the manufacturers instructions. The targets (Table 1) were designed using the Ambion online resource (www.ambion.com/techlib/misc/siRNA_finder.html), and primer oligonucleotides were obtained from TAGN (Newcastle, U.K.). Pre-validated glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific sense and antisense templates provided with the Ambion Silencer kits were used to generate a control ds-siRNA unrelated to the target mRNA to control for nonspecific effects of the gene knockdown process. Each ds-siRNA (final concentration = 5 nM) was transiently transfected into 1 x 106 SH-SY5Y cells by using Lipofectamine 2000 (Life Technologies) for 24 hours, according to the manufacturers instructions. Fetal calf serum was added to a final concentration of 10% 6 hours after the addition of Lipofectamine and ds-siRNA. After 24 hours, the medium was replaced with an equal volume of complete culture medium containing 5 µM fenretinide or the appropriate volume of ethanol (control vehicle) for 624 hours. Cells were subsequently harvested with trypsin for analysis. For experiments in which ceramide or sphingomyelin levels were measured after RNA interference, the medium was replaced after the 24-hour transfection period with fresh medium containing 0.1% fetal calf serum, 5 µM fenretinide, or ethanol control, as appropriate, and 1 µCi/mL [3H]-palmitic acid (Amersham Biosciences, Chalfont St. Giles, U.K.) for 18 hours.
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Although RNA interference produces robust silencing of targeted mRNA, off-target (i.e., nonspecific) effects can occur with sequences of 1115 contiguous nucleotides (26). From BLAST (27) searches of the GenBank nonredundant nucleotide sequence database for the acidic sphingomyelinase target 18 sequence, 11 and 12 contiguous nucleotides matched cAMP-specific phosphodiesterase 8B and cGMP phosphodiesterase, respectively, but there were no matches of greater length to other known phosphodiesterases. For the GD3 synthase target sequence 43, there were no contiguous sequence matches of more than nine nucleotides to known sialyltransferases other than to GD3 synthase. With respect to potential matches of the acidic sphingomyelinase (target 18) and GD3 synthase (target 43) sequences to other genes, there were no matches of more than 15 contiguous nucleotides to known transcripts. Therefore, although off-target effects cannot be discounted, the ds-siRNA probes used here were of high specificity to the selected targets.
Western Blotting
Total proteins (25 µg) were extracted from SH-SY5Y control cells, SH-SY5Y cells transfected with ds-siRNA for acid sphingomyelinase, or SH-SY5Y cells transfected with ds-siRNA for acid sphingomyelinase, treated with fenretinide for 24 hours, and separated by electrophoresis through 12.5% sodium dodecyl sulfatepolyacrylamide gel electrophoresis gels and electroblotted onto nitrocellulose membranes (11). Acid sphingomyelinase was detected by incubating the membranes with a polyclonal goat anti-acid sphingomyelinase antibody (a kind gift from Professor Sandhoff, Department of Chemistry and Biochemistry, University of Bonn, Bonn, Germany) diluted 1 : 2500 in blocking solution (5% nonfat dry milk in PBS containing 0.1% vol/vol Tween 20) for 1 hour at room temperature. Antibody binding was visualized with a peroxidase-conjugated affinity-purified rabbit anti-goat IgG (Jackson Laboratories, West Grove, PA) that was diluted 1 : 10 000 in blocking solution and incubated with the membrane for 1 hour at room temperature; the secondary antibody was detected by enhanced chemiluminescence. -Tubulin was detected with a monoclonal mouse anti-
-tubulin antibody (Sigma; diluted 1 : 1000) and a peroxidase-conjugated affinity-purified goat anti-mouse IgG (Bio-Rad, Hemel Hempstead, U.K.; diluted 1 : 5000) as a control for protein loading.
Measurement of Acidic Sphingomyelinase Activity
Acidic sphingomyelinase activity was assayed as described by Wiegmann et al. (28). Briefly, cells were harvested by scraping, washed twice with PBS, and suspended in 200 µL of 20 mM HEPES buffer (pH 7.4) containing 0.2% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail. After incubation for 15 minutes at 4 °C, cells were homogenized by passing them through an 18-gauge needle, nuclei and debris were removed by centrifugation at 800g, and 50 µg of protein was subsequently incubated for 2 hours at 37 °C in a buffer containing 250 mM sodium acetate, 1 mM EDTA (pH 5.0), and 2 µL of [N-methyl-14C]-sphingomyelin (specific activity, 55 mCi/mMol; Amersham Biosciences). Phosphorylcholine was then extracted with 800 µL of chloroform : methanol [2 : 1 (vol/vol)], and 250 µL of water. Radioactive phosphorylcholine produced from [14C]-sphingomyelin was measured by liquid scintillation counting. Acidic sphingomyelinase activity was calculated as picomoles per milligram per hour, and results were expressed as a percentage of the activity in control uninduced cells.
Measurement of Glucosylceramide Synthase Activity
Glucosylceramide synthase (ceramide glucosyltransferase, EC 2.4.1.80) activity was measured as described previously (29). Briefly, 1 x 106 cells in 5 mL were incubated with 5 µM 6-(N-(7-nitrobenzen-2-oxa-1,3-diazol-4-yl)-amino)hexanoylsphingosine (NBD; Molecular Probes, Eugene, OR). Lipids were extracted and separated by high-performance thin-layer chromatography. GlucosylceramideNBD was detected under UV illumination and extracted from the silica gel; fluorescence was measured with a Perkin-Elmer LS-5 luminescence spectrophotometer (Perkin-Elmer, Monza, Italy). Glucosylceramide synthase activity was then expressed relative to a standard glucosylceramideNBD fluorescence calibration curve.
Cellular Ceramide and Sphingomyelin Content
To determine cellular ceramide and sphingomyelin content, cells were treated with [3H]-palmitic acid (Amersham Biosciences) for 18 hours, as described previously (30), detached with trypsin and washed twice with PBS; total cellular lipids were then extracted. The lipids were subjected to mild alkaline hydrolysis with 0.1 M methanolic KOH for 1 hour at 37 °C and re-extracted with chloroform. The chloroform phase was then analyzed by thin-layer chromatography using different solvent systems for ceramide and sphingomyelin (16). Lipids were visualized by incubating the plates in 3% cupric acetate in 8% phosphoric acid and then heating them in an oven for 15 minutes at 180 °C, according to Spinedi et al. (30). Tritium in the thin-layer chromatographyresolved lipid band and total tritium in equal aliquots of the extracted lipids were quantified by liquid scintillation counting. The amount of ceramide and sphingomyelin was expressed as a percentage of the total lipid tritium in the sample.
Statistical Analysis
Data were analyzed by one- and two-way analysis of variance (ANOVA), and specific hypotheses were tested by the F statistic using pooled variances. Time and dose effects were tested by first-order polynomial contrasts. All statistical tests were two-sided. Statistical procedures were done using Systat, version 10 (SPSS, Chicago, IL). Data shown in the figures are means with 95% confidence intervals. The majority of experiments were done in triplicate, but some ds-siRNA experiments were done in duplicate only; data for these are presented as means with ranges.
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RESULTS |
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Fumonisin B1, an inhibitor of ceramide synthase (31), reduces differentiation of SH-SY5Y cells (32) and inhibits apoptosis of other neuronal cells (33). To assess whether ceramide synthase mediates fenretinide-induced ROS generation and apoptosis in neuroblastoma cells, SH-SY5Y and HTLA230 cells were incubated with fumonisin B1 for 1 hour before they were incubated with fenretinide. In SH-SY5Y cells, 20 µM fumonisin B1 did not block fenretinide-induced ROS generation (Fig. 2, A) or apoptosis (Fig. 2, B). By contrast, fumonisin B1 increased ROS and apoptosis in control cells (ANOVA, ROS, F3,8 = 171.7, P<.001; fumonisin B1 versus control, F1,8 = 10.1, P = .013; apoptosis, F5,12 = 118.1, P<.001; fumonisin B1 versus control, F1,12 = 10.6, P = .007). Fumonisin B1 also increased ROS in combination with fenretinide (F1,8 = 16.6, P<.05) but had no effect on apoptosis in combination with fenretinide, regardless of fenretinide concentration (F1,83.05, P>.1). Similar results were obtained with the HTLA230 cells (data not shown).
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A study of hippocampal neurons has suggested that ceramide generated by neutral sphingomyelinase may be important for neuronal apoptosis (34). To distinguish between neutral and acidic sphingomyelinase as mediators of fenretinide-induced apoptosis, SH-SY5Y cells were incubated with a neutral sphingomyelinase spirepoxide inhibitor for 1 hour before they were incubated with fenretinide. Treatment with the neutral sphingomyelinase spirepoxide inhibitor, unlike treatment with desipramine, did not block fenretinide-induced ROS (Fig. 2, E; ANOVA, F3,8 = 125.2, P<.001; effect of neutral sphingomyelinase spirepoxide inhibitor on fenretinide-induced ROS, F1,8 = 0.02, P>.8) or fenretinide-induced apoptosis (Fig. 2, F; ANOVA, F3,8 = 94.5, P<.001; effect of neutral sphingomyelinase spirepoxide inhibitor on fenretinide-induced apoptosis, F1,8 = 0.06, P>.8) of SH-SY5Y cells. Thus acidic sphingomyelinase, but not neutral sphingomyelinase, is important in fenretinide-induced apoptosis of SH-SY5Y cells.
Acidic Sphingomyelinase Activity in Relation to Fenretinide-Induced Ceramide, Apoptosis, and ROS
To investigate further the role of acidic sphingomyelinase in fenretinide-induced apoptosis, RNA interference was used to reduce the expression of acidic sphingomyelinase. Compared with activity in untransfected SH-SY5Y cells or SH-SY5Y cells transfected with a control ds-siRNA, acidic sphingomyelinase activity was reduced by at least 50% in cells transfected with the acidic sphingomyelinase ds-siRNA (target sequence 18; Table 1 and Fig. 3, A). Western blotting of protein extracts from these cells confirmed a reduction in acidic sphingomyelinase protein in both control and fenretinide-treated SH-SY5Y cells transfected with the acidic sphingomyelinase ds-siRNA (Fig. 3, B). The western blot also revealed that acidic sphingomyelinase protein levels increased in response to treatment with 5 µM fenretinide for 24 hours in control cells and cells transfected with the control ds-siRNA (Fig. 3, B). Thus, the western blot data (Fig. 3, B) and the slight increase in acidic sphingomyelinase activity in response to a 4-hour treatment with 5 µM fenretinide (Fig. 3, A) suggest that fenretinide increased acidic sphingomyelinase levels in SH-SY5Y cells and that the acidic sphingomyelinase ds-siRNA effectively reduced the expression of both constitutive and fenretinide-induced acidic sphingomyelinase.
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Levels of fenretinide-induced apoptosis in cells transfected with the control ds-siRNA did not differ from levels in fenretinide-treated control cells (ANOVA, F5,18 = 39.0, P<.001; fenretinide versus fenretinide with control ds-siRNA, F1,18 = 1.7, P = .215). However, levels of fenretinide-induced apoptosis in cells transfected with acidic sphingomyelinase ds-siRNA were similar to levels in untreated (i.e., no fenretinide) control cells (F1,18 = 49.5, P<.001).
Glucosylceramide Synthase Activity and Gangliosides GD3 and GD2 After Fenretinide Treatment
Sphingomyelinase-generated ceramide is a substrate for glucosylceramide synthase, the activity of which results in the formation of glycosphingolipids and gangliosides, including the disialogangliosides GD3 and GD2. To test whether GD3 and GD2 may be downstream effectors of fenretinide-induced apoptosis, we assessed whether glucosylceramide synthase activity changed over time in response to fenretinide treatment and measured the effects of a glucosylceramide synthase inhibitor, PDMP, on fenretinide-induced apoptosis. Glucosylceramide synthase activity increased in a time-dependent manner during the first 6 hours after the addition of fenretinide (ANOVA, F3,8 = 126.7, P<.001; effect of time: first-order polynomial contrast, F1,8 = 342.1, P<.001) (Fig. 4, A).
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Evidence from other studies (21,35) has suggested that the hydrolysis of sphingomyelin via sphingomyelinase increases levels of GD3 and that GD3 is a critical signaling intermediate in the apoptosis of lymphoid and neuronal cells. Therefore, it is possible that GD3 may increase in SH-SY5Y neuroblastoma cells as a result of a fenretinide-induced increase in glucosylceramide synthase activity, and that this GD3 rise is responsible for subsequent events leading to apoptosis. To test this possibility, we used immunofluorescence flow cytometry to measure levels of GD3 in SH-SY5Y cells after treatment with fenretinide. As shown in Fig. 4, C, we found that GD3 levels increased twofold in response to fenretinide treatment (F1,11 = 12.6, P = .005). GD3 is the first member of the b-series gangliosides and generates GD2 via the addition of N-acetylgalactosamine (36). Therefore, immunofluorescence flow cytometry with an anti-GD2 antibody was used to determine whether GD2 also increases in response to fenretinide. Compared with GD2 levels in untreated SH-SY5Y cells, levels of GD2 in SH-SY5Y cells increased 2.5-fold in response to fenretinide under the same conditions used for the GD3 experiments (Fig. 5).
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GD3 is generated from the monosialoganglioside GM3 by the enzyme -N-acetylneuraminate
-2,8-sialyltransferase (GD3 synthase). To determine whether this enzyme is necessary for fenretinide-induced apoptosis, ds-siRNAs were designed to reduce the expression of GD3 synthase in SH-SY5Y cells. All three ds-siRNAs, targeting different sequences of GD3 synthase mRNA (Table 1), were effective in blocking fenretinide-induced apoptosis after transfection into SH-SY5Y cells (Fig. 6, A). One of these (ds-siRNA43) was used for further experiments. The fenretinide-induced increase in cellular GD3 levels was effectively blocked in cells transfected with ds-siRNA43 (ANOVA, F5,11 = 43.4, P<.001; GD3 synthase siRNA and fenretinide treatment versus control SH-SY5Y cells and fenretinide, F1,11 = 70.0, P<.001; Fig. 6, B and C). This result is indirect evidence that the ds-siRNA43 probe was effective in reducing GD3 synthase activity. Compared with untransfected cells treated with fenretinide, cells transfected with the control ds-siRNA had increased GD3 levels in response to fenretinide (F1,11 = 14.9, P = .003). Levels of fenretinide-induced apoptosis in cells transfected with the GD3 synthase ds-siRNA43 were reduced to the levels seen in untreated control cells (Fig. 6, D and F; ANOVA, F5,12 = 33.2, P<.001; effect of control ds-siRNA on fenretinide-induced apoptosis, F1,12 = 3.1, P>.1; effect of GD3 synthase-specific ds-siRNA on fenretinide-induced apoptosis, F1,12 = 86.9, P<.001). Furthermore, the fenretinide-dependent increase in ROS was blocked in cells transfected with ds-siRNA43 but not in cells transfected with control ds-siRNA (Fig. 6, E; ANOVA, F5,12 = 267.3, P<.001; fenretinide versus fenretinide plus control ds-siRNA, F1,12 = 0.3, P>.5; fenretinide versus fenretinide plus GD3 synthase-specific ds-siRNA, F1,12 = 388.8, P<.001). These data suggest that GD3 synthase mediates fenretinide-induced apoptosis and ROS generation via the production of increased levels of GD3.
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The addition of exogenous GD3 at concentrations up to 10 µM to cultures of smooth muscle cells stimulates ROS and apoptosis (37). Neuroblastoma cells have high levels of endogenous GD2 (38). We added exogenous GD3 or GD2 (at concentrations of 1, 5, and 10 µM) to SH-SY5Y cultures to determine whether these gangliosides would mimic the effects of fenretinide with respect to the induction of ROS and apoptosis. Apoptosis increased dose-dependently after treatment with GD3 (two-way ANOVA, control and GD3 treatments, effects of time and treatment, F1,16 = 26.5, P<.001 and F3,16 = 261.1, P<.001, respectively; first-order polynomial contrast on GD3 dose, F1,16 = 763.1, P<.001), with 10 µM GD3 inducing 40% apoptosis and 5 µM fenretinide inducing 55% apoptosis at 24 hours (Fig. 7, A). Similar results were obtained with GD3-induced ROS (first-order polynomial contrast, effect of GD3 dose, F1,16 = 136.4, P<.001; Fig. 7, B).
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In contrast to the effects of GD3, GD2 did not induce apoptosis of SH-SY5Y cells; indeed, there was an overall reduction in the percentage of apoptosis with increased time of treatment (Fig. 7, E; two-way ANOVA on GD2 and control treatments: effect of time, F1,24 = 31.3, P<.001; effect of GD2, F5,24 = 1.4, P>0.2; no timeGD2 interaction, F5,24 = 0.5, P>.7). Baicalein had no effect on apoptosis in cells treated with GD2 (Fig. 7, E). These results suggest that the apoptotic effects of GD3 were not a result of subsequent metabolism to GD2.
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DISCUSSION |
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The mechanism of either acidic sphingomyelinase or serinepalmitoyltransferase activation in response to fenretinide in any cell type is currently unknown. In addition to inducing oxidative stress, or ROS, fenretinide is an RAR/
agonist. We have argued that both ROS-dependent and RAR-dependent pathways are necessary for fenretinide-induced apoptosis of SH-SY5Y cells on the basis of evidence that RAR
/
antagonists block fenretinide-induced apoptosis but do not block ROS induction (10). In NB4 acute promyelocytic leukemia cells, retinoic acid induces acidic sphingomyelinase via retinoic acid response elements within the acidic sphingomyelinase promoter (41). The rate-limiting enzyme in the de novo synthesis pathway, serinepalmitoyltransferase, can also be activated, albeit at a post-translational level, by retinoic acid during neuronal differentiation (33). Therefore, the induction of acidic sphingomyelinase by fenretinide in SH-SY5Y cells may be mediated by RARs. Alternatively, acidic sphingomyelinase activation could be dependent on Fas-associated protein with death domain and result from an increase in the rate of substrate hydrolysis, as in embryonic fibroblasts (42).
Evidence suggests that ceramide can induce apoptosis in cells through different mechanisms, either directly as a lipid-signaling molecule interacting with enzyme systems that participate in apoptosis regulation or by further metabolism to other lipid mediators, such as gangliosides (43). The data for SH-SY5Y cells imply that glucosylceramide synthase and GD3 synthase, key elements in the synthesis of gangliosides from ceramide, are essential steps in apoptosis and ROS generation in response to fenretinide. Glucosylceramide synthase activity showed a linear, time-dependent increase in response to fenretinide, and this activity may result from the transcriptional induction of glucosylceramide synthase, possibly mediated by nuclear factor-B (44), and/or by post-translational activation (45,46) in response to increasing ceramide levels. In CHP-100 neuroepithelioma cells, blocking glucosylceramide synthase expression with an antisense construct decreases sensitivity to fenretinide-induced apoptosis (29). This supports the role of glucosylceramide synthase in mediating fenretinide-induced apoptosis of SH-SY5Y cells and suggests that this ceramide signaling pathway also mediates fenretinide-induced cell death in other cell types. By contrast, the overexpression of glucosylceramide synthase has been associated with resistance to chemotherapeutic drugs in neuroblastoma and other tumors (47,48). Although inhibitors of glucosylceramide synthase reportedly act synergistically with fenretinide in CHLA-90 neuroblastoma cells (14), such chemosensitizing effects may be independent of the activity of these compounds as glucosylceramide synthase inhibitors (47). Nevertheless, the complexity of these lipid signaling pathways is highlighted by the fact that inhibition of glucosylceramide synthase can increase apoptosis in CHP-100 cells but that blocking this enzyme with an antisense construct does not affect the response to p53-dependent apoptotic drugs (49). Clearly, altering the metabolic flux of sphingolipid metabolism can induce apoptosis by a variety of mechanisms, including ceramide accumulation, perhaps leading to necrosis (14) or ganglioside synthesis.
Glucosylceramide is metabolized via galactosyl transferase to lactosylceramide, and the subsequent activity of different sialyltransferases generates the first intermediates of the a-, b- and c-series gangliosides (Fig. 1) (36). The abrogation of fenretinide-induced apoptosis and ROS generation by knockdown of GD3 synthase suggests that the b- and c-series gangliosides are signaling intermediates of ceramide metabolism. Although the ganglioside GT3, the first member of the c-series gangliosides, has growth-regulatory properties, it may be synthesized within a different compartment of the Golgi than GD3, giving greater weight to the idea that GD3, or a subsequent member of the b-series gangliosides, such as GD2, is the primary messenger. However, although GD2 levels were also increased by fenretinide, this b-series ganglioside did not induce apoptosis. Therefore, GD3 is probably the main ganglioside of the apoptotic signaling pathway induced by fenretinide. Although the effects of exogenous GD3 may be different from those of GD3 generated by intracellular metabolism (37), two recent studies illustrate the importance of GD3 in apoptotic signaling. Bhunia et al. (37) demonstrated that the induction of high levels of GD3 induced ROS and triggered signaling events leading to apoptosis, and Copani et al. (35) suggested that GD3 contributes to the apoptosis of neuronal cells.
Substantial evidence indicates that GD3 can interact directly with mitochondria, stimulating apoptosis as a result of opening the mitochondrial permeability transition pore (36). However, in SH-SY5Y neuroblastoma cells, fenretinide-dependent apoptosis is not mediated by the opening of the mitochondrial permeability transition pore, at least in the early stages of apoptosis (10). Because the effects of exogenous GD3 on ROS generation and apoptosis were, as with fenretinide (11), inhibited by the 12-lipoxygenase inhibitor baicalein, GD3 may be a signaling intermediate upstream of lipoxygenase activation. Although a direct link between gangliosides and lipoxygenase activation has not, to our knowledge, been established, GD3 can modulate 12-lipoxygenase activity in human peripheral blood lymphocytes (39). Clearly, the relationships between gangliosides, lipoxygenase activity, and apoptotic control pathways deserve further investigation.
In summary, our results suggest that acidic sphingomyelinase mediates fenretinide-induced apoptosis in neuroblastoma cells and that the induction of the ganglioside GD3, resulting from glucosylceramide synthase and GD3 synthase activities, may link increased ceramide levels with 12-lipoxygenase activation and subsequent apoptosis. These data raise new questions about the role of gangliosides in fenretinide-induced apoptosis and in regulating lipoxygenase activity, and they identify new targets for the development of drugs to improve current therapy for neuroblastoma. Furthermore, the observation that GD2 levels increase in response to fenretinide suggests that the response of neuroblastoma to therapy with anti-GD2 antibodies might be increased by co-treatment with fenretinide.
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NOTES |
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Supported by funding from Cancer and Leukemia in Childhood in the United Kingdom and by Associazione Italiana per la Ricerca sul Cancro, Fondo per gli Investimenti della Ricerca di Base, and Ricerca Corrente e Finalizza, Ministero della Salute in Italy. F. Di Sano and B. Fazi were supported by fellowships from Fondazione Italiana per la Ricerca sul Cancro.
We thank Prof. Dr. Konrad Sandhoff, Kekule-Institut f. Organische Chemie und Biochemie, Bonn, Germany, for the ASMase antibody, and Dr. Holger Lode, Charite Childrens Hospital, Humboldt University, Berlin, Germany, for the GD2 antibody.
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Manuscript received December 24, 2003; revised June 29, 2004; accepted July 8, 2004.
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