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• W2023595695 abstract "Sphingosine kinase inhibitor (SKI) II has been reported as a dual inhibitor of sphingosine kinases (SKs) 1 and 2 and has been extensively used to prove the involvement of SKs and sphingosine-1-phosphate (S1P) in cellular processes. Dihydroceramide desaturase (Des1), the last enzyme in the de novo synthesis of ceramide (Cer), regulates the balance between dihydroceramides (dhCers) and Cers. Both SKs and Des1 have interest as therapeutic targets. Here we show that SKI II is a noncompetitive inhibitor (Ki = 0.3 μM) of Des1 activity with effect also in intact cells without modifying Des1 protein levels. Molecular modeling studies support that the SKI II-induced decrease in Des1 activity could result from inhibition of NADH-cytochrome b5 reductase. SKI II, but not the SK1-specific inhibitor PF-543, provoked a remarkable accumulation of dhCers and their metabolites, while both SKI II and PF-543 reduced S1P to almost undetectable levels. SKI II, but not PF543, reduced cell proliferation with accumulation of cells in the G0/G1 phase. SKI II, but not PF543, induced autophagy. These overall findings should be taken into account when using SKI II as a pharmacological tool, as some of the effects attributed to decreased S1P may actually be caused by augmented dhCers and/or their metabolites. Sphingosine kinase inhibitor (SKI) II has been reported as a dual inhibitor of sphingosine kinases (SKs) 1 and 2 and has been extensively used to prove the involvement of SKs and sphingosine-1-phosphate (S1P) in cellular processes. Dihydroceramide desaturase (Des1), the last enzyme in the de novo synthesis of ceramide (Cer), regulates the balance between dihydroceramides (dhCers) and Cers. Both SKs and Des1 have interest as therapeutic targets. Here we show that SKI II is a noncompetitive inhibitor (Ki = 0.3 μM) of Des1 activity with effect also in intact cells without modifying Des1 protein levels. Molecular modeling studies support that the SKI II-induced decrease in Des1 activity could result from inhibition of NADH-cytochrome b5 reductase. SKI II, but not the SK1-specific inhibitor PF-543, provoked a remarkable accumulation of dhCers and their metabolites, while both SKI II and PF-543 reduced S1P to almost undetectable levels. SKI II, but not PF543, reduced cell proliferation with accumulation of cells in the G0/G1 phase. SKI II, but not PF543, induced autophagy. These overall findings should be taken into account when using SKI II as a pharmacological tool, as some of the effects attributed to decreased S1P may actually be caused by augmented dhCers and/or their metabolites. Sphingosine kinases (SKs) are oncogenic lipid kinases that catalyze the formation of the mitogenic second messenger sphingosine-1-phosphate (S1P) at the expense of proapoptotic sphingosine (So) and ceramide (Cer). Thus, as central enzymes in modulating the Cer/S1P balance, SKs are attractive targets for cancer therapy (1Orr Gandy K.A. Obeid L.M. Targeting the sphingosine kinase/sphingosine 1-phosphate pathway in disease: review of sphingosine kinase inhibitors.Biochim. Biophys. Acta. 2013; 1831: 157-166Crossref PubMed Scopus (95) Google Scholar). Two SKs exist in humans, SK1 and SK2. SK1 has been extensively studied and there is a large body of evidence that proves its role in promoting cell survival, proliferation, and neoplastic transformation (2Heffernan-Stroud L.A. Obeid L.M. Sphingosine kinase 1 in cancer.Adv. Cancer Res. 2013; 117: 201-235Crossref PubMed Scopus (78) Google Scholar, 3Alshaker H. Sauer L. Monteil D. Ottaviani S. Srivats S. Böhler T. Pchejetski D. Therapeutic potential of targeting SK1 in human cancers.Adv. Cancer Res. 2013; 117: 143-200Crossref PubMed Scopus (40) Google Scholar, 4Pyne S. Bittman R. Pyne N.J. Sphingosine kinase inhibitors and cancer: seeking the golden sword of Hercules.Cancer Res. 2011; 71: 6576-6582Crossref PubMed Scopus (75) Google Scholar, 5Zhang Y. Wang Y. Wan Z. Liu S. Cao Y. Zeng Z. Sphingosine kinase 1 and cancer: a systematic review and meta-analysis.PLoS ONE. 2014; 9: e90362Crossref PubMed Scopus (70) Google Scholar). SK1 is also elevated in many human cancers, which appears to contribute to carcinogenesis, chemotherapeutic resistance, and poor patient outcome. SK2, however, has not been as well-characterized, and there are contradictions in the key physiological functions that have been proposed for this isoform. Despite this, many studies are now emerging that implicate SK2 in key roles in a variety of diseases, including the development of a range of solid tumors (6Neubauer H.A. Pitson S.M. Roles, regulation and inhibitors of sphingosine kinase 2.FEBS J. 2013; 280: 5317-5336Crossref PubMed Scopus (132) Google Scholar). The potential of SKs as therapeutic targets has boosted the development of small molecule inhibitors (4Pyne S. Bittman R. Pyne N.J. Sphingosine kinase inhibitors and cancer: seeking the golden sword of Hercules.Cancer Res. 2011; 71: 6576-6582Crossref PubMed Scopus (75) Google Scholar, 7Lim K.G. Gray A.I. Pyne S. Pyne N.J. Resveratrol dimers are novel sphingosine kinase 1 inhibitors and affect sphingosine kinase 1 expression and cancer cell growth and survival.Br. J. Pharmacol. 2012; 166: 1605-1616Crossref PubMed Scopus (50) Google Scholar, 8Schnute M.E. McReynolds M.D. Kasten T. Yates M. Jerome G. Rains J.W. Hall T. Chrencik J. Kraus M. Cronin C.N. et al.Modulation of cellular S1P levels with a novel, potent and specific inhibitor of sphingosine kinase-1.Biochem. J. 2012; 444: 79-88Crossref PubMed Scopus (209) Google Scholar, 9Gustin D.J. Li Y. Brown M.L. Min X. Schmitt M.J. Wanska M. Wang X. Connors R. Johnstone S. Cardozo M. et al.Structure guided design of a series of sphingosine kinase (SphK) inhibitors.Bioorg. Med. Chem. Lett. 2013; 23: 4608-4616Crossref PubMed Scopus (75) Google Scholar, 10Baek D.J. MacRitchie N. Pyne N.J. Pyne S. Bittman R. Synthesis of selective inhibitors of sphingosine kinase 1.Chem. Commun. (Camb.). 2013; 49: 2136-2138Crossref PubMed Scopus (47) Google Scholar, 11Byun H-S. Pyne S. MacRitchie N. Pyne N.J. Bittman R. Novel sphingosine-containing analogues selectively inhibit sphingosine kinase (SK) isozymes, induce SK1 proteasomal degradation and reduce DNA synthesis in human pulmonary arterial smooth muscle cells.Medchemcomm. 2013; 4: 1394-1399Crossref Scopus (56) Google Scholar, 12Raje M.R. Knott K. Kharel Y. Bissel P. Lynch K.R. Santos W.L. Design, synthesis and biological activity of sphingosine kinase 2 selective inhibitors.Bioorg. Med. Chem. 2012; 20: 183-194Crossref PubMed Scopus (35) Google Scholar, 13Liu K. Guo T.L. Hait N.C. Allegood J. Parikh H.I. Xu W. Kellogg G.E. Grant S. Spiegel S. Zhang S. Biological characterization of 3-(2-amino-ethyl)-5-[3-(4-butoxyl-phenyl)-propylidene]-thiazolidine-2,4-dione (K145) as a selective sphingosine kinase-2 inhibitor and anticancer agent.PLoS ONE. 2013; 8: e56471Crossref PubMed Scopus (67) Google Scholar, 14Wang Z. Min X. Xiao S-H. Johnstone S. Romanow W. Meininger D. Xu H. Liu J. Dai J. An S. et al.Molecular basis of sphingosine kinase 1 substrate recognition and catalysis.Structure. 2013; 21: 798-809Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). A detailed characterization of their pharmacology, particularly their selectivity against human SK1 and SK2, has been published (15Gao P. Peterson Y.K. Smith R.A. Smith C.D. Characterization of isoenzyme-selective inhibitors of human sphingosine kinases.PLoS ONE. 2012; 7: e44543Crossref PubMed Scopus (87) Google Scholar). The first known SK inhibitors were long chain base analogs such as N,N-dimethyl-D-erythro-sphingosine (DMS) (16Yatomi Y. Ruan F. Megidish T. Toyokuni T. Hakomori S. Igarashi Y. N,N-dimethylsphingosine inhibition of sphingosine kinase and sphingosine 1-phosphate activity in human platelets.Biochemistry. 1996; 35: 626-633Crossref PubMed Scopus (160) Google Scholar, 17Edsall L.C. Van Brocklyn J.R. Cuvillier O. Kleuser B. Spiegel S. N,N-Dimethylsphingosine is a potent competitive inhibitor of sphingosine kinase but not of protein kinase C: modulation of cellular levels of sphingosine 1-phosphate and ceramide.Biochemistry. 1998; 37: 12892-12898Crossref PubMed Scopus (216) Google Scholar) and L-threo-dihydrosphingosine (safingol) (18Olivera A. Kohama T. Tu Z. Milstien S. Spiegel S. Purification and characterization of rat kidney sphingosine kinase.J. Biol. Chem. 1998; 273: 12576-12583Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 19Maurer B.J. Melton L. Billups C. Cabot M.C. Reynolds C.P. Synergistic cytotoxicity in solid tumor cell lines between N-(4-hydroxyphenyl)retinamide and modulators of ceramide metabolism.J. Natl. Cancer Inst. 2000; 92: 1897-1909Crossref PubMed Scopus (188) Google Scholar, 20Kolesnick R. The therapeutic potential of modulating the ceramide/sphingomyelin pathway.J. Clin. Invest. 2002; 110: 3-8Crossref PubMed Scopus (381) Google Scholar, 21Dickson M.A. Carvajal R.D. Merrill A.H. Gonen M. Cane L.M. Schwartz G.K. A phase I clinical trial of safingol in combination with cisplatin in advanced solid tumors.Clin. Cancer Res. 2011; 17: 2484-2492Crossref PubMed Scopus (113) Google Scholar). While DMS inhibits both SK1 and SK2 by competing with the natural substrate So, safingol is a competitive inhibitor of SK1 (18Olivera A. Kohama T. Tu Z. Milstien S. Spiegel S. Purification and characterization of rat kidney sphingosine kinase.J. Biol. Chem. 1998; 273: 12576-12583Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar), but unlike DMS, it is a substrate of SK2 (22Liu H. Sugiura M. Nava V.E. Edsall L.C. Kono K. Poulton S. Milstien S. Kohama T. Spiegel S. Molecular cloning and functional characterization of a novel mammalian sphingosine kinase type 2 isoform.J. Biol. Chem. 2000; 275: 19513-19520Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar). A similar behavior is exhibited by FTY720 (23Tonelli F. Lim K.G. Loveridge C. Long J. Pitson S.M. Tigyi G. Bittman R. Pyne S. Pyne N.J. FTY720 and (S)-FTY720 vinylphosphonate inhibit sphingosine kinase 1 and promote its proteasomal degradation in human pulmonary artery smooth muscle, breast cancer and androgen-independent prostate cancer cells.Cell. Signal. 2010; 22: 1536-1542Crossref PubMed Scopus (157) Google Scholar). However, protein kinase C and other kinases are also inhibited by safingol (24Schwartz G.K. Jiang J. Kelsen D. Albino A.P. Protein kinase C: a novel target for inhibiting gastric cancer cell invasion.J. Natl. Cancer Inst. 1993; 85: 402-407Crossref PubMed Scopus (132) Google Scholar), DMS (25Igarashi Y. Hakomori S. Toyokuni T. Dean B. Fujita S. Sugimoto M. Ogawa T. el-Ghendy K. Racker E. Effect of chemically well-defined sphingosine and its N-methyl derivatives on protein kinase C and src kinase activities.Biochemistry. 1989; 28: 6796-6800Crossref PubMed Scopus (240) Google Scholar, 26Igarashi Y. Kitamura K. Toyokuni T. Dean B. Fenderson B. Ogawass T. Hakomori S. A specific enhancing effect of N,N-dimethylsphingosine on epidermal growth factor receptor autophosphorylation. Demonstration of its endogenous occurrence (and the virtual absence of unsubstituted sphingosine) in human epidermoid carcinoma A431 cells.J. Biol. Chem. 1990; 265: 5385-5389Abstract Full Text PDF PubMed Google Scholar), and FTY720 (27Sensken S-C. Gräler M.H. Down-regulation of S1P1 receptor surface expression by protein kinase C inhibition.J. Biol. Chem. 2010; 285: 6298-6307Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar), which are not, therefore, considered to be SK-specific inhibitors. A few compounds have been described as SK1-selective inhibitors, including sphingosine kinase inhibitor (SKI) I (28Paugh S.W. Paugh B.S. Rahmani M. Kapitonov D. Almenara J.A. Kordula T. Milstien S. Adams J.K. Zipkin R.E. Grant S. et al.A selective sphingosine kinase 1 inhibitor integrates multiple molecular therapeutic targets in human leukemia.Blood. 2008; 112: 1382-1391Crossref PubMed Scopus (204) Google Scholar, 29Kapitonov D. Allegood J.C. Mitchell C. Hait N.C. Almenara J.A. Adams J.K. Zipkin R.E. Dent P. Kordula T. Milstien S. et al.Targeting sphingosine kinase 1 inhibits Akt signaling, induces apoptosis, and suppresses growth of human glioblastoma cells and xenografts.Cancer Res. 2009; 69: 6915-6923Crossref PubMed Scopus (155) Google Scholar) and SKI 178 (30Hengst J.A. Wang X. Sk U.H. Sharma A.K. Amin S. Yun J.K. Development of a sphingosine kinase 1 specific small-molecule inhibitor.Bioorg. Med. Chem. Lett. 2010; 20: 7498-7502Crossref PubMed Scopus (48) Google Scholar). Another compound, SKI II, has been widely used as a SK1 inhibitor (31French K.J. Schrecengost R.S. Lee B.D. Zhuang Y. Smith S.N. Eberly J.L. Yun J.K. Smith C.D. Discovery and evaluation of inhibitors of human sphingosine kinase.Cancer Res. 2003; 63: 5962-5969PubMed Google Scholar, 32Truman J-P. García-Barros M. Obeid L.M. Hannun Y.A. Evolving concepts in cancer therapy through targeting sphingolipid metabolism.Biochim. Biophys. Acta. Epub ahead of print, 2013doi:10.1016/j.bbalip.2013.12.013Google Scholar). Nevertheless, SKI II is a dual SK1 and SK2 inhibitor, although it is inactive against other kinases. SKI II is a mixed inhibitor of So and ATP binding to SK1 (33Lim K.G. Tonelli F. Berdyshev E. Gorshkova I. Leclercq T. Pitson S.M. Bittman R. Pyne S. Pyne N.J. Inhibition kinetics and regulation of sphingosine kinase 1 expression in prostate cancer cells: functional differences between sphingosine kinase 1a and 1b.Int. J. Biochem. Cell Biol. 2012; 44: 1457-1464Crossref PubMed Scopus (32) Google Scholar), while the type of inhibition of SK2 has not been reported. Furthermore, SKI II (and also other inhibitors) has also been reported to induce proteasomal degradation of SK (4Pyne S. Bittman R. Pyne N.J. Sphingosine kinase inhibitors and cancer: seeking the golden sword of Hercules.Cancer Res. 2011; 71: 6576-6582Crossref PubMed Scopus (75) Google Scholar). The use of SKI II in the context of cancer therapy has been recently reviewed (32Truman J-P. García-Barros M. Obeid L.M. Hannun Y.A. Evolving concepts in cancer therapy through targeting sphingolipid metabolism.Biochim. Biophys. Acta. Epub ahead of print, 2013doi:10.1016/j.bbalip.2013.12.013Google Scholar). Dihydroceramide desaturase (Des1) is the last enzyme in the de novo synthesis of Cer. Blockade of Des1 produces an increase in dihydroceramides (dhCers), which have emerged as bioactive lipids and the target of several drugs (34Fabrias G. Muñoz-Olaya J. Cingolani F. Signorelli P. Casas J. Gagliostro V. Ghidoni R. Dihydroceramide desaturase and dihydrosphingolipids: debutant players in the sphingolipid arena.Prog. Lipid Res. 2012; 51: 82-94Crossref PubMed Scopus (74) Google Scholar), including fenretinide. Several studies have shown that cells respond to fenretinide treatment with a robust production of dhCers (35Kraveka J.M. Li L. Szulc Z.M. Bielawski J. Ogretmen B. Hannun Y.A. Obeid L.M. Bielawska A. Involvement of dihydroceramide desaturase in cell cycle progression in human neuroblastoma cells.J. Biol. Chem. 2007; 282: 16718-16728Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 36Wang H. Maurer B.J. Liu Y-Y. Wang E. Allegood J.C. Kelly S. Symolon H. Liu Y. Merrill A.H. Gouazé-Andersson V. et al.N-(4-hydroxyphenyl)retinamide increases dihydroceramide and synergizes with dimethylsphingosine to enhance cancer cell killing.Mol. Cancer Ther. 2008; 7: 2967-2976Crossref PubMed Scopus (87) Google Scholar, 37Valsecchi M. Aureli M. Mauri L. Illuzzi G. Chigorno V. Prinetti A. Sonnino S. Sphingolipidomics of A2780 human ovarian carcinoma cells treated with synthetic retinoids.J. Lipid Res. 2010; 51: 1832-1840Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 38Illuzzi G. Bernacchioni C. Aureli M. Prioni S. Frera G. Donati C. Valsecchi M. Chigorno V. Bruni P. Sonnino S. et al.Sphingosine kinase mediates resistance to the synthetic retinoid N-(4-hydroxyphenyl)retinamide in human ovarian cancer cells.J. Biol. Chem. 2010; 285: 18594-18602Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 39Mao Z. Sun W. Xu R. Novgorodov S. Szulc Z.M. Bielawski J. Obeid L.M. Mao C. . Alkaline ceramidase 2 (ACER2) and its product dihydrosphingosine mediate the cytotoxicity of N-(4-hydroxyphenyl)retinamide in tumor cells.J. Biol. Chem. 2010; 285: 29078-29090Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 40Rahmaniyan M. Curley R.W. Obeid L.M. Hannun Y.A. Kraveka J.M. Identification of dihydroceramide desaturase as a direct in vitro target for fenretinide.J. Biol. Chem. 2011; 286: 24754-24764Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 41Apraiz A. Idkowiak-Baldys J.K. Boyano M.D. Pérez-Yarza G. Hannun Y.A. Asumendi A. Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells.BMC Cancer. 2011; 11: 477Crossref PubMed Scopus (11) Google Scholar, 42Apraiz A. Idkowiak-Baldys J. Nieto-Rementería N. Boyano M.D. Hannun Y.A. Asumendi A. Dihydroceramide accumulation and reactive oxygen species are distinct and nonessential events in 4-HPR-mediated leukemia cell death.Biochem. Cell Biol. 2012; 90: 209-223Crossref PubMed Scopus (27) Google Scholar, 43Bikman B.T. Guan Y. Shui G. Siddique M.M. Holland W.L. Kim J.Y. Fabriàs G. Wenk M.R. Summers S.A. Fenretinide prevents lipid-induced insulin resistance by blocking ceramide biosynthesis.J. Biol. Chem. 2012; 287: 17426-17437Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 44Yasuo M. Mizuno S. Allegood J. Kraskauskas D. Bogaard H.J. Spiegel S. Voelkel N.F. Fenretinide causes emphysema, which is prevented by sphingosine 1-phoshate.PLoS ONE. 2013; 8: e53927Crossref PubMed Scopus (22) Google Scholar), and that this increase induces autophagy (45Zheng W. Kollmeyer J. Symolon H. Momin A. Munter E. Wang E. Kelly S. Allegood J.C. Liu Y. Peng Q. et al.Ceramides and other bioactive sphingolipid backbones in health and disease: lipidomic analysis, metabolism and roles in membrane structure, dynamics, signaling and autophagy.Biochim. Biophys. Acta. 2006; 1758: 1864-1884Crossref PubMed Scopus (443) Google Scholar, 46Holliday M.W. Cox S.B. Kang M.H. Maurer B.J. C22:0- and C24:0-dihydroceramides confer mixed cytotoxicity in T-cell acute lymphoblastic leukemia cell lines.PLoS ONE. 2013; 8: e74768Crossref PubMed Scopus (34) Google Scholar). Furthermore, experimental evidence exists on the connection between resistance to fenretinide and increased S1P production (38Illuzzi G. Bernacchioni C. Aureli M. Prioni S. Frera G. Donati C. Valsecchi M. Chigorno V. Bruni P. Sonnino S. et al.Sphingosine kinase mediates resistance to the synthetic retinoid N-(4-hydroxyphenyl)retinamide in human ovarian cancer cells.J. Biol. Chem. 2010; 285: 18594-18602Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 44Yasuo M. Mizuno S. Allegood J. Kraskauskas D. Bogaard H.J. Spiegel S. Voelkel N.F. Fenretinide causes emphysema, which is prevented by sphingosine 1-phoshate.PLoS ONE. 2013; 8: e53927Crossref PubMed Scopus (22) Google Scholar) due to an increased SK activity and SK1 mRNA and protein levels (38Illuzzi G. Bernacchioni C. Aureli M. Prioni S. Frera G. Donati C. Valsecchi M. Chigorno V. Bruni P. Sonnino S. et al.Sphingosine kinase mediates resistance to the synthetic retinoid N-(4-hydroxyphenyl)retinamide in human ovarian cancer cells.J. Biol. Chem. 2010; 285: 18594-18602Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). In agreement, it has been reported that SK inhibitors abolish fenretinide resistance or synergize with fenretinide to enhance cancer cell death (36Wang H. Maurer B.J. Liu Y-Y. Wang E. Allegood J.C. Kelly S. Symolon H. Liu Y. Merrill A.H. Gouazé-Andersson V. et al.N-(4-hydroxyphenyl)retinamide increases dihydroceramide and synergizes with dimethylsphingosine to enhance cancer cell killing.Mol. Cancer Ther. 2008; 7: 2967-2976Crossref PubMed Scopus (87) Google Scholar, 38Illuzzi G. Bernacchioni C. Aureli M. Prioni S. Frera G. Donati C. Valsecchi M. Chigorno V. Bruni P. Sonnino S. et al.Sphingosine kinase mediates resistance to the synthetic retinoid N-(4-hydroxyphenyl)retinamide in human ovarian cancer cells.J. Biol. Chem. 2010; 285: 18594-18602Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 41Apraiz A. Idkowiak-Baldys J.K. Boyano M.D. Pérez-Yarza G. Hannun Y.A. Asumendi A. Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells.BMC Cancer. 2011; 11: 477Crossref PubMed Scopus (11) Google Scholar). In one of the above studies, the SK inhibitor, SKI II, was used to show the involvement of SK1 in the resistance of A2780 ovary cancer cells to fenretinide (38Illuzzi G. Bernacchioni C. Aureli M. Prioni S. Frera G. Donati C. Valsecchi M. Chigorno V. Bruni P. Sonnino S. et al.Sphingosine kinase mediates resistance to the synthetic retinoid N-(4-hydroxyphenyl)retinamide in human ovarian cancer cells.J. Biol. Chem. 2010; 285: 18594-18602Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The authors showed that treatment of fenretinide-resistant cells with SKI II effectively reduced S1P production and sensitized cells to the cytotoxic effect of fenretinide, and that cells treated with the combination fenretinide/SKI II experienced a dramatic increase in dhCer and sphinganine. Of note, treatment with SKI II alone significantly elevated cellular dhCer levels, but not Cer levels, although this increase was much lower than that observed in fenretinide and fenretinide/SKI II treatments. In another report (15Gao P. Peterson Y.K. Smith R.A. Smith C.D. Characterization of isoenzyme-selective inhibitors of human sphingosine kinases.PLoS ONE. 2012; 7: e44543Crossref PubMed Scopus (87) Google Scholar), SKI II was also found to produce an increase in N-hexadecanoyl­dihydrosphingosine (C16dhCer) in A498 kidney adenocarcinoma cells. Surprisingly, none of the articles addressed and discussed these observations. In this work, we show that besides inhibiting SK, SKI II reduces the activity of Des1 and increases the levels of dhCers and their metabolic products. This finding should be taken into account when using SKI II to investigate the role of SK in cell biology, as some of the effects attributed to increased S1P may actually be caused by augmented dhCers and/or their metabolites. Conclusions drawn based on the exclusive use of SKI II as a pharmacological tool should be revisited. SKI II (Chemical Abstracts Registry number 312636-16-1) and PF-543 (Chemical Abstracts Registry number 1415562-82-1) were from Calbiochem. The compounds N-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-D-erythro-dihydrosphin­gosine (dhCerC6NBD) and XM462 (47Munoz-Olaya J.M. Matabosch X. Bedia C. Egido-Gabás M. Casas J. Llebaria A. Delgado A. Fabriàs G. Synthesis and biological activity of a novel inhibitor of dihydroceramide desaturase.ChemMedChem. 2008; 3: 946-953Crossref PubMed Scopus (62) Google Scholar) were synthesized in our laboratories. Internal standards for lipidomics were from Avanti Polar Lipids. Minimum essential media, fetal bovine serum, nonessential amino acids, penicillin/streptomycin, 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT), BSA, NADH, Tween 20, trypsin-EDTA, and the protease inhibitors were from Sigma. Laemmli buffer and acrylamide were from BioRad, SDS from Fluka, and the microBCA protein assay kit from Thermo Scientific. Protease inhibitor cocktail contained 2 μg/ml aprotinin, 5 μg/ml leupeptin, and 1 mM phenylmethylsulphonyl fluoride. Antibodies: anti-SK1 (rabbit) was from Cell Signaling; anti-Des1 (rabbit) and microtubule-associated protein 1-light chain 3 (LC3) II (rabbit) was from Abcam; and β-actin (mouse) was from Sigma. HRP secondary antibodies were from GE Healthcare. The human gastric cancer cell line, HGC 27, was cultured at 37°C in 5% CO2 in minimum essential medium supplemented with 10% fetal bovine serum, 1% nonessential amino acids, and 100 ng/ml each of penicillin and streptomycin. Cells were routinely grown at a 60% maximum confluence. Glioblastoma T98G and HeLa cells were maintained at 37°C in 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 ng/ml each of penicillin and streptomycin. Cell viability was examined in triplicate samples by the MTT method. Cells were seeded at a density of 0.1 × 106 cells/ml, 0.1 ml/well (96-well plates). Twenty-four hours after seeding, seven 2:3 serial dilutions of SKI II, from 200 to 18 μM, and vehicle (100% activity) were added and viability was determined 24 h later. Cells were seeded at 150,000 cells/ml into 6-well plates (1 ml/well). Cells were allowed to adhere for 24 h, and then they were treated with vehicle (0.1% ethanol) or SKI II (10 μM in 0.1% ethanol in medium). After exposure for 24 h, cell media were discarded and cells were washed with 400 μl PBS-EDTA 1% BSA and harvested with 400 μl trypsin-EDTA 1% BSA (37°C/2 min). Cells were pulled down by centrifugation at 1,300 rpm/3 min; cell pellet was washed once with 400 μl PBS-EDTA 1% BSA and again centrifuged at the same speed/time. Cells were fixed with a −20°C overnight incubation with a 70% ethanol (9.5 ml) in 1× PBS solution (0.5 ml). Fixed cells were pulled down, washed once with PBS-EDTA 1% BSA and stained at 37°C for 2 h with propidium iodide solution (0.1 mg/ml in PBS) and RNase, DNase-free (10 μg/ml). Stained cells were analyzed by using a Guava EasyCyte™ flow cytometer (Merck Millipore, Billerica, MA). Data analysis was performed using the Multicycle AV program (Phoenix Flow Systems, San Diego, CA). To prepare the cell lysate for Des1 activity determination in vitro, a suspension of 106 cells/ml per sample was centrifuged (1,400 rpm/3 min); the pellets were washed twice with PBS and resuspended in 0.1 ml of 0.2 M phosphate buffer (pH 7.4). The ice-cooled suspension was submitted to one round of bath sonication (30 s)/rest on ice (30 s), five rounds of bath sonication (15 s)/rest on ice (15 s), and one final round of bath sonication (30 s)/rest on ice (30 s). A 3.5% (v/v) solution of the required amount of stock substrate solution (0.5, 1.0, 1.5, and 2 mM in ethanol) in a BSA solution [3.3 mg/ml in 0.2 M phosphate buffer (pH 7.4)] was prepared to have the needed substrate concentrations (inhibition experiments, 35 μM; kinetics experiment: 17.5, 35, 52, and 70 μM). To each tube containing lysate from 106 cells was added: 85 μl of BSA-substrate mix (final substrate concentrations: inhibition experiments, 10 μM; kinetics experiment: 5, 10, 15, and 20 μM), 3 μl of SKI II stock solution in ethanol (final concentrations: inhibition experiments, 10 μM; kinetics experiment: 2.5 and 0.6 μM), 3 μl of XM462 stock solution in ethanol (10 μM final concentration), or 3 μl ethanol (vehicle control). Then 30 μl of NADH [20 mg/ml in 0.2 M phosphate buffer (pH 7.4)] and 82 μl of 0.2 M phosphate buffer (pH 7.4) were added to have a final volume of 300 μl. The reaction mixture was incubated at 37°C for 4 h. To stop the reaction, 0.7 ml/sample of methanol was added to each tube, mixed by vortex, and kept at 4°C overnight. The mixture was centrifuged (10,000 rpm/3 min), the clear supernatants were transferred to HPLC vials and 25 μl were injected. Instrumental analysis was carried out by HPLC with a fluorimetric detector as reported (47Munoz-Olaya J.M. Matabosch X. Bedia C. Egido-Gabás M. Casas J. Llebaria A. Delgado A. Fabriàs G. Synthesis and biological activity of a novel inhibitor of dihydroceramide desaturase.ChemMedChem. 2008; 3: 946-953Crossref PubMed Scopus (62) Google Scholar). To determine the compound's activity on Des1 in intact cells, cells were seeded in 24-well plates (106 cell/ml, 0.4 ml/well). Twenty-four hours after seeding, the medium was replaced by fresh complete medium containing substrate and either SKI II or XM462, which was used as a positive control (vehicle in controls) (0.4 ml/well). This solution was prepared as follows: 8 μl each of substrate and test compound solutions (10 mM in ethanol) were taken and diluted with medium to 1 ml and then 50 μl each of substrate and test compound solutions were added to each well (substrate = SKI II = XM462 = 10 μM) prior to addition of 0.3 ml/well of medium. After incubation at 37°C for 4 h, the media were collected, cells were washed with PBS (0.2 ml/well), and the washing solution was mixed with the collected media. Cells were harvested by trypsinization (trypsin/EDTA, 0.2 ml/well), washed with PBS, and the pellet was resuspended in water (0.1 ml) and sonicated (water bath) for 30 s. Methanol (media, 0.4 ml; cell lysate, 0.9 ml) was added to each tube and the mixture was stirred and kept at 4°C overnight. Then the suspension was centrifuged (10,000 rpm for 3 min), the solution was transferred to HPLC vials, and either 25 μl (media) or 0.1 ml (cells) was injected. For protein analysis, 1–2 × 104 cells were plated in 6-well plates and were allowed to adhere for 24 h. Cells were treated with 10 μM of SKI II, 8 μM of XM462, 1 μM of PF-543, or ethanol as control for 4 or 24 h, collected with trypsin, and then pellets were washed twice with cold PBS. Cell lysis was performed with 30–40 μl of lysis buffer [150 mM NaCl, 1% Igepal-CA630, 50 mM Tris-HCl (pH 8), 2 μg/ml aprotinin, 5 μg/ml leupeptin, and 1 mM PMSF] by three cycles of bath sonication (5 s)/rest on ice (10 s). Then samples were kept on ice for 30 min and centrifuged for 3 min at 10,000 rpm. Supernatants were collected and protein determination was performed using the Micro BCA™ protein assay kit. Supernatants were combined with Laemmli sample buffer and boiled for 5 min. Equal amounts of proteins (Des1 and SK1, 30 μg; LC3, 20 μg) were loaded onto a 12% polyacrylamide gel, separated by electrophoresis at 100 V/90 min, and transferred onto a polyvinylidene fluoride membrane (100 V/1 h). Unspecific binding sites were then blocked with 5% milk in TBS with 0.1% Twe" @default.
• W2023595695 created "2016-06-24" @default.
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• W2023595695 date "2014-08-01" @default.
• W2023595695 modified "2023-10-17" @default.
• W2023595695 title "Inhibition of dihydroceramide desaturase activity by the sphingosine kinase inhibitor SKI II" @default.
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• W2023595695 doi "https://doi.org/10.1194/jlr.m049759" @default.
• W2023595695 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4109765" @default.
• W2023595695 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/24875537" @default.
• W2023595695 hasPublicationYear "2014" @default.
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