Diterpenoid DGA induces apoptosis via endoplasmic reticulum stress caused by changes in glycosphingolipid composition and inhibition of STAT3 in glioma cells.

Glioma is one of the most common malignant primary brain tumors, with poor prognosis and high recurrence. There are currently few drugs approved for brain tumors; thus, it is necessary to develop new effective drugs. Natural diterpenoids have important biological activities, including antiinflammatory, antioxidative, and antitumor effects. In this study, 7α,14ß-dihydroxy-ent-kaur-17-dimethylamino-3,15-dione (DGA), a diterpenoid compound modified from glaucocalyxin A, inhibited the proliferation of many tumor cells, especially glioma. Flow cytometry analysis showed that DGA induced apoptosis in glioma cells. DGA also inhibited xenograft tumors in nude mice. It affected the expression of ceramide synthases (CerS) in glioma cells; CerS1 decreased, and CerS2 and CerS5 increased, resulting in a change in the composition of glycosphingolipids containing varying acyl chain lengths. In glioma cells treated with DGA, the gene transcription of activating transcription factor 4 (ATF4), X-box binding protein-1 (XBP1), and C/EBP-homologous protein (CHOP) in unfolded protein response pathways was upregulated. Meanwhile, the ratio of proapoptotic protein Bcl-2-associated X protein (BAX) to antiapoptotic protein B-cell lymphoma 2 (Bcl-2) also increased. This suggested that an imbalance of glycosphingolipids caused by DGA induced severe endoplasmic reticulum stress and triggered cell apoptosis. Moreover, Western blotting showed DGA inhibited the signal transducers and activators of transcription 3 (STAT3) signaling pathway by reducing the phosphorylation of STAT3 and its upstream kinases, which also promoted the apoptosis of glioma cells. Together, these results explored the anticancer activities of DGA and highlighted it as a potential candidate for treating glioma.

The combination of microbiome and metabolome to analyze the cross-cooperation mechanism of Echinacea purpurea polysaccharide with the gut microbiota in vitro and in vivo.

Echinacea purpurea polysaccharide (EPP) is a functional compound in Echinacea purpurea. At present, it is generally recognized that plant polysaccharides can regulate the intestinal microecology, but there are few studies on EPP. In this study, we used the digestive model (stomach-small intestine-colon) and a mouse model to study the effect of EPP on intestinal microecology and the mechanism. Also, combined with the microbiome and metabolome analysis methods, the interaction network mechanism of EPP-gut microbiota-metabolites-metabolism was investigated. After EPP was digested by human intestinal microbiota, the microbial diversity changed, with an increase in the relative abundance of Bifidobacterium and a decrease in the abundance of Prevotella, Catenibacterium and Ruminococcus torques. After metabolism in mice, the concentration of short-chain fatty acids increased, the abundances of Muribaculaceae and Alloprevotella increased, and those of Lachnospiraceae and Butyricicoccus decreased. Both in vivo and in vitro experiments revealed that EPP can downregulate the expression of 15 enzymes involved in porphyrin metabolism. In addition, the metabolome results also confirmed that alanine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, and glycine, serine and threonine metabolism are regulatory pathways of EPP. Tryptophan, ornithine, tyrosine, leucine, alanine and serine are hallmark metabolites. The cross-cooperation network greatly influenced the microbiota (Lactobacillus, Lachnospiraceae), metabolites (tryptophan, beta-D-fructose 1,6-bisphosphate), and metabolism (glycosphingolipid biosynthesis), suggesting that they may be the key factors mediating the metabolic function of EPP. Therefore, EPP has the effect of enhancing the proliferation of gut-beneficial bacteria that metabolize polysaccharides and produce valuable metabolites.

Lewis glycosphingolipids as critical determinants of TRAIL sensitivity in cancer cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cancer cell death and contributes to tumor rejection by cytotoxic lymphocytes in cancer immunosurveillance and immunotherapy. TRAIL and TRAIL receptor agonists have garnered wide popularity as promising agents for cancer therapy. We previously demonstrated that the loss of fucosylation in cancer cells impairs TRAIL sensitivity; however, the precise structures of the fucosylated glycans that regulate TRAIL sensitivity and their carrier molecules remain elusive. Herein, we observed that Lewis glycans among various fucosylated glycans positively regulate TRAIL-induced cell death. Specifically, Lewis glycans on lacto/neolacto glycosphingolipids, but not glycoproteins including TRAIL receptors, enhanced TRAIL-induced formation of the cytosolic caspase 8 complex, without affecting the formation of the membranous receptor complex. Furthermore, type I Lewis glycan expression in colon cancer cell lines and patient-derived cancer organoids was positively correlated with TRAIL sensitivity. These findings provide novel insights into the regulatory mechanism of TRAIL-induced cell death and facilitate the identification of novel predictive biomarkers for TRAIL-related cancer therapies in future.

Marine Fungal Cerebroside Flavuside B Protects HaCaT Keratinocytes against Staphylococcus aureus Induced Damage.

Cerebrosides are glycosylated sphingolipids, and in mammals they contribute to the pro-/anti-inflammatory properties and innate antimicrobial activity of the skin and mucosal surfaces. Staphylococcus aureus infection can develop, not only from minor scratches of the skin, but this pathogen can also actively promote epithelial breach. The effect of cerebroside flavuside B from marine sediment-derived fungus Penicillium islandicum (Aniva Bay, the Sea of Okhotsk) on viability, apoptosis, total caspase activity, and cell cycle in human epidermal keratinocytes HaCaT line co-cultivated with S. aureus, as well as influence of flavuside B on LPS-treated HaCaT cells were studied. Influence of flavuside B on bacterial growth and biofilm formation of S. aureus and its effect on the enzymatic activity of sortase A was also investigated. It was found S. aureus co-cultivated with keratinocytes induces caspase-depended apoptosis and cell death, arrest cell cycle in the G0/G1 phase, and increases in cellular immune inflammation. Cerebroside flavuside B has demonstrated its antimicrobial and anti-inflammatory properties, substantially eliminating all the negative consequences caused by co-cultivation of keratinocytes with S. aureus or bacterial LPS. The dual action of flavuside B may be highly effective in the treatment of bacterial skin lesions and will be studied in the future in in vivo experiments.

Cytoprotective Activity of p-Terphenyl Polyketides and Flavuside B from Marine-Derived Fungi against Oxidative Stress in Neuro-2a Cells.

The influence of p-terphenyl polyketides 1-3 from Aspergillus candidus KMM 4676 and cerebroside flavuside B (4) from Penicillium islandicum (=Talaromyces islandicus) against the effect of neurotoxins, rotenone and paraquat, on Neuro-2a cell viability by MTT and LDH release assays and intracellular ROS level, as well as DPPH radical scavenging activity, was investigated. Pre-incubation with compounds significantly diminished the ROS level in rotenone- and paraquat-treated cells. It was shown that the investigated polyketides 1-3 significantly increased the viability of rotenone- and paraquat-treated cells in two of the used assays but they affected only the viability of paraquat-treated cells in the LDH release assay. Flavuside B statistically increased the viability of paraquat-treated cells in both MTT and LDH release assays, however, it increased the viability of rotenone-treated cells in the LDH release assay. Structure-activity relationships for p-terphenyl derivatives, as well as possible mechanisms of cytoprotective action of all studied compounds, were discussed.

Structure Elucidation of Calyxoside B, a Bipolar Sphingolipid from a Marine Sponge Cladocroce sp. through the Use of Beckmann Rearrangement.

Marine sponges are an excellent source of biologically active secondary metabolites. We focus on deep-sea sponges for our discovery study. A marine sponge Cladocroce sp. exhibited cytotoxic activity in the bioactivity screening. From this sponge a previously unreported cytotoxic glycosphingolipid, calyxoside B, was isolated and the structure of this compound was elucidated by analyses of MS and NMR spectra and chemical derivatization. We converted the ketone in the middle of a long aliphatic chain into an oxime to which was applied Beckmann rearrangement to afford two positional isomers of amides. The products were subjected to acidic hydrolysis followed by LC-MS analysis, permitting us to assign unequivocally the position of the ketone. Calyxoside B shows cytotoxicity against HeLa cells with an IC50 value of 31 µM and also weakly stimulated the production of cytokines in mice.

Application of the Antibody-Inducing Activity of Glycosphingolipids to Human Diseases.

Glycosphingolipids (GSLs) are composed of a mono-, di-, or oligosaccharide and a ceramide and function as constituents of cell membranes. Various molecular species of GSLs have been identified in mammalian cells due to differences in the structures of oligosaccharides. The oligosaccharide structure can vary depending on cell lineage, differentiation stage, and pathology; this property can be used as a cell identification marker. Furthermore, GSLs are involved in various aspects of the immune response, such as cytokine production, immune signaling, migration of immune cells, and antibody production. GSLs containing certain structures exhibit strong immunogenicity in immunized animals and promote the production of anti-GSL antibodies. By exploiting this property, it is possible to generate antibodies that recognize the fine oligosaccharide structure of specific GSLs or glycoproteins. In our study using artificially synthesized GSLs (artGSLs), we found that several structural features are correlated with the antibody-inducing activity of GSLs. Based on these findings, we designed artGSLs that efficiently induce the production of antibodies accompanied by class switching and developed several antibodies that recognize not only certain glycan structures of GSLs but also those of glycoproteins. This review comprehensively introduces the immune activities of GSLs and their application as pharmaceuticals.

New glycosylsphingolipids from Psychotria serpens L.

In clinical, Psychotria serpens L. was often substitute for Caulis trachelospermi to treat cancer in China. Meanwhile, EtOAc and n-BuOH fractions of MeOH extract of P. serpens L. show power activity against H460, HepG2, Hela, and PC9/GR cell lines, and no toxic effects against normal 16HBE cell lines. In our ongoing search for bioactive novel compounds from Chinese material medica, one new type of glycosylsphingolipids Psychotramide (1a-1c) were isolated from P. serpens L., and their structures were identified through spectroscopic techniques including NMR (1D and 2D) and MS (LC-MS, and GC-MS).

Neurostatin and other O-acetylated gangliosides show anti-neuroinflammatory activity involving the NFκB pathway.

In many neuropathologies activated microglia and macrophages cause neurotoxicity and prolong the inflammatory response. We have previously characterized the glycosphingolipid Neurostatin (Nst), which potentially reduces these detrimental mechanisms. Nst, isolated from mammalian brain, is the GD1b ganglioside with O-acetylation of the outer sialic acid residue. Using the enzyme sialate-O-acetyltransferase (SOAT), we obtained several O-acetylated gangliosides and O-propionylated GD1b (PrGD1b). In the present study we investigated the anti-inflammatory effects of these compounds. Nst and other O-acetylated gangliosides reduced nitrite production in microglial cells which were activated with lipopolysaccharide (LPS), but did not affect nitrite production after their stimulation with interferon gamma (IFNγ). Structure-activity relationship analysis showed that Nst was the most active ganglioside as inhibitor of nitrite production. Its ceramide moiety is essential for this, and both, the O-acetylation and the monosaccharide chain are important for the anti-inflammatory activity of the gangliosides. We also found that Nst reduced iNOS, IL-6 and IL-12 transcription in LPS-induced microglia, likely by inhibiting nuclear localization of NFκB. In co-cultures, Nst reduced neuronal cell death caused by LPS-activated microglia. In vivo, Nst diminished microglia activation in a mouse model of acute neuroinflammation. We propose that Nst and other O-acetylated gangliosides are neuroprotective regulators of microglia activity under both physiological and pathological conditions.

Occurrence of Melibiose-Containing Glycosphingolipids in a Sample of a Sponge-Coral Association (Desmapsamma anchorata/Carijoa riisei).

In our research on biologically active compounds from Vietnamese marine invertebrates, rare melibiose-containing glycosphingolipids were found in a sample of a sponge-coral association (Desmapsamma anchorata/Carijoa riisei). Melibiosylceramides were analyzed as constituents of some multi-component RP-HPLC fractions, and the structures of 14 new (1b, 3b, 4a-4c, 6a-6c, 8b, 9a, 9b, 10b, 11a, 11b) and five known (2b, 5a-5c, 7b) natural compounds were elucidated using NMR, mass spectrometry, optical rotation, and chemical transformations. These α-d-Galp-(1→6)-ß-d-Glcp-(1 ↔ 1)-ceramides (presumably sponge-derived compounds) were shown to contain phytosphingosine-type n-t17:0 (1), (6E)-n-t17:1 (2), i-t17:0 (3), n-t18:0 (4), (6E)-n-t18:1 (5), i-t18:0 (6), (6E)-i-t18:1 (7), i-t19:0 (8), (6E)-i-t19:1 (9), ai-t19:0 (10), and (6E)-ai-t19:1 (11) backbones N-acylated with saturated straight-chain (2R)-2-hydroxy C21 (a), C22 (b), and C23 (c) acids. Characteristic trends in the fragmentations of the terminal parts of tetraacetylated normal-chain and iso- and anteiso-branched sphingoid bases were observed using GC/MS. The total sum of melibiosylceramides and compound 5b caused a reduction in colony formation of human melanoma cells.

Deep Profiling of Immunosuppressive Glycosphingolipids and Sphingomyelins in Wild Cordyceps.

Deep profiling of glycosphingolipids and sphingomyelins in wild Cordyceps was carried out by using offline chromatographic enrichment followed by ultrahigh performance liquid chromatography-ultrahigh definition-quadrupole time-of-flight mass spectrometry (UHPLC-UHD-Q-TOF-MS). A total of 119 glycosphingolipids (72 new ones) and 87 sphingomyelins (43 new ones) were identified from wild Cordyceps on the basis of the accurate mass and MS/MS fragmentations, isotope patterns, sphingolipid (SPL) database matching, confirmation by SPL standards, and the reversed-phase liquid chromatographic retention rule. This study is the most comprehensive report on the identification of glycosphingolipids and sphingomyelins from fungus. A subsequent lipopolysaccharide-induced mouse splenic lymphocyte proliferation assay showed that the Cordyceps glycosphingolipid fraction exhibits higher immunosuppressive activity compared to that of Cordyceps sphingomyelins. Our findings provided insight into the chemical diversity of sphingolipids in Cordyceps and chemical evidence for the therapeutic application of wild Cordyceps.

Glycosphingolipid metabolic reprogramming drives neural differentiation.

Neural development is accomplished by differentiation events leading to metabolic reprogramming. Glycosphingolipid metabolism is reprogrammed during neural development with a switch from globo- to ganglio-series glycosphingolipid production. Failure to execute this glycosphingolipid switch leads to neurodevelopmental disorders in humans, indicating that glycosphingolipids are key players in this process. Nevertheless, both the molecular mechanisms that control the glycosphingolipid switch and its function in neurodevelopment are poorly understood. Here, we describe a self-contained circuit that controls glycosphingolipid reprogramming and neural differentiation. We find that globo-series glycosphingolipids repress the epigenetic regulator of neuronal gene expression AUTS2. AUTS2 in turn binds and activates the promoter of the first and rate-limiting ganglioside-producing enzyme GM3 synthase, thus fostering the synthesis of gangliosides. By this mechanism, the globo-AUTS2 axis controls glycosphingolipid reprogramming and neural gene expression during neural differentiation, which involves this circuit in neurodevelopment and its defects in neuropathology.

[Neuronal glycolipids regulate glial cell division negatively during development and following a lesion]. / Glicolipidos neuronales regulan negativamente la division glial durante el desarrollo y tras una lesion.

Glial cells in the central nervous system of adult mammals outnumber neurons 10-fold. Their number remains stationary throughout adulthood, controlled by the concomitant presence of mitogens and mitogen inhibitors. The most abundant inhibitor, neurostatin, is ganglioside GD1b O-acetylated on hydroxyl 9 of its outermost sialic acid. Neurostatin inhibited the proliferation of primary microglia and astroblasts in culture (cytostatic) as well as both rodent and human glioma cells (cytotoxic) at nanomolar concentrations. At those concentrations neurostatin had no effect on non-glial lineage cells or differentiated glia. Neurostatin shows direct antimitotic activity on tumoral cells, interfering with multiple signals regulating cell cycle progression. But it also promotes indirectly total destruction of experimental rat brain glioma, presumably by making it visible to the host immune system and activating CD4+ and CD8+ lymphocytes. Neurostatin could be a new anti-inflammatory agent, with multiple convergent direct and indirect actions on glioma growth, a pathology without satisfactory clinical treatment. Neurostatin is produced by neurons but its expression is up-regulated by neuron-astrocyte contact. The action of neurostatin could be mediated by a number of receptor proteins, including integrins, Toll-like receptors and siglecs.

TITLE: Glicolipidos neuronales regulan negativamente la division glial durante el desarrollo y tras una lesion.En el sistema nervioso central de los mamiferos, las celulas gliales superan diez veces en numero a las neuronas. Su numero permanente estacionario durante la edad adulta, controlado por la presencia simultanea de mitogenos gliales e inhibidores de esos mitogenos. El inhibidor mas abundante, la neurostatina, es el gangliosido GD1b O-acetilado en el grupo 9 del acido sialico mas externo. La neurostatina y los oligosacaridos sinteticos inhiben la proliferacion de astroblastos en cultivo primario (citostaticos) y de celulas de gliomas (citotoxicos), tanto de roedores como de humanos, en concentracion nanomolar. A esas concentraciones, la neurostatina no tuvo efecto sobre celulas de linaje no glial ni sobre glia madura. La neurostatina y sus analogos mostraron actividad antimitotica directa sobre las celulas tumorales, interfiriendo con la progresion del ciclo celular en multiples sitios, pero tambien actuaron indirectamente, haciendo visibles las celulas tumorales al sistema inmune del huesped y activando linfocitos CD4+ y CD8+. Analogos de neurostatina podrian generar nuevos farmacos antiinflamatorios, con multiples acciones directas e indirectas contra el crecimiento de gliomas, una patologia todavia sin tratamiento clinico satisfactorio. La neurostatina es producida por las neuronas, pero el contacto de estas con astrocitos estimula notablemente su expresion. La accion de la neurostatina puede estar mediada por numerosas proteinas receptoras, incluyendo integrinas, siglecs y receptores Toll-like.

Synthesis and biological evaluation of neoglycosphingolipids.

Various neoglycosphingolipids were efficiently synthesized in a one-step reaction by the coupling of free sugars with an N-alkylaminooxy-functionalized ceramide analogue. The bioactivity studies demonstrated that most of these compounds could upregulate the expression of matrix metalloproteinase-9 (MMP-9, extracellular matrix proteins associated with tumor migration) in murine melanoma B16 cells in a similar manner to the natural ganglioside monosialodihexosylganglioside (GM3), which highlights the potential use of these neoglycosphingolipids as inhibitors of tumor migration.

T Helper 1 (Th1), Th2, and Th17 Responses to Leishmania major Lipophosphoglycan 3.

Leishmania major is the main causal agent of cutaneous leishmaniasis (CL) that remains a serious public health concern in many tropical and subtropical countries. A long-lasting protective vaccine against leishmaniasis remains as a medical unmet need. Lipophosphoglycan 3 (LPG3) is one of the class II LPG genes from HSP90 family involved in the host immune responses. The aim of the present study is to investigate the capability of recombinant LPG3 (rLPG3) to induce Th1, Th2, Th17 responses. The results showed that rLPG3 in moderate and high concentrations significantly induced expression of Th1 lineage-specific transcription factor (T-bet) and cytokine (IFN-γ)(P < 0.05). Moreover, the Th1-stimulating effect of rLPG3 was confirmed by significant induction of IFN-γ secretion from treated T cells (P < 0.01). However, no significant effect of rLPG3 on Th2 and Th17 lineage cells was observed even in high concentration. Our findings demonstrate that rLPG3 induces Th1, but not Th2 and Th17, lineage responses. Further studies are needed to investigate adjuvant properties of rLPG3 for leishmania therapy.

Lipid Rafts Assemble Dynein Ensembles.

New work by Rai et al. identifies a novel mechanism regulating phagosome transport in cells: the clustering of dynein motors into lipid microdomains, leading to enhanced unidirectional motility. Clustering may be especially important for dynein, a motor that works most efficiently in teams.

New norterpenoids and a sphingolipid from Carissa opaca.

Chemical investigations on the aerial parts of Carissa opaca resulted in the isolation and characterization of two new nor-triterpenoids (compounds 1 and 2) and a new sphingolipid (compound 3) together with six known compounds. The structures of all the isolates were established using spectral data. All the isolated compounds showed DPPH radical scavenging and enzyme inhibitory activities against enzymes acetylcholinesterase, butyrylcholinesterase, and lipoxygenase.

Design and synthesis of a novel glycosphingolipid derived from polyhydroxy 2-pyrrolidinone and phytoceramide appended by a 1,​2,​3-​triazole linker.

Synthetic analogs of glycosphingolipids (GSLs) have been demonstrated as potential therapeutic interventions in certain patho-physiological conditions. This article reviews reports of various bioactive synthetic GSLs, emanated from the Bittman laboratory. KRN7000, a synthetic GSL which is a α-galactosylceramide (α-GalCer) is a potent immunomodulatory agent. Bittman et al. reported several modifications of C-glycosides of KRN7000 with an eye towards achieving selective cytokine response during iNKT cell activation. However, GSLs with azasugar variants were not explored which inspired us to derive a polyhydroxy 2-pyrrolidinone azasugar from d-galactose and append to the phytoceramide via a 1,2,3-triazole linker to afford GSL analog 12. This novel GSL analog 12 may be used to explore the immunomodulatory activity, and other biological activities against targets involving iminosugar or azasugar based therapeutics.

Recombinant Leishmania major lipophosphoglycan 3 activates human T-lymphocytes via TLR2-independent pathway.

Leishmaniasis is one of the most common infectious diseases transmitted by an obligate intracellular genus Leishmania. As there is no efficient vaccination strategy for leishmaniasis, new immunostimulatory components may enhance protective immune responses against this parasite. Lipophosphoglycan 3 (LPG3) is an essential protein required for LPG assembling. In this study, the ability of recombinant LPG3 (rLPG) and its fragments to activate isolated healthy human T-cells and cytokine secretion was evaluated in vitro. The results showed that rLPG3 and its N-terminal fragment (rNT-LPG3) enhanced expression of CD69 on the surface of T-cells and promoted differentiation of CD4(+) T-lymphocytes toward a T-helper 1 (T(H)1) phenotype, in part, through up-regulation of interferon (IFN)-γ expression in a TLR2-independent manner. These results indicated the protective effects of LPG3 (particularly NT-LPG3 fragment) as a potent immunostimulatory component of leishmania in vaccination against leishmaniasis. Further investigations in in vivo assays are clearly warranted.

Promotion of bone morphogenetic protein signaling by tetraspanins and glycosphingolipids.

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor ß (TGFß) superfamily of secreted molecules. BMPs play essential roles in multiple developmental and homeostatic processes in metazoans. Malfunction of the BMP pathway can cause a variety of diseases in humans, including cancer, skeletal disorders and cardiovascular diseases. Identification of factors that ensure proper spatiotemporal control of BMP signaling is critical for understanding how this pathway is regulated. We have used a unique and sensitive genetic screen to identify the plasma membrane-localized tetraspanin TSP-21 as a key new factor in the C. elegans BMP-like "Sma/Mab" signaling pathway that controls body size and postembryonic M lineage development. We showed that TSP-21 acts in the signal-receiving cells and genetically functions at the ligand-receptor level. We further showed that TSP-21 can associate with itself and with two additional tetraspanins, TSP-12 and TSP-14, which also promote Sma/Mab signaling. TSP-12 and TSP-14 can also associate with SMA-6, the type I receptor of the Sma/Mab pathway. Finally, we found that glycosphingolipids, major components of the tetraspanin-enriched microdomains, are required for Sma/Mab signaling. Our findings suggest that the tetraspanin-enriched membrane microdomains are important for proper BMP signaling. As tetraspanins have emerged as diagnostic and prognostic markers for tumor progression, and TSP-21, TSP-12 and TSP-14 are all conserved in humans, we speculate that abnormal BMP signaling due to altered expression or function of certain tetraspanins may be a contributing factor to cancer development.