sphingosine-kinase and Neuroblastoma

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

Although disruptions in the maintenance of iron and cholesterol metabolism have been implicated in several cancers, the association between variants in the HFE gene that is associated with cellular iron uptake and cholesterol metabolism has not been studied. The C282Y-HFE variant is a risk factor for different cancers, is known to affect sphingolipid metabolism, and to result in increased cellular iron uptake. The effect of this variant on cholesterol metabolism and its possible relevance to cancer phenotype was investigated using wild type (WT) and C282Y-HFE transfected human neuroblastoma SH-SY5Y cells. Expression of C282Y-HFE in SH-SY5Y cells resulted in a significant increase in total cholesterol as well as increased transcription of a number of genes involved in its metabolism compared to cells expressing WT-HFE. The marked increase in expression of NPC1L1 relative to that of most other genes, was accompanied by a significant increase in expression of NPC1, a protein that functions in cholesterol uptake by cells. Because inhibitors of cholesterol metabolism have been proposed to be beneficial for treating certain cancers, their effect on the viability of C282Y-HFE neuroblastoma cells was ascertained. C282Y-HFE cells were significantly more sensitive than WT-HFE cells to U18666A, an inhibitor of desmosterol Δ24-reductase the enzyme catalyzing the last step in cholesterol biosynthesis. This was not seen for simvastatin, ezetimibe, or a sphingosine kinase inhibitor. These studies indicate that cancers presenting in carriers of the C282Y-HFE allele might be responsive to treatment designed to selectively reduce cholesterol content in their tumor cells.

Topics: Alleles; Androstenes; Azetidines; Cell Line, Tumor; Cell Survival; Cholesterol; Ezetimibe; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genetic Variation; Hemochromatosis Protein; Histocompatibility Antigens Class I; Humans; Iron; Membrane Proteins; Neuroblastoma; Oxidoreductases; Phenotype; Phosphotransferases (Alcohol Group Acceptor); Risk Factors; Simvastatin; Sphingolipids

Neuroblastoma (NB) is the most common extra-cranial solid tumor in childhood. Poor outcomes for children with advanced disease underscore the need for novel therapeutic strategies. FTY720, an immunomodulating drug approved for multiple sclerosis, has been investigated in oncology with promising preclinical activities. To date, its effect in NB has not been explored. Herein we describe our preclinical experience with FTY720, alone or in combination with topotecan, and its putative mechanism of action in NB.. MTT assay was performed to assess the effect of FTY720 on cell viability. A NB xenograft model was employed to assess the efficacy of FTY720 on tumor growth. Quantitative real-time PCR and Western blot were employed to determine changes of mRNA and protein expression, respectively. Liquid chromatography/tandem mass spectrometry was used to measure sphingolipid levels.. FTY720, but not FTY720-P induced NB cell death. FTY720 inhibited the growth of NB xenografts and enhanced the tumor-suppressive effect of topotecan both in vitro and in vivo. FTY720 significantly inhibited sphingosine kinase 2 (SphK2) mRNA and protein expression in NB cells. Pro-apoptotic sphingosine levels were increased in NB cells and NB xenografts treated with FTY720. FTY720-induced cell death was caspase-independent and involved the dephosphorylation of Akt and BAD at Ser136.. Our data demonstrate that FTY720 has potent preclinical anti-cancer activity in NB. Its unique death signaling mechanism, interference with the sphingolipid pathway, acts cooperatively with that of topotecan, suggesting that FTY720 related molecules may be useful in NB treatment.

Topics: Animals; Cell Death; Child; Child, Preschool; Drug Synergism; Fingolimod Hydrochloride; Humans; Immunosuppressive Agents; Infant; Male; Mice; Mice, Nude; Neuroblastoma; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Signal Transduction; Sphingolipids; Sphingosine; Topoisomerase I Inhibitors; Topotecan; Xenograft Model Antitumor Assays

Inflammation accompanied by severe oxidative stress plays a vital role in the orchestration and progression of neurodegeneration prevalent in chronic and acute central nervous system pathologies as well as in aging. The proinflammatory cytokine tumor necrosis factor-α (TNFα) elicits the formation of the bioactive ceramide by stimulating the hydrolysis of the membrane lipid sphingomyelin by sphingomyelinase activities. Ceramide stimulates the formation of reactive oxygen species (ROS) and apoptotic mechanisms in both neurons and nonneuronal cells, establishing a link between sphingolipid metabolism and oxidative stress. We demonstrated in SH-SY5Y human neuroblastoma cells and primary cortical neurons that TNFα is a potent stimulator of Mg(2+) -dependent neutral sphingomyelinase (Mg(2+) -nSMase) activity, and sphingomyelin hydrolysis, rather than de novo synthesis, was the predominant source of ceramide increases. Mg(2+) -nSMase activity preceded an accumulation of ROS by a neuronal NADPH oxidase (NOX). Notably, TNFα provoked an NOX-dependent oxidative damage to sphingosine kinase-1, which generates sphingosine-1-phosphate, a ceramide metabolite associated with neurite outgrowth. Indeed, ceramide and ROS inhibited neurite outgrowth of dorsal root ganglion neurons by disrupting growth cone motility. Blunting ceramide and ROS formation both rescued sphingosine kinase-1 activity and neurite outgrowth. Our studies suggest that TNFα-mediated activation of Mg(2+) -nSMase and NOX in neuronal cells not only produced the neurotoxic intermediates ceramide and ROS but also directly antagonized neuronal survival mechanisms, thus accelerating neurodegeneration.

Topics: Animals; Apoptosis; Cell Membrane; Ceramides; Cerebral Cortex; Chick Embryo; Drug Interactions; Enzyme Inhibitors; Growth Cones; Humans; Magnesium; NADPH Oxidases; Neurites; Neuroblastoma; Neurons; Palmitates; Phosphotransferases (Alcohol Group Acceptor); Reactive Oxygen Species; RNA, Small Interfering; Sphingomyelin Phosphodiesterase; Tumor Necrosis Factor-alpha

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

Several studies have linked estrogens with sphingosine kinase (SphK) activity, enzyme responsible of sphingosine-1-phosphate synthesis (S-1P), however their possible interaction in the nervous system is not documented yet. In the present study, we developed a glutamate toxicity model in SH-SY5Y cells to evaluate the possible effect of the inhibition of SphK activity on the protective capability of 17β-estradiol (E2). Glutamate induced cytoskeletal actin changes associated to cytotoxic stress, significant increase of apoptotic-like nuclear fragmentation, Tau hyperphosphorylation and increase of p25/p35 cleavage. These effects were prevented by E2 pre-treatment during 24 h. Although the inhibition of SphK did not block this protective effect, significantly increased Tau hyperphosphorylation by glutamate, in a way that was not reverted by E2. Our results suggest that the decrease of glutamate-induced Tau hyperphosphorylation by 17β-estradiol requires SphK.

Topics: Actins; Apoptosis; Cell Line, Tumor; DNA Fragmentation; Estradiol; Glutamic Acid; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neuroblastoma; Phosphatidylinositol 3-Kinases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-akt; tau Proteins

We examined the role of sphingosine kinase-1 (SphK1), a critical regulator of the ceramide/sphingosine 1-phosphate (S1P) biostat, in the regulation of death and survival of SH-SY5Y neuroblastoma cells in response to amyloid beta (Abeta) peptide (25-35). Upon incubation with Abeta, SH-SY5Y cells displayed a marked down-regulation of SphK1 activity coupled with an increase in the ceramide/S1P ratio followed by cell death. This mechanism was redox-sensitive; N-acetylcysteine totally abrogated the down-regulation of SphK1 activity and strongly inhibited Abeta-induced cell death. SphK1 overexpression impaired the cytotoxicity of Abeta, whereas SphK1 silencing by RNA interference mimicked Abeta-induced cell death, thereby establishing a critical role for SphK1. We further demonstrated that SphK1 could mediate the well established cytoprotective action of insulin-like growth factor (IGF-I) against Abeta toxicity. A dominant-negative form of SphK1 or its pharmacological inhibition not only abrogated IGF-I-triggered stimulation of SphK1 but also hampered IGF-I protective effect. Similarly to IGF-I, the neuroprotective action of TGF-beta1 was also dependent on SphK1 activity; activation of SphK1 as well as cell survival were impeded by a dominant-negative form of SphK1. Taken together, these results provide the first illustration of SphK1 role as a critical regulator of death and survival of Abeta-treated cells.

Topics: Amyloid beta-Peptides; Cell Line; Cell Survival; Humans; Insulin-Like Growth Factor I; Neuroblastoma; Peptide Fragments; Phosphotransferases (Alcohol Group Acceptor); Transforming Growth Factor beta1

RET, the receptor of glial cell line-derived neurotrophic factor (GDNF) family ligands, is important for the development of kidney and peripheral neurons. GDNF promotes survival and differentiation of neurons. Mutation of RET leads to the constitutive signal activation causing papillary thyroid carcinoma and multiple endocrine neoplasia type 2 (MEN2). In this study, we report that GDNF/RET signaling up-regulates sphingosine kinase (SPHK) enzyme activity, SPHK1 protein and SPHK1 message in TGW human neuroblastoma cells. Silencing of SPHK1 using siRNA inhibited GDNF-induced neurite formation, GAP43 expression, and cell growth, suggesting the important role of SPHK1 in GDNF signal transduction. Furthermore, NIH3T3 cells transfected with MEN2A type mutated RET but not c-RET demonstrated the up-regulation of SPHK activity, SPHK1 protein and SPHK1 message compared with NIH3T3 cells. The cell growth and anchorage-independent colony formation of MEN2A-NIH3T3 was inhibited with siRNA of SPHK1, while no effect of scramble siRNA was observed. These results suggest the oncogenic role of SPHK1 in MEN2A type tumor. Promoter analysis showed that activator protein 2 and specificity protein 1 binding motif of the 5' promoter region of SPHK1 gene is important for its induction by GDNF. Furthermore, we demonstrated that ERK1/2 and PI3 kinase are involved in GDNF-induced SPHK1 transcription by using specific inhibitors.

Topics: Animals; Cell Differentiation; Cell Line; Cell Proliferation; Electrophoretic Mobility Shift Assay; Glial Cell Line-Derived Neurotrophic Factor; Humans; Mice; Multiple Endocrine Neoplasia Type 2a; Mutation; Neuroblastoma; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-ret; Signal Transduction; Time Factors; Transfection; Up-Regulation

In adherent SH-SY5Y human neuroblastoma cells, activation of G-protein-coupled muscarinic M3 receptors evoked a biphasic elevation of both intracellular [Ca(2+)] ([Ca(2+)]i) and inositol-1,4,5-trisphosphate (D-Ins(1,4,5)P3) mass. In both cases, temporal profiles consisted of rapid transient elevations followed by a decline to a lower, yet sustained level. In contrast, platelet-derived growth factor (PDGF), a receptor tyrosine kinase agonist acting via PDGF receptor b chains in these cells, elicited a slow and transient elevation of [Ca(2+)]i that returned to basal levels within 5 to 10 min with no evidence of inositol phosphate generation. Full responses for either receptor type required intracellular and extracellular Ca(2+) and mobilization of a shared thapsigargin-sensitive intracellular Ca(2+) store. Strategies that affected the ability of D-Ins(1,4,5)P3 to interact with the Ins(1,4,5)P3-receptor demonstrated an Ins(1,4,5)P3-dependency of the muscarinic receptor-mediated elevation of [Ca(2+)]i but showed that PDGF-mediated elevations of [Ca(2+)]i are Ins(1,4,5)P3-independent in these cells.

Topics: Calcium; Cell Adhesion; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Humans; Inositol 1,4,5-Trisphosphate; Neuroblastoma; Phosphotransferases (Alcohol Group Acceptor); Platelet-Derived Growth Factor; Receptors, Muscarinic; Signal Transduction; Thapsigargin; Thimerosal; Time Factors; Tumor Cells, Cultured

Extracellular application of lysophosphatidic acid (LPA) elevated intracellular Ca(2+) concentration ([Ca(2+)](i)) in human SH-SY5Y neuroblastoma cells. The maximal response to LPA occurred between 0. 1 and 1 microM, at which point [Ca(2+)](i) was increased by approx. 500 nM. This increase was of similar magnitude to that caused by the muscarinic acetylcholine receptor agonist methacholine (MCh), although the initial rate of release by LPA was slower. Both LPA and MCh released Ca(2+) from intracellular stores, as assessed by inhibition of their effects by thapsigargin, a blocker of endoplasmic reticular Ca(2+) uptake, and by the persistence of their action in nominally Ca(2+)-free extracellular medium. Similarly, both agonists appeared to stimulate store-refilling Ca(2+) entry. MCh produced a marked elevation in cellular Ins(1,4,5)P(3) and stimulated [(3)H]InsP accumulation in the presence of Li(+). In contrast, LPA failed to stimulate detectable phosphoinositide turnover. Chronic down-regulation of Ins(1,4,5)P(3) receptor (InsP(3)R) proteins with MCh did not affect Ca(2+) responses to LPA. In addition, heparin, a competitive antagonist of InsP(3)Rs, blocked Ca(2+)-mobilization in permeabilized SH-SY5Y cells in response to MCh or exogenously added Ins(1,4,5)P(3), but failed to inhibit Ca(2+)-release induced by LPA. Elevation of [Ca(2+)](i) elicited by LPA was blocked by guanosine 5'-[beta-thio]-diphosphate, indicating that this agonist acts via a G-protein-coupled receptor. However, pertussis toxin was without effect on LPA-evoked [Ca(2+)](i) responses, suggesting that G(i/o)-proteins were not involved. In the absence of extracellular Ca(2+), N,N-dimethylsphingosine (DMS, 30 microM), a competitive inhibitor of sphingosine kinase, blocked LPA-induced Ca(2+) responses by almost 90%. In addition, MCh-induced Ca(2+) responses were also diminished by the addition of DMS, although to a lesser extent than with LPA. We conclude that LPA mobilizes intracellular Ca(2+)-stores in SH-SY5Y cells independently of the generation and action of Ins(1,4,5)P(3). Furthermore, the Ca(2+)-response to LPA appears to be dependent on sphingosine kinase activation and the potential generation of the putative second messenger sphingosine 1-phosphate.

Topics: Calcium; Down-Regulation; Enzyme Activation; Guanosine Diphosphate; Heparin; Humans; Inositol 1,4,5-Trisphosphate; Ionomycin; Lysophospholipids; Methacholine Chloride; Neuroblastoma; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; Tumor Cells, Cultured