sphingosine-1-phosphate and Neuroblastoma

Soluble oligomers arising from the aggregation of the amyloid beta peptide (Aβ) have been identified as the main pathogenic agents in Alzheimer's disease (AD). Prefibrillar oligomers of the 42-residue form of Aβ (Aβ

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Humans; Neuroblastoma; Neurons; Rats; Receptors, N-Methyl-D-Aspartate

Although implicated in neurodegeneration, autophagy has been characterized mostly in yeast and mammalian non-neuronal cells. In a recent study, we sought to determine if SPHK1 (sphingosine kinase 1), implicated previously in macroautophagy/autophagy in cancer cells, regulates autophagy in neurons. SPHK1 synthesizes sphingosine-1-phosphate (S1P), a bioactive lipid involved in cell survival. In our study, we discovered that, when neuronal autophagy is pharmacologically stimulated, SPHK1 relocalizes to the endocytic and autophagic organelles. Interestingly, in non-neuronal cells stimulated with growth factors, SPHK1 translocates to the plasma membrane, where it phosphorylates sphingosine to produce S1P. Whether SPHK1 also binds to the endocytic and autophagic organelles in non-neuronal cells upon induction of autophagy has not been demonstrated. Here, we determined if the effect in neurons is operant in the SH-SY5Y neuroblastoma cell line. In both non-differentiated and differentiated SH-SY5Y cells, a short incubation of cells in amino acid-free medium stimulated the formation of SPHK1-positive puncta, as in neurons. We also found that, unlike neurons in which these puncta represent endosomes, autophagosomes, and amphisomes, in SH-SY5Y cells SPHK1 is bound only to the endosomes. In addition, a dominant negative form of SPHK1 was very toxic to SH-SY5Y cells, but cultured primary cortical neurons tolerated it significantly better. These results suggest that autophagy in neurons is regulated by mechanisms that differ, at least in part, from those in SH-SY5Y cells.

Topics: Animals; Apoptosis; Autophagosomes; Autophagy; Cell Differentiation; Cell Line; Cell Line, Tumor; Cell Membrane; Cell Survival; Cells, Cultured; Endocytosis; Endosomes; Green Fluorescent Proteins; Humans; Light; Lipids; Lysophospholipids; Lysosomes; Neuroblastoma; Neurons; Phagosomes; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Rats; Signal Transduction; Sphingosine

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. Preliminary data derived from a human angiogenesis array in NB showed that the bioactive lipid sphingosine-1-phosphate (S1P) induced the secretion of several angiogenesis-related proteins including the important inflammatory factor chemokine (C-C motif) ligand 2 (CCL2). In the present study, we investigated the mechanism of S1P-induced CCL2 expression in NB.. Quantitative real-time PCR and CCL2 ELISA were conducted to detect the mRNA expression and protein secretion of CCL2 in NB cells. Gain and loss of function studies were performed by using specific S1PR antagonists, adenoviral transduction and siRNA transfection. Macrophage F4/80 receptor in NB xenografts was detected by quantitative real-time PCR and immunohistochemistry staining.. S1P induced CCL2 mRNA expression and protein secretion in a time- and concentration-dependent manner in NB cells. Blockade of S1P2 signaling using the selective S1P2 antagonist JTE-013 inhibited S1P-induced CCL2 expression. Overexpression of S1P2 by adenoviral transduction increased CCL2 secretion while knockdown of S1P2 by siRNA transfection decreased S1P-induced CCL2 secretion in NB cells. Macrophage infiltration, as detected by F4/80 staining, was significantly decreased in JTE-013-treated NB xenografts.. Taken together, our data for the first time demonstrate that S1P induced the macrophage-recruiting factor CCL2 expression in NB cells via S1P2, providing new insights into the complicated functions of S1P2 in cancer.

Topics: Chemokines, C; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Lysophospholipids; Neuroblastoma; Real-Time Polymerase Chain Reaction; RNA, Neoplasm; Signal Transduction; Sphingosine; Tumor Cells, Cultured

Neuroblastoma is the most common extracranial solid tumor in the pediatric population. Sorafenib (Nexavar), a multikinase inhibitor, blocks cell proliferation and induces apoptosis in certain types of cancers. Here, we tested antitumor effects of sorafenib (≤ 10 µM) on four human neuroblastoma cell lines, CHLA255, CHLA171, CHLA90 and SK-N-AS. Sorafenib inhibited cell proliferation and induced apoptosis of neuroblastoma tumor cells in a dose-dependent manner. Sorafenib inhibited phosphorylation of Signal Transducer and Activator of Transcription 3 (STAT3) proteins at Tyr705 in these cells, associated with inhibition of phosphorylated JAK2, an upstream kinase that mediates STAT3 phosphorylation. Expression of a constitutively-activated STAT3 mutant (pSTAT3-C) partially blocked the antitumor effects of sorafenib on neuroblastoma cells. Sorafenib also inhibited the phosphorylation of STAT3 induced by IL-6 and sphingosine-1-phosphate (S1P), a recently identified regulator for STAT3, in these tumor cells. Moreover, sorafenib downregulated phosphorylation of MAPK (p44/42) in neuroblastoma cells, consistent with inhibition of their upstream regulators MEK1/2. Sorafenib inhibited expression of cyclin E, cyclin D1/D2/D3, key regulators for cell cycle, and the antiapoptotic proteins Mcl-1 and survivin. Finally, sorafenib suppressed the growth of human neuroblastoma cells in a mouse xenograft model. Taken together, these findings suggest the potential use of sorafenib for the treatment of pediatric neuroblastomas.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Benzenesulfonates; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Interleukin-6; Janus Kinase 2; Lysophospholipids; Mice; Mice, Inbred NOD; Mice, SCID; Mitogen-Activated Protein Kinases; Neuroblastoma; Niacinamide; Phenylurea Compounds; Phosphorylation; Pyridines; Signal Transduction; Sorafenib; Sphingosine; STAT3 Transcription Factor; Xenograft Model Antitumor Assays

Metabolism of sphingolipids into downstream lipid mediators followed by signaling modulates tumor microenvironment and the cancer cells to influence tumor progression. As such, sphingolipid signaling represents a novel way to modulate tumor biology. Neuroblastoma (NB), the most common extracranial solid tumor of childhood, is highly angiogenic and often displays poor prognosis. However, the role of sphingolipid mediators is not known in NB. We found that NB expresses high levels of sphingosine kinase-2, which is essential for the formation of sphingosine-1-phosphate (S1P). S1P induced VEGF expression in SK-N-AS NB cells. The effect occurred at the transcriptional level. Hypoxia in combination with S1P had a synergistic effect on VEGF expression. Strong correlation was detected between S1P receptor-2 (S1P(2)) and VEGF mRNAs in 11 different cell lines and 17 NB tissues. Blockade of S1P(2) with the selective antagonist JTE-013 significantly inhibited S1P-induced VEGF expression. Overexpression and knockdown of S1P(2) in SK-N-AS cells increased or inhibited S1P-induced VEGF secretion, respectively. Interestingly, JTE-013 significantly inhibited tumor growth, VEGF mRNA expression, and induced apoptosis in the NB tumor xenografts. Taken together, our data suggest that enhanced formation of sphingolipid mediator S1P in NB profoundly influences tumor microenvironment by inducing VEGF expression via S1P(2). Modulation of sphingolipid signaling by inhibiting S1P(2) may constitute a novel strategy to control NB.

Topics: Angiogenesis Inducing Agents; Animals; Brain Neoplasms; Cell Line, Tumor; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Neoplastic; Humans; Hypoxia; In Situ Nick-End Labeling; Lysophospholipids; Male; Mice; Mice, Nude; Neuroblastoma; Phosphotransferases (Alcohol Group Acceptor); Platelet Endothelial Cell Adhesion Molecule-1; Sphingolipids; Sphingosine; Vascular Endothelial Growth Factor A

The ability of G protein-coupled receptors to regulate osmosensitive uptake of the organic osmolyte, taurine, into human SH-SY5Y neuroblastoma cells has been examined. When monitored under isotonic conditions and in the presence of physiologically relevant taurine concentrations (1-100 microM), taurine influx was mediated exclusively by a Na(+)-dependent, high-affinity (K(m) = 2.5 microM) saturable transport mechanism (V(max) = 0.087 nmol/mg protein/min). Reductions in osmolarity of > 20% (attained under conditions of a constant NaCl concentration) resulted in an inhibition of taurine influx (> 30%) that could be attributed to a reduction in V(max), whereas the K(m) for uptake remained unchanged. Inclusion of the muscarinic cholinergic agonist, oxotremorine-M (Oxo-M), also resulted in an attenuation of taurine influx (EC(50) approximately 0.7 microM). Although Oxo-M-mediated inhibition of taurine uptake could be observed under isotonic conditions (approximately 25-30%), the magnitude of inhibition was significantly enhanced by hypotonicity (approximately 55-60%), a result that also reflected a reduction in the V(max), but not the K(m), for taurine transport. Oxo-M-mediated inhibition of taurine uptake was dependent upon the availability of extracellular Ca(2+) but was independent of protein kinase C activity. In addition to Oxo-M, inclusion of either thrombin or sphingosine 1-phosphate also attenuated volume-dependent taurine uptake. The ability of Oxo-M to inhibit the influx of taurine was attenuated by 4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid, an inhibitor of the volume-sensitive organic osmolyte and anion channel. 4-[(2-Butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid also prevented receptor-mediated changes in the efflux and influx of K(+) under hypoosmotic conditions. The results suggest that muscarinic receptor activation can regulate both the volume-dependent efflux and uptake of taurine and that these events may be functionally coupled.

Topics: Alanine; Antioxidants; Biological Transport; Calcium; Cell Line, Tumor; Cyclopentanes; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guanidine; Humans; Indans; Lysophospholipids; Muscarinic Agonists; Neuroblastoma; Osmolar Concentration; Oxotremorine; Receptors, Muscarinic; Saline Solution, Hypertonic; Sphingosine; Taurine; Thapsigargin; Thrombin; Tritium

Sphingosine 1-phosphate (S1P) has assumed great importance within neuroscience research because of putative links between S1P-sensitive Edg receptors and neuroregeneration, cell survival, and alterations in neurite outgrowth. In the present study, we examined the mechanisms by which the endogenous complement of S1P-sensitive human Edg receptors can elevate Ca(2+) in the human neuroblastoma cell line, SH-SY5Y. Reverse transcriptase-polymersase chain reaction (RT-PCR) confirmed the expression of mRNA for Edg 3, 5, and 8 subtypes of S1P-responsive Edg receptors in SH-SY5Y cells. Neither S1P nor the muscarinic agonist methacholine were able to cause a change in SH-SY5Y cell morphology, whereas retinoic acid caused a range of changes, including an increase in neurite outgrowth, under similar test conditions. Stimulation with S1P resulted in a slowly rising increase in cytosolic Ca(2+) levels. These responses were dependent upon inositol-1,4,5-trisphosphate receptors, thapsigargin-sensitive endoplasmic reticulum, and also intact functional mitochondria. S1P-evoked Ca(2+) responses were similar in mechanism to those of methacholine, which activated a much faster responding, larger amplitude Ca(2+) response. These studies indicate that in an endogenous human expression system, S1P appears to be an efficacious agonist of Edg receptors. Despite its slow time course of response, S1P appears to activate the same single Ca(2+) store in SH-SY5Y cells as is activated by methacholine and other G protein coupled receptors.

Topics: Calcium Signaling; Dose-Response Relationship, Drug; Gene Expression Regulation; Humans; Lysophospholipids; Neuroblastoma; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysophospholipid; RNA, Messenger; Sphingosine; Tumor Cells, Cultured

The sphingosine kinase inhibitor, dimethylsphingosine, is an important tool for investigating intracellular effects of the putative second messenger compound, sphingosine 1-phosphate. However, the specificity of action of dimethylsphingosine has not been fully investigated. In human SH-SY5Y neuroblastoma cells, dimethylsphingosine (30 microM), produced a 25-fold increase in the EC(50) for methacholine-induced Ca(2+) mobilisation, and reduced the maximum response by 57+/-5%, suggesting the involvement of sphingosine 1-phosphate production in the Ca(2+) signal. However, dimethylsphingosine also inhibited [3H]N-methylscopolamine binding to whole SH-SY5Y cells and reduced methacholine-induced phosphoinositide turnover. Thus, this compound must be used with caution when investigating the role of sphingosine kinase in G-protein coupled receptor-mediated Ca(2+) mobilisation responses.

Topics: Brain Neoplasms; Calcium; Cell Line; Enzyme Inhibitors; GTP-Binding Proteins; Humans; Image Processing, Computer-Assisted; Inositol 1,4,5-Trisphosphate; Lysophospholipids; Methacholine Chloride; Muscarinic Agonists; N-Methylscopolamine; Neuroblastoma; Receptor, Muscarinic M3; Receptors, Muscarinic; Signal Transduction; Sphingosine

Lysophosphatidic acid (LPA)-mediated Ca(2+) mobilization in human SH-SY5Y neuroblastoma cells does not involve either inositol 1,4, 5-trisphosphate (Ins(1,4,5)P(3))- or ryanodine-receptor pathways, but is sensitive to inhibitors of sphingosine kinase. This present study identifies Edg-4 as the receptor subtype involved and investigates the presence of a Ca(2+) signaling cascade based upon the lipid second messenger molecule, sphingosine 1-phosphate. Both LPA and direct G-protein activation increase [(3)H]sphingosine 1-phosphate levels in SH-SY5Y cells. Measurements of (45)Ca(2+) release in premeabilized SH-SY5Y cells indicates that sphingosine 1-phosphate, sphingosine, and sphingosylphosphorylcholine, but not N-acetylsphingosine are capable of mobilizing intracellular Ca(2+). Furthermore, the effect of sphingosine was attenuated by the sphingosine kinase inhibitor dimethylsphingosine, or removal of ATP. Confocal microscopy demonstrated that LPA stimulated intracellular Ca(2+) "puffs," which resulted from an interaction between the sphingolipid Ca(2+) release pathway and Ins(1,4,5)P(3) receptors. Down-regulation of Ins(1,4,5)P(3) receptors uncovered a Ca(2+) response to LPA, which was manifest as a progressive increase in global cellular Ca(2+) with no discernible foci. We suggest that activation of an LPA-sensitive Edg-4 receptor solely utilizes the production of intracellular sphingosine 1-phosphate to stimulate Ca(2+) mobilization in SH-SY5Y cells. Unlike traditional Ca(2+) release processes, this novel pathway does not require the progressive recruitment of elementary Ca(2+) events.

Topics: Caffeine; Calcium; Calcium Channels; Calcium Signaling; Guanosine 5'-O-(3-Thiotriphosphate); Heterotrimeric GTP-Binding Proteins; Humans; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Kinetics; Lysophospholipids; Neuroblastoma; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; Receptors, G-Protein-Coupled; Receptors, Lysophosphatidic Acid; Reverse Transcriptase Polymerase Chain Reaction; Sphingosine; Tumor Cells, Cultured

Treatment of rat glioma C6 cells with the beta-adrenergic agonist L-isoproterenol leads to a rise in cAMP level and a subsequent change in cell morphology from an epithelial to an astrocytic type of appearance. This morphological response is reverted by the addition of sphingosine-1-phosphate (S1P) with an EC50 of 10 nM. In rat glioma C6 cells loaded with the Ca2+ indicator Fura-2, S1P evoked Ca2+ release from internal stores and Ca2+ influx from the external medium. Half-maximal stimulation of the Ca2+ increase was 10-20 nM. A similar Ca2+ signal was observed in primary rat astrocytes loaded with the Ca2+ indicator fluo-3. Pretreatment of the C6 cells with PMA (162 nM) prevented both the S1P-induced Ca2+ increase and the morphological reversion. Ca2+ ions therefore seem essential for the morphological reversion by S1P. Pretreatment of the cells with the Clostridium botulinum C3 exoenzyme did not affect the reversion of the morphological response by S1P, indicating that the small GTP-binding protein Rho is not involved in the S1P-induced reversion.

Topics: Adrenergic beta-Agonists; Animals; Astrocytes; Calcium; Cell Size; Cells, Cultured; Glioma; Lysophospholipids; Mice; Neuroblastoma; Rats; Signal Transduction; Sphingosine; Thrombin; Tumor Cells, Cultured