RESUMEN
We report here the virtual screening design, synthesis and activity of eight new inhibitors of SphK1. For this study we used a pre-trained Graph Convolutional Network (GCN) combined with docking calculations. This exploratory analysis proposed nine compounds from which eight displayed significant inhibitory effect against sphingosine kinase 1 (SphK1) demonstrating a high level of efficacy for this approach. Four of these compounds also displayed anticancer activity against different tumor cell lines, and three of them (5), (6) and (7) have shown a wide inhibitory action against many of the cancer cell line tested, with GI50 below 5 µM, being (5) the most promising with TGI below 10 µM for the half of cell lines. Our results suggest that the three most promising compounds reported here are the pyrimidine-quinolone hybrids (1) and (6) linked by p-aminophenylsulfanyl and o-aminophenol fragments respectively, and (8) without such aryl linker. We also performed an exhaustive study about the molecular interactions that stabilize the different ligands at the binding site of SphK1. This molecular modeling analysis was carried out by using combined techniques: docking calculations, MD simulations and QTAIM analysis. In this study we also included PF543, as reference compound, in order to better understand the molecular behavior of these ligands at the binding site of SphK1.These results provide useful information for the design of new inhibitors of SphK1 possessing these structural scaffolds.
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Antineoplásicos , Fosfotransferasas (Aceptor de Grupo Alcohol) , Quinolonas , Quinolonas/farmacología , Inhibidores de Proteínas Quinasas , Antineoplásicos/química , Modelos Moleculares , Línea Celular Tumoral , Simulación del Acoplamiento Molecular , Ensayos de Selección de Medicamentos Antitumorales , Proliferación Celular , Relación Estructura-Actividad , Estructura MolecularRESUMEN
The oncogenic mutated kinase BRAFV600E is an attractive molecular target because it is expressed in several human cancers, including melanoma. To present, only three BRAF small inhibitors are approved by the FDA for the treatment of patients with metastatic melanoma: Vemurafenib, Dabrafenib and Encorafenib. Although many protocol treatments have been probed in clinical trials, BRAF inhibition has a limited effectiveness because patients invariably develop resistance and secondary toxic effects associated with the therapy. These limitations highlight the importance of designing new and better inhibitors with different structures that could establish different interactions in the active site of the enzyme and therefore decrease resistance progress. Considering the data from our previous report, here we studied two series of derivatives of structural scaffolds as potential BRAF inhibitors: hydroxynaphthalenecarboxamides and substituted piperazinylpropandiols. Our results indicate that structural analogues of substituted piperazinylpropandiols do not show significantly better activities to that previously reported. In contrast, the hydroxynaphthalenecarboxamides derivatives significantly inhibited cell viability and ERK phosphorylation, a measure of BRAF activity, in Lu1205 BRAFV600E melanoma cells. In order to better understand these experimental results, we carried out a molecular modeling study using different combined techniques: docking, MD simulations and quantum theory of atoms in molecules (QTAIM) calculations. Thus, by using this approach we determined that the molecular interactions that stabilize the different molecular complexes are closely related to Vemurafenib, a well-documented BRAF inhibitor. Furthermore, we found that bi-substituted compounds may interact more strongly respect to the mono-substituted analogues, by establishing additional interactions with the DFG-loop at the BRAF-active site. On the bases of these results we synthesized and tested a new series of hydroxynaphthalenecarboxamides bi-substituted. Remarkably, all these compounds displayed significant inhibitory effects on the bioassays performed. Thus, the structural information reported here is important for the design of new BRAFV600E inhibitors possessing this type of structural scaffold.
Asunto(s)
Antineoplásicos/uso terapéutico , Melanoma/tratamiento farmacológico , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Antineoplásicos/farmacología , Humanos , Modelos Moleculares , FosforilaciónRESUMEN
The identification of the V600E activating mutation in the protein kinase BRAF in around 50% of melanoma patients has driven the development of highly potent small inhibitors (BRAFi) of the mutated protein. To date, Dabrafenib and Vemurafenib, two specific BRAFi, have been clinically approved for the treatment of metastatic melanoma. Unfortunately, after the initial response, tumors become resistant and patients develop a progressive and lethal disease, making imperative the development of new therapeutic options. The main objective of this work was to find new BRAF inhibitors with different structural scaffolds than those of the known inhibitors. Our study was carried out in different stages; in the first step we performed a virtual screening that allowed us to identify potential new inhibitors. In the second step, we synthesized and tested the inhibitory activity of the novel compounds founded. Finally, we conducted a molecular modelling study that allowed us to understand interactions at the molecular level that stabilize the formation of the different molecular complexes. Our theoretical and experimental study allowed the identification of four new structural scaffolds, which could be used as starting structures for the design and development of new inhibitors of BRAF. Our experimental data indicate that the most active compounds reduced significantly ERK½ phosphorylation, a measure of BRAF inhibition, and cell viability. Thus, from our theoretical and experimental results, we propose new substituted hydroxynaphthalenecarboxamides, N-(hetero)aryl-piperazinylhydroxyalkylphenylcarbamates, substituted piperazinylethanols and substituted piperazinylpropandiols as initial structures for the development of new inhibitors for BRAF. Moreover, by performing QTAIM analysis, we are able to describe in detail the molecular interactions that stabilize the different Ligand-Receptor complexes. Such analysis indicates which portion of the different molecules must be changed in order to obtain an increase in the binding affinity of these new ligands.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Vemurafenib/farmacologíaRESUMEN
Tumour-necrosis factor (TNF) receptor-associated factor 2 (TRAF2) is a key component in NF-kappaB signalling triggered by TNF-alpha. Genetic evidence indicates that TRAF2 is necessary for the polyubiquitination of receptor interacting protein 1 (RIP1) that then serves as a platform for recruitment and stimulation of IkappaB kinase, leading to activation of the transcription factor NF-kappaB. Although TRAF2 is a RING domain ubiquitin ligase, direct evidence that TRAF2 catalyses the ubiquitination of RIP1 is lacking. TRAF2 binds to sphingosine kinase 1 (SphK1), one of the isoenzymes that generates the pro-survival lipid mediator sphingosine-1-phosphate (S1P) inside cells. Here we show that SphK1 and the production of S1P is necessary for lysine-63-linked polyubiquitination of RIP1, phosphorylation of IkappaB kinase and IkappaBalpha, and IkappaBalpha degradation, leading to NF-kappaB activation. These responses were mediated by intracellular S1P independently of its cell surface G-protein-coupled receptors. S1P specifically binds to TRAF2 at the amino-terminal RING domain and stimulates its E3 ligase activity. S1P, but not dihydro-S1P, markedly increased recombinant TRAF2-catalysed lysine-63-linked, but not lysine-48-linked, polyubiquitination of RIP1 in vitro in the presence of the ubiquitin conjugating enzymes (E2) UbcH13 or UbcH5a. Our data show that TRAF2 is a novel intracellular target of S1P, and that S1P is the missing cofactor for TRAF2 E3 ubiquitin ligase activity, indicating a new paradigm for the regulation of lysine-63-linked polyubiquitination. These results also highlight the key role of SphK1 and its product S1P in TNF-alpha signalling and the canonical NF-kappaB activation pathway important in inflammatory, antiapoptotic and immune processes.
Asunto(s)
Lisofosfolípidos/metabolismo , Esfingosina/análogos & derivados , Factor 2 Asociado a Receptor de TNF/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Biocatálisis , Línea Celular , Activación Enzimática , Humanos , Quinasa I-kappa B/metabolismo , Proteínas I-kappa B/metabolismo , Lisina/metabolismo , Lisofosfolípidos/biosíntesis , Lisofosfolípidos/química , Ratones , Modelos Moleculares , Inhibidor NF-kappaB alfa , FN-kappa B/metabolismo , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Esfingosina/biosíntesis , Esfingosina/química , Esfingosina/metabolismo , Especificidad por Sustrato , Factor 2 Asociado a Receptor de TNF/química , Factor de Necrosis Tumoral alfa/farmacología , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
BACKGROUND: Sphingosine-1-phosphate (S1P), which is produced by 2 sphingosine kinase (SphK) isoenzymes, SphK1 and SphK2, has been implicated in IgE-mediated mast cell responses. However, studies of allergic inflammation in isotype-specific SphK knockout mice have not clarified their contribution, and the role that S1P plays in vivo in a mast cell- and IgE-dependent murine model of allergic asthma has not yet been examined. OBJECTIVE: We used an isoenzyme-specific SphK1 inhibitor, SK1-I, to investigate the contributions of S1P and SphK1 to mast cell-dependent airway hyperresponsiveness (AHR) and airway inflammation in mice. METHODS: Allergic airway inflammation and AHR were examined in a mast cell-dependent murine model of ovalbumin (OVA)-induced asthma. C57BL/6 mice received intranasal delivery of SK1-I before sensitization and challenge with OVA or only before challenge. RESULTS: SK1-I inhibited antigen-dependent activation of human and murine mast cells and suppressed activation of nuclear factor κB (NF-κB), a master transcription factor that regulates the expression of proinflammatory cytokines. SK1-I treatment of mice sensitized to OVA in the absence of adjuvant, in which mast cell-dependent allergic inflammation develops, significantly reduced OVA-induced AHR to methacholine; decreased numbers of eosinophils and levels of the cytokines IL-4, IL-5, IL-6, IL-13, IFN-γ, and TNF-α and the chemokines eotaxin and CCL2 in bronchoalveolar lavage fluid; and decreased pulmonary inflammation, as well as activation of NF-κB in the lungs. CONCLUSION: S1P and SphK1 play important roles in mast cell-dependent, OVA-induced allergic inflammation and AHR, in part by regulating the NF-κB pathway.
Asunto(s)
Amino Alcoholes/farmacología , Asma/tratamiento farmacológico , Hiperreactividad Bronquial/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Mastocitos/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Animales , Asma/inducido químicamente , Asma/enzimología , Asma/metabolismo , Hiperreactividad Bronquial/enzimología , Hiperreactividad Bronquial/metabolismo , Hiperreactividad Bronquial/patología , Líquido del Lavado Bronquioalveolar/química , Células Cultivadas , Quimiocina CCL2/metabolismo , Femenino , Células Caliciformes/efectos de los fármacos , Células Caliciformes/metabolismo , Humanos , Hiperplasia/tratamiento farmacológico , Hiperplasia/metabolismo , Inmunoglobulina E/metabolismo , Inflamación/metabolismo , Interferón gamma/metabolismo , Interleucinas/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Lisofosfolípidos/metabolismo , Mastocitos/metabolismo , Cloruro de Metacolina/farmacología , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Ovalbúmina/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Neuroblastoma, the most common extracranial solid tumor occurring in childhood, originates from the aberrant proliferation of neural crest cells. Accordingly, the mechanism underling neuronal differentiation could provide new strategies for neuroblastoma treatment. It is well known that neurite outgrowth could be induced by Angiotensin II (Ang II) AT2 receptors; however, the signaling mechanism and its possible interaction with NGF (neural growth factor) receptors remain unclear. Here, we show that Ang II and CGP42112A (AT2 receptor agonist) promote neuronal differentiation by inducing neurite outgrowth and ßIII-tubulin expression in SH-SY5Y neuroblastoma cells. In addition, we demonstrate that treatment with PD123319 (AT2 receptor antagonist) reverts Ang II or CGP42112A-induced differentiation. By using specific pharmacological inhibitors we established that neurite outgrowth induced by CGP42112A requires the activation of MEK (mitogen-activated protein kinase kinase), SphK (sphingosine kinase) and c-Src but not PI3K (phosphatidylinositol 3-kinase). Certainly, CGP42112A stimulated a rapid and transient (30 s, 1 min) phosphorylation of c-Src at residue Y416 (indicative of activation), following by a Src deactivation as indicated by phosphorylation of Y527. Moreover, inhibition of the NGF receptor tyrosine kinase A (TrkA) reduced neurite outgrowth induced by Ang II and CGP42112A. In summary, we demonstrated that AT2 receptor-stimulated neurite outgrowth in SH-SY5Y cells involves the induction of MEK, SphK and c-Src and suggests a possible transactivation of TrkA. In that regard, AT2 signaling pathway is a key player in neuronal differentiation and might be a potential target for therapeutic treatments.
RESUMEN
Over the last years, the incidence of melanoma, the deadliest form of skin cancer, has risen significantly. Nearly half of the melanoma patients exhibit the BRAFV600E mutation. Although the use of BRAF and MEK inhibitors (BRAFi and MEKi) showed an impressive success rate in melanoma patients, durability of response remains an issue because tumor quickly becomes resistant. Here, we generated and characterized Lu1205 and A375 melanoma cells resistant to vemurafenib (BRAFi). Resistant cells (Lu1205R and A375R) exhibit higher IC50 (5-6 fold increase) and phospho-ERK levels and 2-3 times reduced apoptosis than their sensitive parents (Lu1205S and A375S). Moreover, resistant cells are 2-3 times bigger, display a more elongated morphology and have a modulation of migration capacity. Interestingly, pharmacological inhibition of sphingosine kinases, that prevents sphingosine-1-phosphate production, reduces migration of Lu1205R cells by 50 %. In addition, although Lu1205R cells showed increased basal levels of the autophagy markers LC3II and p62, they have decreased autophagosome degradation and autophagy flux. Remarkably, expression of Rab27A and Rab27B, which are involved in the release of extracellular vesicles are dramatically augmented in resistant cells (i.e. 5-7 fold increase). Indeed, conditioned media obtained from Lu1205R cells increased the resistance to vemurafenib of sensitive cells. Hence, these results support that resistance to vemurafenib modulates migration and the autophagic flux and may be transferred to nearby sensitive melanoma cells by factors that are released to the extracellular milieu by resistant cells.
Asunto(s)
Melanoma , Proteínas Proto-Oncogénicas B-raf , Humanos , Vemurafenib/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Sulfonamidas/farmacología , Indoles/farmacología , Resistencia a Antineoplásicos/genética , Línea Celular Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , AutofagiaRESUMEN
The role of tumor necrosis factor-α (TNF-α) in shaping the tumor microenvironment is ambiguous. Consistent with its uncertain role in melanoma, TNF-α plays a dual role, either acting as a cytotoxic cytokine or favoring a tumorigenic inflammatory microenvironment. TNF-α signals via two cognate receptors, namely TNFR1 (p55) and TNFR2 (p75), which mediate divergent biological activities. Here, we analyzed the impact of TNFR1 deficiency in tumor progression in the B16.F1 melanoma model. Tumors developed in mice lacking TNFR1 (TNFR1 knock-out; KO) were smaller and displayed lower proliferation compared to their wild type (WT) counterpart. Moreover, TNFR1 KO mice showed reduced tumor angiogenesis. Although no evidence of spontaneous metastases was observed, conditioned media obtained from TNFR1 KO tumors increased tumor cell migration. Whereas the analysis of tumor-associated immune cell infiltrates showed similar frequency of total and M2-polarized tumor-associated macrophages (TAMs), the percentage of CD8+ T cells was augmented in TNFR1 KO tumors. Indeed, functional ex vivo assays demonstrated that CD8+ T cells obtained from TNFR1KO mice displayed an increased cytotoxic function. Thus, lack of TNFR1 attenuates melanoma growth by modulating tumor cell proliferation, migration, angiogenesis and CD8+ T cell accumulation and activation, suggesting that interruption of TNF-TNFR1 signaling may contribute to control tumor burden.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/inmunología , Neovascularización Patológica/inmunología , Receptores Tipo I de Factores de Necrosis Tumoral/deficiencia , Animales , Proliferación Celular , Activación de Linfocitos/inmunología , Melaninas/metabolismo , Melanoma Experimental/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Invasividad Neoplásica , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Transducción de Señal , Microambiente Tumoral/inmunologíaRESUMEN
Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates myriad important cellular processes, including growth, survival, cytoskeleton rearrangements, motility, and immunity. Here we report that treatment of Jurkat and U937 leukemia cells with the pan-sphingosine kinase (SphK) inhibitor N,N-dimethylsphingosine to block S1P formation surprisingly caused a large increase in expression of SphK1 concomitant with induction of apoptosis. Another SphK inhibitor, D,L-threo-dihydrosphingosine, also induced apoptosis and produced dramatic increases in SphK1 expression. However, up-regulation of SphK1 was not a specific effect of its inhibition but rather was a consequence of apoptotic stress. The chemotherapeutic drug doxorubicin, a potent inducer of apoptosis in these cells, also stimulated SphK1 expression and activity and promoted S1P secretion. The caspase inhibitor ZVAD reduced not only doxorubicin-induced lethality but also the increased expression of SphK1 and secretion of S1P. Apoptotic cells secrete chemotactic factors to attract phagocytic cells, and we found that S1P potently stimulated chemotaxis of monocytic THP-1 and U937 cells and primary monocytes and macrophages. Collectively, our data suggest that apoptotic cells may up-regulate SphK1 to produce and secrete S1P that serves as a "come-and-get-me" signal for scavenger cells to engulf them in order to prevent necrosis.
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Apoptosis/fisiología , Lisofosfolípidos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Esfingosina/análogos & derivados , Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Quimiotaxis de Leucocito/efectos de los fármacos , Quimiotaxis de Leucocito/fisiología , Doxorrubicina/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Técnicas In Vitro , Células Jurkat , Monocitos/efectos de los fármacos , Monocitos/fisiología , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Esfingosina/metabolismo , Esfingosina/farmacología , Células U937 , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Angiotensin II (Ang II) AT(2) receptors are abundantly expressed in rat fetal tissues where they probably contribute to development. In the present study we examine the effects of Ang II type 2 receptor stimulation on SHP-1 activation. Ang II (10(-7) M) elicits a rapid and transient tyrosine phosphorylation of SHP-1, maximal at 1 min, in a dose-dependent form, blocked by the AT(2) antagonist, PD123319. SHP-1 phosphorylation is followed in time by tyrosine dephosphorylation of different proteins, suggesting a sequence of events. Ang II induces association of SHP-1 to AT(2) receptors as shown by co-immunoprecipitation, Western blot and binding assays. SHP-1 activity was determined in immunocomplexes obtained with either anti-AT(2) or anti-SHP-1 antibodies, after Ang II stimulation (1 min), in correlation with the maximal level of SHP-1 phosphorylation. Interestingly, following receptor stimulation (1 min) c-Src was associated to AT(2) or SHP-1 immunocomplexes. Preincubation with the c-Src inhibitor PP2 inhibited SHP-1 activation and c-Src association, thus confirming the participation of c-Src in this pathway. We demonstrated here for the first time the involvement of c-Src in SHP-1 activation via AT(2) receptors present in an ex vivo model expressing both receptor subtypes. In this model, AT(2) receptors are not constitutively associated to SHP-1 and SHP-1 is not constitutively activated. Thus, we clearly establish that SHP-1 activation, mediated by the AT(2) subtype, involves c-Src and precedes protein tyrosine dephosphorylation, in rat fetal membranes.
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Feto/enzimología , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Receptor de Angiotensina Tipo 2/metabolismo , Animales , Femenino , Fosforilación , Ratas , Ratas Wistar , Transducción de SeñalRESUMEN
Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that has been implicated in many biological processes, including cell migration, survival, proliferation, angiogenesis and immune and allergic responses. S1P levels inside cells are regulated tightly by the balance between its synthesis by sphingosine kinases and degradation by S1P lyases and S1P phosphatases. Activation of sphingosine kinase by any of a variety of agonists increases S1P levels, which in turn can function intracellularly as a second messenger or in an autocrine and/or paracrine fashion to activate and signal through S1P receptors present on the surface of the cell. This review summarizes recent findings on the roles of S1P as a mediator of the actions of cytokines, growth factors and hormones.
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Comunicación Autocrina/fisiología , Lisofosfolípidos/fisiología , Comunicación Paracrina/fisiología , Esfingosina/análogos & derivados , Animales , Neoplasias de la Mama/metabolismo , Hormonas Esteroides Gonadales/biosíntesis , Hormonas Esteroides Gonadales/fisiología , Humanos , Mediadores de Inflamación/metabolismo , Mediadores de Inflamación/fisiología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/fisiología , Lisofosfolípidos/metabolismo , Modelos Biológicos , Neovascularización Patológica/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Transducción de Señal/fisiología , Esfingosina/metabolismo , Esfingosina/fisiologíaRESUMEN
Sphingosine-1-phosphate (S1P) is a bioactive lipid that modulates migratory behavior of cells during embryonic development. In addition, S1P might promote tumor progression by enhancing migratory ability and invasiveness of tumor cells. Migration is a complex process that implies cytoskeletal reorganization and formation of structures that enable cell movement. Besides having similar requirements than migration, invasion also involves proteolytic degradation of extracellular matrix (ECM). Matrix metalloproteases (MMPs) have been identified to break down components of the ECM, allowing cancer cells to spread out of the primary tumor. In this chapter, we will describe different techniques to study migration and invasion induced by S1P. To this end, we include detailed protocols of end-point assays to study migration/invasion, and zymography assay to analyze MMP-2 and MMP-9 activity that were standardized in our laboratory in human melanoma cell lines.
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Lisofosfolípidos/farmacología , Metaloproteinasas de la Matriz/metabolismo , Melanoma/metabolismo , Esfingosina/análogos & derivados , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Técnicas In Vitro , Invasividad Neoplásica , Esfingosina/farmacologíaRESUMEN
In the last 15 years, increasing evidences demonstrate a strong link between sphingosine-1-phosphate (S1P) and both normal physiology and progression of different diseases, including cancer and inflammation. Indeed, numerous studies show that tissue levels of this sphingolipid metabolite are augmented in many cancers, affecting survival, proliferation, angiogenesis, and metastatic spread. Recent insights into the possible role of S1P as a therapeutic target has attracted enormous attention and opened new opportunities in this evolving field. In this review, we will focus on the role of S1P in cancer, with particular emphasis in new developments that highlight the many functions of this sphingolipid in the tumor microenvironment. We will discuss how S1P modulates phenotypic plasticity of macrophages and mast cells, tumor-induced immune evasion, differentiation and survival of immune cells in the tumor milieu, interaction between cancer and stromal cells, and hypoxic response.
RESUMEN
Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator that regulates many processes in inflammation and cancer. S1P is a ligand for five G-protein-coupled receptors, S1PR1 to -5, and also has important intracellular actions. Previously, we showed that intracellular S1P is involved in tumor necrosis factor alpha (TNF)-induced NF-κB activation in melanoma cell lines that express filamin A (FLNA). Here, we show that extracellular S1P activates NF-κB only in melanoma cells that lack FLNA. In these cells, S1P, but not TNF, promotes IκB kinase (IKK) and p65 phosphorylation, IκBα degradation, p65 nuclear translocation, and NF-κB reporter activity. NF-κB activation induced by S1P was mediated via S1PR1 and S1PR2. Exogenous S1P enhanced the phosphorylation of protein kinase Cδ (PKCδ), and its downregulation reduced S1P-induced the phosphorylation of IKK and p65. In addition, silencing of Bcl10 also inhibited S1P-induced IKK phosphorylation. Surprisingly, S1P reduced Akt activation in melanoma cells that express FLNA, whereas in the absence of FLNA, high phosphorylation levels of Akt were maintained, enabling S1P-mediated NF-κB signaling. In accord, inhibition of Akt suppressed S1P-mediated IKK and p65 phosphorylation and degradation of IκBα. Hence, these results support a negative role of FLNA in S1P-mediated NF-κB activation in melanoma cells through modulation of Akt.
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Filaminas/inmunología , Lisofosfolípidos/inmunología , Melanoma/inmunología , FN-kappa B/inmunología , Proteínas Proto-Oncogénicas c-akt/inmunología , Transducción de Señal , Esfingosina/análogos & derivados , Línea Celular Tumoral , Humanos , Proteína Quinasa C-delta/inmunología , Receptores de Lisoesfingolípidos/inmunología , Esfingosina/inmunología , Receptores de Esfingosina-1-FosfatoRESUMEN
Transforming growth factor beta (TGFbeta) plays a dual role in oncogenesis, acting as both a tumor suppressor and a tumor promoter. These disparate processes of suppression and promotion are mediated primarily by Smad and non-Smad signaling, respectively. A central issue in understanding the role of TGFbeta in the progression of epithelial cancers is the elucidation of the mechanisms underlying activation of non-Smad signaling cascades. Because the potent lipid mediator sphingosine-1-phosphate (S1P) has been shown to transactivate the TGFbeta receptor and activate Smad3, we examined its role in TGFbeta activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and promotion of migration and invasion of esophageal cancer cells. Both S1P and TGFbeta activate ERK1/2, but only TGFbeta activates Smad3. Both ligands promoted ERK1/2-dependent migration and invasion. Furthermore, TGFbeta rapidly increased S1P, which was required for TGFbeta-induced ERK1/2 activation, as well as migration and invasion, since downregulation of sphingosine kinases, the enzymes that produce S1P, inhibited these responses. Finally, our data demonstrate that TGFbeta activation of ERK1/2, as well as induction of migration and invasion, is mediated at least in part by ligation of the S1P receptor, S1PR2. Thus, these studies provide the first evidence that TGFbeta activation of sphingosine kinases and formation of S1P contribute to non-Smad signaling and could be important for progression of esophageal cancer.
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Neoplasias Esofágicas/metabolismo , Regulación Neoplásica de la Expresión Génica , Lisofosfolípidos/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Esfingosina/análogos & derivados , Factor de Crecimiento Transformador beta/metabolismo , Línea Celular Tumoral , Movimiento Celular , Quimiotaxis , Progresión de la Enfermedad , Activación Enzimática , Neoplasias Esofágicas/patología , Humanos , Lisofosfolípidos/química , Modelos Biológicos , Invasividad Neoplásica , Esfingosina/química , Esfingosina/fisiologíaRESUMEN
Sphingosine kinase 1 (SphK1) catalyzes the phosphorylation of sphingosine to produce the potent lipid mediator sphingosine-1-phosphate (S1P), which plays a critical role in cell motility via its cell surface receptors. Here, we have identified filamin A (FLNa), an actin-cross-linking protein involved in cell movement, as a bona fide SphK1-interacting protein. Heregulin stimulated SphK1 activity only in FLNa-expressing A7 melanoma cells but not in FLNa-deficient cells and induced its translocation and colocalization with FLNa at lamellipodia. SphK1 was required for heregulin-induced migration, lamellipodia formation, activation of PAK1, and subsequent FLNa phosphorylation. S1P directly stimulated PAK1 kinase, suggesting that it may be a target of intracellularly generated S1P. Heregulin also induced colocalization of S1P(1) (promotility S1P receptor) but not S1P(2), with SphK1 and FLNa at membrane ruffles. Moreover, an S1P(1) antagonist inhibited the lamellipodia formation induced by heregulin. Hence, FLNa links SphK1 and S1P(1) to locally influence the dynamics of actin cytoskeletal structures by orchestrating the concerted actions of the triumvirate of SphK1, FLNa, and PAK1, each of which requires and/or regulates the actions of the others, at lamellipodia to promote cell movement.
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Movimiento Celular/fisiología , Proteínas Contráctiles/metabolismo , Proteínas de Microfilamentos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Seudópodos/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Actinas/metabolismo , Línea Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Proteínas Contráctiles/genética , Citoesqueleto/metabolismo , Filaminas , Humanos , Proteínas de Microfilamentos/genética , Neurregulina-1/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores de Lisoesfingolípidos/genética , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato , Técnicas del Sistema de Dos Híbridos , Quinasas p21 Activadas/genética , Quinasas p21 Activadas/metabolismoRESUMEN
Sphingosine-1-phosphate (S1P) is now emerging as a potent lipid mediator produced by mast cells that contributes to inflammatory and allergic responses. In contrast to its weak effect on degranulation of murine mast cells, S1P potently induced degranulation of the human LAD2 mast-cell line and cord blood-derived human mast cells (hMCs). S1P also stimulated production and secretion of cytokines, TNF-alpha and IL-6, and markedly enhanced secretion of a chemokine, CCL2/MCP-1, important modulators of inflammation. S1P is produced in mast cells by the 2 sphingosine kinases, SphK1 and SphK2. SphK1 but not SphK2 plays a critical role in IgE/Ag-induced degranulation, migration toward antigen, and CCL2 secretion from hMCs, as determined by specifically down-regulating their expression. However, both isoenzymes were required for efficient TNF-alpha secretion. Taken together, our data suggest that differential formation of S1P by SphK1 and SphK2 has distinct and important actions in hMCs.
Asunto(s)
Mastocitos/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Antígenos/farmacología , Degranulación de la Célula/efectos de los fármacos , Línea Celular , Quimiocina CCL2/metabolismo , Quimiotaxis/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Humanos , Lisofosfolípidos/farmacología , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Mastocitos/fisiología , Toxina del Pertussis/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Esfingosina/análogos & derivados , Esfingosina/farmacología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are lysophospholipid mediators of diverse cellular processes important for cancer progression. S1P is produced by two sphingosine kinases, SphK1 and SphK2. Expression of SphK1 is elevated in many cancers. Here, we report that LPA markedly enhanced SphK1 mRNA and protein in gastric cancer MKN1 cells but had no effect on SphK2. LPA also up-regulated SphK1 expression in other human cancer cells that endogenously express the LPA(1) receptor, such as DLD1 colon cancer cells and MDA-MB-231 breast cancer cells, but not in HT29 colon cancer cells or MDA-MB-453 breast cancer cells, which do not express the LPA(1) receptor. An LPA(1) receptor antagonist or down-regulation of its expression prevented SphK1 and S1P(3) receptor up-regulation by LPA. LPA transactivated the epidermal growth factor receptor (EGFR) in these cells, and the EGFR inhibitor AG1478 attenuated the increased SphK1 and S1P(3) expression induced by LPA. Moreover, down-regulation of SphK1 attenuated LPA-stimulated migration and invasion of MNK1 cells yet had no effect on expression of neovascularizing factors, such as interleukin (IL)-8, IL-6, urokinase-type plasminogen activator (uPA), or uPA receptor induced by LPA. Finally, down-regulation of S1P(3), but not S1P(1), also reduced LPA-stimulated migration and invasion of MKN1 cells. Collectively, our results suggest that SphK1 is a convergence point of multiple cell surface receptors for three different ligands, LPA, EGF, and S1P, which have all been implicated in regulation of motility and invasiveness of cancer cells.
Asunto(s)
Movimiento Celular/fisiología , Receptores ErbB/metabolismo , Lisofosfolípidos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismo , Neoplasias Gástricas/patología , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular , Quimiotaxis , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Invasividad Neoplásica , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , ARN Mensajero , Receptores de Lisoesfingolípidos/genética , Receptores de Lisoesfingolípidos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Activación Transcripcional , Células Tumorales Cultivadas , Regulación hacia Arriba , Activador de Plasminógeno de Tipo Uroquinasa/genética , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
Angiotensin II (Ang II), a major regulator of blood pressure, is also involved in the control of cellular proliferation and hypertrophy and might exhibit additional actions in vivo by modulating the signaling of other hormones. As hypertension and Insulin (Ins) resistance often coexist and are risk factors for cardiovascular diseases, Ang II and Insulin signaling cross-talk may have an important role in hypertension development. The effect of Ins on protein tyrosine phosphorylation was assayed in rat liver membrane preparations, a rich source of Ins receptors. Following stimulation, Ins (10(-7) M) induced tyr-phosphorylation of different proteins. Insulin consistently induced tyr-phosphorylation of a 160 kDa protein (pp160) with maximum effect between 1 and 3 min. The pp160 protein was identified by anti-IRS-4 but not by anti-IRS-1 antibody. Pre-stimulation with Ang II (10(-7) M) diminishes tyr-phosphorylation level of pp160/IRS-4 in a dose-dependent manner. Okadaic acid, the PP1A and PP2A Ser/Thr phosphatase inhibitor, increases pp160 phosphorylation induced by Ins and prevents the inhibitory effect of Ang II pre-stimulation. Genistein, a tyrosine kinase inhibitor, diminishes tyr-phosphorylation level of IRS-4. PI3K inhibitors Wortmanin and LY294002, both increase tyr-phosphorylation of IRS-4, either in the presence of Ins alone or combined with Ang II. These results suggest that Ins and Ang II modulate IRS-4 tyr-phosphorylation in a PI3K-dependent manner. In summary, we showed that Ins induces tyr-phosphorylation of IRS-4, an effect modulated by Ang II. Assays performed in the presence of different inhibitors points toward a PI3K involvement in this signaling pathway.
Asunto(s)
Angiotensina II/farmacología , Membrana Celular/enzimología , Hígado/metabolismo , Fosfoproteínas/metabolismo , Tirosina/metabolismo , Vasoconstrictores/farmacología , Androstadienos/farmacología , Angiotensina II/metabolismo , Animales , Membrana Celular/efectos de los fármacos , Genisteína/farmacología , Insulina/farmacología , Proteínas Sustrato del Receptor de Insulina , Hígado/efectos de los fármacos , Masculino , Ácido Ocadaico/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosfoproteínas/antagonistas & inhibidores , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Wistar , Vasoconstrictores/metabolismo , WortmaninaRESUMEN
Angiotensin II (Ang II) elicits a variety of physiological effects through specific Ang II receptors in numerous tissues. In addition, Ang II is a modulator of cellular growth and exerts a positive or negative effect on cell growth depending on which receptor subtype is activated. Expression of the intrarenal AT2 receptors occurs at its highest levels in the fetal kidney, with a rapid decline after birth. In the present paper, we performed a study on the signaling mechanism of Ang II receptors in rat fetal (E20) kidney, a rich source of AT2 receptors, where both Ang II receptor subtypes are present. Ang II induces Tyr-dephosphorylation of proteins in rat fetal kidney membranes. The response is dose-dependent, with a reduction of 20% with respect to the control (100%), signal that is completely reversed by Ang IIAT2 competitor PD123319. Orthovanadate, the inhibitor of phospho-Tyr-phosphatases (PTPase), reverts Ang II effect, suggesting the involvement of a protein tyrosine phosphatase. The peptide analog of Ang II, CGP42112, exhibits an agonist effect, which is dose-dependent. Thus, in rat fetal (E20) kidney, the Ang-induced protein Tyr-dephosphorylation of several proteins is mediated by AT2 receptors, mechanism that involves an orthovanadate sensitive PTPase.