Dexamethasone-Induced Skeletal Muscle Atrophy Increases O-GlcNAcylation in C2C12 Cells.

Skeletal muscle atrophy is a well-known adverse effect of chronic treatment with glucocorticoids and it also occurs when stress conditions such as sepsis and cachexia increase the release of endogenous glucocorticoids. Although the mechanisms of action of these hormones have been elucidated, the possible molecular mechanisms causing atrophy are not yet fully understood. The involvement of the O-GlcNAcylation process has recently been reported in disuse atrophy. O-GlcNAcylation, a regulatory post-translational modification of nuclear and cytoplasmic proteins consists in the attachment of O-GlcNAc residues on cell proteins and is regulated by two enzymes: O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA). O-GlcNAcylation plays a crucial role in many cellular processes and it seems to be related to skeletal muscle physiological function. The aim of this study is to investigate the involvement of O-GlcNAcylation in glucocorticoid-induced atrophy by using an "in vitro" model, achieved by treatment of C2C12 with 10 µM dexamethasone for 48 h. In atrophic condition, we observed that O-GlcNAc levels in cell proteins increased and concomitantly protein phosphorylation on serine and threonine residues decreased. Analysis of OGA expression at mRNA and protein levels showed a reduction in this enzyme in atrophic myotubes, whereas no significant changes of OGT expression were found. Furthermore, inhibition of OGA activity by Thiamet G induced atrophy marker expression. Our current findings suggest that O-GlcNAcylation is involved in dexamethasone-induced atrophy. In particular, we propose that the decrease in OGA content causes an excessive and mostly durable level of O-GlcNAc residues on sarcomeric proteins that might modify their function and stability. J. Cell. Biochem. 117: 1833-1842, 2016. © 2016 Wiley Periodicals, Inc.

HSPH1 inhibition downregulates Bcl-6 and c-Myc and hampers the growth of human aggressive B-cell non-Hodgkin lymphoma.

We have shown that human B-cell non-Hodgkin lymphomas (B-NHLs) express heat shock protein (HSP)H1/105 in function of their aggressiveness. Here, we now clarify its role as a functional B-NHL target by testing the hypothesis that it promotes the stabilization of key lymphoma oncoproteins. HSPH1 silencing in 4 models of aggressive B-NHLs was paralleled by Bcl-6 and c-Myc downregulation. In vitro and in vivo analysis of HSPH1-silenced Namalwa cells showed that this effect was associated with a significant growth delay and the loss of tumorigenicity when 10(4) cells were injected into mice. Interestingly, we found that HSPH1 physically interacts with c-Myc and Bcl-6 in both Namalwa cells and primary aggressive B-NHLs. Accordingly, expression of HSPH1 and either c-Myc or Bcl-6 positively correlated in these diseases. Our study indicates that HSPH1 concurrently favors the expression of 2 key lymphoma oncoproteins, thus confirming its candidacy as a valuable therapeutic target of aggressive B-NHLs.

Molecular subtyping of metastatic melanoma based on cell ganglioside metabolism profiles.

BACKGROUND: In addition to alterations concerning the expression of oncogenes and onco-suppressors, melanoma is characterized by the presence of distinctive gangliosides (sialic acid carrying glycosphingolipids). Gangliosides strongly control cell surface dynamics and signaling; therefore, it could be assumed that these alterations are linked to modifications of cell behavior acquired by the tumor. On these bases, this work investigated the correlations between melanoma cell ganglioside metabolism profiles and the biological features of the tumor and the survival of patients. METHODS: Melanoma cell lines were established from surgical specimens of AJCC stage III and IV melanoma patients. Sphingolipid analysis was carried out on melanoma cell lines and melanocytes through cell metabolic labeling employing [3-3H]sphingosine and by FACS. N-glycolyl GM3 was identified employing the 14 F7 antibody. Gene expression was assayed by Real Time PCR. Cell invasiveness was assayed through a Matrigel invasion assay; cell proliferation was determined through the soft agar assay, MTT, and [3H] thymidine incorporation. Statistical analysis was performed using XLSTAT software for melanoma hierarchical clustering based on ganglioside profile, the Kaplan-Meier method, the log-rank (Mantel-Cox) test, and the Mantel-Haenszel test for survival analysis. RESULTS: Based on the ganglioside profiles, through a hierarchical clustering, we classified melanoma cells isolated from patients into three clusters: 1) cluster 1, characterized by high content of GM3, mainly in the form of N-glycolyl GM3, and GD3; 2) cluster 2, characterized by the appearance of complex gangliosides and by a low content of GM3; 3) cluster 3, which showed an intermediate phenotype between cluster 1 and cluster 3. Moreover, our data demonstrated that: a) a correlation could be traced between patients' survival and clusters based on ganglioside profiles, with cluster 1 showing the worst survival; b) the expression of several enzymes (sialidase NEU3, GM2 and GM1 synthases) involved in ganglioside metabolism was associated with patients' survival; c) melanoma clusters showed different malignant features such as growth in soft agar, invasiveness, expression of anti-apoptotic proteins. CONCLUSIONS: Ganglioside profile and metabolism is strictly interconnected with melanoma aggressiveness. Therefore, the profiling of melanoma gangliosides and enzymes involved in their metabolism could represent a useful prognostic and diagnostic tool.

Sialidase NEU3 dynamically associates to different membrane domains specifically modifying their ganglioside pattern and triggering Akt phosphorylation.

Lipid rafts are known to regulate several membrane functions such as signaling, trafficking and cellular adhesion. The local enrichment in sphingolipids and cholesterol together with the low protein content allows their separation by density gradient flotation after extraction with non-ionic detergent at low temperature. These structures are also referred to as detergent resistant membranes (DRM). Among sphingolipids, gangliosides play important roles in different biological events, including signal transduction and tumorigenesis. Sialidase NEU3 shows high enzymatic specificity toward gangliosides. Moreover, the enzyme is present both at the cell surface and in endosomal structures and cofractionates with caveolin. Although changes in the expression level of NEU3 have been correlated to different tumors, little is known about the precise distribution of the protein and its ability in modifying the ganglioside composition of DRM and non-DRM, thus regulating intracellular events. By means of inducible expression cell system we found that i) newly synthesized NEU3 is initially associated to non-DRM; ii) at steady state the protein is equally distributed between the two membrane subcompartments, i.e., DRM and non-DRM; iii) NEU3 is degraded via the proteasomal pathway; iv) the enzyme specifically modifies the ganglioside composition of the membrane areas where it resides; and v) NEU3 triggers phosphorylation of Akt, even in absence of exogenously administered EGF. Taken together our data demonstrate that NEU3 regulates the DRM ganglioside content and it can be considered as a modulator of Akt phosphorylation, further supporting the role of this enzyme in cancer and tumorigenesis.

Prevalence of a characteristic gene profile in high-level rhythmic gymnasts.

High-level physical performance in rhythmic gymnastics is influenced by numerous skills and anthropometric factors. In order to understand if genetic predisposition could play a role to define the elite rhythmic gymnast phenotype, we analysed the frequency of common polymorphisms linked to genes correlated with body mass (ADRB2 and FTO), explosive strength (ACTN3 and ACE), and joint mobility (COL5A1), in 42 gymnasts involved in National and International events, and in 42 control girls. Our results demonstrated that high-level rhythmic gymnasts constituted a genetically selected population showing higher frequency of: (a) ADRB2 and FTO alleles linked to low body mass index and low fat mass; (b) COL5A1 CT genotype linked to high joint mobility and to the occurrence of genu recurvatum, but also to a higher incidence of injuries. ACTN3 and ACE polymorphisms did not appear to be connected with the phenotype of high-level rhythmic gymnast. Based on these data, it can be assumed that these polymorphisms could positively affect the phenotype and performance of gymnasts.

Sphingolipids: key regulators of apoptosis and pivotal players in cancer drug resistance.

Drug resistance elicited by cancer cells still constitutes a huge problem that frequently impairs the efficacy of both conventional and novel molecular therapies. Chemotherapy usually acts to induce apoptosis in cancer cells; therefore, the investigation of apoptosis control and of the mechanisms used by cancer cells to evade apoptosis could be translated in an improvement of therapies. Among many tools acquired by cancer cells to this end, the de-regulated synthesis and metabolism of sphingolipids have been well documented. Sphingolipids are known to play many structural and signalling roles in cells, as they are involved in the control of growth, survival, adhesion, and motility. In particular, in order to increase survival, cancer cells: (a) counteract the accumulation of ceramide that is endowed with pro-apoptotic potential and is induced by many drugs; (b) increase the synthesis of sphingosine-1-phosphate and glucosylceramide that are pro-survivals signals; (c) modify the synthesis and the metabolism of complex glycosphingolipids, particularly increasing the levels of modified species of gangliosides such as 9-O acetylated GD3 (αNeu5Ac(2-8)αNeu5Ac(2-3)ßGal(1-4)ßGlc(1-1)Cer) or N-glycolyl GM3 (αNeu5Ac (2-3)ßGal(1-4)ßGlc(1-1)Cer) and de-N-acetyl GM3 (NeuNH(2)ßGal(1-4)ßGlc(1-1)Cer) endowed with anti-apoptotic roles and of globoside Gb3 related to a higher expression of the multidrug resistance gene MDR1. In light of this evidence, the employment of chemical or genetic approaches specifically targeting sphingolipid dysregulations appears a promising tool for the improvement of current chemotherapy efficacy.

Gangliosides as a potential new class of stem cell markers: the case of GD1a in human bone marrow mesenchymal stem cells.

Owing to their exposure on the cell surface and the possibility of being directly recognized with specific antibodies, glycosphingolipids have aroused great interest in the field of stem cell biology. In the search for specific markers of the differentiation of human bone marrow mesenchymal stem cells (hBMSCs) toward osteoblasts, we studied their glycosphingolipid pattern, with particular attention to gangliosides. After lipid extraction and fractionation, gangliosides, metabolically (3)H-labeled in the sphingosine moiety, were separated by high-performance TLC and chemically characterized by MALDI MS. Upon induction of osteogenic differentiation, a 3-fold increase of ganglioside GD1a was observed. Therefore, the hypothesis of GD1a involvement in hBMSCs commitment toward the osteogenic phenotype was tested by comparison of the osteogenic propensity of GD1a-highly expressing versus GD1a-low expressing hBMSCs and direct addition of GD1a in the differentiation medium. It was found that either the high expression of GD1a in hBMSCs or the addition of GD1a in the differentiation medium favored osteogenesis, providing a remarkable increase of alkaline phosphatase. It was also observed that ganglioside GD2, although detectable in hBMSCs by immunohistochemistry with an anti-GD2 antibody, could not be recognized by chemical analysis, likely reflecting a case, not uncommon, of molecular mimicry.

Protective role of 17-ß-estradiol towards IL-6 leukocyte expression induced by intense training in young female athletes.

Exercise performed at a competitive level could deeply modify the immune system and the cytokine response of athletes. In this report, we demonstrated that young elite female artistic gymnasts (n = 16; age: 9-15 years) showed an increase of interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-α) mRNA expression in blood mononuclear cells (PBMCs), in comparison to girls performing the same sport at a recreational level (n = 16; age: 10-15 years). The increase of IL-6 and TNF-α mRNAs appeared to be directly linked to the intensity and duration of the training. Moreover, in elite athletes engaged in artistic gymnastics or in synchronised swimming (n =34; age: 9-15 years), IL-6 gene expression appeared to be modulated by the levels of circulating oestrogens: pre-pubertal athletes (n = 20; age: 11 ± 1 years) revealed a higher increase in IL-6 than pubertal athletes (n = 14; age: 14 ± 1.6 years). In pre-pubertal athletes, body mass index (BMI) percentile was inversely correlated with the increase of both IL-6 and TNF-α. The consequence of these events was the shift of the cytokine profile towards a pro-inflammatory status. These modifications, induced by training performed at an elite level, might negatively affect the growth of female children athletes.

A proline-rich loop mediates specific functions of human sialidase NEU4 in SK-N-BE neuronal differentiation.

Human sialidase NEU4 long (N4L) is a membrane-associated enzyme that has been shown to be localized in the outer mitochondrial membrane. A role in different cellular processes has been suggested for this enzyme, such as apoptosis, neuronal differentiation and tumorigenesis. However, the molecular bases for these roles, not found in any of the other highly similar human sialidases, are not understood. We have found that a proline-rich sequence of 81 amino acids, unique to NEU4 sequence, contains potential Akt and Erk1 kinase motifs. Molecular modeling, based on the experimentally determined three-dimensional structure of cytosolic human NEU2, showed that the proline-rich sequence is accommodated in a loop, thus preserving the typical beta-barrel structure of sialidases. In order to investigate the role of this loop in neuronal differentiation, we obtained SK-N-BE neuroblastoma cells stably overexpressing either human wild-type N4L or a deletion mutant lacking the proline-rich loop. Our results demonstrate that the proline-rich region can also enhance cell proliferation and retinoic acid (RA)-induced neuronal differentiation and it is also involved in NEU4 interaction with Akt, as well as in substrate recognition, modifying directly or through the interaction with other protein(s) the enzyme specificity toward sialylated glycoprotein(s). On the whole, our results suggest that N4L could be a downstream component of the PI3K/Akt signaling pathway required for RA-induced differentiation of neuroblastoma SK-N-BE cells.

Identification of lysosomal sialidase NEU1 and plasma membrane sialidase NEU3 in human erythrocytes.

The sialylation level of molecules, sialoglycoproteins and gangliosides, protruding from plasma membranes regulates multiple facets of erythrocyte function, from interaction with endothelium to cell lifespan. Our results demonstrate that: (a) Both sialidases NEU1 and NEU3 are present on erythrocyte plasma membrane; (b) NEU1 is kept on the plasma membrane in absence of the protective protein/cathepsin A (PPCA); (c) NEU1 and NEU3 are retained on the plasma membrane, as peripheral proteins, associated to the external leaflet and released by alkaline treatments; (d) NEU1 and NEU3 are segregated in Triton X-100 detergent-resistant membrane domains (DRMs); (e) NEU3 shows activity also at neutral pH; and (f) NEU1 and NEU3 are progressively lost during erythrocyte life. Interestingly, sialidase activity released from erythrocyte membranes after an alkaline treatment preserves its functionality and recognizes sialoglycoproteins and gangliosides. On the other hand, the weak anchorage of sialidases to the plasma membrane and their loss during erythrocyte life could be a tool to preserve the cellular sialic acid content in order to avoid the early ageing of erythrocyte and processes of cell aggregation in the capillaries.

NEU3 sialidase is activated under hypoxia and protects skeletal muscle cells from apoptosis through the activation of the epidermal growth factor receptor signaling pathway and the hypoxia-inducible factor (HIF)-1α.

NEU3 sialidase, a key enzyme in ganglioside metabolism, is activated under hypoxic conditions in cultured skeletal muscle cells (C2C12). NEU3 up-regulation stimulates the EGF receptor signaling pathway, which in turn activates the hypoxia-inducible factor (HIF-1α), resulting in a final increase of cell survival and proliferation. In the same cells, stable overexpression of sialidase NEU3 significantly enhances cell resistance to hypoxia, whereas stable silencing of the enzyme renders cells more susceptible to apoptosis. These data support the working hypothesis of a physiological role played by NEU3 sialidase in protecting cells from hypoxic stress and may suggest new directions in the development of therapeutic strategies against ischemic diseases, particularly of the cerebro-cardiovascular system.

The plasma membrane sialidase NEU3 regulates the malignancy of renal carcinoma cells by controlling ß1 integrin internalization and recycling.

The human plasma membrane sialidase NEU3 is a key enzyme in the catabolism of membrane gangliosides, is crucial in the regulation of cell surface processes, and has been demonstrated to be significantly up-regulated in renal cell carcinomas (RCCs). In this report, we show that NEU3 regulates ß1 integrin trafficking in RCC cells by controlling ß1 integrin recycling to the plasma membrane and controlling activation of the epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK)/protein kinase B (AKT) signaling. NEU3 silencing in RCC cells increased the membrane ganglioside content, in particular the GD1a content, and changed the expression of key regulators of the integrin recycling pathway. In addition, NEU3 silencing up-regulated the Ras-related protein RAB25, which directs internalized integrins to lysosomes, and down-regulated the chloride intracellular channel protein 3 (CLIC3), which induces the recycling of internalized integrins to the plasma membrane. In this manner, NEU3 silencing enhanced the caveolar endocytosis of ß1 integrin, blocked its recycling and reduced its levels at the plasma membrane, and, consequently, inhibited EGFR and FAK/AKT. These events had the following effects on the behavior of RCC cells: they (a) decreased drug resistance mediated by the block of autophagy and the induction of apoptosis; (b) decreased metastatic potential mediated by down-regulation of the metalloproteinases MMP1 and MMP7; and (c) decreased adhesion to collagen and fibronectin. Therefore, our data identify NEU3 as a key regulator of the ß1 integrin-recycling pathway and FAK/AKT signaling and demonstrate its crucial role in RCC malignancy.

Implications for the mammalian sialidases in the physiopathology of skeletal muscle.

The family of mammalian sialidases is composed of four distinct versatile enzymes that remove negatively charged terminal sialic acid residues from gangliosides and glycoproteins in different subcellular areas and organelles, including lysosomes, cytosol, plasma membrane and mitochondria. In this review we summarize the growing body of data describing the important role of sialidases in skeletal muscle, a complex apparatus involved in numerous key functions and whose functional integrity can be affected by various conditions, such as aging, chronic diseases, cancer and neuromuscular disorders. In addition to supporting the proper catabolism of glycoconjugates, sialidases can affect different signaling pathways by desialylation of many receptors and modulation of ganglioside content in cell membranes, thus actively participating in myoblast proliferation, differentiation and hypertrophy, insulin responsiveness and skeletal muscle architecture.

The synthetic purine reversine selectively induces cell death of cancer cells.

The synthetic purine reversine has been shown to possess a dual activity as it promotes the de-differentiation of adult cells, including fibroblasts, into stem-cell-like progenitors, but it also induces cell growth arrest and ultimately cell death of cancer cells, suggesting its possible application as an anti-cancer agent. Aim of this study was to investigate the mechanism underneath reversine selectivity in inducing cell death of cancer cells by a comparative analysis of its effects on several tumor cells and normal dermal fibroblasts. We found that reversine is lethal for all cancer cells studied as it induces cell endoreplication, a process that malignant cells cannot effectively oppose due to aberrations in cell cycle checkpoints. On the other hand, normal cells, like dermal fibroblasts, can control reversine activity by blocking the cell cycle, entering a reversible quiescent state. However, they can be induced to become sensitive to the molecule when key cell cycle proteins, e.g., p53, are silenced.

MmNEU3 sialidase over-expression in C2C12 myoblasts delays differentiation and induces hypertrophic myotube formation.

Several factors affect the skeletal muscle differentiation process, in particular modifications of cell-cell contact, cell adhesion, and plasma membrane characteristics. In order to support the role of the plasma membrane-associated sialidase NEU3 in skeletal muscle differentiation and to analyse which events of the process are mainly affected by this sialidase, we decided to stably over-express MmNEU3 in C2C12 cells by a lentiviral vector and to investigate cell behavior during the differentiation process. Vitally stained C2C12 and NEU3 over-expressing cells were counted to reveal modifications in differentiation induction. We found that NEU3 over-expressing cells remained proliferative longer than control cells and delayed the onset of differentiation. Expression of p21, myogenic transcription factors, and myosin heavy chain (MHC), assessed by real time PCR, confirmed this behavior. In particular, no MHC-positive myotubes were present in NEU3 over-expressing cells as compared to wild type C2C12 cells at day 3 of differentiation. Moreover, NEU3 over-expressing cells completed the differentiation process very quickly and formed hypertrophic myotubes. Analysis of MAPK/ERK pathway activation showed an increased ERK 1/2 phosphorylation in NEU3 over-expressing cells at the beginning of differentiation. We postulate that sialidase NEU3, decreasing plasma membrane ganglioside GM3 content, affects the EGF receptor and the downstream signaling pathways, promoting proliferation and delaying differentiation. Furthermore NEU3 improves myoblast fusion probably via neural-cell adhesion molecule (NCAM) desialylation. Therefore, this work further supports the central role of NEU3 as a key modulator in skeletal muscle differentiation, particularly in the myoblast fusion step.

NEU4L sialidase overexpression promotes ß-catenin signaling in neuroblastoma cells, enhancing stem-like malignant cell growth.

Neuroblastoma (NB) is a frequently lethal tumor that occurs in childhood and originates from embryonic neural crest cells. The malignant and aggressive phenotype of NB is strictly related to the deregulation of pivotal pathways governing the proliferation/differentiation status of neural crest precursor cells, such as MYCN, Delta/Notch and Wnt/ß-catenin (CTNNB1) signaling. In this article, we demonstrate that sialidase NEU4 long (NEU4L) influences the differentiation/proliferation behavior of NB SK-N-BE cells by determining hyperactivation of the Wnt/ß-catenin signaling pathway. NEU4L overexpression in SK-N-BE cells induced significant increases in active, nonphosphorylated ß-catenin content, ß-catenin/TCF transcriptional activity and ß-catenin gene target expression including MYCN, MYC, CCND2 (cyclin D2) and CDC25A. In turn, these molecular features strongly modified the behavior of NEU4L SK-N-BE overexpressing cells, promoting the following: (1) an enhanced proliferation rate, mainly due to a faster transition from G1 to S phase in the cell cycle; (2) a more undifferentiated cell phenotype, which was similar to stem-like NB cells and possibly mediated by an increase of the expression of the pluripotency genes, MYC, NANOG, OCT-4, CD133 and NES (nestin); (3) the failure of NB cell differentiation after serum withdrawal. The molecular link between NEU4L and Wnt/ß-catenin signaling appeared to rely most likely on the capability of the enzyme to modify the sialylation level of cell glycoproteins. These findings could provide a new candidate for therapeutic treatment.

Gallus gallus NEU3 sialidase as model to study protein evolution mechanism based on rapid evolving loops.

BACKGROUND: Large surface loops contained within compact protein structures and not involved in catalytic process have been proposed as preferred regions for protein family evolution. These loops are subjected to lower sequence constraints and can evolve rapidly in novel structural variants. A good model to study this hypothesis is represented by sialidase enzymes. Indeed, the structure of sialidases is a ß-propeller composed by anti-parallel ß-sheets connected by loops that suit well with the rapid evolving loop hypothesis. These features prompted us to extend our studies on this protein family in birds, to get insights on the evolution of this class of glycohydrolases. RESULTS: Gallus gallus (Gg) genome contains one NEU3 gene encoding a protein with a unique 188 amino acid sequence mainly constituted by a peptide motif repeated six times in tandem with no homology with any other known protein sequence. The repeat region is located at the same position as the roughly 80 amino acid loop characteristic of mammalian NEU4. Based on molecular modeling, all these sequences represent a connecting loop between the first two highly conserved ß-strands of the fifth blade of the sialidase ß-propeller. Moreover this loop is highly variable in sequence and size in NEU3 sialidases from other vertebrates. Finally, we found that the general enzymatic properties and subcellular localization of Gg NEU3 are not influenced by the deletion of the repeat sequence. CONCLUSION: In this study we demonstrated that sialidase protein structure contains a surface loop, highly variable both in sequence and size, connecting two conserved ß-sheets and emerging on the opposite site of the catalytic crevice. These data confirm that sialidase family can serve as suitable model for the study of the evolutionary process based on rapid evolving loops, which may had occurred in sialidases. Giving the peculiar organization of the loop region identified in Gg NEU3, this protein can be considered of particular interest in such evolutionary studies and to get deeper insights in sialidase evolution.

Glycoglycerolipid analogues inhibit PKC translocation to the plasma membrane and downstream signaling pathways in PMA-treated fibroblasts and human glioblastoma cells, U87MG.

Glycoglycerolipid analogues, derived from 2-O-ß-D-galactosylglycerol, have been synthesized on the base of the structure of natural glycoglycerolipids showing anti-tumor and anti-inflammatory efficacy. These compounds have been previously demonstrated to inhibit phorbol 12-myristate-13-acetate (PMA) induced tumor promotion in mouse skin, but their mechanism of action has never been elucidated. In this work, we studied the effects of glycoglycerolipid analogues on PKC activation induced by PMA and its downstream target molecules, in human fibroblasts. Our results proved that: a) the tested compounds were able to block PKC translocation to the plasma membrane, promoted by PMA, in a dose-dependent manner (IC50: 0.48 µM for the most active compound 2); b) the efficacy of these compounds was strongly connected to their acyl chain linked to galactose; in particular, the addition of hexanoyl and branched chains enhanced PKC inhibition, the presence of a cyclohexane ring and an excessive length of the acyl chain, or its lack, exerted a negative effect; c) the inhibition of PKC translocation blocked enzyme activation and downstream signaling pathways, MAPK and FAK, involved in proliferation and adhesion/migration control. In addition, the branched glycoglycerolipid (compound 2) was able to inhibit PKC translocation and activation in naturally highly PKC activating glioblastoma cells, U87MG. As consequence, U87MG cell proliferation and, especially, migration potential resulted to be markedly reduced (-30% and -84%, respectively). Thus, these results reveal the role of a PKC-dependent mechanism in glycoglycerolipid analogues mediated protective effects and highlight their possible employment in the field of prevention/treatment of cancer.

Sialidases in vertebrates: a family of enzymes tailored for several cell functions.

This review summarizes the recent research development on vertebrate sialidase biology. Sialic acid-containing compounds play important roles in many physiological processes, including cell proliferation, apoptosis and differentiation, control of cell adhesion, immune surveillance, and clearance of plasma proteins. In this context, sialidases, the glycohydrolases that remove the terminal sialic acid at the non-reducing end of various glycoconjugates, perform an equally pivotal function. Sialidases in higher organisms are differentially expressed in cells and tissues/organs, with particular subcellular distribution and substrate specificity: they are the lysosomal (NEU1), the cytosolic (NEU2), and plasma membrane- and intracellular-associated sialidases (NEU3 and NEU4). The molecular cloning of several mammalian sialidases since 1993 has boosted research in this field. Here we summarize the results obtained since 2002, when the last general review on the molecular biology of mammalian sialidases was written. In those few years many original papers dealing with different aspects of sialidase biology have been published, highlighting the increasing relevance of these enzymes in glycobiology. Attention has also been paid to the trans-sialidases, which transfer sialic acid residues from a donor sialoconjugate to an acceptor asialo substrate. These enzymes are abundantly distributed in trypanosomes and employed to express pathogenicity, also in humans. There are structural similarities and strategic differences at the level of the active site between the mammalian sialidases and trans-sialidases. A better knowledge of these properties may permit the design of better anti-pathogen drugs.

LPS-induced cytokine production in human dendritic cells is regulated by sialidase activity.

Removal of sialic acid from glycoconjugates on the surface of monocytes enhances their response to bacterial LPS. We tested the hypothesis that endogenous sialidase activity creates a permissive state for LPS-induced cytokine production in human monocyte-derived DCs. Of the four genetically distinct sialidases (Neu1-4), Neu1, Neu3, and Neu4 are expressed in human monocytes, but only Neu1 and Neu3 are up-regulated as cells differentiate into DCs. Neu1 and Neu3 are present on the surface of monocytes and DCs and are also present intracellularly. DCs contain a greater amount of sialic acid than monocytes, but the amount of sialic acid/mg total protein declines during differentiation to DCs. This relative hyposialylation of cells does not occur in mature DCs grown in the presence of zanamivir, a pharmacologic inhibitor of Neu3 but not Neu1, or DANA, an inhibitor of Neu1 and Neu3. Inhibition of sialidase activity during differentiation to DCs causes no detectable change in cell viability or expression of DC surface markers. Differentiation of monocytes into DCs in the presence of zanamivir results in reduced LPS- induced expression of IL-6, IL-12p40, and TNF-α by mature DCs, demonstrating a role for Neu3 in cytokine production. A role for Neu3 is supported by inhibition of cytokine production by DANA in DCs from Neu1⁻/⁻ and WT mice. We conclude that sialidase-mediated change in sialic acid content of specific cell surface glycoconjugates in DCs regulates LPS-induced cytokine production, thereby contributing to development of adaptive immune responses.