Abstract: 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. 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. Sphingolipid metabolism plays a pivotal role in the mechanism of apoptosis induced in tumor cells. Ceramide, produced under physiological (tumor necrosis factor α, γ-interferon, and interleukins) and pharmacological (anticancer drugs, including daunorubicin, vincristine, 1-α-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. 1Obeid L.M. Linardic C.M. Karolak L.A. Hannun Y.A. Science. 1993; 259: 1769-1771Google Scholar and 2Perry D.K. Hannun Y.A. Biochim. Biophys. Acta. 1998; 1436: 233-243Google Scholar).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 (3Sietsma H. Veldman R.J. Kok J.W. J. Membr. Biol. 2001; 181: 153-162Google Scholar, 4Senchenkov A. Litwak D.A. Cabot M.C. J. Natl. Cancer Inst. 2001; 93: 347-357Google Scholar, 5Cabot M.C. Giuliano A.E. Volner A. Han T.Y. FEBS Lett. 1996; 394: 129-131Google Scholar, 6Lavie Y. Cao H. Bursten S.L. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1996; 271: 19530-19536Google Scholar, 7Lavie Y. Cao H. Volner A. Lucci A. Han T.Y. Geffen V. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1997; 272: 1682-1687Google Scholar, 8Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1999; 274: 1140-1146Google Scholar, 9Liu Y.Y. Han T.Y. Giuliano A.E. Ichikawa S. Hirabayashi Y. Cabot M.C. Exp. Cell Res. 1999; 252: 464-470Google Scholar, 10Liu Y.Y. Han T.Y. Giuliano A.E. Hansen N. Cabot M.C. J. Biol. Chem. 2000; 275: 7138-7143Google Scholar, 11Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. FASEB J. 2001; 15: 719-730Google Scholar, 12Veldman R.J. Klappe K. Hinrichs J. Hummel I. van der Schaaf G. Sietsma A. Kok J.W. FASEB J. 2002; 16: 1111-1113Google Scholar). High levels of GlcCer 1The abbreviations used are: GlcCer, β-Glc-(1–1)-Cer; LacCer, β-Gal-(1–4)-β-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 1The abbreviations used are: GlcCer, β-Glc-(1–1)-Cer; LacCer, β-Gal-(1–4)-β-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 (6Lavie Y. Cao H. Bursten S.L. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1996; 271: 19530-19536Google Scholar, 7Lavie Y. Cao H. Volner A. Lucci A. Han T.Y. Geffen V. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1997; 272: 1682-1687Google Scholar, 8Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1999; 274: 1140-1146Google Scholar, 9Liu Y.Y. Han T.Y. Giuliano A.E. Ichikawa S. Hirabayashi Y. Cabot M.C. Exp. Cell Res. 1999; 252: 464-470Google Scholar, 11Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. FASEB J. 2001; 15: 719-730Google Scholar, 12Veldman R.J. Klappe K. Hinrichs J. Hummel I. van der Schaaf G. Sietsma A. Kok J.W. FASEB J. 2002; 16: 1111-1113Google Scholar) and of glucosylceramide synthase activity (5Cabot M.C. Giuliano A.E. Volner A. Han T.Y. FEBS Lett. 1996; 394: 129-131Google Scholar, 8Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1999; 274: 1140-1146Google Scholar, 9Liu Y.Y. Han T.Y. Giuliano A.E. Ichikawa S. Hirabayashi Y. Cabot M.C. Exp. Cell Res. 1999; 252: 464-470Google Scholar) and/or expression (8Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1999; 274: 1140-1146Google Scholar, 9Liu Y.Y. Han T.Y. Giuliano A.E. Ichikawa S. Hirabayashi Y. Cabot M.C. Exp. Cell Res. 1999; 252: 464-470Google Scholar,11Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. FASEB J. 2001; 15: 719-730Google Scholar, 12Veldman R.J. Klappe K. Hinrichs J. Hummel I. van der Schaaf G. Sietsma A. Kok J.W. FASEB J. 2002; 16: 1111-1113Google Scholar) were detected in a number of drug-resistant cancer cell lines and in specimens from cancer patients not responding to chemotherapy treatment (13Lucci A. Cho W.I. Han T.Y. Giuliano A.E. Morton D.L. Cabot M.C. Anticancer Res. 1998; 18: 475-480Google Scholar). 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) (3Sietsma H. Veldman R.J. Kok J.W. J. Membr. Biol. 2001; 181: 153-162Google Scholar, 14Gottesmann M.M. Pastan I. Annu. Rev. Biochem. 1993; 62: 385-427Google Scholar). 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 (4Senchenkov A. Litwak D.A. Cabot M.C. J. Natl. Cancer Inst. 2001; 93: 347-357Google Scholar, 5Cabot M.C. Giuliano A.E. Volner A. Han T.Y. FEBS Lett. 1996; 394: 129-131Google Scholar, 6Lavie Y. Cao H. Bursten S.L. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1996; 271: 19530-19536Google Scholar, 7Lavie Y. Cao H. Volner A. Lucci A. Han T.Y. Geffen V. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1997; 272: 1682-1687Google Scholar, 8Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1999; 274: 1140-1146Google Scholar, 9Liu Y.Y. Han T.Y. Giuliano A.E. Ichikawa S. Hirabayashi Y. Cabot M.C. Exp. Cell Res. 1999; 252: 464-470Google Scholar, 10Liu Y.Y. Han T.Y. Giuliano A.E. Hansen N. Cabot M.C. J. Biol. Chem. 2000; 275: 7138-7143Google Scholar, 11Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. FASEB J. 2001; 15: 719-730Google Scholar).On the other hand, it has been shown (12Veldman R.J. Klappe K. Hinrichs J. Hummel I. van der Schaaf G. Sietsma A. Kok J.W. FASEB J. 2002; 16: 1111-1113Google Scholar, 15Kok J.W. Veldman R.J. Klappe K. Konino H. Filipeanu C.M. Muller M. Int. J. Cancer. 2000; 87: 172-178Google Scholar) 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 (16Hakomori S. Cancer Res. 1996; 56: 5309-5318Google Scholar, 17Riboni L. Viani P. Bassi R. Prinetti A. Tettamanti G. Prog. Lipid Res. 1997; 36: 153-195Google Scholar, 18Radin N.S. Eur. J. Biochem. 2001; 268: 193-204Google Scholar).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 (19Nagy L. Thomazy V.A. Heyman R.A. Davies P.J. Cell. Death Differ. 1998; 5: 11-19Google Scholar). Retinoids exert their effects by regulating gene expression through two classes of nuclear receptors: retinoic acid (RA) receptors α, β, and γ and retinoid X receptors α, β, and γ. 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 (20Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Google Scholar). 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 (21Lotan R. FASEB J. 1996; 10: 1031-1039Google Scholar).4-[3,7-Dimethyl-9-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2,4, 6,8- nonatetraenamido]-1-hydroxybenzene, known asN-(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 (22Formelli F. Barua A.B. Olson J.A. FASEB J. 1996; 10: 1014-1024Google Scholar). 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 (23De Palo G. Veronesi U. Camerini T. Formelli F. Mascotti G. Boni C. Fosser V. Del Vecchio M. Campa T. Costa A. Marubini E. J. Natl. Cancer Inst. 1995; 87: 146-147Google Scholar, 24Veronesi U. De Palo G. Marubini E. Costa A. Formelli F. Mariani L. Decensi A. Camerini T. Del Turco M.R. Di Mauro M.G. Muraca M.G. Del Vecchio M. Pinto C. D'Aiuto G. Boni C. Campa T. Magni A. Miceli R. Perloff M. Malone W.F. Sporn M.B. Natl. Cancer Inst. 1999; 91: 1847-1856Google Scholar) as well as chemopreventive and therapeutic efficacy against different tumors in animal models (22Formelli F. Barua A.B. Olson J.A. FASEB J. 1996; 10: 1014-1024Google Scholar). In vitro studies have demonstrated that HPR has significant antiproliferative activity associated with induction of apoptosis in several tumor cell types (22Formelli F. Barua A.B. Olson J.A. FASEB J. 1996; 10: 1014-1024Google Scholar). 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 (25Swisshelm K. Ryan K. Lee X. Tsou H.C. Peacocke M. Sager R. Cell Growth Differ. 1994; 5: 133-141Google Scholar, 26Fanjul A. Delia D. Pierotti M.A. Rideout D. Qiu J. Pfahl M. J. Biol. Chem. 1996; 271: 22441-22446Google Scholar, 27Sabichi A.L. Hendricks D.T. Bober M.A. Birrer M.J. J Natl. Cancer Inst. 1998; 90: 597-605Google Scholar, 28Pergolizzi R. Appierto V. Crosti M. Cavadini E. Cleris L. Guffanti A. Formelli F. Int. J. Cancer. 1999; 81: 829-834Google Scholar). 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 (29Delia D. Aiello A. Meroni L. Nicolini M. Reed J.C. Pierotti M.A. Carcinogenesis. 1997; 18: 943-948Google Scholar, 30Oridate N. Suzuki S. Higuchi M. Mitchell M.F. Hong W.K. Lotan R. J. Natl. Cancer Inst. 1997; 89: 1191-1198Google Scholar) or increases in ceramide (31Wang H. Maurer B.J. Reynolds C.P. Cabot M.C. Cancer Res. 2001; 61: 5102-5105Google Scholar, 32Panigone S. Bergomas R. Fontanella E. Prinetti A. Sandhoff K. Grabowski G.A. Delia D. FASEB J. 2001; 15: 1475-1477Google Scholar).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 (33Ponzanelli I. Giannı̀ M. Giavazzi R. Garofalo A. Nicoletti I. Reichert U. Erba E. Rambaldi A. Terao M. Garattini E. Blood. 2000; 95: 2672-2682Google Scholar, 34Appierto V. Cavadini E. Pergolizzi R. Cleris L. Lotan R. Canevari S. Formelli F. Br. J. Cancer. 2001; 84: 1528-1534Google Scholar). 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 (35Supino R. Crosti M. Clerici M. Warlters A. Cleris L. Zunino F. Formelli F. Int J Cancer. 1996; 65: 491-497Google Scholar), were continuously exposed to the drug, they developed a 10-fold resistance to the drug (34Appierto V. Cavadini E. Pergolizzi R. Cleris L. Lotan R. Canevari S. Formelli F. Br. J. Cancer. 2001; 84: 1528-1534Google Scholar). Differences in HPR uptake and metabolism were observed between sensitive and resistant cells (34Appierto V. Cavadini E. Pergolizzi R. Cleris L. Lotan R. Canevari S. Formelli F. Br. J. Cancer. 2001; 84: 1528-1534Google Scholar). 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 (34Appierto V. Cavadini E. Pergolizzi R. Cleris L. Lotan R. Canevari S. Formelli F. Br. J. Cancer. 2001; 84: 1528-1534Google Scholar). The expression of RA receptor β, a putative tumor suppressor (36Houle B. Rochette-Egly C. Bradley W.E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 985-989Google Scholar), was markedly increased, whereas the expression of cell surface molecules associated with tumor progression including HER-2 laminin receptor and β1 integrin was markedly reduced.An increase in the cellular levels of ceramide upon HPR treatment was reported in neuroblastoma (31Wang H. Maurer B.J. Reynolds C.P. Cabot M.C. Cancer Res. 2001; 61: 5102-5105Google Scholar, 37Maurer B.J. Metelitsa L.S. Seeger R.C. Cabot M.C. Reynolds C.P. J. Natl. Cancer Inst. 1999; 91: 1138-1146Google Scholar) and breast cancer cell lines (32Panigone S. Bergomas R. Fontanella E. Prinetti A. Sandhoff K. Grabowski G.A. Delia D. FASEB J. 2001; 15: 1475-1477Google Scholar). 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. Sphingolipid metabolism plays a pivotal role in the mechanism of apoptosis induced in tumor cells. Ceramide, produced under physiological (tumor necrosis factor α, γ-interferon, and interleukins) and pharmacological (anticancer drugs, including daunorubicin, vincristine, 1-α-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. 1Obeid L.M. Linardic C.M. Karolak L.A. Hannun Y.A. Science. 1993; 259: 1769-1771Google Scholar and 2Perry D.K. Hannun Y.A. Biochim. Biophys. Acta. 1998; 1436: 233-243Google Scholar). 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 (3Sietsma H. Veldman R.J. Kok J.W. J. Membr. Biol. 2001; 181: 153-162Google Scholar, 4Senchenkov A. Litwak D.A. Cabot M.C. J. Natl. Cancer Inst. 2001; 93: 347-357Google Scholar, 5Cabot M.C. Giuliano A.E. Volner A. Han T.Y. FEBS Lett. 1996; 394: 129-131Google Scholar, 6Lavie Y. Cao H. Bursten S.L. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1996; 271: 19530-19536Google Scholar, 7Lavie Y. Cao H. Volner A. Lucci A. Han T.Y. Geffen V. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1997; 272: 1682-1687Google Scholar, 8Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1999; 274: 1140-1146Google Scholar, 9Liu Y.Y. Han T.Y. Giuliano A.E. Ichikawa S. Hirabayashi Y. Cabot M.C. Exp. Cell Res. 1999; 252: 464-470Google Scholar, 10Liu Y.Y. Han T.Y. Giuliano A.E. Hansen N. Cabot M.C. J. Biol. Chem. 2000; 275: 7138-7143Google Scholar, 11Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. FASEB J. 2001; 15: 719-730Google Scholar, 12Veldman R.J. Klappe K. Hinrichs J. Hummel I. van der Schaaf G. Sietsma A. Kok J.W. FASEB J. 2002; 16: 1111-1113Google Scholar). High levels of GlcCer 1The abbreviations used are: GlcCer, β-Glc-(1–1)-Cer; LacCer, β-Gal-(1–4)-β-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 1The abbreviations used are: GlcCer, β-Glc-(1–1)-Cer; LacCer, β-Gal-(1–4)-β-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 (6Lavie Y. Cao H. Bursten S.L. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1996; 271: 19530-19536Google Scholar, 7Lavie Y. Cao H. Volner A. Lucci A. Han T.Y. Geffen V. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1997; 272: 1682-1687Google Scholar, 8Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1999; 274: 1140-1146Google Scholar, 9Liu Y.Y. Han T.Y. Giuliano A.E. Ichikawa S. Hirabayashi Y. Cabot M.C. Exp. Cell Res. 1999; 252: 464-470Google Scholar, 11Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. FASEB J. 2001; 15: 719-730Google Scholar, 12Veldman R.J. Klappe K. Hinrichs J. Hummel I. van der Schaaf G. Sietsma A. Kok J.W. FASEB J. 2002; 16: 1111-1113Google Scholar) and of glucosylceramide synthase activity (5Cabot M.C. Giuliano A.E. Volner A. Han T.Y. FEBS Lett. 1996; 394: 129-131Google Scholar, 8Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1999; 274: 1140-1146Google Scholar, 9Liu Y.Y. Han T.Y. Giuliano A.E. Ichikawa S. Hirabayashi Y. Cabot M.C. Exp. Cell Res. 1999; 252: 464-470Google Scholar) and/or expression (8Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1999; 274: 1140-1146Google Scholar, 9Liu Y.Y. Han T.Y. Giuliano A.E. Ichikawa S. Hirabayashi Y. Cabot M.C. Exp. Cell Res. 1999; 252: 464-470Google Scholar,11Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. FASEB J. 2001; 15: 719-730Google Scholar, 12Veldman R.J. Klappe K. Hinrichs J. Hummel I. van der Schaaf G. Sietsma A. Kok J.W. FASEB J. 2002; 16: 1111-1113Google Scholar) were detected in a number of drug-resistant cancer cell lines and in specimens from cancer patients not responding to chemotherapy treatment (13Lucci A. Cho W.I. Han T.Y. Giuliano A.E. Morton D.L. Cabot M.C. Anticancer Res. 1998; 18: 475-480Google Scholar). 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) (3Sietsma H. Veldman R.J. Kok J.W. J. Membr. Biol. 2001; 181: 153-162Google Scholar, 14Gottesmann M.M. Pastan I. Annu. Rev. Biochem. 1993; 62: 385-427Google Scholar). 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 (4Senchenkov A. Litwak D.A. Cabot M.C. J. Natl. Cancer Inst. 2001; 93: 347-357Google Scholar, 5Cabot M.C. Giuliano A.E. Volner A. Han T.Y. FEBS Lett. 1996; 394: 129-131Google Scholar, 6Lavie Y. Cao H. Bursten S.L. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1996; 271: 19530-19536Google Scholar, 7Lavie Y. Cao H. Volner A. Lucci A. Han T.Y. Geffen V. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1997; 272: 1682-1687Google Scholar, 8Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1999; 274: 1140-1146Google Scholar, 9Liu Y.Y. Han T.Y. Giuliano A.E. Ichikawa S. Hirabayashi Y. Cabot M.C. Exp. Cell Res. 1999; 252: 464-470Google Scholar, 10Liu Y.Y. Han T.Y. Giuliano A.E. Hansen N. Cabot M.C. J. Biol. Chem. 2000; 275: 7138-7143Google Scholar, 11Liu Y.Y. Han T.Y. Giuliano A.E. Cabot M.C. FASEB J. 2001; 15: 719-730Google Scholar). On the other hand, it has been shown (12Veldman R.J. Klappe K. Hinrichs J. Hummel I. van der Schaaf G. Sietsma A. Kok J.W. FASEB J. 2002; 16: 1111-1113Google Scholar, 15Kok J.W. Veldman R.J. Klappe K. Konino H. Filipeanu C.M. Muller M. Int. J. 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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 (19Nagy L. Thomazy V.A. Heyman R.A. Davies P.J. Cell. Death Differ. 1998; 5: 11-19Google Scholar). Retinoids exert their effects by regulating gene expression through two classes of nuclear receptors: retinoic acid (RA) receptors α, β, and γ and retinoid X receptors α, β, and γ. 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 (20Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Google Scholar). 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 (21Lotan R. FASEB J. 1996; 10: 1031-1039Google Scholar). 4-[3,7-Dimethyl-9-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2,4, 6,8- nonatetraenamido]-1-hydroxybenzene, known asN-(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 (22Formelli F. Barua A.B. Olson J.A. FASEB J. 1996; 10: 1014-1024Google Scholar). 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 (23De Palo G. Veronesi U. Camerini T. Formelli F. Mascotti G. Boni C. Fosser V. Del Vecchio M. Campa T. Costa A. Marubini E. J. Natl. 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Sandhoff K. Grabowski G.A. Delia D. FASEB J. 2001; 15: 1475-1477Google Scholar). 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 (33Ponzanelli I. Giannı̀ M. Giavazzi R. Garofalo A. Nicoletti I. Reichert U. Erba E. Rambaldi A. Terao M. Garattini E. Blood. 2000; 95: 2672-2682Google Scholar, 34Appierto V. Cavadini E. Pergolizzi R. Cleris L. Lotan R. Canevari S. Formelli F. Br. J. Cancer. 2001; 84: 1528-1534Google Scholar). 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 (35Supino R. Crosti M. Clerici M. Warlters A. Cleris L. Zunino F. Formelli F. Int J Cancer. 1996; 65: 491-497Google Scholar), were continuously exposed to the drug, they developed a 10-fold resistance to the drug (34Appierto V. Cavadini E. Pergolizzi R. Cleris L. Lotan R. Canevari S. Formelli F. Br. J. Cancer. 2001; 84: 1528-1534Google Scholar). Differences in HPR uptake and metabolism were observed between sensitive and resistant cells (34Appierto V. Cavadini E. Pergolizzi R. Cleris L. Lotan R. Canevari S. Formelli F. Br. J. Cancer. 2001; 84: 1528-1534Google Scholar). 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 (34Appierto V. Cavadini E. Pergolizzi R. Cleris L. Lotan R. Canevari S. Formelli F. Br. J. Cancer. 2001; 84: 1528-1534Google Scholar). The expression of RA receptor β, a putative tumor suppressor (36Houle B. Rochette-Egly C. Bradley W.E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 985-989Google Scholar), was markedly increased, whereas the expression of cell surface molecules associated with tumor progression including HER-2 laminin receptor and β1 integrin was markedly reduced. An increase in the cellular levels of ceramide upon HPR treatment was reported in neuroblastoma (31Wang H. Maurer B.J. Reynolds C.P. Cabot M.C. Cancer Res. 2001; 61: 5102-5105Google Scholar, 37Maurer B.J. Metelitsa L.S. Seeger R.C. Cabot M.C. Reynolds C.P. J. Natl. Cancer Inst. 1999; 91: 1138-1146Google Scholar) and breast cancer cell lines (32Panigone S. Bergomas R. Fontanella E. Prinetti A. Sandhoff K. Grabowski G.A. Delia D. FASEB J. 2001; 15: 1475-1477Google Scholar). 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.