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1.
Biochem Biophys Res Commun ; 500(3): 597-602, 2018 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-29673590

RESUMEN

Sphingosine-1-phosphate is synthesized by two sphingosine kinases, cytosolic SK1 and nuclear SK2 but SK2 expression was much higher than SK1in mouse skin fibroblasts. However, in SK2-/- cells, SK1 expression was markedly increased to SK2 levels whereas in SK1-/- cells, SK2 expression was unaffected. Ceramide, glucosylceramide and sphingosine levels were all increased in SK1-/- but less so in SK2-/- cells and S1P levels were not significantly reduced in either SK1-/- or SK2-/- cells. Greatly increased Ceramide glucosyltransferase expression was observed in SK1-/- cells but less so in SK2-/- cells suggested a role in drug resistance. SK2-/- cells grew faster than control and SK1-/-. The cell division gene PCNA was significantly overexpressed in SK2-/- cells, suggesting a cross regulation between SKs and Ceramide glucosyltransferase.


Asunto(s)
Fibroblastos/citología , Fibroblastos/enzimología , Glucosiltransferasas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Muerte Celular , Línea Celular , Proliferación Celular , Cromatografía Líquida de Alta Presión , Glucosilceramidas/metabolismo , Ratones , Piel/citología , Esfingolípidos/metabolismo , Espectrometría de Masas en Tándem
2.
Biochim Biophys Acta ; 1861(2): 78-90, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26548718

RESUMEN

Increased synthesis of hyaluronic acid (HA) is often associated with increased metastatic potential and invasivity of tumor cells. 4-Methylumbelliferone (MU) is an inhibitor of HA synthesis, and has been studied as a potential anti-tumor drug to inhibit the growth of primary tumors and distant metastasis of tumor cells. Although several studies reported that the anticancer effects of MU are mediated by inhibition of HA signaling, the mechanism still needs to be clarified. In a previous study we demonstrated the regulation of HA synthesis by ceramide, and now show how MU activated neutral sphingomyelinase2 (NSMase2) generates ceramides and mediates MU induced inhibition of HA synthesis, cell migration and invasion, and apoptosis of tumor cells. Using a HA enriched mouse oligodendroglioma cell line G26-24 we found that MU elevated the activity of NSMase2 and increased ceramide levels, which in turn increased phosphatase PP2A activity. Further, the activated PP2A reduced phosphorylation of Akt, decreased activities of HA synthase2 (HAS2) and calpains, and inhibited both the synthesis of HA, and the migration and invasion of G26-24 tumor cells. In addition, MU mediated ceramide stimulated activation of p53 and caspase-3, reduced SIRT1 expression and decreased G26-24 viability. The mechanism of the MU anticancer therefore initially involves NSMase2/ceramide/PP2A/AKT/HAS2/caspase-3/p53/SIRT1 and the calpain signaling pathway, suggesting that ceramides play a key role in the ability of a tumor to become aggressively metastatic and grow.


Asunto(s)
Antineoplásicos/farmacología , Ceramidas/biosíntesis , Regulación Neoplásica de la Expresión Génica , Ácido Hialurónico/antagonistas & inhibidores , Himecromona/farmacología , Neuroglía/efectos de los fármacos , Esfingomielina Fosfodiesterasa/genética , Animales , Apoptosis/efectos de los fármacos , Calpaína/genética , Calpaína/metabolismo , Caspasa 3/genética , Caspasa 3/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Activación Enzimática , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Humanos , Hialuronano Sintasas , Ácido Hialurónico/metabolismo , Ratones , Neuroglía/metabolismo , Neuroglía/patología , Fosforilación/efectos de los fármacos , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Sirtuina 1/genética , Sirtuina 1/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo
3.
J Neurochem ; 139(5): 872-885, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27622309

RESUMEN

The use of RNAi to suppress protein synthesis offers a potential way of reducing the level of enzymes or the synthesis of mutant toxic proteins but there are few tools currently available for their delivery. To address this problem, bioconjugated quantum dots (QDs) containing a hydrophobic component (N-palmitate) and a sequence VKIKK designed to traverse across cell membranes and visualize drug delivery were developed and tested on cell lines of brain origin. We used the Zn outer shell of the QD to bind HIS6 in JB577 (W•G•Dap(N-Palmitoyl)•VKIKK•P9 •G2 •H6 ) and by a gel-shift assay showed that siRNAs would bind to the positively charged KIKK sequence. By comparing many peptides and QD coatings, we showed that the QD-JB577-siRNA construct was taken up by cells of nervous system origin, distributed throughout the cytosol, and inhibited protein synthesis, implying that JB577 was also promoting endosome egress. By attaching siRNA for luciferase in a cell line over-expressing luciferase, we showed 70% inhibition of mRNA after 24-48 h. To show more specific effects, we synthesized siRNA for neutral (NSMase2), acid (lysosomal ASMase) sphingomyelinase, and sphingosine kinase 1 (SK1), we demonstrated a dose-dependent inhibition of activity. These data suggest that QDs are a useful siRNA delivery tool and QD-siRNA could be a potential theranostic for a variety of diseases.


Asunto(s)
Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Puntos Cuánticos/administración & dosificación , ARN Interferente Pequeño/administración & dosificación , Esfingomielina Fosfodiesterasa/antagonistas & inhibidores , Esfingomielina Fosfodiesterasa/metabolismo , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Inhibidores Enzimáticos/administración & dosificación , Técnicas de Transferencia de Gen , Humanos , Ratones , ARN Interferente Pequeño/genética
4.
J Biol Chem ; 287(17): 13620-32, 2012 Apr 20.
Artículo en Inglés | MEDLINE | ID: mdl-22383528

RESUMEN

Fibroblasts from the fro/fro mouse, with a deletion in the Smpd3 gene coding for the active site of neutral sphingomyelinase 2 (NSMase2), secreted increased amounts of hyaluronan (HA). This was reversed by transfection with the Smpd3 gene, suggesting a connection between sphingolipid and glycosaminoglycan metabolism. The deficiency of NSMase2 resulted in storage of sphingomyelin (SM) and cholesterol with a 50% reduction in ceramides (Cer). RT-PCR and Western blot analysis showed that increased HA secretion resulted from increased hyaluronan synthase 2 (HAS2) activity localized to sphingolipid-enriched lipid rafts. Although cholesterol levels were also elevated in lipid rafts from mouse fibroblasts deficient in lysosomal acid SMase activity (deletion of the Smpd1(-/-) gene), there was no increase in HA secretion. We then showed that in fro/fro fibroblasts, the reduced ceramide was associated with decreased phosphorylation of protein phosphatase 2A (PP2A) and increased phosphorylation of its substrate Akt-p, together with PI3K, PDK1, mTOR (mammalian target of rapamycin), and p70S6K, although PTEN was unaffected. Exogenous ceramide, as well as inhibitors of Akt (Akt inhibitor VIII), PI 3-kinase (LY294002 and wortmannin), and mTOR (rapamycin) reduced secretion of HA, whereas the NSMase2 inhibitor GW4869 increased HA synthesis and secretion. We propose that NSMase2/Cer are the key mediators of the regulation of HA synthesis, via microdomains and the Akt/mTOR pathway.


Asunto(s)
Ceramidas/metabolismo , Glucuronosiltransferasa/metabolismo , Ácido Hialurónico/biosíntesis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Esfingomielina Fosfodiesterasa/biosíntesis , Esfingomielina Fosfodiesterasa/deficiencia , Animales , Encéfalo/metabolismo , Ceramidas/química , Activación Enzimática , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Glicosaminoglicanos/química , Hialuronano Sintasas , Microdominios de Membrana/química , Ratones , Osteogénesis Imperfecta/metabolismo , Fosforilación , Transducción de Señal , Esfingolípidos/química , Serina-Treonina Quinasas TOR/metabolismo , Regulación hacia Arriba
5.
J Biol Chem ; 285(19): 14134-43, 2010 May 07.
Artículo en Inglés | MEDLINE | ID: mdl-20215115

RESUMEN

Both cultured neonatal rat hippocampal neurons and differentiated oligodendrocytes rapidly metabolized exogenous C(2)- and C(6)-ceramides to sphingosine (Sph) and sphingosine 1-phosphate (S1P) but only minimally to C(16-24)-ceramides. Dihydrosphinolipids were unaffected but were increased by exogenous C(6)-dihydroceramide. Conversely, quantitative liquid chromatography-tandem mass spectrometry technology showed that exogenous S1P (0.25-10 microm) was rapidly metabolized to both Sph (a >200-fold increase) and predominantly C(18)-ceramide (a >2-fold increase). Longer treatments with either C(2)-ceramide (>2.5 microm) or S1P (10 microm) led to apoptotic cell death. Thus, there is an active sphingolipid salvage pathway in both neurons and oligodendrocytes. Staurosporine-induced cell death was shown to be associated with decreased S1P and increased Sph and C(16/18)-ceramide levels. The physiological significance of this observation was confirmed by the analysis of affected white matter and plaques from brains of multiple sclerosis patients in which reduced S1P and increased Sph and C(16/18)-ceramides were observed.


Asunto(s)
Apoptosis , Ceramidas/metabolismo , Lisofosfolípidos/metabolismo , Esclerosis Múltiple/metabolismo , Neuronas/metabolismo , Oligodendroglía/metabolismo , Esfingosina/análogos & derivados , Animales , Animales Recién Nacidos , Autopsia , Encéfalo/metabolismo , Estudios de Casos y Controles , Hipocampo/metabolismo , Humanos , Esclerosis Múltiple/patología , Ratas , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Esfingosina/metabolismo
6.
Biochem Biophys Res Commun ; 404(1): 321-3, 2011 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-21130737

RESUMEN

The immunomodulator drug Gilenya (FTY720), marketed as the first oral sphingosine-1-phosphate receptor (S1P-R) modulator for treatment of Multiple Sclerosis (MS) also inhibits lysosomal acid sphingomyelinase (ASMase). Treatment of cultured cells for 24 h with FTY720 (up to 10 µM) inhibited ASMase by >80% and this could be reversed by pre-treatment with the cathepsin protease inhibitor leupeptin (5 µM). In contrast, neutral sphingomyelinase activity was unaffected and sphingosine-1-phosphate treatment had no effect on ASMase. RT-PCR revealed no inhibition of ASMase mRNA and there was no direct (in vitro) inhibition of ASMase by either FTY720 or FTY720-phosphate. This suggests that its mechanism of inhibition is similar to that of tricyclic anti-depressants such as desipramine, which are also amphiphilic cationic drugs. Both Desipramine and FTY720 treatment reduced ASMase without significant inhibition of other lysosomal hydrolases but most hydrolases showed increased secretion (up to a 50% increase) providing more evidence of lysosomal disruption by these drugs.


Asunto(s)
Antidepresivos Tricíclicos/farmacología , Desipramina/farmacología , Inhibidores Enzimáticos/farmacología , Glicoles de Propileno/farmacología , Esfingomielina Fosfodiesterasa/antagonistas & inhibidores , Esfingosina/análogos & derivados , Animales , Catepsinas/antagonistas & inhibidores , Línea Celular Tumoral , Células Cultivadas , Inhibidores de Cisteína Proteinasa/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Clorhidrato de Fingolimod , Humanos , Hidrolasas/antagonistas & inhibidores , Inmunosupresores/farmacología , Leupeptinas/farmacología , Ratones , Neuronas/efectos de los fármacos , Neuronas/enzimología , Esfingosina/farmacología
7.
J Neurochem ; 110(5): 1388-99, 2009 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19545281

RESUMEN

Reactive oxygen species play a major role in neurodegeneration. Increasing concentrations of peroxide induce neural cell death through activation of pro-apoptotic pathways. We now report that hydrogen peroxide generated sn-2 oxidized phosphatidylcholine (OxPC) in neonatal rat oligodendrocytes and that synthetic OxPC [1-palmitoyl-2-(5'-oxo)valeryl-sn-glycero-3 phosphorylcholine, POVPC] also induced apoptosis in neonatal rat oligodendrocytes. POVPC activated caspases 3 and 8, and neutral sphingomyelinase (NSMase) but not acid sphingomyelinase. Downstream pro-apoptotic pathways activated by POVPC treatment included the Jun N-terminal kinase proapoptotic cascade and the degradation of phospho-Akt. Activation of NSMase occurred within 1 h, was blocked by inhibitors of caspase 8, increased mainly C18 and C24:1 ceramides, and appeared to be concentrated in detergent-resistant microdomains (Rafts). We concluded that OxPC initially activated NSMase and converted sphingomyelin into ceramide to mediate a series of downstream pro-apoptotic events in oligodendrocytes.


Asunto(s)
Oligodendroglía/metabolismo , Fosfatidilcolinas/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Humanos , Peróxido de Hidrógeno/farmacología , Oligodendroglía/efectos de los fármacos , Oxidación-Reducción/efectos de los fármacos , Éteres Fosfolípidos/farmacología , Ratas
8.
J Neurosci Res ; 86(11): 2414-22, 2008 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-18438937

RESUMEN

Demyelination is a common result of oxidative stress in the nervous system, and we report here that the response of oligodendrocytes to oxidative stress involves the receptor for advanced glycation end products (RAGE). RAGE has not previously been reported in neonatal rat oligodendrocytes (NRO), but, by using primers specific for rat RAGE, we were able to show expression of messenger RNA (mRNA) for RAGE in NRO, and a 55-kDa protein was detected by Western blotting with antibodies to RAGE. Neonatal rat oligodendrocytes stained strongly for RAGE, suggesting membrane localization of RAGE. Addition of low concentrations of hydrogen peroxide (100 microM) initiated 55-kDa RAGE shedding from the cell membrane and the appearance of "soluble" 45-kDa RAGE in the culture medium, followed by restoration of RAGE expression to normal levels. Increasing hydrogen peroxide concentration (>200 microM) resulted in no restoration of RAGE, and the cells underwent apoptosis and necrosis. We further confirmed the observation in a human oligodendroglioma-derived (HOG) cell line. Both the antioxidant N-acetyl-L-cysteine and the broad-spectrum metalloproteases inhibitor TAPI0 were able partially to inhibit shedding of RAGE, suggesting involvement of metalloproteases in cleavage to produce soluble RAGE. The level of 55-kDa RAGE in autopsy brain of patients undergoing neurodegeneration with accompanying inflammation [multiple sclerosis and neuronal ceroid-lipofuscinosis (Batten's disease)] was much lower than that in age-matched controls, suggesting that shedding of RAGE might occur as reactive oxygen species accumulate in brain cells and be part of the process of neurodegeneration.


Asunto(s)
Degeneración Nerviosa/metabolismo , Oligodendroglía/metabolismo , Estrés Oxidativo/fisiología , Receptores Inmunológicos/metabolismo , Animales , Antioxidantes/farmacología , Western Blotting , Encéfalo/metabolismo , Encéfalo/patología , Inhibidores Enzimáticos/farmacología , Humanos , Peróxido de Hidrógeno/farmacología , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , Lipofuscinosis Ceroideas Neuronales/metabolismo , Lipofuscinosis Ceroideas Neuronales/patología , Oxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Ratas , Receptor para Productos Finales de Glicación Avanzada , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
9.
FEBS Lett ; 586(22): 4002-9, 2012 Nov 16.
Artículo en Inglés | MEDLINE | ID: mdl-23046545

RESUMEN

NSMase2 is associated to the plasma membrane, whereas ASMase is predominantly lysosomal; both hydrolyze sphingomyelin (SM) to ceramide and phosphocholine. Although SM accumulated in both ASMase(-/-) and fro/fro (NSMase2(-/-)) fibroblasts, the reduction of ceramides was more dramatic in fro/fro cells. ASMase mRNA, protein and enzyme activity were substantially elevated in fro/fro fibroblasts. In contrast, NSMase2 activity was unaffected in ASMase(-/-) fibroblasts. ASMase(-/-) cells showed normal cell cycling whereas fro/fro cells grew slowly and were arrested in G1/G0 and could be corrected by transfection with smpd3 gene. This suggests two distinct subcellular pathways for SM catabolism with distinct functions.


Asunto(s)
Membrana Celular/enzimología , Fibroblastos/metabolismo , Lisosomas/enzimología , Esfingomielina Fosfodiesterasa/genética , Animales , Western Blotting , Puntos de Control del Ciclo Celular/genética , Células Cultivadas , Ceramidas/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/enzimología , Citometría de Flujo , Fase G1/genética , Perfilación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Noqueados , Fosforilcolina/metabolismo , Fase de Descanso del Ciclo Celular/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esfingomielina Fosfodiesterasa/metabolismo , Esfingomielinas/metabolismo
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