Altered Sphingolipid Metabolism in N-(4-Hydroxyphenyl)- retinamide-resistant A2780 Human Ovarian Carcinoma Cells*

Alessandro Prinetti, Luisa Basso, Valentina AppiertoDagger , Maria Grazia VillaniDagger , Manuela Valsecchi, Nicoletta Loberto, Simona Prioni, Vanna Chigorno, Elena CavadiniDagger , Franca FormelliDagger , and Sandro Sonnino§

From the Center of Excellence on Neurodegenerative Diseases, Study Center for the Biochemistry and Biotechnology of Glycolipids, Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milan, 20090 Segrate, Italy and the Dagger  Department of Experimental Oncology, Istituto Nazionale Tumori, 20133 Milan, Italy

Received for publication, July 19, 2002, and in revised form, December 13, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

In the present work, we studied the effects of fenretinide (N-(4-hydroxyphenyl)retinamide (HPR)), a hydroxyphenyl derivative of all-trans-retinoic acid, on sphingolipid metabolism and expression in human ovarian carcinoma A2780 cells. A2780 cells, which are sensitive to a pharmacologically achievable HPR concentration, become 10-fold more resistant after exposure to increasing HPR concentrations. Our results showed that HPR was able to induce a dose- and time-dependent increase in cellular ceramide levels in sensitive but not in resistant cells. This form of resistance in A2780 cells was not accompanied by the overexpression of multidrug resistance-specific proteins MDR1 P-glycoprotein and multidrug resistance-associated protein, whose mRNA levels did not differ in sensitive and resistant A2780 cells. HPR-resistant cells were characterized by an overall altered sphingolipid metabolism. The overall content in glycosphingolipids was similar in both cell types, but the expression of specific glycosphingolipids was different. Specifically, our findings indicated that glucosylceramide levels were similar in sensitive and resistant cells, but resistant cells were characterized by a 6-fold lower expression of lactosylceramide levels and by a 6-fold higher expression of ganglioside levels than sensitive cells. The main gangliosides from resistant A2780 cells were identified as GM3 and GM2. The possible metabolic mechanisms leading to this difference were investigated. Interestingly, the mRNA levels of glucosylceramide and lactosylceramide synthases were similar in sensitive and resistant cells, whereas GM3 synthase mRNA level and GM3 synthase activity were remarkably higher in resistant cells.

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Sphingolipid metabolism plays a pivotal role in the mechanism of apoptosis induced in tumor cells. Ceramide, produced under physiological (tumor necrosis factor alpha , gamma -interferon, and interleukins) and pharmacological (anticancer drugs, including daunorubicin, vincristine, 1-alpha -D-arabinofuranosylcytosine, and retinoids) stimuli by sphingomyelin hydrolysis or by de novo biosynthesis, is a mediator of apoptosis and an inhibitor of cell proliferation in a variety of tumor cell lines (reviewed in Refs. 1 and 2).

Interestingly, in tumor cell lines, resistance to chemotherapeutic treatments is often associated with an increased ability of the cell to glycosylate ceramide, as a consequence of a higher activity of glucosylceramide synthase (3-12). High levels of GlcCer1 (6-9, 11, 12) and of glucosylceramide synthase activity (5, 8, 9) and/or expression (8, 9, 11, 12) were detected in a number of drug-resistant cancer cell lines and in specimens from cancer patients not responding to chemotherapy treatment (13). GlcCer accumulation results in the ability of drug-resistant cells to scavenge ceramide, thus preventing ceramide-induced apoptotic death. Multidrug resistance (MDR), a drug-resistant phenotype characterized by the ability of tumor cells to become insensitive to a variety of chemically unrelated chemotherapeutics after exposition to one single drug, is hallmarked by the overexpression of energy-dependent drug efflux pump proteins, such as MDR1 P-glycoprotein (MDR1) and multidrug resistance-associated protein (MRP) (3, 14). Extensive studies by Cabot's group lead to the conclusion that elevated GlcCer levels are a specific marker for MDR phenotype in cancer cells and that modifying ceramide metabolism might represent a winning strategy to overcome drug resistance in tumor cells (4-11).

On the other hand, it has been shown (12, 15) that GlcCer accumulation is not the only consequence of an altered sphingolipid metabolism in MDR cancer cells. In MDR human ovarian carcinoma cells, SM and galactosylceramide levels are also higher respect to parental sensitive cells, whereas LacCer and all of the more complex GSLs are present in lower amounts. These data can be at least in part interpreted in the scenario described above. However, in addition to this it is necessary to recall that sphingolipids and sphingolipid metabolites other than ceramide have biological activities that could be responsible for the acquisition of a drug resistance phenotype (16-18).

Retinoids are natural and synthetic derivatives of vitamin A that play a critical role in different biological processes including morphogenesis in the embryo, cell proliferation, differentiation, and apoptosis (19). Retinoids exert their effects by regulating gene expression through two classes of nuclear receptors: retinoic acid (RA) receptors alpha , beta , and gamma  and retinoid X receptors alpha , beta , and gamma . RA receptors and retinoid X receptors activate gene transcription by binding as homo- or heterodimers to specific DNA sequences, the retinoic acid-responsive elements, and the retinoid X-responsive elements, usually found in the 5'-flanking regions of responsive genes (20). Retinoids have been shown to have differentiating and antitumor activities in several experimental models, and their effectiveness in the treatment and prevention of human cancer has already been established (21).

4-[3,7-Dimethyl-9-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2,4, 6,8- nonatetraenamido]-1-hydroxybenzene, known as N-(4-hydroxyphenyl)retinamide (HPR), pharmacologically known as fenretinide, is a synthetic amide of all-trans-RA, which has shown reduced toxicity relative to RA while maintaining a significant biological activity (22). HPR is under investigation in clinical trials as preventive and therapeutic agent and has already shown a preventive effect for ovarian and breast cancers in premenopausal women (23, 24) as well as chemopreventive and therapeutic efficacy against different tumors in animal models (22). In vitro studies have demonstrated that HPR has significant antiproliferative activity associated with induction of apoptosis in several tumor cell types (22). To date, the mechanism of action of HPR is poorly understood. Some studies suggest that the effects of this retinoid are mediated through RA receptor and retinoid X receptor signaling (25-28). Other studies have shown that apoptosis in response to HPR primarily occurs by a receptor-independent mechanism, which is accompanied by generation of reactive oxygen species (29, 30) or increases in ceramide (31, 32).

Discontinuation of retinoid treatment leads to recurrence of the lesion. However, as occurs with chemotherapeutic agents, continuous retinoid exposure might cause development of drug resistance. So far, only few in vitro models had been developed to investigate mechanisms and molecular characteristics associated with retinoid resistance (33, 34). In a recent study, we hypothesized that continuous HPR treatment might lead to resistance to the retinoid. We showed that when A2780 human ovarian carcinoma cells, which are very sensitive to HPR (35), were continuously exposed to the drug, they developed a 10-fold resistance to the drug (34). Differences in HPR uptake and metabolism were observed between sensitive and resistant cells (34). HPR intracellular peak levels were 2 times lower, and a polar metabolite, not detected in sensitive cells, was found in cell extracts from resistant cells. Moreover, the development of HPR resistance was associated with changes in marker expression, suggestive of a more differentiated status (34). The expression of RA receptor beta , a putative tumor suppressor (36), was markedly increased, whereas the expression of cell surface molecules associated with tumor progression including HER-2 laminin receptor and beta 1 integrin was markedly reduced.

An increase in the cellular levels of ceramide upon HPR treatment was reported in neuroblastoma (31, 37) and breast cancer cell lines (32). In the present work, we studied the possible involvement of ceramide in HPR-induced apoptosis in A2780 human ovarian carcinoma cells. Moreover, we assessed whether resistance to HPR was associated with modifications of sphingolipid patterns and metabolism in these cells.

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Chemicals-- Commercial chemicals were the purest available, common solvents were distilled before use, and water was doubly distilled in a glass apparatus. HPR, obtained from Dr. J. A. Crowell, Division of Cancer Prevention, NCI, National Institutes of Health (Bethesda, MD) was dissolved in Me2SO at a concentration of 10 mM and stored at -80 °C under N2 in the dark. Sodium boro[3H]hydride was from Amersham Biosciences (specific radioactivity 12.0 Ci/mmol). Clostridium perfringens sialidase was from Roche Molecular Biochemicals, and Vibrio cholerae sialidase was from Sigma.

Lipids and Radioactive Lipids-- Sphingolipids and glycerolipids to be used as standards were extracted from rat brain, purified, and characterized (39). Gangliosides were extracted from bovine brain and purified by partitioning (39). GM12 was prepared from the bovine brain ganglioside mixture by enzymatic treatment with C. perfringens sialidase (40). GM3 was prepared from GM1 using the GM1-lactone hydrolysis procedure (41). Lactosylceramide was prepared from GM3 by enzymatic treatment with Vibrio cholerae sialidase (42). Sphingosine was prepared from cerebroside (43). [1-3H]sphingosine was prepared by specific chemical oxidation of the primary hydroxyl group of sphingosine followed by reduction with sodium boro[3H]hydride (44) (radiochemical purity over 98%; specific radioactivity 2.2 Ci/mmol). N-Acetylsphingosine (C2Cer) and N-palmitoylsphingosine (C16Cer) were prepared by N-acylation of sphingosine using acetic anhydride and hexadecanoic anhydride, respectively (45). The same procedure applied to [1-3H]sphingosine was used to synthesize tritium-labeled N-palmitoylsphingosine (radiochemical purity over 99%; specific radioactivity 2.2 Ci/mmol). GM3 tritium labeled at the position 3 of sphingosine, [3-3H(sphingosine)]GM3, was prepared by the dichlorodicyanobenzoquinone-sodium borohydride method (46), and the natural erythro diastereoisomer was purified by reverse phase HPLC as described (47) (radiochemical purity 98%, specific radioactivity 1.2 Ci/mmol). [3-3H(sphingosine)]LacCer was prepared from [3-3H(sphingosine)]GM3 by enzymatic treatment with V. cholerae sialidase and purified by silica gel column chromatography (radiochemical purity over 99%, specific radioactivity 1.2 Ci/mmol). [3H]lipids used as chromatographic standards were prepared from [1-3H]sphingosine-fed cell cultures as previously described (48).

Cell Cultures-- An HPR-resistant cell line, A2780/HPR, was developed from parental A2780 cells by in vitro incubation of A2780 cells with increasing concentrations of HPR as previously described (34). Briefly, cells surviving 60 rounds of selections in HPR-containing medium (3 rounds at 1 µM, 11 rounds at 2 µM, 8 rounds at 3 µM, and 38 rounds at 5 µM) were cloned by limiting dilution. One clone (A2780/HPR) was expanded and when tested for HPR sensitivity demonstrated a 10-fold resistance to HPR, which was slightly reversible upon drug removal for five rounds (34). Therefore, A2780/HPR cells were continuously maintained in 5 µM HPR and seeded without the drug in all of the experiments. A2780 and A2780/HPR cells were grown in monolayer in RPMI 1640 medium (Biowhittaker) containing 10% fetal bovine serum (Invitrogen) in 5% CO2 at 37 °C.

Effects of C2Cer on A2780 and A2780/HPR Cells-- Cells were seeded at density of 10,000 cells/well in 96-well tissue culture plates, treated on the next day with different concentrations of C2Cer in the absence of fetal bovine serum, and incubated for one additional day. Control cultures received the same amount of ethanol as the treated cultures (0.1%). Cell number was estimated by using the sulforhodamine B assay (49). Two analyses were performed, and four replicate wells were used for each analysis. Apoptotic cells were identified by the terminal dUTP nick-end labeling (TUNEL) method using an in situ cell death detection kit (Roche Diagnostics) according to the manufacturer's instructions. Briefly, A2780 and A2780/HPR cells were treated 24 h after seeding with 2.5 µM C2Cer in the absence of fetal bovine serum, incubated for one additional day, harvested, and washed twice in phosphate-buffered saline. After centrifugation, cells were fixed in 2% paraformaldehyde for 1 h at 26 °C and permeabilized with 0.1% Triton X-100 and 0.1% sodium citrate in phosphate-buffered saline. After washing, the cells were resuspended in TUNEL reaction mixture containing fluorescein isothiocyanate-dUTP and terminal deoxynucleotidyltransferase. Control cells were suspended in the TUNEL reaction mixture containing fluorescein isothiocyanate-dUTP without terminal deoxynucleotidyltransferase, and incubations were performed for 1 h at 37 °C before washing the cells twice. The number of TUNEL-positive cells, as detected by fluorescent microscopy, was assessed on at least 100 cells in two different smears and referred to the whole cell population.

Northern Analysis-- Total cellular RNA was isolated using the TriReagent method (Molecular Research Center, Cincinnati, OH). Twenty µg of total RNA were fractionated in formaldehyde/Mops-agarose gel and blotted on Hybond N+ nylon membranes (Amersham Biosciences). Filters were hybridized with cDNA probes for MDR1, MRP, GlcCer synthase, LacCer synthase, and GM3 synthase, obtained by RT-PCR using A2780/HPR cDNA as template. Briefly, single-stranded cDNA was synthesized from 2 µg of RNA by reverse transcription and then amplified by PCR. The primer pairs used for PCR amplification are the following: MDR1, sense (5'-AAAAAGATCAACTCGTAGGAGTG-3') and antisense (5'-GCACAAAATACACCAACAA-3') (161-bp amplified product); MRP, sense (5'-CCACCTCCTCATTCGCATCCACCTTG-3') and antisense (5'-GGAAACCATCCACGACCCTAATCCCT-3') (296-bp amplified product); GlcCer synthase, sense (5'-TCTACACCCGATTACACCTC-3') and antisense (5'-TGGGATAATCCAATTCAAAGAAT-3') (147-bp amplified product); LacCer synthase, sense (5'-AAAAACAGTACGCTCAACGG-3') and antisense (5'-ATCATCTTCTCCTCCCCATC-3') (676-bp amplified product); GM3 synthase, sense (5'-AAAAATGCATTATGTGGACCC-3') and antisense (5'-GTCTTGGCTTTCAAGTGTTCA-3') (303-bp amplified product); human glyceraldehyde-3-phosphate dehydrogenase, sense (5'-CCATGGAGAAGGCTGCGC-3') and antisense (5'-CAAAGTTGTCATGGATGACC-3') (195-bp amplified product). The cycling program was 94 °C for 30 s, 53 °C for 30 s, and 72 °C for 60 s for 35 cycles. PCR fragments were labeled with [alpha -32P]dCTP (3,000 Ci/nmol; ICN, Milan, Italy) using a multiprime DNA labeling system (Amersham Biosciences) to a specific activity of ~2 × 109 cpm/µg of cDNA. Following hybridization for 3-12 h at 66 °C in Rapid-Hyb buffer (Amersham Biosciences), the blots were washed twice with 2× SSC, 0.1% SDS at room temperature for 10 min. The final washes were done with 0.1× SSC, 0.1% SDS at 65 °C for 20 min at least two times. Membranes were exposed to x-ray film (Hyperfilm-MP; Amersham Biosciences) using double intensifying screens. RNA from human colon cancer LOVO-DX cells (kindly provided by Monica Binaschi) was used as positive control for MDR1 (50).

Treatment of Cell Cultures with [3H]Sphingosine-- 24 h after seeding, cells were incubated in the presence of 3 × 10-8 M [1-3H]sphingosine (5 ml/dish) in culture medium for 2 h (pulse). After the pulse, the medium was replaced with fresh medium without radioactive sphingosine, and cells were further incubated for up to 4 days (chase). Under these conditions, all sphingolipids (including ceramide, SM, neutral glycolipids, and gangliosides) and phosphatidylethanolamine (PE) (obtained by recycling of radioactive ethanolamine formed in the catabolism of [1-3H]sphingosine) were metabolically radiolabeled (32, 48, 51).

Treatment of [3H]Sphingosine-labeled Cell Cultures with HPR-- After metabolic labeling of cell lipids with [1-3H]sphingosine, at the end of a 24-h chase, the medium was replaced with medium containing HPR at final concentrations of 5 and 10 µM for up to 72 h. Time-matched control cells received the same amount of Me2SO as HPR-treated cells (0.1%).

Lipid Extraction and Determination-- In the experiments described above, at the end of the treatment periods, cells adherent to the dishes were harvested in ice-cold water (2 ml) by scraping with a rubber policeman. Cells floating in the culture medium were collected by centrifugation. Adherent and floating cells were analyzed to determine the content of radioactivity associated with lipids. Samples were lyophilized, and lipids were extracted twice with chloroform/methanol 2:1 by volume (first extraction, 1.5 ml; second extraction, 0.25 ml) (51). The total lipid extracts were subjected to a two-phase partitioning as previously described (52), resulting in the separation of an aqueous phase containing gangliosides and in an organic phase containing all other lipids. Aliquots of total lipid extracts, aqueous and organic phases were analyzed by HPTLC as described below, followed by radioactivity imaging for quantification of radioactivity.

The identity of radioactive lipids separated by HPTLC (using HPTLC Silica Gel 60 plates from Merck) was assessed by comigration with standard lipids and confirmed by susceptibility of compounds to the following enzymatic and chemical treatments (51). A sample of the aqueous phase was treated at 37 °C for 2 h, in 50 µl of water, in the presence of 1 milliunit of V. cholerae sialidase, to yield GM1. For the identification of SM, a sample of the organic phase was treated at 37 °C overnight in 30 µl of 100 mM Tris-HCl, pH 7.4, 0.5 mM MgCl2, 0.05% sodium deoxycholate in the presence of 11 milliunits of Staphylococcus aureus sphingomyelinase to yield ceramide; PE contained in the organic phase was characterized following its degradation under alkaline conditions. The enzymatic or chemical reaction mixtures were separated by HPTLC, and the reaction products were identified by chromatographic comparison with standard lipids.

The total lipid extract from about 50 mg of cell proteins was analyzed for mass lipid content. The phospholipid content was determined in the organic phase as phosphate after perchloric acid digestion by the method of Bartlett (53). Gangliosides from the aqueous phase and phospholipids and cholesterol from the organic phase were separated by HPTLC as described below. Identification of lipids after separation was assessed by comigration with standard lipids and confirmed by their susceptibility to enzymatic and chemical treatments as previously described (51). After chromatographic separation, compounds were chemically detected, and their amount was determined by densitometry as described below. Gangliosides from the aqueous phase were characterized by HPLC-ESI-MS (46) (see below).

GM3 Synthase Assay-- Cells cultured in 100-mm dishes as described above were harvested using a plastic scraper and washed two times with phosphate-buffered saline. GM3 synthase activity was assayed as previously described (54, 55) with some modifications. Cells were resuspended in 150 mM sodium cacodylate-HCl buffer, pH 6.6 (20 mg of cell protein/ml) with protease inhibitors (2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.0016 mM aprotinin, 0.044 mM leupeptin, 0.08 mM bestatin, 0.03 mM pepstatin A, 0.028 mM E-64) (Sigma) and homogenized with a Dounce homogenizer (10 strokes, tight). In each reaction tube, 10 µl of Triton CF-54 1.5% (v/v) in chloroform/methanol (2:1) were mixed with 0.5-50 nmol of [3-3H(sphingosine)]LacCer (corresponding to 45 nCi) from a stock solution in chloroform/methanol (2:1) and dried under N2. To this mixture, 8 µl of 750 mM sodium cacodylate-HCl buffer, pH 6.6, 4 µl of 125 mM MgCl2, 4 µl of 125 mM 2-mercaptoethanol, 10 µl of 5 mM CMP-NeuAc, and 10 µl of cell homogenate (containing 200 µg of protein) were added in a total reaction volume of 50 µl. Negative controls were performed using heat-inactivated cell homogenates (100 °C for 3 min). The incubation was performed at 37 °C for 3 h with continuous shaking. The reaction was stopped by adding 1.5 ml of chloroform/methanol (2:1). The reaction mixture was analyzed by HPTLC using the solvent system chloroform/methanol/water (55:20:3 by volume). Radioactive lipids were detected and quantified by radioactivity imaging as described below.

Ceramidase Assay-- Cell homogenates were prepared as described for the GM3 synthase assay, using 20 mM Tris-HCl, pH 7.4, as homogenization buffer. The activity of acidic, neutral, and alkaline ceramidase was assayed as previously described (56, 57) with some modifications. In each reaction tube, 25 µl of 0.1% Triton-X 100 (v/v) in chloroform/methanol (2:1) and 25 µl of 0.2% sodium cholate (w/v) in chloroform/methanol (2:1) were mixed with 2-100 pmol of [1-3H(sphingosine)]C16Cer (corresponding to 4.5 nCi) from a stock solution in chloroform/methanol (2:1) and dried under N2. To this mixture, 7.5 µl of water was added, and the tubes were sonicated for 3 min, heated for 5 s at 80 °C, and then put on ice. Ceramidase activities were assayed by adding 12.5 µl of 400 mM sodium acetate buffer, pH 4.5 (for the acidic enzyme), 12.5 µl of 400 mM Tris-HCl, pH 7.4 (for the neutral enzyme), or 12.5 µl of 400 mM glycine-NaOH, pH 9.0 (for the alkaline enzyme), 5 µl 50 mM MgCl2, and 10 µl of cell homogenate (containing 200 µg of protein) in a total reaction volume of 50 µl. Negative controls were performed using heat-inactivated cell homogenates (100 °C for 3 min). The incubation was performed at 37 °C for 1 h with continuous shaking. The reaction was stopped by adding 1.5 ml of chloroform/methanol (2:1). The reaction mixture was analyzed as described for GM3 synthase assay.

Analytical Procedures-- Lipids were separated by monodimensional and two-dimensional HPTLC carried out with the following solvent systems: chloroform/methanol/0.2% aqueous CaCl2 (55:45:10 by volume) to separate ceramide from sphingosine; chloroform/methanol/0.2% aqueous CaCl2 (50:42:11 or 55:45:10 by volume) and chloroform/methanol/0.2% aqueous CaCl2/32% NH3 (60:50:9:1 by volume) to analyze total lipids and gangliosides; chloroform/methanol/water (55:20:3 by volume) to analyze total lipids and lipids from the organic phase and to separate GM3 from LacCer; and 1-butanol/acetic acid/water (3:1:1 by volume) and hexane/chloroform/acetone/acetic acid (10:35:10:1 by volume) to analyze ceramide. Endogenous phospholipids from organic phase were separated by mono- or bidimensional HPTLC using the following solvent systems and conditions (51): chloroform/methanol/acetic acid/water (30:20:2:1 by volume) to analyze glycerophospholipids and SM; chloroform/methanol/acetic acid/water (30:20:2:1 by volume) first run and second run with intermediate exposure to HCl vapors for 15 min to analyze plasmalogens of glycerophospholipids. The solvent system chloroform/methanol/water (55:40:3 by volume) was used to analyze neutral GSLs and SM from organic phase after alkaline treatment. Hexane/diethylether/acetic acid (80:20:1 by volume) and hexane/ethyl acetate (3:2 by volume) were used to analyze cholesterol.

Ganglioside, neutral GSL, and phospholipid species were quantified after separation on HPTLC followed by specific detection with a p-dimethylaminobenzaldehyde reagent (58), an aniline/diphenylamine reagent (59), or a molybdate reagent (60), respectively. The relative amounts of each ganglioside, neutral GSL, or phospholipid were determined by densitometry. The mass content of each phospholipid was calculated on the basis of the percentage distribution and total phospholipid content, determined as described above. The quantity of each ganglioside, neutral GSL, or SM was determined by densitometry and comparison with known amounts of standard compounds using the Molecular Analyst program (Bio-Rad). Cholesterol was quantified by visualization with 15% concentrated sulfuric acid in 1-butanol (48). The quantity of cholesterol was determined by densitometry and comparison with 0.1-2 µg of standard compounds.

Radioactive lipids were detected and quantified by radioactivity imaging performed with a Beta-Imager 2000 instrument (Biospace, Paris, France) using an acquisition time of about 48 h. The radioactivity associated with individual lipids was determined with the specific beta -Vision software provided by Biospace.

The radioactivity associated with cells, lipids, lipid extracts, and aqueous or organic phases was determined by liquid scintillation counting.

Structural characterization of gangliosides has been carried out using a ThermoQuest Finnigan LCQDeca mass spectrometer (FINNIGAN MAT, San Jose, CA) equipped with an ESI ion source and a Xcalibur data system and connected to an HPLC TSP P4000 quaternary pump and an automatic solvent degasser. Optimum conditions included sheath gas flow of 70 arbitrary units, auxiliary gas flow of 10 arbitrary units, spray voltage of 4 kV, capillary voltage of -42 V, capillary temperature of 260 °C, fragmentor voltage (used for collision induced dissociation) of 40-80%. Mass spectra were acquired over an m/z range 200-2000.

HPLC separation of gangliosides dissolved in CH3CN/5 mM ammonium acetate buffer, pH 7 (64:36 by volume), was carried out on a 5-µm LiChrospher C8 reverse-phase column (250 × 4 mm) (Merck) at a flow rate of 0.5 ml/min using a gradient formed by the solvent system A, consisting of CH3CN, 5 mM ammonium acetate buffer, pH 7 (15:85 by volume), and solvent system B containing CH3CN/H2O (85:15 by volume). The gradient was linear from 30:70 to 20:80, by volume, of A:B over 25 min and then to 100 of B in 5 min.

The protein content was determined according to Lowry (61), using bovine serum albumin as the reference standard.

    RESULTS AND DISCUSSION
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Effect of HPR on Ceramide Levels in A2780 Cells-- The possible involvement of ceramide in the effect of HPR in A2780 human ovarian carcinoma cells was investigated. To this purpose, A2780 and A2780/HPR cells were incubated in the presence of [1-3H]sphingosine for 2 h, followed by a 24-h chase. [1-3H]sphingosine is efficiently incorporated by cultured cells and converted into ceramide, which is further utilized for the synthesis of more complex sphingolipids. This metabolic radiolabeling approach was successfully used in the past as a valuable tool to quantitatively and sensitively determine changes in cellular ceramide levels elicited by different treatments, including RA (62) and HPR (32). Cells were then treated with 5 and 10 µM HPR for 48 and 72 h. As previously reported (35), treatment of A2780 cells with HPR under these experimental conditions was able to induce apoptosis, and a relevant number of cells (25.0 and 60.6% of total cells after treatment with 5 µM HPR for 48 and 72 h, respectively) were detached from the culture dishes and floating in the culture medium. The same treatment had only weak effect (3.8 and 5.5% of total cells were floating after treatment with 5 µM HPR for 48 and 72 h, respectively) on A2780/HPR cells (34). Cell lipids were extracted from both cell types, separated by HPTLC and analyzed by digital autoradiography. The values for radioactivity incorporation into ceramide in control and HPR-treated cells are reported in Fig. 1. Under these experimental conditions, treatment of A2780 cells with HPR increased the incorporation of radioactivity from [1-3H]sphingosine into ceramide in floating cells respect to untreated cells or adherent cells (Fig. 1, A and B). On the other hand, in the case of A2780/HPR cells, the incorporation of [1-3H]sphingosine into ceramide was very similar in adherent and floating cells, treated with HPR or not (Fig. 1, C and D). HPR treatment of A2780/HPR cells is thus not able to determine a rise in the production of radioactive ceramide.


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Fig. 1.   Ceramide detection in A2780 and A2780/HPR cells. After cell lipid metabolic labeling with [1-3H]sphingosine as described under "Experimental Procedures," A2780 (A and B) and A2780/HPR (C and D) cells were treated with vehicle (white) or 5 µM (gray) and 10 µM HPR (black) for 48 or 72 h. A, A2780, adherent cells; B, A2780, floating cells; C, A2780/HPR, adherent cells; D, A2780/HPR, floating cells. Radioactive lipids were extracted, separated by HPTLC, and detected by digital autoradiography (250 dpm applied on a 3-mm line). Time of acquisition was 48 h, and radioactivity associated with ceramide was quantitatively determined. Data are expressed as nCi/mg of cell protein and are the means of three different experiments, with the S.D. never exceeding 10% of the mean values.

Effects of C2Cer on A2780 and A2780/HPR Cells-- To further examine the involvement of ceramide in the HPR effect in A2780 cells, we investigated whether cells resistant to HPR were also resistant to ceramide. To this aim, the antiproliferative and apoptotic effect of C2Cer was evaluated in parental and HPR-resistant cells. After 24 h of exposure to the drug, the sensitivity of the two cell lines to C2Cer was different (Fig. 2A); doses of C2Cer ranging from 1 to 5 µM caused ~60% growth inhibition in A2780, whereas the same doses were ineffective in A2780/HPR cells. No difference in sensitivity to C2Cer between A2780 and A2780/HPR cells was observed at doses higher than 7.5 µM, which caused a strong dose-dependent growth inhibition in both cell lines. TUNEL analysis performed in cells treated with 2.5 µM C2Cer showed an increase in the percentage of apoptotic cells, compared with untreated cells, in A2780 cells, whereas no increase was observed in A2780/HPR cells (Fig. 2B).


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Fig. 2.   C2Cer effects on A2780 and A2780/HPR cells. A, C2Cer antiproliferative activity on A2780 (open circle) and A2780/HPR (solid circle) cells. Cells were treated 24 h after seeding, and surviving cell number was evaluated 1 day later. Data are expressed as percentage of control untreated cells. Results are the mean ± S.D. of two independent experiments. B, detection of DNA fragmentation induced by C2Cer. A2780 (lane 1) and A2780/HPR (lane 2) cells were treated with vehicle (white) or 2.5 µM C2Cer (gray) for 24 h 1 day after seeding. Cells were then harvested, processed for TUNEL reaction, and observed using a fluorescence microscope. The results are in reference to the whole population and represent the mean ± S.D. of two independent experiments.

Expression Levels of MDR1 and MRP-- In several tumor cell lines, the loss of ability to respond to anticancer drugs with the rise of cellular ceramide levels hallmarks the acquisition of the MDR phenotype. To understand whether HPR resistance in A2780 cells is accompanied by the overexpression of multidrug resistance-specific proteins, MDR1 and MRP mRNA expression levels were analyzed in parental and resistant cells (Fig. 3). No differences were observed in gene expression between sensitive and resistant cells; MDR1 mRNA was not detected in A2780 and A2780/HPR cells, whereas MRP1 was expressed in both cell lines at similar levels.


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Fig. 3.   Constitutive expression of MDR1 and MRP mRNA in A2780 and A2780/HPR cells. Northern blot analysis was performed as described under "Experimental Procedures". Each lane contains 20 µg of total RNA. Filters were hybridized with cDNA probes for MDR1 (A) and MRP (B), and as a control for loading, the filters were stripped and rehybridized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (lower part of each panel). Lane 1, A2780 cells; lane 2, A2780/HPR cells; lane 3, RNA from LOVO-DX cells was used as a positive control for MDR1.

Sphingolipid Metabolism in A2780 and A2780/HPR Cells-- To study possible differences in sphingolipid patterns and metabolism, A2780 cells and A2780/HPR cells were incubated in the presence of [1-3H]sphingosine for 2 h, followed by different chase times (up to 4 days). As reported for other cell types (48, 51), under these conditions, all sphingolipids (including ceramide, SM, neutral glycolipids, and gangliosides) were efficiently metabolically radiolabeled, reaching a steady-state distribution of radioactivity after 48 h (data not shown). The use of [1-3H]sphingosine as metabolic precursor allows the quantitative analysis of cell sphingolipids with very high sensitivity. Moreover, during the degradation of the exogenous [1-3H]sphingosine taken up by the cells or of [1-3H]sphingosine-containing sphingolipids, radioactive ethanolamine is formed, which is recycled for the synthesis of PE. The formation of [3H]PE is thus a very useful and sensitive tool to quantify sphingolipid catabolism.

Under these experimental conditions, at all investigated chase times, A2780 and A2780/HPR cells incorporated comparable amounts of radioactivity (Table I). Cell lipids were extracted, separated by HPTLC, and analyzed by digital autoradiography. Fig. 4 shows the patterns of radioactive lipids extracted from A2780 and A2780/HPR cells after HPTLC separation. In both cell lines, radioactive bands co-migrating with standard ceramide, glucosylceramide, PE, lactosylceramide, and SM were detectable. In the case of A2780/HPR cells, at least two intense radioactive bands migrating below SM were also present. To allow a better resolution of these lipid profiles, the total lipid extracts were subjected to a two-phase partitioning, resulting in the separation of an aqueous phase containing gangliosides and an organic phase containing all less polar lipids (including radioactive ceramide, PE, neutral glycosphingolipids, and SM) that were further analyzed. As reported in Table I, most radioactive lipids were associated with the organic phase in both A2780 and A2780/HPR cells. Remarkably, independently from the chase time, the radioactivity associated with the aqueous phase in the case of resistant A2780/HPR cells was 3 times higher than in sensitive A2780 cells.

                              
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Table I
Radioactivity incorporation into total lipid extracts and aqueous and organic phases in A2780 and A2780/HPR cells after a 2-h pulse with [1-3H]sphingosine followed by 48, 72, and 96 h of chase


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Fig. 4.   Radioactive lipid patterns in total lipid extracts from A2780 and A2780/HPR cells. After metabolic labeling with [1-3H]sphingosine for a 2-h pulse followed by 48-h chase, lipids were extracted from A2780 (lane 1) and A2780/HPR (lane 2) cells and separated by HPTLC in the solvent system chloroform/methanol/water (55:20:3 by volume). Radioactive lipids were detected by digital autoradiography (250 dpm applied on a 3-mm line). Time of acquisition was 48 h. The position of pure standard lipids is indicated on the left. The pattern is representative of that obtained in three different experiments.

Fig. 5 shows the patterns of radioactive lipids present in aqueous (Fig. 5A) and organic phases (Fig. 5B) obtained from A2780 and A2780/HPR cell lipid extracts after HPTLC separation. The major radioactive bands present in the aqueous phase from both cell types showed a similar chromatographic behavior. On the basis of their chromatographic migration and sensitivity to V. cholerae sialidase treatment, the two upper bands were identified as GM3, and the two lower bands, accounting for about 60% of the aqueous phase radioactivity in the case of A2780/HPR cells, were identified as GM2. In the organic phase from both cell types (Fig. 5B), ceramide, glucosylceramide, lactosylceramide, PE, and SM were identified. The quantitative data, summarized in Table II as radioactivity incorporation into different lipids, indicate that the metabolic use of [1-3H]sphingosine is different in A2780 and A2780/HPR cells, thus resulting in quantitatively different [3H]lipid patterns. In both cell types, a significant portion of [1-3H]sphingosine-containing lipids underwent complete degradation, as indicated by the formation of high levels of radioactive PE (particularly in the case of A2780/HPR cells). In both cell types, more than 50% of radioactivity from [1-3H]sphingosine was associated with complex sphingolipids, including SM and glycosphingolipids. Although the total radioactivity associated with glycosphingolipids was similar in A2780 and A2780/HPR cells, its distribution among different glycosphingolipids was radically different in these two cell lines. In particular, A2780 cells were characterized by high levels of LacCer (about 6-fold higher than in A2780/HPR), whereas A2780/HPR express remarkably more elevated (6-fold higher) ganglioside levels than the parental cell line.


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Fig. 5.   Radioactive lipid patterns in aqueous and organic phases obtained after phase separation of total lipid extracts from A2780 and A2780/HPR cells. After metabolic labeling with [1-3H]sphingosine for a 2-h pulse followed by 48-h chase, lipids were extracted from A2780 (lanes 1 and 3) and A2780/HPR (lanes 2 and 4) cells. Total lipid extracts were subjected to phase separation as described under "Experimental Procedures." Lipids from the aqueous (A) and organic (B) phases were separated by HPTLC using solvent systems chloroform/methanol/0.2% aqueous CaCl2 (50:42:11 by volume) and chloroform/methanol/water (55:20:3 by volume), respectively. To selectively remove PE and allow the quantitative detection of LacCer, aliquots of the organic phases were subjected to chemical treatment under alkaline conditions (lanes 3 and 4). Aliquots of aqueous and organic phases corresponding to identical amounts of cell proteins were analyzed. Radioactive lipids were detected by digital autoradiography (250-1000 dpm applied on a 3-mm line). Time of acquisition was 48 h. The position of pure standard lipids is indicated on the left. The pattern is representative of that obtained in three different experiments.

                              
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Table II
Radioactivity incorporation into different lipids after metabolic labeling of A2780 and A2780/HPR cells with [1-3H]sphingosine (2-h pulse followed by 48 h of chase)

Lipid Composition of A2780 and A2780/HPR Cells-- The data obtained using metabolic labeling with [1-3H]sphingosine strongly indicate that, in A2780 cells, resistance to HPR is linked to an alteration in sphingolipid metabolism leading to an increased expression of gangliosides. To evaluate possible alterations in the metabolism of other lipid classes, we analyzed the mass content of cholesterol and phospholipids in A2780 and A2780/HPR cells. Cholesterol content was 45.20 ± 3.12 and 45.20 ± 2.97 nmol/mg of cell protein in A2780 and A2780/HPR cells, respectively (Fig. 6A). Phospholipid phosphorous content was 130.60 ± 5.21 and 118.30 ± 6.13 nmol/mg of cell protein in A2780 and A2780/HPR cells, respectively, and the phospholipid pattern was very similar in both cell types (Fig. 6B). Thus, A2780 and A2780/HPR cells were almost identical in their bulk lipid composition.


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Fig. 6.   Patterns of endogenous lipids from A2780 and A2780/HPR cells. Cholesterol (Chol) and phospholipids from A2780 (lane 1) and A2780/HPR cells (lane 2) were analyzed by HPTLC followed by chemical detection as described under "Experimental Procedures." Aliquots of samples corresponding to identical amounts of cell proteins were analyzed for both cell types. A, cholesterol was separated from organic phases (corresponding to 0.4 mg of cell proteins). The solvent system was hexane/ethyl acetate (3:2 by volume), and visualization was by charring with 15% concentrated sulfuric acid in 1-butanol. B, phospholipids were separated from organic phases (corresponding to 0.6 mg of cell proteins). The solvent system was chloroform/methanol/acetic acid/water (30:20:2:1 by volume), and visualization was by detection with a molybdate reagent. PC, phosphatidylcholine; PS, phosphatidylserine; PI, phosphatidylinositol.

In the case of sphingolipids, colorimetric chemical detection did not allow an accurate quantification, due to their low cellular content. However, the qualitative comparison of endogenous neutral glycosphingolipid and ganglioside patterns confirmed the radiolabeling data. Mass spectrometry analyses showed that GM2 and GM3 were the main components of the ganglioside mixture extracted from A2780/HPR cells. Both gangliosides were present as molecular species containing d18:1-16:0, d18:1-18:0, d18:1-24:1, and d18:1-24:0 ceramide. As shown in Fig. 7, the GM2 and GM3 species were characterized by the deprotonated molecular ions [M-H]- at m/z 1354, 1382, 1464, and 1466 and at m/z 1151, 1179, 1261, and 1263, respectively. Fig. 7 shows as an example the characterization of GM2(d18:1-16:0) and GM3(d18:1-16:0). MS2 of molecular ion [M-H]- gave a typical fragmentation pattern due to the sequential loss of sugar units, providing information about the carbohydrate structure and the total ceramide mass; GM3 and GM2 molecular species contained N-acetylneuraminic acid, whereas species containing N-glycolylneuraminic acid were undetectable.


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Fig. 7.   Mass spectra of main gangliosides from A2780 and A2780/HPR cells. Lipids were extracted from A2780 and A2780/HPR cells, total lipid extracts were subjected to phase separation as described under "Experimental Procedures," and gangliosides present in the aqueous phases were analyzed by HPLC-ESI-MS. A, total negative ESI-MS spectrum of the gangliosides from HPR-resistant cells, with indication of the main GM3 and GM2 molecular species differing in the acyl structure. B, HPLC-ESI-MS mass spectrum of the GM3(d18:1-16:0) molecular species together with the MS2 spectrum derived from the ion at m/z 1151 and the MS3 spectrum derived from the ion at m/z 536. C, HPLC-ESI-MS mass spectrum of the GM2(d18:1-16:0) molecular species together with the MS2 spectrum derived from the ion at m/z 1354 and the MS3 spectrum derived from the ion at m/z 536.

Expression Levels of GlcCer Synthase, LacCer Synthase, and GM3 Synthase in A2780 and A2780/HPR Cells-- To investigate potential mechanisms underlying the observed differences in sphingolipid levels in A2780 and A2780/HPR cells, the mRNA expression of the enzymes GlcCer synthase, LacCer synthase, and GM3 synthase was evaluated in parental and resistant cells. A2780/HPR cells showed GlcCer synthase and LacCer synthase mRNA levels similar to those of A2780 cells, whereas GM3 synthase mRNA was markedly increased (Fig. 8). In A2780 cells, the mRNA levels of GM3 synthase were below the detection limit of our Northern blot analysis. Using RT-PCR, we were able to detect in A2780 cells a faint band relative to GM3 synthase.


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Fig. 8.   Constitutive expression for GlcCer synthase, LacCer synthase, and GM3 synthase mRNA in A2780 and A2780/HPR cells. Northern blot analysis was performed as described under "Experimental Procedures." Each lane contains 20 µg of total RNA. Filters were hybridized with cDNA probes for GlcCer synthase (A), LacCer synthase (B), and GM3 synthase (C), and as a control for loading, the filters were stripped and rehybridized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (lower part of each panel). Lane 1, A2780 cells; lane 2, A2780/HPR cells.

To assess whether observed changes in the mRNA levels of GM3 synthase were paralleled by differences in the enzyme levels, we measured the activity of GM3 synthase in A2780 and A2780/HPR cells by means of a cell-free assay using radioactive LacCer as substrate. As indicated in Fig. 9, the in vitro activity of GM3 synthase was 20-fold higher in resistant A2780/HPR cells than in sensitive A2780 cells.


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Fig. 9.   GM3 synthase activity in A2780 and A2780/HPR cells. GM3 synthase activity was assayed on cell homogenates from A2780 (1) and A2780/HPR cells (2) as described under "Experimental Procedures" in the presence of 10 µM (dark gray) or 100 µM (light gray) [3-3H(sphingosine)]LacCer (corresponding to 45 nCi/assay). Aliquots of cell homogenates containing the same amount of proteins (200 µg) were added in a total reaction volume of 50 µl. The incubation was performed at 37 °C for 3 h. Negative controls were performed using heat-inactivated cell homogenates. The reaction mixture was analyzed by HPTLC using the solvent system chloroform/methanol/water (55:20:3 by volume). Radioactive lipids were detected and quantified by radioactivity imaging as described in the legend to Fig. 1. Data are expressed as pmol of formed GM3/h/mg of cell protein and are the means of three different experiments, with the S.D. never exceeding 10% of the mean values.

Ceramidase Activities in A2780 and A2780/HPR Cells-- Among the metabolic mechanisms that contribute to the regulation of cellular ceramide levels, a possible role of ceramidases (enzymes that cleave the N-acyl bond of ceramide) was recently highlighted (reviewed in Ref. 63). To determine whether differences in the activity of ceramidases were associated with the resistance to HPR in human ovarian carcinoma cells, we measured the in vitro activity of ceramidase at acidic, neutral, and alkaline pH in A2780 and A2780/HPR cells using [1-3H(sphingosine)]C16Cer as substrate. As reported in Table III, the in vitro activities of acid, neutral and alkaline ceramidases were similar in sensitive and resistant cells.

                              
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Table III
Activity of acid, alkaline, and neutral ceramidase in A2780 and A2780/HPR cells
Ceramidase activity was measured by the mean of a cell-free assay at pH 4.5, 9.0, and 7.4 using radioactive ceramide as substrate at three different concentrations. The activities are expressed as pmol of hydrolyzed ceramide/h/mg of cell protein and are the mean of three different experiments, with the S.D. never exceeding 15% of the mean values.

Conclusions-- The synthetic retinoid HPR exerts an apoptotic effect in several tumor cell lines, including A2780 (35). In the case of neuroblastoma (37), breast carcinoma (32), and acute lymphoblastic leukemia cell lines (64), HPR-induced apoptosis is accompanied by the elevation of ceramide cellular levels. In the present paper, we show that this is the case also for human ovarian carcinoma A2780 cells. The administration of proapoptotic concentrations of HPR to A2780 cells resulted in an up to 10-fold increase of cellular ceramide levels. The rise of cellular ceramide levels was not observed when A2780/HPR cells, resistant to HPR-induced apoptosis, were treated with HPR under the same experimental conditions. These data indicate that (a) ceramide is a possible mediator in HPR-induced apoptosis in parental A2780 cells and (b) the different sensitivity of parental A2780 and A2780/HPR cells to HPR might be linked to their different ability to respond with an increase in the cellular levels of the proapoptotic sphingolipid ceramide. Interestingly, a parallel between ceramide generation in response to HPR treatment and sensitivity to HPR was observed in cells of lymphoid origin (64). Different acute lymphoblastic leukemia cell lines respond to cytotoxic HPR treatment with an increase in ceramide. On the other hand, HPR is not cytotoxic and has no effect on ceramide levels in nonmalignant lymphoid cell types (64).

In the well studied case of multidrug resistance (3-12, 14), MDR cells are able to escape the drug-induced apoptotic death elicited in their sensitive counterparts by a rise in the cellular ceramide levels through two distinctive features. MDR cells express high levels of efflux pump proteins belonging to the ATP-binding cassette superfamily of membrane transport proteins, such as MDR1 and MRP. The overexpression of energy-dependent drug efflux pump proteins may lead to lowering intracellular drug levels to sublethal concentrations. MDR cells show increased levels of GlcCer and GlcCer synthase expression and/or activity with respect to their sensitive counterparts. The ability to scavenge ceramide by converting it to GlcCer could represent an efficient way to reduce the drug effects at the intracellular level. In addition, in 2780AD cells (an MDR cell line deriving from the parental A2780 line), in addition to enhanced levels of glucosylceramide, more complex alteration of sphingolipid metabolism with respect to the parental A2780 cells was reported (12). In these cells, a reduced synthesis of LacCer was observed, which resulted in lower levels of LacCer, GM3, and GM2 and higher levels of galactosylceramide and SM.

According to this finding, the resistance to a retinoid in A2780 cells herein reported does not seem to be related to MDR. In fact, our data indicate that levels of MDR1 mRNA and MRP mRNA as well as those of GlcCer and GlcCer synthase mRNA were substantially unchanged in A2780/HPR cells with respect to parental A2780 cells.

Instead, striking differences were observed between A2780 and A2780/HPR cells in the metabolism of more complex glycosphingolipids. In A2780/HPR cells, the sphingolipid metabolism is markedly oriented toward the synthesis of gangliosides. We characterized the main gangliosides of A2780/HPR cells as GM3 and GM2, and their levels were 6-fold higher than in parental cells, whereas LacCer, the direct neutral glycolipid precursor of gangliosides, was proportionally lower. Finally, differences were observed in the mRNA levels of the enzyme GM3 synthase, which is more expressed in A2780/HPR than in sensitive A2780 cells. This difference was reflected by the in vitro activity of GM3 synthase, which was 20-fold higher in resistant A2780/HPR cells than in sensitive A2780 cells.

Thus, the data presented in this paper indicate that all of the biosynthetic pathway downstream of ceramide is more active in A2780/HPR cells than in the parental sensitive cell line, leading to the expression of higher levels of GM3 and GM2 gangliosides. This particular alteration in sphingolipid metabolism associated with the development of resistance to an apoptosis inducing drug is reported here for the first time.

It could be argued that an overall increased flow through the ganglioside biosynthetic pathway is also a mechanism, even if quite elaborate, to scavenge ceramide, thus counteracting the apoptotic effect of HPR, in analogy with what is observed in MDR cells synthesizing high GlcCer levels. However, the role acknowledged for complex sphingolipids in modulating the properties of tumor cells (such as adhesion, motility, invasiveness, and proliferation) is well documented (reviewed in Ref. 16) even if less codified that the role of ceramide in tumor cell apoptosis. In bladder cancer, a specific role for GM3 ganglioside as a negative modulator of malignant potential of this tumor was hypothesized. In fact, superficial bladder tumors express higher levels of GM3 and GM3 synthase compared with invasive tumors (65), and exogenous GM3 treatment (65) or GM3 synthase overexpression (66) reduced invasion potential and induced apoptosis in bladder tumor cell lines. Even in the case of MDR cells (where the link between sensitivity to drug and generation of ceramide as described above seems extremely well established), a possible role for GlcCer as substrate or modulator of the pump activity of MDR-related proteins (that are interestingly localized at the level of plasma membrane in sphingolipid-rich environments) was suggested (3). As presented in this paper, the evidence we obtained using A2780/HPR cells suggests that, also in the case of retinoids, differences in sphingolipid metabolism are related to differences in the sensitivity of these cells to the drug, as previously reported for chemotherapeutic drugs. However, in our case, these differences might be not simply related to the ability of cells to neutralize the proapoptotic action of ceramide. In particular, our data suggest that changes in the expression of gangliosides could contribute to the onset of a resistant phenotype in tumor cells.

    FOOTNOTES

* This work was supported by COFIN-PRIN Grants 2000 and 2001, Consiglio Nazionale delle Ricerche (PF Biotechnology), Italy, and Mizutani Foundation for Glycoscience Grant 2002 (to S. S.), and by Associazione Italiana per la Ricerca sul Cancro (to F. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Via Fratelli Cervi 93, 20090 Segrate, Italy. Fax: 39-0250330365; E-mail: Sandro.Sonnino@unimi.it.

Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M207269200

2 Ganglioside and glycosphingolipid nomenclature is in accordance with Svennerholm (67) and the IUPAC-IUBMB recommendations (38, 68).

    ABBREVIATIONS

The abbreviations used are: GlcCer, beta -Glc-(1-1)-Cer; LacCer, beta -Gal-(1-4)-beta -Glc-(1-1)-Cer; Cer, ceramide, N-acyl-sphingosine; C2Cer, N-acetylsphingosine; C16Cer, N-palmitoylsphingosine; sphingosine, (2S,3R,4E)-2-amino-1,3-dihydroxyoctadecene; PE, phosphatidylethanolamine; SM, sphingomyelin; ESI, electrospray ionization; MS, mass spectrometry; GSL(s), glycosphingolipid(s); HPR, N-(4-hydroxyphenyl)retinamide; HPTLC, high performance thin layer chromatography; HPLC, high performance liquid chromatography; MDR, multidrug resistance; MDR1, MDR1 P-glycoprotein; MRP, multidrug resistance-associated protein; RA, retinoic acid; RT, reverse transcription; SSC, standard sodium citrate; TUNEL, terminal dUTP nick-end labeling; Mops, 4-morpholinepropanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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