Tamara Mirzapoiazova

BACKGROUND: The possibility that μ opioid agonists can influence cancer recurrence is a subject of recent interest. Epidemiologic studies suggested that there were differences in cancer recurrence in breast and prostate cancer contingent on anesthetic regimens. In this study, we identify a possible mechanism for these epidemiologic findings on the basis of μ opioid receptor (MOR) regulation of Lewis lung carcinoma (LLC) tumorigenicity in cell and animal models. METHODS: We used human lung tissue and human non-small cell lung cancer (NSCLC) cell lines and evaluated MOR expression using immunoblot and immunohistochemical analysis. LLC cells were treated with the peripheral opioid antagonist methylnaltrexone (MNTX) or MOR shRNA and evaluated for proliferation, invasion, and soft agar colony formation in vitro and primary tumor growth and lung metastasis in C57BL/6 and MOR knockout mice using VisEn fluorescence mediated tomography imaging and immunohistochemical analysis. RESULTS: We provide several lines of evidence that the MOR may be a potential target for lung cancer, a disease with high mortality and few treatment options. We first observed that there is ∼5- to 10-fold increase in MOR expression in lung samples from patients with NSCLC and in several human NSCLC cell lines. The MOR agonists morphine and [D-Ala(2), N-MePhe(4), Gly-ol]-enkephalin (DAMGO) increased in vitro LLC cell growth. Treatment with MNTX or silencing MOR expression inhibited LLC invasion and anchorage-independent growth by 50%-80%. Injection of MOR silenced LLC lead to a ∼65% reduction in mouse lung metastasis. In addition, MOR knockout mice do not develop significant tumors when injected with LLC in comparison with wild-type controls. Finally, continuous infusion of the peripheral opioid antagonist MNTX attenuates primary LLC tumor growth and reduces lung metastasis. CONCLUSIONS: Taken together, our data suggest a possible direct effect of opiates on lung cancer progression, and provide a plausible explanation for the epidemiologic findings. Our observations further suggest a possible therapeutic role for opioid antagonists.

Recent epidemiologic studies implying differences in cancer recurrence based on anesthetic regimens raise the possibility that the mu opioid receptor (MOR) can influence cancer progression. Based on our previous observations that overexpression of MOR in human non-small cell lung cancer (NSCLC) cells increased tumor growth and metastasis, this study examined whether MOR regulates growth factor receptor signaling and epithelial mesenchymal transition (EMT) in human NSCLC cells. We utilized specific siRNA, shRNA, chemical inhibitors and overexpression vectors in human H358 NSCLC cells that were either untreated or treated with various concentrations of DAMGO, morphine, fentanyl, EGF or IGF. Cell function assays, immunoblot and immunoprecipitation assays were then performed. Our results indicate MOR regulates opioid and growth factor-induced EGF receptor signaling (Src, Gab-1, PI3K, Akt and STAT3 activation) which is crucial for consequent human NSCLC cell proliferation and migration. In addition, human NSCLC cells treated with opioids, growth factors or MOR overexpression exhibited an increase in snail, slug and vimentin and decrease ZO-1 and claudin-1 protein levels, results consistent with an EMT phenotype. Further, these effects were reversed with silencing (shRNA) or chemical inhibition of MOR, Src, Gab-1, PI3K, Akt and STAT3 (p<0.05). Our data suggest a possible direct effect of MOR on opioid and growth factor-signaling and consequent proliferation, migration and EMT transition during lung cancer progression. Such an effect provides a plausible explanation for the epidemiologic findings.

BACKGROUND: Recent epidemiologic studies suggesting that there were differences in cancer recurrence contingent on anesthetic regimens have raised the possibility that μ-opioid agonists can influence cancer progression. Based on our previous studies indicating the μ-opioid receptor (MOR) is up-regulated in several types of non-small cell lung cancer, this study examined the functional significance of MOR overexpression to elucidate a possible mechanism for the epidemiologic findings. METHODS: Stable vector control and MOR1 overexpressing human bronchioloalveolar carcinoma cells were evaluated using immunoblot analysis, proliferation and transendothelial extravasation assays with or without Akt inhibitor, mTOR inhibitor (temsirolimus), or the peripheral MOR antagonist, methylnaltrexone. In human lung cancer xenograft models, primary tumor growth rates and lung metastasis were analyzed using consecutive tumor volume measurements and nestin immunoreactivity in lungs of the nude mouse model. RESULTS: The authors provide evidence that MOR is an important regulator of lung cancer progression. MOR overexpression increased Akt and mTOR activation, proliferation, and extravasation in human bronchioloalveolar carcinoma cells. In vivo, overexpression of MOR in human bronchoalveolar carcinoma cells increased primary tumor growth rates in nude mice by approximately 2.5-fold and lung metastasis by approximately 20-fold compared with vector control cells (n = 4 per condition). CONCLUSIONS: The overexpression data suggest a possible direct effect of MOR on Akt and mTOR activation and lung cancer progression. Such an effect provides a plausible explanation for the epidemiologic findings. The authors' observations further suggest that exploration of MOR in non-small cell lung carcinoma merits further study both as a diagnostic and therapeutic option.

The therapeutic options for ameliorating the profound vascular permeability, alveolar flooding, and organ dysfunction that accompanies acute inflammatory lung injury (ALI) remain limited. Extending our previous finding that the intravenous administration of the sphingolipid angiogenic factor, sphingosine 1-phosphate (S1P), attenuates inflammatory lung injury and vascular permeability via ligation of S1PR(1), we determine that a direct intratracheal or intravenous administration of S1P, or a selective S1P receptor (S1PR(1)) agonist (SEW-2871), produces highly concentration-dependent barrier-regulatory responses in the murine lung. The intratracheal or intravenous administration of S1P or SEW-2871 at < 0.3 mg/kg was protective against LPS-induced murine lung inflammation and permeability. However, intratracheal delivery of S1P at 0.5 mg/kg (for 2 h) resulted in significant alveolar-capillary barrier disruption (with a 42% increase in bronchoalveolar lavage protein), and produced rapid lethality when delivered at 2 mg/kg. Despite the greater selectivity for S1PR(1), intratracheally delivered SEW-2871 at 0.5 mg/kg also resulted in significant alveolar-capillary barrier disruption, but was not lethal at 2 mg/kg. Consistent with the S1PR(1) regulation of alveolar/vascular barrier function, wild-type mice pretreated with the S1PR(1) inverse agonist, SB-649146, or S1PR(1)(+/-) mice exhibited reduced S1P/SEW-2871-mediated barrier protection after challenge with LPS. In contrast, S1PR(2)(-/-) knockout mice as well as mice with reduced S1PR(3) expression (via silencing S1PR3-containing nanocarriers) were protected against LPS-induced barrier disruption compared with control mice. These studies underscore the potential therapeutic effects of highly selective S1PR(1) receptor agonists in reducing inflammatory lung injury, and highlight the critical role of the S1P delivery route, S1PR(1) agonist concentration, and S1PR(1) expression in target tissues.

Novel immunomodulatory molecule FTY720 is a synthetic analog of myriocin, but unlike myriocin FTY720 does not inhibit serine palmitoyltransferase. Although many of the effects of FTY720 are ascribed to its phosphorylation and subsequent sphingosine 1-phosphate (S1P)-like action through S1P1,3–5 receptors, studies on modulation of intracellular balance of signaling sphingolipids by FTY720 are limited. In this study, we used stable isotope pulse labeling of human pulmonary artery endothelial cells with l-[U-13C, 15N]serine as well as in vitro enzymatic assays and liquid chromatography-tandem mass spectrometry methodology to characterize FTY720 interference with sphingolipid de novo biosynthesis. In human pulmonary artery endothelial cells, FTY720 inhibited ceramide synthases, resulting in decreased cellular levels of dihydroceramides, ceramides, sphingosine, and S1P but increased levels of dihydrosphingosine and dihydrosphingosine 1-phosphate (DHS1P). The FTY720-induced modulation of sphingolipid de novo biosynthesis was similar to that of fumonisin B1, a classical inhibitor of ceramide synthases, but differed in the efficiency to inhibit biosynthesis of short-chain versus long-chain ceramides. In vitro kinetic studies revealed that FTY720 is a competitive inhibitor of ceramide synthase 2 toward dihydrosphingosine with an apparent Ki of 2.15 μm. FTY720-induced up-regulation of DHS1P level was mediated by sphingosine kinase (SphK) 1, but not SphK2, as confirmed by experiments using SphK1/2 silencing with small interfering RNA. Our data demonstrate for the first time the ability of FTY720 to inhibit ceramide synthases and modulate the intracellular balance of signaling sphingolipids. These findings open a novel direction for therapeutic applications of FTY720 that focuses on inhibition of ceramide biosynthesis, ceramide-dependent signaling, and the up-regulation of DHS1P generation in cells. Novel immunomodulatory molecule FTY720 is a synthetic analog of myriocin, but unlike myriocin FTY720 does not inhibit serine palmitoyltransferase. Although many of the effects of FTY720 are ascribed to its phosphorylation and subsequent sphingosine 1-phosphate (S1P)-like action through S1P1,3–5 receptors, studies on modulation of intracellular balance of signaling sphingolipids by FTY720 are limited. In this study, we used stable isotope pulse labeling of human pulmonary artery endothelial cells with l-[U-13C, 15N]serine as well as in vitro enzymatic assays and liquid chromatography-tandem mass spectrometry methodology to characterize FTY720 interference with sphingolipid de novo biosynthesis. In human pulmonary artery endothelial cells, FTY720 inhibited ceramide synthases, resulting in decreased cellular levels of dihydroceramides, ceramides, sphingosine, and S1P but increased levels of dihydrosphingosine and dihydrosphingosine 1-phosphate (DHS1P). The FTY720-induced modulation of sphingolipid de novo biosynthesis was similar to that of fumonisin B1, a classical inhibitor of ceramide synthases, but differed in the efficiency to inhibit biosynthesis of short-chain versus long-chain ceramides. In vitro kinetic studies revealed that FTY720 is a competitive inhibitor of ceramide synthase 2 toward dihydrosphingosine with an apparent Ki of 2.15 μm. FTY720-induced up-regulation of DHS1P level was mediated by sphingosine kinase (SphK) 1, but not SphK2, as confirmed by experiments using SphK1/2 silencing with small interfering RNA. Our data demonstrate for the first time the ability of FTY720 to inhibit ceramide synthases and modulate the intracellular balance of signaling sphingolipids. These findings open a novel direction for therapeutic applications of FTY720 that focuses on inhibition of ceramide biosynthesis, ceramide-dependent signaling, and the up-regulation of DHS1P generation in cells. FTY720 2The abbreviations used are: FTY720, 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol; FTY720-P, (S)-FTY720 phosphate; SphK, sphingosine kinase; Sph, sphingosine; DHSph, dihydrosphingosine; S1P, sphingosine 1-phosphate; DHS1P, dihydrosphingosine 1-phosphate; Cer, ceramides; DHCer, dihydroceramides; SPT, serine palmitoyltransferase; CerS, ceramide synthase; cPLA2, cytosolic phospholipase A2; LC/MS/MS, liquid chromatography-tandem mass spectrometry; HPAEC, human pulmonary artery endothelial cell; FB1, fumonisin B1; siRNA, small interfering RNA; ESI, electrospray ionization; MRM, multiple reaction monitoring; BSA, bovine serum albumin.2The abbreviations used are: FTY720, 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol; FTY720-P, (S)-FTY720 phosphate; SphK, sphingosine kinase; Sph, sphingosine; DHSph, dihydrosphingosine; S1P, sphingosine 1-phosphate; DHS1P, dihydrosphingosine 1-phosphate; Cer, ceramides; DHCer, dihydroceramides; SPT, serine palmitoyltransferase; CerS, ceramide synthase; cPLA2, cytosolic phospholipase A2; LC/MS/MS, liquid chromatography-tandem mass spectrometry; HPAEC, human pulmonary artery endothelial cell; FB1, fumonisin B1; siRNA, small interfering RNA; ESI, electrospray ionization; MRM, multiple reaction monitoring; BSA, bovine serum albumin. is a synthetic analog of sphingosine and is currently being studied as a potent immunosuppressive and immunomodulatory agent (1Zhang Z. Schluesener H.J. Mini Rev. Med. Chem. 2007; 7: 845-850Crossref PubMed Scopus (26) Google Scholar, 2Hiestand P.C. Rausch M. Meier D.P. Foster C.A. Prog. Drug Res. 2008; 66: 363-381Google Scholar, 3Mansoor M. Melendez A.J. Rev. Recent Clin. Trials. 2008; 3: 62-69Crossref PubMed Scopus (32) Google Scholar). FTY720-induced immunosuppression is ascribed, in part, to its protective effect on endothelial cell barrier function that results in inhibition of lymphocyte egress from lymph nodes and down-regulation of innate and adaptive immune responses (4Schwab S.R. Cyster J.G. Nat. Immunol. 2007; 8: 1295-1301Crossref PubMed Scopus (481) Google Scholar). As endothelial cells predominantly express the sphingosine 1-phosphate 1 (S1P1) receptor and its activation initiates signaling that results in the assembly of VE-cadherin-based adherens junctions (5Lee M.J. Thangada S. Claffey K.P. Ancellin N. Liu C.H. Kluk M. Volpi M. Sha'afi R.I. Hla T. Cell. 1999; 99: 301-312Abstract Full Text Full Text PDF PubMed Scopus (863) Google Scholar), it is thought that the phosphorylation of FTY720 and the binding of FTY720-P to the S1P1 receptor determine its effect on vasculature (1Zhang Z. Schluesener H.J. Mini Rev. Med. Chem. 2007; 7: 845-850Crossref PubMed Scopus (26) Google Scholar). Recently it became evident that the action of FTY720 is more complex as several other direct protein targets were identified. Thus, FTY720 was found to bind to and inhibit the cannabinoid CB1 receptor (6Paugh S.W. Cassidy M.P. He H. Milstien S. Sim-Selley L.J. Spiegel S. Selley D.E. Mol. Pharmacol. 2006; 70: 41-50Crossref PubMed Scopus (65) Google Scholar), to inhibit cytosolic phospholipase A2 (cPLA2), and to counteract ceramide 1-phosphate-induced cPLA2 activation (7Payne S.G. Oskeritzian C.A. Griffiths R. Subramanian P. Barbour S.E. Chalfant C.E. Milstien S. Spiegel S. Blood. 2007; 109: 1077-1085Crossref PubMed Scopus (143) Google Scholar). Additionally FTY720 but not FTY720-P was shown to inhibit S1P lyase (8Bandhuvula P. Tam Y.Y. Oskouian B. Saba J.D. J. Biol. Chem. 2005; 280: 33697-33700Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), which degrades S1P to ethanolamine phosphate and (E)-2-hexadecenal and regulates the removal of sphingoid bases from the cumulative pool of sphingolipids. These findings characterize FTY720 as a molecule with a multitargeted mode of action whose cellular effects are complicated by its metabolic transformation to FTY720-P, a structural and functional analog of S1P. Phosphorylation of FTY720 to FTY720-P by sphingosine kinases (SphKs) is the only reported metabolic transformation of FTY720 and has been actively explored because of its link to S1P-mediated signaling (1Zhang Z. Schluesener H.J. Mini Rev. Med. Chem. 2007; 7: 845-850Crossref PubMed Scopus (26) Google Scholar, 2Hiestand P.C. Rausch M. Meier D.P. Foster C.A. Prog. Drug Res. 2008; 66: 363-381Google Scholar, 9Oo M.L. Thangada S. Wu M.T. Liu C.H. Macdonald T.L. Lynch K.R. Lin C.Y. Hla T. J. Biol. Chem. 2007; 282: 9082-9089Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, 10Kharel Y. Lee S. Snyder A.H. Sheasley-O'neill S.L. Morris M.A. Setiady Y. Zhu R. Zigler M.A. Burcin T.L. Ley K. Tung K.S. Engelhard V.H. Macdonald T.L. Pearson-White S. Lynch K.R. J. Biol. Chem. 2005; 280: 36865-36872Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Recent studies suggest that the endogenous balance between S1P and ceramide molecules regulates prosurvival and proapoptotic signaling cascades, which determine the outcome of cellular response to different stress conditions (11Petrache I. Natarajan V. Zhen L. Medler T.R. Richter A.T. Cho C. Hubbard W.C. Berdyshev E.V. Tuder R.M. Nat. Med. 2005; 11: 491-498Crossref PubMed Scopus (419) Google Scholar, 12Morales A. Fernandez-Checa J.C. Mini Rev. Med. Chem. 2007; 7: 371-382Crossref PubMed Scopus (33) Google the efficiency of A. Fernandez-Checa J.C. Mini Rev. Med. Chem. 2007; 7: 371-382Crossref PubMed Scopus (33) Google Scholar, Y. A. C.E. J. B. J. Biol. Chem. 2007; 282: Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. 2006; PubMed Scopus Google Scholar). the that FTY720 sphingosine and is a of B. B. M. R. N. T. A. Blood. 2006; PubMed Scopus Google Scholar, S.W. S.G. Barbour S.E. Milstien S. Spiegel S. PubMed Scopus Google Scholar, A. P. N. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, in Scholar), are reported studies on the effect of FTY720 on the balance of signaling sphingolipids. between proapoptotic and prosurvival 1-phosphate molecules are because it is that fumonisin an inhibitor of synthases, not only the of and the intracellular of dihydrosphingosine but the cellular level of DHS1P H. 2007; PubMed Scopus Google Scholar, Lee S. S. J. A. 2006; PubMed Scopus (26) Google Scholar). In of it is to with a ability to with the the balance in cells. this we the effect of FTY720 on metabolic to ceramide and sphingoid 1-phosphate generation in human pulmonary artery endothelial cells by using a stable isotope pulse labeling and liquid chromatography-tandem mass spectrometry of signaling sphingolipids. demonstrate that of with FTY720 results in the inhibition of de novo ceramide with a in and DHS1P in cells. FTY720 a direct inhibition of ceramide synthases in an in vitro it was with the classical inhibitor of ceramide synthases, Our findings ceramide synthase as a novel for FTY720 a direction for its therapeutic through the inhibition of ceramide biosynthesis, ceramide-dependent signaling, and the up-regulation of DHS1P generation in cells. and DHSph, a analog of S1P, DHS1P, a analog of S1P sphingosine and the and were from was from The were in the sphingoid were in a of and were FTY720 and FTY720-P were as R. 2006; Scopus Google Scholar). was from were from were by were from and were in between and were used for were in a with of with and pool and and and were from to in were with agent with in to the 1 of bovine serum was and the cells were for an with FTY720 and by The efficiency of silencing was confirmed by time and by and was using a and time and were to of and using for the human The were used for and in was to its The of in was as 2 to the of its being to the of its of reaction were with that were from and not for were in the of the first being to for and for were by a and J. PubMed Scopus Google with the of for and were used as and were the of The were in and were to determine the as J. PubMed Scopus Google Scholar). were a of in to and to of sphingoid ceramides, and sphingoid of FTY720, and FTY720 of the sphingolipids were by The used was an mass Foster with a with an liquid and The sphingolipids were electrospray with multiple reaction of sphingoid bases and the of used in with using a of J. J. Chem. Rev. PubMed Scopus Google Scholar, A.H. K. J. PubMed Google Scholar). of sphingoid bases was with a and a from with to with a of The used for of sphingoid bases were as and were using a and a from with to with a of for the of ceramide were as and S1P and DHS1P were as with as the using liquid ESI, and of this were E.V. J.G. Natarajan V. Hubbard W.C. 2005; PubMed Scopus Google Scholar). FTY720 was with sphingoid bases using and of the from to was used as the for the of FTY720-P was with S1P and DHS1P as a using and as the The was used to and for of the sphingoid sphingoid ceramide FTY720, and FTY720-P were the of of the to of the structural of the sphingolipid used as the and the of the were a The were the of of of the sphingolipid with were with FTY720 in for the cells were for 2 with l-[U-13C, 15N]serine in the of FTY720 The was and the were as of the the sphingolipids was by the of by the in and used in to sphingoid ceramides, and sphingoid as E.V. P. Y. B. Hubbard Natarajan V. Cell. 2006; PubMed Scopus Google Scholar). and ceramide synthase was using the as L. I. S. H. A.H. A.H. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google with and different assays were with as a in and of for in a reaction of 2 and FTY720 was as a in to the reaction 2 the of in The reaction was by with as the and and was by The serine was as A. A. Y. Y. T. 2005; PubMed Scopus Google with 1 stable l-[U-13C, 15N]serine and as The reaction was with of for in a of and The reaction was by with as the The stable analog of was by by a from to which to the isotope of and of The of response of of versus of was to a of the was in experiments were and the data for are as the S.E. were as of of the inhibition of ceramide synthases by FTY720 was using the kinetic of FTY720 and FTY720-P and as the of FTY720 and FTY720-P the and used of of sphingoid bases and sphingoid we an with synthetic and as the we the of FTY720 and found that the from to to the of molecules and the is the for FTY720 in the mode we confirmed that FTY720 from sphingoid bases using the and we used for the sphingoid we the of the with of FTY720 and a of and with the of from of on the The data demonstrate a of the response a of with we found that FTY720-P in the mode as its with S1P and The and the of FTY720-P are and the of an to a to FTY720-P to the S1P and DHS1P and the of the response of of FTY720-P versus a of is a of FTY720-P in the of 1, and of FTY720 and FTY720-P the of of sphingoid bases and sphingoid is a and FTY720 and FTY720-P are similar to and DHS1P, and and are similar to of and the of synthetic of and S1P as the for the of FTY720 and FTY720-P is a of the structural between FTY720-P and DHS1P, of and and in the found that and are for stable of FTY720 and FTY720-P, which but are not analog of FTY720 and its were used as to FTY720 and FTY720-P, B. B. M. R. N. T. A. Blood. 2006; PubMed Scopus Google Scholar, P. Lynch J. 2006; PubMed Scopus Google Scholar). because are not the of and as for of FTY720 and FTY720-P is a and FTY720 DHS1P through the of of with FTY720 intracellular levels of of the signaling sphingolipids. Thus, of FTY720 decreased S1P the DHS1P in the cells The in was by similar in the of sphingoid bases that FTY720 with ceramide biosynthesis FTY720 decreased the ceramide level in cells but effect on not a inhibition of to by the inhibition of we the of the FTY720 to of FTY720 and FTY720-P in revealed that of the FTY720 was to FTY720-P, of the FTY720 was from cells 2 the of the metabolic in the sphingolipid by FTY720, we used stable isotope labeling in with of an by for sphingolipid metabolic studies to DHS1P and S1P in cells E.V. P. Y. B. Hubbard Natarajan V. Cell. 2006; PubMed Scopus Google Scholar). of pulse labeling of cells with l-[U-13C, 15N]serine and of the mass of sphingolipids that to which metabolic of sphingolipid de novo biosynthesis is As shown in of with FTY720 to a of stable in cells, but in the of was FB1, a classical inhibitor of ceramide synthases P. Lynch J. 2006; PubMed Scopus Google Scholar, K. J. M. A.H. PubMed Scopus Google Scholar, A.H. T.R. C. J. Med. Biol. PubMed Scopus Google Scholar), a similar effect on the of sphingoid bases FTY720 was as as in the of l-[U-13C, 15N]serine and a efficiency for in ceramide biosynthesis in of l-[U-13C, 15N]serine ceramide in the of FTY720 and was more in l-[U-13C, 15N]serine and FTY720 was more potent in de novo biosynthesis of and FTY720 and DHS1P FTY720 was with FB1, but not FTY720, increased the cellular levels of and not effects of FTY720 and on the de novo generation of ceramide were with FTY720 for and the cells were with l-[U-13C, 15N]serine for 2 and were by are as of the ceramide in cells. the inhibition of ceramide synthases by FTY720, we an in vitro of ceramide synthase as L. I. S. H. A.H. A.H. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google using the cell and as and to we the of inhibition of ceramide synthase 2 by FTY720 using as of its L. I. S. H. A.H. A.H. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). FTY720 to an inhibitor of with an apparent of was used as the The inhibition of was as the of inhibition with the of the The of the kinetic of inhibition with revealed that FTY720 does not the apparent of the reaction of but the apparent from to a competitive of inhibition by FTY720 with an apparent Ki of 2.15 we the of FTY720 to inhibit the of the of in the cell by using for and As shown in FTY720 a similar in of the ceramide synthase with being the The ability of FTY720 to inhibit l-[U-13C, 15N]serine and in was similar to that of we in an in vitro using and as the In to the labeling studies with l-[U-13C, 15N]serine in the experiments with the cell revealed that was more potent FTY720 in the of and the of modulation of ceramide biosynthesis and in cells. FTY720 and in does not inhibit M. K. T. M. T. Y. T. M. N. M. Y. K. K. S. T. J. Med. Chem. PubMed Scopus Google Scholar), but is to as a of H. N. 2006; PubMed Scopus Google Scholar). FTY720 and modulate in we the effects of FTY720 and myriocin a classical on in the and stable l-[U-13C, The methodology of of was FTY720 effect on myriocin the of not These data that FTY720 and not modulate in but SphK2, in FTY720-induced DHS1P FTY720 increased DHS1P we which of sphingosine kinase is in the FTY720-induced DHS1P up-regulation as well as in FTY720-P in of endogenous and was by to and of but not SphK2, the effect of FTY720 on DHS1P but effect on FTY720 to the of SphK2, but not decreased FTY720-P in The of FTY720 to FTY720-P was of the FTY720 in cells 2 and this is with of in is in the Y. I. He T. C. R. J.G. Berdyshev E.V. Natarajan V. J. Biol. Chem. 2007; 282: Full Text Full Text PDF PubMed Scopus Google Scholar). These results for and in and FTY720 as for FTY720 is an immunomodulatory molecule and is currently in as an immunosuppressive agent and for the of multiple P.C. Rausch M. Meier D.P. Foster C.A. Prog. Drug Res. 2008; 66: 363-381Google Scholar, 3Mansoor M. Melendez A.J. Rev. Recent Clin. Trials. 2008; 3: 62-69Crossref PubMed Scopus (32) Google Scholar). Additionally FTY720 its been to inhibit M. A. I. M. C. 2005; PubMed Scopus Google Scholar, M. I. A. M. S. C. C. J. Cell. 2007; PubMed Scopus Google and T. T. Wu M.T. K. V. Claffey K. Hla T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K. A. C. T. L. T. B. J. A. A. Hla T. J. Res. 2006; 66: PubMed Scopus Google Scholar), that FTY720 has in and The of FTY720 to FTY720-P and its action through S1P1,3–5 has a of because of the potent effects of the functional of the intracellular effects of FTY720 through the modulation of the balance of signaling sphingolipids not been as of its of in a FTY720 was not to with sphingolipid biosynthesis P.C. Rausch M. Meier D.P. Foster C.A. Prog. Drug Res. 2008; 66: 363-381Google Scholar). the inhibition of S1P lyase (8Bandhuvula P. Tam Y.Y. Oskouian B. Saba J.D. J. Biol. Chem. 2005; 280: 33697-33700Abstract Full Text Full Text PDF PubMed Scopus (122) Google and the reported inhibition of S.W. S.G. Barbour S.E. Milstien S. Spiegel S. PubMed Scopus Google Scholar, M. J. L. R. J. Mol. 2007; PubMed Scopus Google by FTY720 suggest that effects of FTY720 to its action on intracellular S1P novel data on the modulation of ceramide and levels by FTY720 by inhibition of ceramide synthases in Our results demonstrate for the first time that FTY720 ceramide synthase in endothelial cells as well as in of sphingoid sphingoid ceramides, and a effect of FTY720 on intracellular that from the methodology was a effect of FTY720 on the of DHS1P and S1P that was by similar in and These findings suggest that FTY720 inhibit the of to in cells. was by the of methodology in with a pulse labeling of cells with l-[U-13C, ceramide inhibition by FTY720 in cells and similar to that of FB1, a inhibitor of ceramide synthases P. Lynch J. 2006; PubMed Scopus Google Scholar), but with a different efficiency of inhibition of biosynthesis of short-chain versus long-chain ceramides. Our kinetic of inhibition in cell that the FTY720-induced inhibition was and competitive toward FTY720 inhibited in vitro with an of and inhibited of the other ceramide synthase with a similar as by using different and novel and that FTY720 its in ceramide and signaling in endothelial cell are in the biosynthesis of complex sphingolipids and signaling molecules multiple cellular function and the of cell versus 2006; PubMed Scopus Google Scholar, L.J. J. 2005; PubMed Scopus Google Scholar). conditions been reported to to increased ceramide de novo biosynthesis to ceramide generation (11Petrache I. Natarajan V. Zhen L. Medler T.R. Richter A.T. Cho C. Hubbard W.C. Berdyshev E.V. Tuder R.M. Nat. Med. 2005; 11: 491-498Crossref PubMed Scopus (419) Google Scholar, 2006; PubMed Scopus Google Scholar, L.J. 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Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). direct between FTY720 and that a similar for the of long-chain and by but is more potent in the of by and in in cell was found to a more potent inhibitor of as well as a of with other of sphingolipid biosynthesis and that is in by the up-regulation of by in H. N. 2006; PubMed Scopus Google Scholar). of ceramide synthases by FTY720 an but of to the action of of ceramide biosynthesis in decreased ceramide ceramide 1-phosphate in cells. 1-phosphate is an of cPLA2 P. Z. A. Cho Chalfant C.E. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar, K. Chalfant C.E. T. J. Mol. Pharmacol. 2005; PubMed Scopus Google Scholar, Subramanian P. M. Cho Chalfant C.E. J. Biol. Chem. 2007; 282: Full Text Full Text PDF PubMed Scopus Google Scholar). from and is in biosynthesis. FTY720 has been shown to and generation in cells through a direct inhibition of cPLA2 and with ceramide 1-phosphate of cPLA2 but not through an S1P (7Payne S.G. Oskeritzian C.A. Griffiths R. Subramanian P. Barbour S.E. Chalfant C.E. Milstien S. Spiegel S. Blood. 2007; 109: 1077-1085Crossref PubMed Scopus (143) Google Scholar). Our data suggest that an of cPLA2 inhibition by FTY720 through the of the de novo biosynthesis of ceramide and ceramide in the was the up-regulation of the intracellular level of DHS1P by Although for intracellular DHS1P is has been shown to for DHS1P biosynthesis. we that of but not SphK2, intracellular of DHS1P through the de novo biosynthesis E.V. P. Y. B. Hubbard Natarajan V. Cell. 2006; PubMed Scopus Google Scholar). Our results suggest that the of for to DHS1P as a of a metabolic the level of ceramide synthases to a for FTY720 and action in cells. In DHS1P was to a of fumonisin Lee S. S. J. A. 2006; PubMed Scopus (26) Google Scholar, Lee Lee S. Res. 2007; PubMed Scopus Google Scholar). Our data the that similar to E.V. P. Y. B. Hubbard Natarajan V. Cell. 2006; PubMed Scopus Google Scholar), has to the de novo to not to in DHS1P generation because silencing of SphK2, in to silencing of effect on FTY720-induced up-regulation of DHS1P in cells Our data suggest that the inhibition of S1P lyase by FTY720 (8Bandhuvula P. Tam Y.Y. Oskouian B. Saba J.D. J. Biol. Chem. 2005; 280: 33697-33700Abstract Full Text Full Text PDF PubMed Scopus (122) Google is not a to DHS1P in cells as the of S1P was of being by of with The is for a inhibition of S1P and phosphate by FTY720 as of between S1P and DHS1P Y. I. He T. C. R. J.G. Berdyshev E.V. Natarajan V. J. Biol. Chem. 2007; 282: Full Text Full Text PDF PubMed Scopus Google Scholar, H. C. R. Milstien S. Spiegel S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The human endothelial cells used in this FTY720 to confirmed that but not phosphorylation of this is with on phosphorylation of FTY720 in cells B. B. M. R. N. T. A. Blood. 2006; PubMed Scopus Google Scholar, S.W. S.G. Barbour S.E. Milstien S. Spiegel S. PubMed Scopus Google Scholar, A. P. N. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of FTY720 to FTY720-P in was and only of FTY720 was in its of the of not FTY720 the of of FTY720-P in data the that effects of FTY720 on sphingolipid are by data not a for FTY720-P in DHS1P in cells because silencing of effect on the intracellular level of FTY720-P but decreased the of DHS1P up-regulation in response to FTY720 the silencing of decreased the intracellular of FTY720-P but effect on DHS1P up-regulation by These results suggest a for intracellular FTY720-P with FTY720 in DHS1P in results the of ceramide synthase inhibition by FTY720 as a of up-regulation of DHS1P in endothelial cells. In we a novel for the of the intracellular balance of signaling sphingolipids ceramide synthases by of FTY720 action to the complex of the of FTY720 in cellular and a of its interference with sphingolipid balance and signaling Our findings a direction for applications of FTY720 and its through the of ceramide de novo biosynthesis. of the by which FTY720 and its sphingoid balance ceramide synthases targets for and therapeutic for the