Sphingosine Kinase Mediates Resistance to the SyntheticRetinoid N-(4-Hydroxyphenyl)retinamide in Human OvarianCancer Cells*□S

Received for publication, October 5, 2009, and in revised form, April 14, 2010 Published, JBC Papers in Press, April 19, 2010, DOI 10.1074/jbc.M109.072801

Giuditta Illuzzi‡, Caterina Bernacchioni§, Massimo Aureli‡, Simona Prioni‡, Gianluca Frera‡, Chiara Donati§,Manuela Valsecchi‡, Vanna Chigorno‡, Paola Bruni§, Sandro Sonnino‡, and Alessandro Prinetti‡1

From the ‡Center of Excellence on Neurodegenerative Diseases, Department of Medical Chemistry, Biochemistry andBiotechnology, University of Milan, 20090 Segrate and the §Department of Biochemical Sciences, University of Florence,50134 Florence, Italy

A2780 human ovarian carcinoma cells respond to treatmentwith the synthetic retinoid N-(4-hydroxyphenyl)retinamide(HPR) with the production of dihydroceramide and with a con-comitant reduction of cell proliferation and induction of apo-ptosis. The derived HPR-resistant clonal cell line, A2780/HPR,is less responsive to HPR in terms of dihydroceramide genera-tion. In this report, we show that the production of sphingosine1-phosphate (S1P) is significantly higher in A2780/HPR versusA2780 cells due to an increased sphingosine kinase (SK) activityand SK-1 mRNA and protein levels. Treatment of A2780 andA2780/HPR cells with a potent and highly selective pharmaco-logical SK inhibitor effectively reduced S1P production andresulted in a marked reduction of cell proliferation. Moreover,A2780/HPR cells treated with a SK inhibitor were sensitized tothe cytotoxic effect of HPR, due to an increased dihydroceram-ide production. On the other hand, the ectopic expression ofSK-1 in A2780 cells was sufficient to induce HPR resistance inthese cells. Challenge of A2780 and A2780/HPR cells with ago-nists and antagonists of S1P receptors had no effects on theirsensitivity to the drug, suggesting that the role of SK in HPRresistance in these cells is not mediated by the S1P receptors.These data clearly demonstrate a role for SK in determining

resistance to HPR in ovarian carcinoma cells, due to its effect inthe regulation of intracellular ceramide/S1P ratio, which is crit-ical in the control of cell death and proliferation.

Ovarian cancer is the fifth most common cancer, and it isthe leading cause of death from all types of gynecologicalcancer. This carcinoma has a high rate of recurrence and sub-sequent mortality after chemotherapy; many patients relapseafter first line treatment, and only the 15% are long survivors.The failure of this kind of treatment is generally caused by theacquisition of drug resistance. Cancer cells develop multiplemechanisms to evade drug toxicity. Several commonly used

anticancer drugs, including daunorubicin, vincristine, and ret-inoids, exert their cytotoxic action at least in part by triggeringthe production of the sphingolipid ceramide, a mediator ofapoptosis and an inhibitor of cell proliferation in a variety oftumor cell lines (1). It has been demonstrated that chemoresis-tant tumor and tumor cell lines are frequently characterized bythe increased glycosylation of ceramide with formation of glu-cosylceramide, due to an increased expression or activation ofglucosylceramide synthase (2). Scavenging ceramide via itsincreased glycosylation would allow tumor cells to escape cer-amide-induced apoptosis, thus contributing to the drug-resis-tant phenotype (3–5). However, it has been shown that GlcCeraccumulation is not the only consequence of an altered sphin-golipidmetabolism in drug-resistant cancer cells. Inmultidrug-resistant human ovarian carcinoma cells, sphingomyelin andgalactosylceramide levels were also higher respect to parentalsensitive cells, whereas lactosylceramide and all more complexglycosphingolipids were present in lower amounts (6). On theother hand, retinoid-resistant ovarian cancer cells had similarlevels of glucosylceramide synthase with respect to their drug-sensitive counterpart, but higher levels of GM32 synthase lead-ing to higher ganglioside content (7). In addition to its use as aprecursor of complex sphingolipids, ceramide can be convertedinto sphingosine, which is then phosphorylated from sphingo-sine kinases (1 and 2) to generate S1P. The formation of S1P isthe requisite for the complete degradation of ceramide andother sphingolipids. On the other hand, S1P is another impor-tant sphingolipid-derived mediator actively participating insignal transduction pathways and regulating many differentcell functions. It is well recognized that a dual mode of S1Paction exists: being the bioactive sphingolipid capable of actingintracellularly but also via the activation of a family of fiveG-protein-coupled receptors (S1P1–5) (7). In this regard insome instances S1P exerts its pro-mitogenic and anti-apoptoticaction as intracellular mediator (8), whereas in other instancesligation to S1P1 and S1P3 is required to convey these two bio-logical effects (9–11). Independently from its action mode, it is* This work was supported by the Mizutani Foundation for Glycoscience

Grant 070002 (to A. P.) and Associazione Italiana per la Ricerca sul CancroGrant 2007 (to S. S.).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. 1– 4, Table 1, text, and additional references.

1 To whom correspondence should be addressed: Dipartimento di Chimica,Biochimica e Biotecnologie per la Medicina, Via Fratelli Cervi 93, 20090Segrate (Milano), Italy. Telefax: 39-025-033-0365; E-mail: alessandro.prinetti@unimi.it.

2 The abbreviations used are: GM3, NeuAc�2,3Gal�1,4Glcceramide (ganglio-side and glycosphingolipid nomenclature is in accordance with the IUPAC-IUBMB recommendations (40)); HPR, 4-[3,7-dimethyl-9-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2,4,6,8-nonatetraenamido]-1-hydroxybenzene; MTT,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; S1P, sphin-gosine 1-phosphate; S1PR, S1P receptor; SK, sphingosine kinase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 24, pp. 18594 –18602, June 11, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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clear that ceramide and S1P exert opposite effects on cell fate,and enzymes contributing to the regulation of the relative con-centrations of these lipids, such as SK, represent an essentialcheckpoint in determining whether a cell proliferates or under-goes apoptosis (12–14). Recently, a role of SK in the resistanceof cancer cells to chemotherapy has been suggested (15–18).The synthetic retinoid HPR is cytotoxic in vitro to a variety

of cancer cell types, including neuroblastoma, breast, lung,prostate, and ovarian cancer, andmight represent a promisingchemopreventive and antitumor agent (18). Currently HPR isunder clinical trials for the cure of prostatic and ovarian can-cers, neuroblastoma, lymphoma, and leukemia. Ceramide-me-diated apoptosis seems to be the major (even if not the sole)cytotoxic mechanism for HPR (reviewed in Ref. 19). HPR-in-duced production of ceramide mainly occurs via de novo syn-thesis, because HPR activates both serine palmitoyltransferaseand dihydroceramide synthase (20, 21) that catalyze the firststeps in sphingolipid biosynthesis (22). Moreover, it has beenrecently shown that HPR concomitantly inhibits dihydrocer-amide desaturase (22, 23), suggesting that dihydroceramiderather than (or in addition to) ceramide might mediate HPR-induced toxicity (24) possibly involving other mechanisms ofdeath in addition to apoptosis. In this work, we investigatewhether the resistance to HPR of human ovarian cancer cellscould be associated with the activation of the SK/S1P leading toan altered dihydroceramide/S1P ratio that could prevent orovercome ceramide-mediated HPR-induced cell death.

EXPERIMENTAL PROCEDURES

Chemicals—HPR was from Sigma. VPC23019, JTE013,CAY10444, W146, SW2871, VPC24191, S1P, and SK inhibitor2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole were obtainedfrom Calbiochem. [1-3H]Sphingosine (radiochemical purityover 98%; specific radioactivity of 2.2 Ci/mmol) and 3H-lipidsused as chromatographic standards were prepared as describedin a previous study (7).Cell Culture andTransfection—A2780 andA2780/HPR cells,

were cultured in RPMI 1640 (Sigma) supplementedwith 10% ofheat-inactivated fetal bovine serum (AmershamBiosciences), 2mM glutamine, 100 units/ml penicillin, and 100 �g/ml strepto-mycin. A2780 and HPR were cultured in the presence of 5 �M

HPR (25). A2780 cells were transfected by FuGENE� (RocheApplied Science) with the pcDNA3-hSK1WTFlag (26) (a gift ofDr. Stuart M. Pitson, Hanson Institute human Immunology,Institute forMedical and Veterinary Science, Adelaide, Austra-lia) or with the empty vector, following themanufacturer’s pro-tocol. Stable transfectants were isolated after selectionwith 500�g/ml Geneticin (G418, Sigma).Lipid Analysis—For the analysis of S1P production, cells

were pulsed with [1-3H]sphingosine for 45 min as previouslydescribed (27). Cell lipids were extracted with chloroform/methanol, and the total lipid extractswere partitionedwith 0.15volume of 0.1 M NH4OH. S1P in the upper alkaline phase wasseparated by high-performance TLC using the solvent sys-tem n-butanol/acetic acid/water (3:1:1). The radioactivity wasdetermined by liquid scintillation counting. Radioactive lipidson high-performance TLC plates were detected and quantifiedby radioactivity imaging performed with a Beta-Imager 2000

instrument (Biospace) using an acquisition time of �48 h. Theradioactivity associated with individual lipids was determinedwith the specific �-Vision software provided by Biospace.Mass Spectrometry—The total lipid extract derived from 4 �

107 A2780/HPR and SK1-overexpressing A2780 cells underbasal conditions or treated with HPR, SK inhibitor, or bothwere subjected to a two-phase partitioning with 0.15 volumeof 0.1 M NH4OH, the organic phases were treated with alkali asdescribed previously (28) and were subjected to mass spec-trometry (MS) analyses, carried out using a Thermo QuestFinnigan LCQDeca ion trap mass spectrometer (FINNIGANMAT, San Jose, CA) equippedwith an electrospray ionization ionsource, an Xcalibur data system, and a TSP P4000 quaternarypump high-performance liquid chromatography. Separations oflong-chain bases and Cer molecular species were obtained on a5-�m, 250 � 4mmLiChrospher 100 RP8 column (Merck).Elution of long-chain bases and Cermolecular species was car-

ried out, at a flow rate of 0.5ml/min,with a gradient formedby thesolvent system A, consisting of methanol/water (90:10, v/v), andsolvent system B, consisting of methanol, both containing 5 mM

ammonium acetate. The gradient elution programwas as follows:5 min with solvent A; 5 min with a linear gradient from 100%solvent A to 100% solvent B; 15 min with 100% solvent B; 5 minwith a linear gradient from 100% solvent B to 100% methanol.Methanol was also used to wash the column for 10 min, followedby equilibration procedure with solvent A for 15min.Optimum conditions for Cer molecular species MS analyses

included sheath gas flow of 50 arbitrary units, spray voltage of 4kV, capillary voltage of �47 V, capillary temperature of 260 °C,fragmentation voltage (used for collision induced dissociation)of 40–60%. Mass spectra were acquired over an m/z range of200–1000 in negative mode.Optimum conditions for long-chain bases MS analyses

included sheath gas flow of 50 arbitrary units, auxiliary gas flowof 5 arbitrary units, spray voltage of 4 kV, capillary voltage of 34V, capillary temperature of 250 °C, and fragmentation voltage(used for collision-induced dissociation) of 60%. Mass spectrawere acquired over a rangem/z 200–1000 in positive mode.For all experiments, source ion optics were adjusted to accom-

plish desolvation of ions while minimizing fragmentation.As internal standards were used uncommon d18:1/17:0 cer-

amide, d18:0/17:0 ceramide, and d20:0 sphinganine. A stocksolution of internal standards in ammonium acetate (5 mM) inmethanol was quantitatively prepared (50 �M) and stored at�20 °C. Serial dilutions were prepared from this stock solutionand utilized for calibration curves.SK Activity Assay—To measure SK activity, cell lysates (60

�g) were incubated (29) in the presence of 50 �M D-erythro-sphingosine dissolved in 4 mg/ml bovine serum albumin and1 mM ATP. Reaction was initiated by addition of [�-32P]ATP(0.5 �Ci, 1 mM) and 10 mM MgCl2 and terminated after30-min incubation at 37 °C by addition of 20 �l of 1 N HCl and900 �l of chloroform/methanol/HCl (100:200:1, v/v). 240 �l ofchloroform and 240 �l of 1 M KCl were added, and phases wereseparated by centrifugation. 500 �l of the lower phase weredried under a stream of nitrogen and dissolved in 100 �l ofchloroform/methanol (2:1). [32P]S1P was separated by TLCwith 1-butanol/methanol/acetic acid/water (80:20:10:20, v/v)

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and visualized by autoradiography. The radioactive spots cor-responding to [32P]S1P were scraped and counted in a scintil-lation counter. SK specific activity was expressed in picomoles/min*mg of protein in experiments performed at least induplicate.Western Blot—Cell homogenates were analyzed by SDS-

PAGE. After separation, proteins were transferred to polyvi-nylidene difluoride membranes. The presence of transfectedSK was assessed by immunoblotting using an anti-flagM2mouse polyclonal antibody (Sigma), SK1 was detected withpolyclonal anti-SK1 antibodies (30) (kindly provided by Dr. Y.Banno, Gifu University School of Medicine, Japan), SK-2 wasimmunorevealed employing rabbit polyclonal anti-SK2 anti-bodies (31), a kind gift of Dr. S. Nakamura (Dept. of MolecularandCellular Biology, KobeUniversityGraduate School ofMed-icine, Kobe, Japan), S1P1 and S1P3 receptors were detectedusing subtype-specific polyclonal antibodies (Abcam). Primaryantibodies were visualized by reaction with secondary horse-radish peroxidase-conjugated antibodies and enhanced chemi-luminescence detection (Pierce Supersignal). �-Actin was usedas loading control (anti-�-actin goat polyclonal from SantaCruz Biotechnology, Santa Cruz, CA). The data acquisitionwasperformed using a GS-700 Imaging Densitometer (Bio-Rad).Acquired blots were elaborated using the Quantity One soft-ware (Bio-Rad).Quantitative Real-time and Reverse Transcription-PCR—

Total RNA (2�g), extractedwithTriReagent, was reverse-tran-scribed using Superscript II reverse transcriptase (Invitrogen)as described in the manufacturer’s protocols. To detect theexpression of SK1 andSK2, cDNA fromA2780 andA2780/HPRcells were subjected to PCR using specific primers listed insupplemental Table 1. Data were normalized on the house-keeping genes glyceraldehyde-3-phosphate dehydrogenase and�-actin. PCR amplification products were separated by electro-phoresis on a 1.2% agarose gel and the exact size evaluated bycomparison with PCR 100 bp Low Ladder (Sigma).The quantification of S1PR mRNA was performed by Real-

time PCR employing TaqMan gene expression assays, usingthe automated ABI Prism 7700 sequence detector system(Applied Biosystems, Foster City, CA) essentially as previ-ously described (32). Each measurement was carried out intriplicate in Micro-Amp optical 96-well plates (Applied Bio-systems) with a TaqMan Universal PCRMaster Mix (AppliedBiosystems). Primers and probe for S1P1, S1P2, S1P3, S1P4, andS1P5 were Assay-On-Demand gene expression products(Applied Biosystems). Simultaneous amplification of the targetsequence together with the housekeeping gene, 18 S rRNA, wascarried out with the following universal profile: initial denatur-ation for 10 min at 95 °C was followed by denaturation for 15 sat 95 °C, primer annealing, and elongation at 60 °C for 1min for40–50 cycles. Results were analyzed by using the ABI PrismSequenceDetection System (version 1.7) (Applied Biosystems).The 2���Ct method was applied as a comparative method ofquantification (33), and datawere normalized to ribosomal 18 SRNA expression.Cell Proliferation and Viability: MTT Reduction and Trypan

Blue Dye Exclusion Assay—For theMTT assay, A2780, A2780/HPR, and A2780 transfectants were plated into 96-well tissue

culture plates and kept in culture for up to 96 h in normal cellculture medium or under different experimental conditions asdescribed below. For the basal proliferation, the same amountof cells were plated (2000 cells for well). After treatments, thecells were incubated at 37 °C for 1 h with 100 �l of cell culturemedium containing 120 �MMTT and then lysed with 100 �l oflysis solution (10% SDS in 10 mM HCl aqueous solution) andmaintained for 12 h at 37 °C. The absorbance was measured at570 nmwith aVictor plate reader instrument (PerkinElmer LifeSciences).The number of living and dead cells has been determined by

counting after Trypan blue staining, as previously described(34). Three thousand A2780, A2780/HPR, and A2780 transfec-tants cells were plated in 60-mm tissue culture plates. Briefly,cells were detached with phosphate-buffered saline containing0.02% EDTA, incubated in 0.25% Trypan blue solution for 2min and counted using a Burker chamber. The extent of celldeath was calculated as the percent of trypan blue-positive cellsin each cell population.Pharmacological Inhibition of SK—Twelve hours after seed-

ing, A2780/HPR cells were treated or not with 10 �M SK inhib-itor solubilized in cell culture medium containing 0, 5, and 10�M of HPR. Cell proliferation and viability were estimated atdifferent times of treatment by the MTT reduction assay andthe Trypan blue dye exclusion assay, respectively, as describedabove.Treatment of A2780 and A2780/HPR Cells with Agonists and

Antagonists of S1P Receptors—1000–2000 A2780 or A2780/HPR cells were plated in 96-well culture plates. After 12 h,A2780 cells were treated with 1�M SEW2871 (S1P1 agonist), orVPC24191 (S1P1/3 agonist), or S1P in culture medium contain-ing or not 1 �M HPR; A2780/HPR cells were treated with 1 �M

VPC23019 (S1P1/3 antagonist), or JTE013 (S1P2 antagonist), orCAY10444 (S1P3 antagonist), or 10�MW146 (S1P1 antagonist)in medium containing or not 10 �M HPR; each treatment wererenewed every 48 h. After 96 h, cell proliferation was assessedby MTT reduction assay as described above.DNA Fragmentation Analysis—Analysis of genomic DNA:

the analysis of genomic DNA, fromA2780/HPR cells, treatedor not with SK inhibitor in the presence of different HPRconcentrations, was carried out using Mammalian GenomicDNA Kit (Invitrogen) according to the manufacturer’sinstructions. The DNA extract was quantified by absorbanceat 260 nm. Equal amounts of total DNA were analyzed by aga-rose gel electrophoresis and detected by ethidium bromidestaining. Analysis of fragmented DNA: 1-ml aliquots of phos-phate-buffered saline suspension containing 3 � 106 A2780/HPR cells treated as described above were centrifuged at 200 �g for 10 min at 4 °C. Cells were lysed by adding to the pellet 0.5ml of 0.2% Triton X-100 in TE buffer, pH 7.4. To separate frag-mented DNA from intact chromatin, the cell lysates were cen-trifuged at 20,000 � g for 10 min at 4 °C. The supernatant wasremoved, and the pellet was resuspended in 0.5 ml of 0.2% Tri-ton X-100 in TE buffer, pH 7.4, and 0.1 ml of ice-cold 5 M NaCland vigorously vortexed. Then 0.7 ml of ice-cold iso-propanolwas added, vortexed vigorously, and DNA was allowed to pre-cipitate overnight at �20 °C. DNA was recovered by centrifu-gation for 10 min at 20,000 � g at 4 °C. The supernatants were

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carefully removed, and the samples were dried. DNA was dis-solved by adding to each tube 20 �l of TE solution and left at37 °C for 12 h. Then DNA were mixed with loading buffer andheated at 65 °C for 10 min. Samples were loaded in 1% agarosegel containing ethidium bromide.Other Procedures—Protein content was determined accord-

ing to Lowry et al. (35), using bovine serum albumin as thereference standard.Statistical Analysis—Experiments were run in triplicate,

unless otherwise stated. Data are expressed as mean value �S.D. and were analyzed by one-way analysis of variance fol-lowed by the Student-Newman-Keuls’ test. p values are indi-cated in the legend of each figure.

RESULTS

A2780 human ovarian carcinoma cells are very sensitiveto a wide array of antitumor drugs, including the syntheticretinoic acid analogue HPR. When these cells were continu-ously exposed to this drug, they developed resistance to it.A2780/HPR cells are a HPR-resistant clonal line obtained byculturing A2780 human ovarian carcinoma cells in the pres-ence of increasing concentrations of HPR. A2780/HPR are notonly characterized by a 10-fold increase in resistance respect toparental sensible A2780 cells but show also several phenotypicdifferences, including altered morphology, reduced colony-forming ability, and differential expression of adhesion, differ-entiation, and tumor progression markers (25) and alteredsphingolipid metabolism (7).In addition, we observed that proliferation of A2780/HPR

cells was significantly higher than that of parental cells (Fig. 1).Our previous data indicated that in A2780/HPR cells the deg-radative pathway of sphingosine is more active than in A2780cells (7). Sphingosine degradation requires its conversion toS1P by SK, a key enzyme in the maintaining of intracellularceramide/S1P ratio, which is critical in the control of cell deathand proliferation.To quantitatively evaluate S1P production in the two cell lines,

cells were pulsed for 45 min with [1-3H]sphingosine, cell lipidswere extracted, and S1Pwas recovered by phase separation underalkaline conditions as previously described and analyzed by high-performance TLC followed by digital autoradiography. With asimilar incorporation of the radioactive label in the two celltypes, we calculated that 0.71% (0.17 � 0.01 nCi/mg of protein)and 1.33% (0.28 � 0.02 nCi/mg of protein) of the lipid radioac-tivity was associated with S1P in A2780 and A2780/HPR cells,respectively. Thus, short pulse labeling experimentswith radio-active sphingosine revealed that the production of S1P is signif-icantly higher inA2780/HPR cells versusA2780 cells (p� 0.01).Next SK activity in HPR-sensitive and -resistant A2780 cellswas examined. In vitro enzyme assay performed in whole celllysates revealed that activity of SK was increased �5-fold inA2780/HPR versus A2780 cells (Fig. 2A). In A2780/HPR cells,the increase in the SK activity was essentially due to the up-reg-ulation of SK1.As shown in Fig. 2,B andC, theA2780/HPRcellsdisplayed increased mRNA expression and protein content ofSK1, whereas SK2 expression was unchanged.To ascertain whether proliferation of sensitive and resistant

A2780 cells was dependent on the production of S1P, A2780,

and A2780/HPR cell proliferation was evaluated under basalconditions or in the presence of 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole, a potent and highly selective pharmaco-logical SK inhibitor. The treatment with this inhibitor effec-tively reduced S1P production: in A2780/HPR cells, conversionof radioactive sphingosine to S1P dropped from 0.28 � 0.02nCi/mg of protein in control cells to 0.06 � 0.01 nCi/mg ofprotein in treated cells (p � 0.01 in inhibitor-treated versusuntreated A2780/HPR cells). Treatment of A2780 and A2780/HPR cells with SK inhibitor resulted in a 3-fold reductionof cell mitochondrial metabolic activity measured by theMTT reduction assay (Fig. 3A), indicating that SK activity isessential for proliferation of both HPR-sensitive and -resis-tant cells. Next we checked the effect of SK inhibition onthe sensitivity of A2780/HPR cells to HPR. A2780/HPR cellswere treated with SK inhibitor in the presence of differentHPR concentrations, and, after 96 h, the mitochondrial met-abolic activity was measured by the MTT reduction assay(Fig. 3B, left), and the cell number was determined by countingtrypsinized cells (Fig. 3B, right). The proliferation of A2780/HPR cells was reduced by the treatment with SK inhibitorboth under basal conditions or in the presence of 5 and 10�MHPR. However, in the presence of 10�MHPR, a significantincrease in the number of death cells (Fig. 4A) was observed incells treated with SK inhibitor. A2780/HPR cell death uponsimultaneous treatment with 10 �M HPR and SK inhibitor washallmarked by a significant reduction of the genomicDNAcon-tent and by the appearance of DNA fragmentation (Fig. 4B). Alltogether these data suggest that SK inhibition sensitized

FIGURE 1. Growth of A2780 and A2780/HPR cells. At different times afterseeding, the mitochondrial metabolic activity of A2780 (triangle) and A2780/HPR (square) cells was measured by the MTT reduction assay (A), and the cellnumber was determined by counting trypsinized cells using a Burker count-ing chamber (B). Data are the means � S.D. of three different experiments. *,p � 0.001 versus A2780 cells.

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A2780/HPR cells to HPR-induced apoptosis. Because the anti-proliferative and cytotoxic effect ofHPRonA2780 cells ismedi-ated by a HPR-induced elevation in cellular dihydroceramidelevels (7, 36), a higher SK activity in resistant cells might beresponsible for a reduced capability to form dihydroceram-ide or ceramide (i.e. to a lower dihydroceramide/S1P ratio withrespect to sensitive cells upon challenge with the drug). Thus,we tested the effect of SK inhibition on the production of cera-mide anddihydroceramide inA2780/HPRcells upon treatmentwith 10 �M HPR. The dihydroceramide and ceramide molecu-lar species of A2780/HPR cells under basal conditions or upontreatment with HPR, SK inhibitor, or both were characterizedby electron spray ionization-MS (Fig. 4C and supplementalFig. 1). As shown in Fig. 4C, treatment with HPR alone was ableto induce a sensitive increase in dihydroceramide, withoutaffecting ceramide. Treatment with SK inhibitor as well signif-icantly elevated cellular dihydroceramide but not ceramide lev-els. On the other hand, simultaneous treatment with both HPRand SK inhibitor led to a dramatic increase in dihydroceramide(significantly higher that the increased observed in the presenceofHPR or SK inhibitor alone), againwith no effect on ceramide.Sphingosine and sphinganine levels were also measured inA2780/HPR cells under the same experimental conditions.Under basal condition, sphingoid base levels in A2780/HPR

cells were 65 � 8 pmol/mg of protein, with sphingosine repre-senting 85% of total sphingoid bases. HPR treatment alone or incombination with the SK inhibitor induced a marked increasein the sphinganine/sphingosine ratio. In treated samples, sph-inganine represented �95% of total sphingoid bases, and itsmass levels were increased in parallel of those of dihydroceram-ide. In samples treated with HPR and SK inhibitor, this corre-sponded to an �100-fold increase in sphinganine levels. Thesedata suggest that SK inhibition sensitized A2780/HPR cells toHPR-induced dihydroceramide and sphinganine production,resulting in increased cell death.Although S1P can act as an intracellularmediator, S1P recep-

tors largely account for itsmultiple biological activities. For thisreason, to obtain more information on the mechanism bywhich altered S1P formationmediates the onset of resistance toHPR inA2780 cells, the S1P receptor (S1PR) expression patternwas examined in parallel in HPR-sensitive and -resistant cells,by quantitative real-time PCR analysis. Data presented in Fig. 5show strikingly different S1PR expressions between the twocell types. Interestingly, by normalizing individual S1PRmRNAcontent of resistant cells onto that of sensitive ones, it wasclearly appreciable that a tremendous increase of S1P1 mRNAoccurred in HPR-resistant cells, which was accompanied by arobust rise of S1P3 mRNA content, whereas the other S1PRsubtypes were slightly reduced. These data were corroboratedby Western blot analysis of S1P1 and S1P3. Indeed, the proteinband corresponding to S1P1 was clearly detectable in chemore-sistant cells, whereas it could not be detected in chemosensitive

FIGURE 2. SK activity and SK isoform expression in A2780 and A2780/HPRcells. A, A2780 and A2780/HPR cells were lysed, and cell extracts wereemployed for SK activity determination as described under “ExperimentalProcedures.” Data represent the mean � S.D. of three independent experi-ments performed in duplicate. The difference between A2780 and A2780/HPR cells was statistically different by the Student’s t test (*, p � 0.01). B, semi-quantitative PCR analysis of SK1 and SK2 mRNA expression levels wasperformed in A2780 and A2780/HPR cells by simultaneous amplification ofthe housekeeping gene �-actin. Data are normalized versus �-actin expres-sion and utilizing individual SK isoforms of the A2780 specimen set as 1. Dataare means � S.D. of three independent experiments performed in triplicate.C, cell extracts from A2780 and A2780/HPR cells were employed for Westernanalysis using anti-SK1 and anti-SK2 antibodies. Left panel: a blot representa-tive of three independent experiments is shown. Right panel: the histogramrepresents mean densitometric quantification (n � 3) of SK1 and SK2 versus�-actin, reported as a percentage relative to the intensity of the band corre-sponding to A2780 specimen set as 100. Statistical significance was deter-mined by the Student’s t test (*, p � 0.01).

FIGURE 3. Effect of SK inhibitor on the proliferation of A2780 and A2780/HPR cells and on HPR sensitivity in A2780/HPR cells. A, 12 h after seeding,A2780 and A2780/HPR cells were treated with SK inhibitor (gray), and cellviability was evaluated after 96 h as mitochondrial metabolic activity mea-sured by the MTT reduction assay. Data are expressed as percentage of thecontrol treated with vehicle (white). B, 12 h after seeding, A2780/HPR cellswere treated with SK inhibitor in the presence of different HPR concentrationsand, after 96 h, the mitochondrial metabolic activity was measured by theMTT reduction assay (B, left), and cell number was determined by countingtrypsinized cells using a Burker counting chamber (B, right). Data are themeans � S.D. of three different experiments. *, p � 0.001 versus controls, cellstreated with vehicle only.

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cells; moreover, the S1P3 protein content was sensibly aug-mented in HPR-resistant cells (Fig. 5A, inset). However, prolif-eration of A2780 and A2780/HPR cells was not affected by thetreatment with different antagonists of S1PR (Fig. 5B), includ-ing antagonists selective for S1P1 (VPC23019 andW146), S1P2(JTE013), and S1P3 (VPC23019 and CAY10444).To determine whether the role of SK in provoking resis-

tance to HPR could be due to an increased activation of S1PR,we checked the effect of S1PR antagonists on HPR-resistantcells treated with 10 �M HPR (Fig. 5C, right), and the effect ofexogenous S1P and S1PR agonists (SEW2871 for S1P1 andVPC24191 for S1P1 and S1P3) on HPR-sensitive cells treatedwith 1 �M HPR (Fig. 5C, left). The effect of HPR on A2780 andA2780/HPR cells was not affected by the treatment with S1PRagonists or antagonists, respectively, suggesting that the role ofSK inHPR resistance in these cells is notmediated by the S1PR.Because the inefficacy of S1PR antagonist treatment on the pro-liferation rate could be due to inactivation of the pharmacolog-ical compounds during prolonged incubation, the ability of the

antagonists to prevent S1P-dependent Akt phosphorylationafter 95-h incubation was examined. Akt phosphorylation byS1Pwasmimicked by specific S1PR agonists such as VPC24191or SEW2871, supporting the view that this signaling is coupledto multiple S1PRs (supplemental Fig. 2). In the same figure isshown that Akt phosphorylation at 10 min of 1 �M S1P treat-ment was abolished by VPC23019, strongly reduced by W146,and attenuated by JTE013, indicating that all the compoundswere active.To further substantiate the hypothesis of a role for SK1 in

determining resistance to HPR in ovarian carcinoma cells,we stably overexpressed a tagged form of SK1 in A2780cells (Fig. 6A). SK1 overexpression was accompanied by a sig-nificant increase of S1P levels, as evaluated by short pulse with[1-3H]sphingosine (radioactivity incorporation in mock trans-fected cells was 0.18 � 0.01 nCi/mg of protein and 0.24 � 0.02nCi/mg of protein in SK1-overexpressing clones; p � 0.01 inSK1-transfected versus mock transfected A2780 cells). Wild-type, mock transfected, and SK1-overexpressing A2780 cellswere treated with 1 �M HPR, and, after 96 h, HPR cytotoxicitywas assessed by determining the number of dead cells (Fig. 6B).As expected, HPR treatment under these conditions caused asignificant increase in cell death in wild-type and mock trans-fected cells. On the other hand, cell viability was not affected byHPR treatment in SK1-overexpressing A2780 cells, suggestingthat SK1 is sufficient to induce HPR resistance in these cells.HPR treatment of SK1-overexpressing A2780 cells resulted in asignificant rise in dihydroceramide levels, even if at a lesserextent than in A2780/HPR cells (Fig. 4C). On the other hand,treatment with HPR in SK1-overexpressing A2780 cells alsoinduced a significant increase in the levels of ceramide, whichwere unaffected inA2780 andA2780/HPR cells under the sametreatment. This could be due to the higher expression levels ofdihydroceramide desaturase 1 observed in the SK1-overex-pressing clone (supplemental Figs. 3 and 4).Finally, to obtain a more complete picture of the sphingo-

lipid metabolism in all the cell lines studied, the gene expres-sion of the major enzymes involved in the sphingolipid met-abolic pathway were examined by reverse transcription-PCR(supplemental Fig. 3) and quantitative real-time reverse tran-scription-PCR (supplemental Fig. 4). Significant changes inthe mRNA expression were detected only for SMS2, that wasmore expressed in A2780/HPR cells, and dihydroceramidedesaturase 1, whose expression was significantly higher in SK1-overexpressing A2780 cells. Signals for neutral ceramidase,alkaline ceramidase 1, and ceramide synthase 3 were hardlydetectable in our samples.

DISCUSSION

The cytotoxic and/or antiproliferative effect of many anti-tumor drugs is at least in part a consequence of the drug-elicited increase in the cellular levels of the sphingolipid cer-amide, a potent mediator of apoptosis and an inhibitor ofcell proliferation in a variety of tumor cell lines. This is thecase also for HPR, whose antiproliferative effect in neuro-blastoma, leukemia, breast, ovarian (including A2780 humanovarian carcinoma cells, the experimental model used in thispaper), prostate, and colon carcinoma cell lines has been asso-

FIGURE 4. Effect of SK inhibition on HPR sensitivity and on ceramide anddihydroceramide production in A2780/HPR cells. 12 h after seeding,A2780/HPR cells were treated with SK inhibitor in the presence of differentHPR concentrations and, after 96 h, the number of dead cells was evaluatedby the Trypan blue exclusion assay (A, left). Data are the means � S.D. of threedifferent experiments. *, p � 0.01 versus controls. Non-fragmented (B, left)and fragmented DNA (B, right) was extracted as described under “Experimen-tal Procedures.” The samples were analyzed by agarose gel electrophoresisand visualized by ethidium bromide staining (in B, right, genomic DNA wasloaded on the same gel as control). C, MS analysis of ceramide and dihydro-ceramide content in A2780 cells resistant to HPR. Ceramide (light gray) anddihydroceramide (dark gray) content expressed as nanomoles/106 cells in 1,A2780/HPR control; 2, A2780/HPR plus HPR; 3, A2780/HPR plus SK inhibitor; 4,A2780/HPR plus HPR plus SK inhibitor; 5, SK1-overexpressing A2780; and 6,SK1-overexpressing A2780 plus HPR. Data are the means � S.D. of three dif-ferent experiments. *, p � 0.01 versus control cells. #, p � 0.025 versus SK1-overexpressing A2780.

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ciated with induction of apoptosis,which is accompanied by increasesin ceramide. Ceramideproduction inHPR-treated tumor cells is some-how peculiar, because it has beensuggested that the most relevantmechanism for HPR-induced cera-mide generation might be the denovo biosynthesis (HPR is indeed apotent activator of serine palmitoyl-transferase and dihydroceramidesynthases, the key enzymes in theearly steps of ceramide biosynthesis),whereas for many other antitumordrugs sphingomyelin hydrolysis dueto the activation of sphingomyeli-nases is the main source of pro-apo-ptotic ceramide.Moreover, it has beendemonstrated that dihydroceramide,rather than ceramide, accumulates inHPR-treated tumor cells, due to theinhibitory effect of this retinoid ondihydroceramide desaturase (22, 24,37). Indeed we recently showed byelectrospray ionization-MS analysisthat dihydroceramide and not cera-mide is increased upon treatment ofA2780 cells with HPR (36). In addi-tion, recent observations suggest thatHPR cytotoxicity might at leastin part due to the elevation of cel-lular sphinganine levels (24). Inde-pendently from the mechanismunderlying the production of cyto-toxic (dihydro)ceramide upon che-motherapeutic treatments, it hasbeen suggested that the acquiredresistance to chemotherapeutic drugsin tumors and tumor cell lines can beascribed to alterations in sphingolipidmetabolism leading to a reducedpotential for dihydroceramide for-mation or an increased clearance ofdihydroceramide. In particular, mul-tidrug-resistant tumors and tumorcell lines are characterized by highlevels of glucosylceramide, becauseof the consequence of the increasedexpression or activation of glucosyl-ceramide synthase, and ceramide gly-cosylation by this enzyme as a wayto thwart drug-induced dihydro-ceramide accumulation and to pre-vent dihydroceramide-induced celldeath has been convincingly docu-mented by many previous studies.On the other hand, dihydroceram-ide is the pivot of a very complex

FIGURE 5. Expression and effect of agonists or antagonists of S1PRs on proliferation and HPR resistancein A2780 and A2780/HPR cells. A, quantitative real-time PCR analysis was performed in A2780 and A2780/HPR by simultaneous amplification of the target S1P1, S1P2, S1P3, S1P4, and S1P5 genes together with thehousekeeping gene 18 S rRNA. The mRNA quantization was based on the comparative 2���Ct method, utilizingfor each receptor its expression in A2780 cells as calibrator. Data are means � S.D. of three independentexperiments performed in triplicate. Inset: cell extracts from A2780 and A2780/HPR were employed for Westernanalysis using anti-S1P1 or anti-S1P3 antibodies. Equally loaded protein was checked by expression of �-actin.B, 12 h after seeding, A2780 and A2780/HPR cells were treated with different antagonists of S1PRs, and cellproliferation was evaluated after 96 h as mitochondrial metabolic activity measured by the MTT reductionassay. Data are the means � S.D. of three different experiments. C, 12 h after seeding, A2780 cells were treatedwith different agonists of S1PRs alone or in the presence of 1 �M HPR (left panel), and A2780/HPR cells weretreated with different antagonists of S1PRs alone or in the presence of 10 �M HPR (right panel); after 96 h, themitochondrial metabolic activity was measured by the MTT reduction assay. Data are the means � S.D. of threedifferent experiments.

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metabolic network, and many different pathways can poten-tially contribute to the regulation of cellular ceramide levels andcould be involved in the increased ability of drug-resistant cellsto scavenge ceramide. Clues supporting this hypothesis comefrom the observation that it is clearly emerging that increasedconversion of ceramide into glucosylceramide is not the onlyalteration in sphingolipid metabolism occurring in tumor cellsupon acquisition of drug resistant phenotypes (2).Short-chain ceramide is cytotoxic for A2780 human ovarian

carcinoma cells (7), and these cells respond to HPR treatmentwith a robust production of dihydroceramide (36), likely due toits increased biosynthesis, with a concomitant reduction of cellproliferation and induction of apoptosis. On the other hand,HPR induced a much lesser response in terms of dihydrocer-amide production in the derived HPR-resistant clonal cell line,A2780/HPR. In these cells, glucosylceramide synthase expres-sion and glucosylceramide levels are not changed with respectto those in sensitive parental cells. However, overall alterationsin sphingolipid metabolism were observed in the resistant cellline, including an increased catabolic turnover of ceramide (7).

It is well established that S1P is endowed with propertiesopposite to those displayed by ceramide and possibly by dihy-droceramide, being able to stimulate cell survival and prolifer-ation. The opposing directions of ceramide- and S1P-mediatedsignaling generated the concept of a ceramide/S1P rheostat inwhich the ratio between these two lipids could play a relevantrole in determining the cell fate. Compelling evidence is in favorof a key role of SK, which catalyzes S1P formation, in the regu-lation of cellular ceramide/S1P balance. Notably, in agreementwith this notion, this study shows that altered sphingolipidmetabolism displayed by A2780/HPR cells, whose proliferationis higher respect to A2780 cells, was characterized by enhancedformation of S1P (Fig. 2). Moreover, the activity of SK wasremarkably augmented in these cells as result of SK1 up-regu-lation (Fig. 2). Together with our previous observation, that inA2780/HPR cells the degradation of sphingosine occurs at ahigher rate than in A2780 cells (7), these data support the viewthat the augmented catabolism of dihydroceramide due to SK1higher activity in A2780/HPR cells impedes the accumulationof the toxic lipid in response to drug treatment. More impor-tantly, pharmacological inhibition of SK strongly reduced S1Pformation in HPR-resistant cells and concurrently was foundcapable of rescuing their sensitivity to HPR treatment due toincreased HPR-mediated dihydroceramide production (Fig. 4),indicating that the shift toward S1P formation, which takesplace in HPR cells, is a critical event for the appearance of che-moresistance in A2780 ovarian cancer cells. This notion wasfurther strengthened by the finding that enhanced S1P forma-tion subsequent to ectopic expression of SK1 rendered A2780cells less sensitive to HPR (Fig. 6).Sphinganine production was also enhanced by HPR treat-

ment, and a possible contribution of sphinganine to HPR-in-duced toxicity has been recently suggested (24). Obviously,increased SK activity associated with HPR resistance would beeffective in scavenging sphinganine as well. Thus, even if therole of different sphingolipid mediators in HPR-induced celldeath deserves further investigation, our data support theimportance of SK in resistance to HPR. The here highlightedcritical reliance on enhanced SK activity of ovarian cancer cellresistance to the chemotherapeutic HPR drug is in full agree-ment with previous reports in camptothecin- and docetaxel-resistant prostate cancer cells (14, 38), oxaliplatin-resistantcolon cancer cells (15), and gemcitabine-resistant pancreaticcancer cells (17), corroborating the notion that up-regulation ofSK and a concomitant decrease of the levels of sphingolipidmediators upstream of SK represent common features of resis-tant tumor cells.In keeping with the pro-growth signal borne by SK (39), its

inhibition reduced the proliferation rate of A2780 cells, inde-pendently of their sensitivity to HPR (Fig. 3). Thus, a pharma-cological intervention that addresses SK in these cells appearsto be especially efficacious, being capable of reducing cell pro-liferation and simultaneously counteracting drug resistance.Inhibition of individual S1PR subtypes by specific antago-

nists did not affect cell sensitivity to HPR, ruling out theirinvolvement in this cell behavior (Fig. 5). Moreover, S1PRwere found to be disengaged also from the regulation of cellproliferation, which was modulated by SK activity (Fig. 5).

FIGURE 6. Effect of SK-1 overexpression on HPR sensitivity in A2780 cells.A2780 cells were transfected with the empty expression vector (mock trans-fected) or with pcDNA3-hSK1WT plasmid (SK1-overexpressing cells). A, West-ern blot analysis of FLAG expression in A2780 transfectants. Equal amount ofproteins of A2780, mock, and SK1-overexpressing cells were loaded ontoSDS-PAGE and then blotted onto a polyvinylidene difluoride membrane, andthe overexpressed SK1 tagged with FLAG M2 epitope was detected by anti-FLAG M2 antibody. B, 12 h after seeding, A2780 and A2780 transfectants (onemock and 3 SK1-overexpressing clones, 1, 2, and 3) were treated with 1 �M

HPR. After 48 h, the total cell number was determined by counting trypsinizedcells using a Burker counting chamber, and the number of dead cells wasevaluated by the Trypan blue exclusion test. Data are presented as the per-centage of the total counted cells and are the means � S.D. of three differentexperiments. *, p � 0.01 versus A2780 cells.

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Hence, these findings strongly support a primary role ofreduced dihydroceramide production in mediating theresistance of A2780 cells to HPR, although the participationof S1P as intracellular mediator capable of addressing directlyor indirectly elements of the anti-apoptotic machinery cannotbe excluded. Despite the function of S1PR lacking in A2780chemoresistance, the pattern of S1PR in A2780/HPR cells wasfound to be profoundly modified in comparison to parentalcells. In particular, S1P1 mRNA was extraordinarily aug-mented, whereas S1P3 was significantly up-regulated (Fig. 5).Intriguingly, the same receptor subtypes were highly increasedin chemotherapy-resistant prostate cancer cells, but, at vari-ance, they were implicated in mediating cell proliferation (38).Because these receptors have been found to be coupled to keybiological functions, such as cell motility and cell adhesion,their differential expression could account for specific behaviorof these cancer cell lines, and it will be important to address thispossibility in future studies.

Acknowledgment—We are indebted to Dr. Stuart M. Pitson (Molec-ular Signaling Laboratory, Hanson Institute and Division of HumanImmunology, Institute of Medical and Veterinary Science, Adelaide,Australia) for providing the pcDNA3-hSphK1-FLAG plasmid.

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Sonnino and Alessandro PrinettiVanna Chigorno, Paola Bruni, Sandro Frera, Chiara Donati, Manuela Valsecchi,Massimo Aureli, Simona Prioni, Gianluca Giuditta Illuzzi, Caterina Bernacchioni,  Ovarian Cancer Cells-(4-Hydroxyphenyl)retinamide in Human

Nthe Synthetic Retinoid Sphingosine Kinase Mediates Resistance toLipids:

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