NKT TCR recognition of CD1d-α-C-galactosylceramide.

NKT cells respond to a variety of CD1d-restricted glycolipid Ags that are structurally related to the prototypic Ag α-galactosylceramide (α-GalCer). A modified analog of α-GalCer with a carbon-based glycosidic linkage (α-C-GalCer) has generated great interest because of its apparent ability to promote prolonged, Th1-biased immune responses. In this study, we report the activation of spleen NKT cells to α-C-GalCer, and related C-glycoside ligands, is weaker than that of α-GalCer. Furthermore, the Vß8.2 and Vß7 NKT TCR affinity for CD1d-α-C-GalCer, and some related analogs, is ∼10-fold lower than that for the NKT TCR-CD1d-α-GalCer interaction. Nevertheless, the crystal structure of the Vß8.2 NKT TCR-CD1d-α-C-GalCer complex is similar to that of the corresponding NKT TCR-CD1d-α-GalCer complex, although subtle differences at the interface provide a basis for understanding the lower affinity of the NKT TCR-CD1d-α-C-GalCer interaction. Our findings support the concept that for CD1d-restricted NKT cells, altered glycolipid ligands can promote markedly different responses while adopting similar TCR-docking topologies.

Chemopreventive sphingadienes downregulate Wnt signaling via a PP2A/Akt/GSK3ß pathway in colon cancer.

Sphingadienes (SDs) derived from soy and other natural sphingolipids are cytotoxic to colon cancer cells via an Akt-dependent mechanism and reduce adenoma formation in Apc(Min/+) mice. Wnt signaling is fundamental to colon carcinogenesis and is the basis for spontaneous tumorigenesis in Apc(Min/+) mice and patients with familial adenomatous polyposis. In the present study, we investigated the impact of SDs on Wnt signaling. Oral SD administration reduced levels of active ß-catenin and Wnt targets c-Myc and cyclin D1 in Apc(Min/+) mouse intestinal tissues. Colon cancer cells treated with SDs exhibited reduced Wnt transcriptional activity, as well as reduced nuclear ß-catenin localization and subsequent reduction in active-ß-catenin levels. Further, we observed a decrease in phosphorylated (inactive) GSK3ß in SD-treated mice and colon cancer cells. Expression of constitutively active myristoylated-Akt or inactivation of GSK3ß using LiCl attenuated SD-mediated inhibition of Wnt transcriptional activity and active-ß-catenin levels. SDs exhibited additive effects with inhibitors of the phosphatidylinositol-3-kinase/Akt/mTOR pathway to induce cytotoxicity. Further, a combination regime of SDs and low-dose rapamycin decreased visible polyps in Apc(Min/+) mice and reduced the levels of Wnt target gene expression and mTOR target activation. SD-mediated inhibition of Akt and Wnt pathways and cytotoxicity in colon cancer cells was dependent upon the activity of protein phosphatase 2A, as shown by reversal of these effects by pretreatment with okadaic acid or calyculin A. Our cumulative findings indicate that SDs inhibit Wnt signaling through a protein phosphatase 2A/Akt/GSK3ß-dependent mechanism that may contribute to their chemopreventive effects in intestinal tumorigenesis.

The sphingolipid degradation product trans-2-hexadecenal forms adducts with DNA.

Sphingosine 1-phosphate, a bioactive signaling molecule with diverse cellular functions, is irreversibly degraded by the endoplasmic reticulum enzyme sphingosine 1-phosphate lyase, generating trans-2-hexadecenal and phosphoethanolamine. We recently demonstrated that trans-2-hexadecenal causes cytoskeletal reorganization, detachment, and apoptosis in multiple cell types via a JNK-dependent pathway. These findings and the known chemistry of related α,ß-unsaturated aldehydes raise the possibility that trans-2-hexadecenal may interact with additional cellular components. In this study, we show that it reacts readily with deoxyguanosine and DNA to produce the diastereomeric cyclic 1,N(2)-deoxyguanosine adducts 3-(2-deoxy-ß-d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8R-hydroxy-6R-tridecylpyrimido[1,2-a]purine-10(3H)one and 3-(2-deoxy-ß-d-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8S-hydroxy-6S-tridecylpyrimido[1,2-a]purine-10(3H)one. Thus, our findings suggest that trans-2-hexadecenal produced endogenously by sphingosine 1-phosphate lyase can react directly with DNA forming aldehyde-derived DNA adducts with potentially mutagenic consequences.

Characterization of sphingosine-1-phosphate lyase activity by electrospray ionization-liquid chromatography/tandem mass spectrometry quantitation of (2E)-hexadecenal.

Sphingosine-1-phosphate (S1P) is a sphingolipid signaling molecule crucial for cell survival and proliferation. S1P-mediated signaling is largely controlled through its biosynthesis and degradation, and S1P lyase (S1PL) is the only known enzyme that irreversibly degrades sphingoid base-1-phosphates to phosphoethanolamine and the corresponding fatty aldehydes. S1PL-mediated degradation of S1P results in the formation of (2E)-hexadecenal, whereas hexadecanal is the product of dihydrosphingosine-1-phosphate (DHS1P) degradation. Fatty aldehydes can undergo biotransformation to fatty acids and/or alcohols, making them elusive and rendering the task of fatty aldehyde quantitation challenging. We have developed a simple, highly sensitive, and high-throughput protocol for (2E)-hexadecenal quantitation as a semicarbazone derivative by liquid chromatography-electrospray ionization-tandem mass spectrometry. The approach was applied to determining S1PL activity in vitro with the ability to use as low as 0.25µg of microsomal protein per assay. The method is also applicable to the use of total tissue homogenate as the source of S1PL. A correction for (2E)-hexadecenal disappearance due to its biotransformation during enzymatic reaction is required, especially at higher protein concentrations. The method was applied to confirm FTY720 as the inhibitor of S1PL with an IC50 value of 52.4µM.

Total synthesis of α-1C-galactosylceramide, an immunostimulatory C-glycosphingolipid, and confirmation of the stereochemistry in the first-generation synthesis.

A nonisosteric α-C-glycoside analogue of KRN7000 (α-1C-GalCer, 1) was reported to induce a selective type of cytokine release in human invariant natural killer cells in vitro. We report here a very concise synthetic route to 1 and its analogue 1'. The key steps include olefin cross-metathesis, Sharpless asymmetric epoxidation, and epoxide opening by NaN(3)/NH(4)Cl. Inversion of configuration at the amide-bearing carbon in the phytosphingosine backbone constructed by epoxide opening in our previous synthesis of 1 was verified, indicating that remote group participation is not involved during the epoxide-opening reaction.

Lysophosphatidic acid induces neointima formation through PPARgamma activation.

Neointimal lesions are characterized by accumulation of cells within the arterial wall and are a prelude to atherosclerotic disease. Here we report that a brief exposure to either alkyl ether analogs of the growth factor-like phospholipid lysophosphatidic acid (LPA), products generated during the oxidative modification of low density lipoprotein, or to unsaturated acyl forms of LPA induce progressive formation of neointima in vivo in a rat carotid artery model. This effect is completely inhibited by the peroxisome proliferator-activated receptor (PPAR)gamma antagonist GW9662 and mimicked by PPARgamma agonists Rosiglitazone and 1-O-hexadecyl-2-azeleoyl-phosphatidylcholine. In contrast, stearoyl-oxovaleryl phosphatidylcholine, a PPARalpha agonist and polypeptide epidermal growth factor, platelet-derived growth factor, and vascular endothelial growth factor failed to elicit neointima. The structure-activity relationship for neointima induction by LPA analogs in vivo is identical to that of PPARgamma activation in vitro and disparate from that of LPA G protein-coupled receptor activation. Neointima-inducing LPA analogs up-regulated the CD36 scavenger receptor in vitro and in vivo and elicited dedifferentiation of cultured vascular smooth muscle cells that was prevented by GW9662. These results suggest that selected LPA analogs are important novel endogenous PPARgamma ligands capable of mediating vascular remodeling and that activation of the nuclear transcription factor PPARgamma is both necessary and sufficient for neointima formation by components of oxidized low density lipoprotein.

Asymmetric synthesis of D-ribo-phytosphingosine from 1-tetradecyne and (4-methoxyphenoxy)acetaldehyde.

An asymmetric synthesis of d-ribo-phytosphingosine (1) was achieved by utilizing the ProPhenol (12)-catalyzed alkynylation of unsaturated aldehyde 8 to afford allylic propargylic alcohol (S)-6 followed by asymmetric epoxidation and opening of propargylic epoxy alcohol anti-5 with NaN(3)/NH(4)Cl. Deprotection and reduction of the resulting acyclic azide 3 then gave 1. Alkyne-azide 3 was subjected to an intramolecular click reaction, generating a bicyclic triazole, which was found to have unexpected vicinal coupling constants. Application of the advanced Mosher method verified the configurations of the three contiguous stereogenic centers of 1. An alkynyl azide analogue of 1, which may be useful as a glycosyl acceptor in the synthesis of alpha-galactosylceramide derivatives, was also readily prepared by this route.

Molecular features of phospholipids that affect glycolipid transfer protein-mediated galactosylceramide transfer between vesicles.

The glycolipid transfer protein (GLTP)-mediated movement of galactosylceramide from model membrane donor vesicles to acceptor vesicles is sensitive to the membrane environment surrounding the glycolipid. GLTP can catalyze the transfer of a fluorescently labeled GSL, anthrylvinyl-galactosylceramide (AV-GalCer), from vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and dipalmitoylphosphatidylcholine matrices, but not from vesicles prepared from N-palmitoylsphingomyelin, regardless of the cholesterol content of the vesicles. In this study, we have examined the structural features of sphingomyelin (SM) that are responsible for its inhibition of the rate of GLTP-catalyzed transfer of AV-GalCer. The rate of glycolipid transfer was enhanced when the N-palmitoyl chain of SM was replaced with an N-oleoyl chain. Analogs of N-palmitoyl-SM in which the 4,5-double bond of the long-chain base is reduced or the 3-hydroxy group is removed did not inhibit GLTP-catalyzed transfer of AV-GalCer. When the donor vesicles were prepared with phosphatidylcholines or ether-linked phosphatidylcholine analogs, the transfer rates of AV-GalCer increased with increasing degree of unsaturation. The rate of AV-GalCer transfer was strongly dependent on the unsaturation degree of the acyl and/or alkyl chains. For ester-linked PCs, the transfer rate increased in the order DPPC

Effect of ether glycerol lipids on interleukin-1ß release and experimental autoimmune encephalomyelitis.

We have assessed the effect of two ether glycerol lipids, 77-6 ((2S, 3R)-4-(Tetradecyloxy)-2-amino-1,3-butanediol) and 56-5 ((S)-2-Amino-3-O-hexadecyl-1-propanol), which are substrates for sphingosine kinases, on inflammatory responses. Treatment of differentiated U937 macrophage-like cells with 77-6 but not 56-5 enhanced IL-1ß release; either alone or in the presence of LPS. The stimulatory effect of sphingosine or 77-6 on LPS-stimulated IL-1ß release was reduced by pretreatment of cells with the caspase-1 inhibitor, Ac-YVAD-CHO, thereby indicating a role for the inflammasome. The enhancement of LPS-stimulated IL-1ß release in response to sphingosine, but not 77-6, was reduced by pretreatment of cells with the cathepsin B inhibitor, CA074Me, indicating a role for lysosomal destabilization in the effect of sphingosine. Administration of 56-5 to mice increased disease progression in an experimental autoimmune encephalomyelitis model and this was associated with a considerable increase in the infiltration of CD4(+) T-cells, CD11b(+) monocytes and F4/80(+) macrophages in the spinal cord. 56-5 and 77-6 were without effect on the degradation of myc-tagged sphingosine 1-phosphate 1 receptor in CCL39 cells. Therefore, the effect of 56-5 on EAE disease progression is likely to be independent of the inflammasome or the sphingosine 1-phosphate 1 receptor. However, 56-5 is chemically similar to platelet activating factor and the exacerbation of EAE disease progression might be linked to platelet activating factor receptor signaling.

Stereochemical properties of lysophosphatidic acid receptor activation and metabolism.

Ligand recognition by G protein-coupled receptors (GPCR), as well as substrate recognition by enzymes, almost always shows a preference for a naturally occurring enantiomer over the unnatural one. Recognition of lysophosphatidic acid (LPA) by its receptors is an exception, as both the natural L (R) and unnatural D (S) stereoisomers of LPA are equally active in bioassays. In contrast to the enantiomers of LPA, analogs of N-acyl-serine phosphoric acid (NASPA) and N-acyl-ethanolamine phosphoric acid (NAEPA), which contain a serine and an ethanolamine backbone, respectively, in place of glycerol, are recognized in a stereoselective manner. This stereoselective interaction may lead to the development of receptor subtype-selective antagonists. In the present study, we review the stereochemical aspects of LPA pharmacology and describe the chemical synthesis of pure LPA enantiomers together with their ligand-binding properties toward the LPA1, LPA2, and LPA3 receptors and their metabolism by lipid phosphate phosphatase 1 (LPP1). Finally, we evaluate the concept of stereopharmacology in developing novel ligands for LPA receptors.

Synthesis and use of novel ether phospholipid enantiomers to probe the molecular basis of the antitumor Effects of alkyllysophospholipids: correlation of differential activation of c-Jun NH(2)-terminal protein kinase with antiproliferative effects in neuronal tumor cells.

The enantiomers of a novel unsaturated phosphonocholine antitumor ether lipid were synthesized and found to have differential antiproliferative effects against epithelial cancer cell lines. The basis of the enantioselective effects on the cells was investigated in SK-N-MC and SK-N-SH neuroblastoma tumor cells. Our results indicate that the enantioselective antiproliferative potency arises primarily from the activation of the JNK signaling pathway by the ether lipids.

Enantioselective Synthesis of 3-Deoxy-(R)-sphingomyelin from (S)-1-(4'-Methoxyphenyl)glycerol.

(R)-3-Deoxysphingomyelin (2) was prepared from (S)-1-(4'-methoxyphenyl)-glycerol (3). The latter was converted into either p-methoxyphenyl (PMP) (S)-oxiranylmethyl ether (5) or (R)-1-(4'-methoxyphenyl)glycerol 2,3-cyclic sulfate (6). Opening of 5 with lithium pentadecyne in the presence of BF(3).Et(2)O gave PMP (S)-2-hydroxy-4-octadecynyl ether (7) in 65% yield. Alternatively, opening of cyclic sulfate 6 with excess lithium pentadecyne in the presence of catalytic cuprous iodide, followed by acidic workup, gave 7 in 90% yield. After introduction of the amide group via azide displacement, reduction, and N-acylation, simultaneous reduction of the triple bond and deprotection of the PMP group by Birch reduction (Li, EtNH(2)) provided 3-deoxy-N-palmitoyl-(R)-ceramide (9). Finally, phosphitylation of 9, oxidation of the cyclic phosphite with bromine, followed by in situ ring opening gave a (2-bromoethyl)phosphate ester, which on quaternization with aqueous trimethylamine afforded 3-deoxy-N-palmitoyl-(R)-sphingomyelin (2) in 49% overall yield from PMP (S)-2-hydroxy-4-octadecynyl ether (7).

A fluorescent alkyllysophospholipid analog exhibits selective cytotoxicity against the hormone-insensitive prostate cancer cell line PC3.

A fluorescent analog of ET-18-OCH3, 1-O-(7'-N,N-dimethylamino-3'-pentadecanoyl-1'-naphthyl)-2-O-methyl-sn-glycerophosphocholine (1), was synthesized and its bioactivity was screened against 12 human cancer cell lines. The bioactivity of 1 was found to differ markedly from that of ET-18-OCH3. Growth of two prostate cell lines (PC3 and DU145) and a glioma cell line (U251) was significantly affected by 1, with IC50 values of 2, 6, and 12 µM, respectively. Compound 1 was cytotoxic to PC3 cells by caspasedependent apoptosis. The subcellular distribution of 1 differed from that reported for a phenyl-polyene analog of ET-18-OCH3; 1 was found to be localized in the endoplasmic reticulum, mitochondria, and lysosomes but not in the plasma membrane or nucleus of PC3 cells. However, no differences in accumulation of 1 were found between PC3 and cells that were not affected by the compound, implying that the selective PC3 cytotoxicity is a consequence of specific molecular components of PC3 cells.

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.

Sphingosine 1-phosphate (S1P) is involved in hyper-proliferative diseases such as cancer and pulmonary arterial hypertension. We have synthesized inhibitors that are selective for the two isoforms of sphingosine kinase (SK1 and SK2) that catalyze the synthesis of S1P. A thiourea adduct of sphinganine (F02) is selective for SK2 whereas the 1-deoxysphinganines 55-21 and 77-7 are selective for SK1. (2S,3R)-1-Deoxysphinganine (55-21) induced the proteasomal degradation of SK1 in human pulmonary arterial smooth muscle cells and inhibited DNA synthesis, while the more potent SK1 inhibitors PF-543 and VPC96091 failed to inhibit DNA synthesis. These findings indicate that moderate potency inhibitors such as 55-21 are likely to have utility in unraveling the functions of SK1 in inflammatory and hyperproliferative disorders.

Practical multigram-scale synthesis of 4,6- and 4,8-sphingadienes, chemopreventive sphingoid bases.

Sphingadienes are chemopreventive agents that act by blocking signaling pathways that are activated in cancer. A practical synthesis of 4,6- and 4,8-sphingadienes on a scale of gram quantities is reported here in order to allow evaluation of the biological properties of these sphingolipids. The key steps in the preparation of 4,6-sphingadiene (1a) are an intramolecular cyclization of N-Boc derivative 5a to oxazolidinone derivative 6a, followed by conversion to carbamate intermediate 7a and base-mediated hydrolysis to afford the product without further purification. 4,8-Sphingadiene (1b) was prepared in a similar fashion; the requisite trans-γ,δ-unsaturated aldehyde 15 was prepared by an ester enolate Ireland-Claisen rearrangement.

An active endocytosis pathway is required for the cytotoxic effects of glycosylated antitumor ether lipids.

BACKGROUND: Glycosylated antitumor ether lipids (GAELs) kill cells by an apoptosis-independent pathway. A hallmark of this pathway is the formation of large acidic vacuoles; however, very little is known about the process. We examined the hypothesis that 1-O-hexadecyl-2-O-methyl-3-O-(2'-amino-2'-deoxy-ß-D-glucopyranosyl)-sn-glycerol (Gln), a potent GAEL, diffuses across cell membranes into lysosomes, where protonation of the amine leads to its accumulation and generation of the vacuoles. MATERIALS AND METHODS: N-Benzylamine analogs with similar pKa values, but with greater hydrophobicity than the parental Gln were synthesized and their activities against epithelial cancer cell lines were compared. The role of endocytosis in Gln action was investigated by inhibiting endocytosis with methyl-ß-cyclodextrin (MCD), and inhibiting the maturation of the endocytic vesicles by low temperature incubation and analyzing their effects on Gln activity. RESULTS: The N-benzylamines were either inactive or less active than Gln, indicating that activity was unrelated to diffusion or protonation. Toxicity was only observed with analogs that generated vacuoles. The incubation of cells with MCD inhibited the generation of the vacuoles and the toxic effects of Gln. The toxic effect of Gln was inhibited when cells were incubated with the drug at 20°C, a temperature that inhibits the maturation of early endosomes. CONCLUSION: The results of the study show that GAELs are taken up by endocytosis and an active endocytic pathway is required for the formation of large acidic vacuoles by GAELs and manifestation of their cytotoxic effects.

The sphingolipid degradation product trans-2-hexadecenal induces cytoskeletal reorganization and apoptosis in a JNK-dependent manner.

The bioactive signaling molecule D-erythro-sphingosine-1-phosphate (S1P) is irreversibly degraded by the enzyme S1P lyase (SPL). The reaction of SPL with C18-S1P generates ethanolamine phosphate and a long-chain fatty aldehyde, trans-2-hexadecenal. Modulation of SPL expression in cells and organisms produces significant phenotypes, most of which have been attributed to corresponding changes in S1P-dependent signaling. However, the physiological functions of SPL products are not well understood. In the present study, we explored the biological activities of trans-2-hexadecenal in human and murine cells. We demonstrate that trans-2-hexadecenal causes cytoskeletal reorganization leading to cell rounding, detachment and eventual cell death by apoptosis in multiple cell types, including HEK293T, NIH3T3 and HeLa cells. Trans-2-hexadecenal stimulated a signaling pathway involving MLK3 and the respective phosphorylation of MKK4/7 and JNK, whereas ERK, AKT and p38 were unaffected. Trans-2-hexadecenal-induced apoptosis was accompanied by activation of downstream targets of JNK including c-Jun phosphorylation, cytochrome c release, Bax activation, Bid cleavage and increased translocation of Bim into mitochondria. The antioxidant N-acetylcysteine prevented JNK activation by trans-2-hexadecenal. Further, inhibition of JNK abrogated the cytoskeletal changes and apoptosis caused by trans-2-hexadecenal, whereas Rac1 and RhoA were not involved. In conclusion, our studies provide a new paradigm of sphingolipid signaling by demonstrating for the first time that S1P metabolism generates a bioactive product that induces cellular effects through oxidant stress-dependent MAP kinase cell signaling.

Selective deuterium labeling of the sphingoid backbone: facile syntheses of 3,4,5-trideuterio-d-erythro-sphingosine and 3-deuterio-d-erythro-sphingomyelin.

Deuteration at C-4 and C-5 of sphingosine was achieved via a hydrogen-deuterium exchange reaction of a ß-ketophosphonate intermediate catalyzed by ND4Cl in D2O/tetrahydrofuran. To install deuterium at C-3 of sphingosine and sphingomyelin, sodium borodeuteride reduction/cerium(III) chloride reduction of an α,ß-enone in perdeuteromethanol was used.

Synthesis and antitumor activity of ether glycerophospholipids bearing a carbamate moiety at the sn-2 position: selective sensitivity against prostate cancer cell lines.

Analogues of 1-O-hexadecyl-sn-3-glycerophosphonocholine (compounds 1-4) or sn-3-glycerophosphocholine (compound 5) bearing a carbamate or dicarbamate moiety at the sn-2 position were synthesized and evaluated for their antiproliferative activity against cancer cells derived from a variety of tissues. Although all of the compounds are antiproliferative, surprisingly the carbamates (1 and 2) are more effective against the hormone-independent cell lines DU145 and PC3 than toward other cancer cell lines we examined. This selectivity was not observed with the dicarbamates (3 and 4). Phosphocholine carbamate analogue 5 is as effective against the prostate cancer cell lines as the corresponding phosphonocholine analogue 1. Cell death induced by 2'-(trimethylammonio)ethyl 4-hexadecyloxy-3(R)-N-methylcarbamoyl-1-butanephosphonate (carbamate analogue 2) appeared to be mediated by apoptosis, as assessed by caspase activation and loss of mitochondrial membrane potential. The in vivo activity of 2 was evaluated in a murine prostate cancer xenograft model. Oral and intravenous administration showed that 2 is effective in inhibiting the growth of PC3 tumors in Rag2M mice. Our studies show that the glycerolipid carbamates reported herein represent a class of prostate-cancer-selective cytotoxic agents.

Synthesis of immunostimulatory alpha-C-galactosylceramide glycolipids via Sonogashira coupling, asymmetric epoxidation, and trichloroacetimidate-mediated epoxide opening.

Stereocontrolled syntheses of alpha-C-GalCer (2) and its alpha-C-acetylenic analogue 6 were accomplished in high efficiency by a convergent construction strategy from 1-hexadecene and d-galactose. The key transformations include Sonogashira coupling, Sharpless asymmetric epoxidation, and Et(2)AlCl-catalyzed cyclization of an epoxytrichloroacetimidate to generate protected dihydrooxazine 21.