RESUMEN
Sphingosine has been previously shown to kill many strains of pathogenic bacteria including Pseudomonas aeruginosa, Staphyloccus aureus, Acinetobacter, and atypical mycobacteria. However, these studies were performed on isolated or extracellular bacteria and it is unknown whether sphingosine also targets intracellular bacteria. Here, we demonstrate that exogenously-added sphingosine directly binds to extracellular P. aeruginosa and S. aureus, but also targets and binds to intracellular bacteria. Intracellular sphingosine and bacteria were identified by sequential immunostainings. We further show that exogenously-added sphingosine also kills intracellular P. aeruginosa and S. aureus using modified gentamycin assays. Intracellular killing of P. aeruginosa and S. aureus by sphingosine is not mediated by improved phagosomal-lysosomal fusion. In summary, our data indicate that sphingosine binds to and most likely also directly kills extra- and intracellular P. aeruginosa and S. aureus.
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Pseudomonas aeruginosa , Esfingosina , Staphylococcus aureus , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/metabolismo , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/metabolismo , Esfingosina/análogos & derivados , Esfingosina/farmacología , Esfingosina/metabolismo , Humanos , Antibacterianos/farmacología , Viabilidad Microbiana/efectos de los fármacos , AnimalesRESUMEN
Sepsis-induced acute lung injury (SALI) is the common complication of sepsis, resulting in high incidence and mortality rates. The primary pathogenesis of SALI is the interplay between acute inflammation and endothelial barrier damage. Studies have shown that kaempferol (KPF) has anti-sepsis properties. Sphingosine kinase 1 (SphK1)/sphingosine-1-phosphate (S1P) signaling pathway's significance in acute lung damage and S1P receptor 1 (S1PR1) agonists potential in myosin light chain 2 (MLC2) phosphorylation are documented. Whether KPF can regulate the SphK1/S1P/S1PR1/MLC2 signaling pathway to protect the lung endothelial barrier remains unclear. This study investigates the KPF's therapeutic effects and molecular mechanisms in repairing endothelial cell barrier damage in both LPS-induced sepsis mice and human umbilical vein endothelial cells (HUVECs). KPF significantly reduced lung tissue damage and showed anti-inflammatory effects by decreasing IL-6 and TNF-α synthesis in the sepsis mice model. Further, KPF administration can reduce the high permeability of the LPS-induced endothelial cell barrier and alleviate lung endothelial cell barrier injury. Mechanistic studies showed that KPF pretreatment can suppress MLC2 hyperphosphorylation and decrease SphK1, S1P, and S1PR1 levels. The SphK1/S1P/S1PR1/MLC2 signaling pathway controls the downstream proteins linked to endothelial barrier damage, and the Western blot (WB) showed that KPF raised the protein levels. These proteins include zonula occludens (ZO)-1, vascular endothelial (VE)-cadherin and Occludin. The present work revealed that in mice exhibiting sepsis triggered by LPS, KPF strengthened the endothelial barrier and reduced the inflammatory response. The SphK1/S1P/S1PR1/MLC2 pathway's modulation is the mechanism underlying this impact.
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Lesión Pulmonar Aguda , Miosinas Cardíacas , Células Endoteliales de la Vena Umbilical Humana , Quempferoles , Pulmón , Lisofosfolípidos , Ratones Endogámicos C57BL , Cadenas Ligeras de Miosina , Sepsis , Transducción de Señal , Esfingosina , Animales , Sepsis/tratamiento farmacológico , Sepsis/complicaciones , Sepsis/metabolismo , Lesión Pulmonar Aguda/tratamiento farmacológico , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/etiología , Lesión Pulmonar Aguda/patología , Humanos , Cadenas Ligeras de Miosina/metabolismo , Transducción de Señal/efectos de los fármacos , Ratones , Lisofosfolípidos/metabolismo , Quempferoles/farmacología , Quempferoles/uso terapéutico , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Esfingosina/farmacología , Masculino , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Miosinas Cardíacas/metabolismo , Pulmón/patología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Lipopolisacáridos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Interleucina-6/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismoRESUMEN
Straightforward access to enantiomerically pure 3,4-diamino-3,4-dideoxyphytosphingosines, as novel analogues of natural d-ribo-phytosphingosine was accomplished, starting from two available chirons: dimethyl l-tartrate and d-isoascorbic acid. A sequential Overman rearrangement followed by late-stage introduction of the alkyl side chain moiety via olefin cross-metathesis is the cornerstone of this approach. The preliminary evaluation study of the synthesised sphingomimetics, based on their ability to inhibit a proliferation of human cancer cells, showed promising cytotoxicity against Jurkat and HeLa cells for (2R,3R,4S)-2,3,4-triaminooctadecan-1-ol trihydrochloride.
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Proliferación Celular , Esfingosina , Esfingosina/análogos & derivados , Esfingosina/química , Esfingosina/farmacología , Esfingosina/síntesis química , Humanos , Células HeLa , Proliferación Celular/efectos de los fármacos , Células Jurkat , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , EstereoisomerismoRESUMEN
Lymphatic endothelial cells (LECs) of the lymph node (LN) parenchyma orchestrate leukocyte trafficking and peripheral T cell dynamics. T cell responses to immunotherapy largely rely on peripheral T cell recruitment in tumors. Yet, a systematic and molecular understanding of how LECs within the LNs control T cell dynamics under steady-state and tumor-bearing conditions is lacking. Intravital imaging combined with immune phenotyping shows that LEC-specific deletion of the essential autophagy gene Atg5 alters intranodal positioning of lymphocytes and accrues their persistence in the LNs by increasing the availability of the main egress signal sphingosine-1-phosphate. Single-cell RNA sequencing of tumor-draining LNs shows that loss of ATG5 remodels niche-specific LEC phenotypes involved in molecular pathways regulating lymphocyte trafficking and LEC-T cell interactions. Functionally, loss of LEC autophagy prevents recruitment of tumor-infiltrating T and natural killer cells and abrogates response to immunotherapy. Thus, an LEC-autophagy program boosts immune-checkpoint responses by guiding systemic T cell dynamics.
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Autofagia , Inhibidores de Puntos de Control Inmunológico , Ganglios Linfáticos , Esfingosina/análogos & derivados , Linfocitos T , Autofagia/efectos de los fármacos , Animales , Ganglios Linfáticos/inmunología , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Ratones , Linfocitos T/inmunología , Linfocitos T/metabolismo , Ratones Endogámicos C57BL , Proteína 5 Relacionada con la Autofagia/metabolismo , Proteína 5 Relacionada con la Autofagia/genética , Células Endoteliales/metabolismo , Esfingosina/farmacología , Esfingosina/metabolismo , Humanos , Lisofosfolípidos/metabolismo , Inmunoterapia/métodos , Movimiento CelularRESUMEN
BACKGROUND: Cancer cells exhibit selective metabolic reprogramming to promote proliferation, invasiveness, and metastasis. Sphingolipids such as sphingosine and sphinganine have been reported to modulate cell death processes in cancer cells. However, the potential of extracellular sphinganine and its mimetic compounds as inducers of cancer cell death has not been thoroughly investigated. METHODS: We obtained extracellular conditioned medium from HCT-116 cells treated with the previously reported anticancer composition, goat urine DMSO fraction (GUDF). The extracellular metabolites were purified using a novel and in-house developed vertical tube gel electrophoresis (VTGE) technique and identified through LC-HRMS. Extracellular metabolites such as sphinganine, sphingosine, C16 sphinganine, and phytosphingosine were screened for their inhibitory role against intracellular kinases using molecular docking. Molecular dynamics (MD) simulations were performed to study the inhibitory potential of a novel designed modified mimetic sphinganine (MMS) (Pubchem CID: 162625115) upon c-Src kinase. Furthermore, inhibitory potential and ADME profile of MMS was compared with luteolin, a known c-Src kinase inhibitor. RESULTS: Data showed accumulation of sphinganine and other sphingolipids such as C16 sphinganine, phytosphingosine, and ceramide (d18:1/14:0) in the extracellular compartment of GUDF-treated HCT-116 cells. Molecular docking projected c-Src kinase as an inhibitory target of sphinganine. MD simulations projected MMS with strong (-7.1 kcal/mol) and specific (MET341, ASP404) binding to the inhibitory pocket of c-Src kinase. The projected MMS showed comparable inhibitory role and acceptable ADME profile over known inhibitors. CONCLUSION: In summary, our findings highlight the significance of extracellular sphinganine and other sphingolipids, including C16 sphinganine, phytosphingosine, and ceramide (d18:1/14:0), in the context of drug-induced cell death in HCT-116 cancer cells. Furthermore, we demonstrated the importance of extracellular sphinganine and its modified mimetic sphinganine (MMS) as a potential inhibitor of c-Src kinase. These findings suggest that MMS holds promise for future applications in targeted and combinatorial anticancer therapy.
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Neoplasias , Esfingosina , Esfingosina/análogos & derivados , Humanos , Esfingosina/farmacología , Esfingosina/metabolismo , Proteína Tirosina Quinasa CSK , Simulación del Acoplamiento Molecular , Esfingolípidos/metabolismo , Ceramidas/farmacología , Neoplasias/patologíaRESUMEN
Celastrol (Cel) shows potent antitumor activity in various experimental models. This study examined the relationship between Cel's antivascular and antitumor effects and sphingolipids. CCK-8 assay, transwell assay, Matrigel, PCR-array/RT-PCR/western blotting/immunohistochemistry assay, ELISA and HE staining were used to detect cell proliferation, migration and invasion, adhesion and angiogenesis, mRNA and protein expression, S1P production and tumor morphology. The results showed that Cel could inhibit proliferation, migration or invasion, adhesion and angiogenesis of human umbilical vein endothelial cells (HUVECs) and MDA-MB-231 cells by downregulating the expression of degenerative spermatocyte homolog 1 (DEGS1). Transfection experiments showed that downregulation of DEGS1 inhibited the above processes and sphingosine-1-phosphate (S1P) production of HUVECs and MDA-MB-231 cells, while upregulation of DEGS1 had the opposite effects. Coculture experiments showed that HUVECs could promote proliferation, migration and invasion of MDA-MB-231 cells through S1P/sphingosine-1-phosphate receptor (S1PR) signaling pathway, while Cel inhibited these processes in MDA-MB-231 cells induced by HUVECs. Animal experiments showed that Cel could inhibit tumor growth in nude mice. Western blotting, immunohistochemistry and ELISA assay showed that Cel downregulated the expression of DEGS1, CD146, S1PR1-3 and S1P production. These data confirm that DEGS1/S1P signaling pathway may be related to the antivascular and antitumor effects of cel.
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Fenómenos Biológicos , Triterpenos Pentacíclicos , Receptores de Lisoesfingolípidos , Esfingosina/análogos & derivados , Ratones , Animales , Humanos , Receptores de Lisoesfingolípidos/genética , Receptores de Lisoesfingolípidos/metabolismo , Células MDA-MB-231 , Angiogénesis , Ratones Desnudos , Transducción de Señal , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Esfingosina/farmacología , Esfingosina/metabolismo , Lisofosfolípidos/farmacología , Lisofosfolípidos/metabolismoRESUMEN
SKI-349 is a novel sphingosine kinases (SPHK) inhibitor with anti-tumor effects. This study aimed to assess the effect of SKI-349 on cell biological behaviors, downstream pathways, and its synergistic effect with sorafenib in hepatocellular carcinoma (HCC). HCC cell lines (Huh7 and Hep3B) were treated with SKI-349 at concentrations of 1, 2, 4, or 8 µM. Then, SPHK1/2 activity, cell viability, proliferation, apoptosis, invasion, and protein expressions of phosphorylated-protein kinase B (p-AKT), AKT, phosphorylated-mammalian target of rapamycin (p-mTOR) and mTOR were detected. Combination index values of SKI-349 (0, 1, 2, 4, or 8 µM) and sorafenib (0, 2.5, 5, 10, or 20 µM) were calculated. SKI-349 decreased the relative SPHK1 and SPHK2 activity compared with blank control in a dose-dependent manner in the Huh7 and Hep3B cell lines. Meanwhile, SKI-349 reduced cell viability, 5-ethynyl-2'-deoxyuridine (EdU) positive cells, and invasive cells, while it increased apoptotic cells compared to blank control in a dose-dependent manner in Huh7 and Hep3B cell lines. Based on the western blot assay, SKI-349 decreased the ratio of p-AKT to AKT and that of p-mTOR to mTOR compared with blank control in a dose-dependent manner in the Huh7 and Hep3B cell lines. Additionally, SKI-349 combined with sorafenib declined cell viability with concentration gradient effects compared to SKI-349 sole treatment, and they had synergistic cytotoxic effects in Huh7 and Hep3B cell lines. SKI-349 suppresses SPHK1 and SPHK2 activity, cell viability, invasion, and AKT/mTOR signaling pathway, as well as exhibits a synergistic cytotoxic effect with sorafenib in HCC.
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Antineoplásicos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Sorafenib/farmacología , Sorafenib/uso terapéutico , Esfingosina/farmacología , Esfingosina/uso terapéutico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Supervivencia Celular , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Niacinamida/farmacología , Compuestos de Fenilurea/farmacología , Compuestos de Fenilurea/uso terapéutico , Línea Celular Tumoral , Transducción de Señal , Antineoplásicos/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Serina-Treonina Quinasas TOR/uso terapéutico , Apoptosis , Proliferación CelularRESUMEN
Mechanical ventilation (MV) is a life-supporting strategy employed in the Intensive Care Unit (ICU). However, MV-associated mechanical stress exacerbates existing lung inflammation in ICU patients, resulting in limited improvement in mortality and a condition known as Ventilator-Induced Lung Injury (VILI). Sphingosine-1-phosphate (S1P) is a circulating bioactive lipid that maintains endothelial integrity primarily through S1P receptor 1 (S1PR1). During VILI, mechanical stress upregulates endothelial S1PR3 levels. Unlike S1PR1, S1PR3 mediates endothelial barrier disruption through Rho-dependent pathways. However, the specific impact of elevated S1PR3 on lung endothelial function, apart from Rho activation, remains poorly understood. In this study, we investigated the effects of S1PR3 in endothelial pathobiology during VILI using an S1PR3 overexpression adenovirus. S1PR3 overexpression caused cytoskeleton rearrangement, formation of paracellular gaps, and a modified endothelial response towards S1P. It resulted in a shift from S1PR1-dependent barrier enhancement to S1PR3-dependent barrier disruption. Moreover, S1PR3 overexpression induced an ADAM10-dependent cleavage of Vascular Endothelial (VE)-cadherin, which hindered endothelial barrier recovery. S1PR3-induced cleavage of VE-cadherin was at least partially regulated by S1PR3-mediated NFκB activation. Additionally, we employed an S1PR3 inhibitor TY-52156 in a murine model of VILI. TY-52156 effectively attenuated VILI-induced increases in bronchoalveolar lavage cell counts and protein concentration, suppressed the release of pro-inflammatory cytokines, and inhibited lung inflammation as assessed via a histological evaluation. These findings confirm that mechanical stress associated with VILI increases S1PR3 levels, thereby altering the pulmonary endothelial response towards S1P and impairing barrier recovery. Inhibiting S1PR3 is validated as an effective therapeutic strategy for VILI.
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Neumonía , Lesión Pulmonar Inducida por Ventilación Mecánica , Humanos , Ratones , Animales , Receptores de Esfingosina-1-Fosfato , Cadherinas , Esfingosina/farmacología , Lesión Pulmonar Inducida por Ventilación Mecánica/metabolismo , Lisofosfolípidos/farmacología , Receptores de Lisoesfingolípidos/metabolismo , Proteína ADAM10 , Proteínas de la Membrana , Secretasas de la Proteína Precursora del AmiloideRESUMEN
Microglia, the resident immune cells of the central nervous system, play important roles in brain homeostasis as well as in neuroinflammation, neurodegeneration, neurovascular diseases, and traumatic brain injury. In this context, components of the endocannabinoid (eCB) system have been shown to shift microglia towards an anti-inflammatory activation state. Instead, much less is known about the functional role of the sphingosine kinase (SphK)/sphingosine-1-phosphate (S1P) system in microglia biology. In the present study, we addressed potential crosstalk of the eCB and the S1P systems in BV2 mouse microglia cells challenged with lipopolysaccharide (LPS). We show that URB597, the selective inhibitor of fatty acid amide hydrolase (FAAH)-the main degradative enzyme of the eCB anandamide-prevented LPS-induced production of tumor necrosis factor-α (TNFα) and interleukin-1ß (IL-1ß), and caused the accumulation of anandamide itself and eCB-like molecules such as oleic acid and cis-vaccenic acid ethanolamide, palmitoylethanolamide, and docosahexaenoyl ethanolamide. Furthermore, treatment with JWH133, a selective agonist of the eCB-binding cannabinoid 2 (CB2) receptor, mimicked the anti-inflammatory effects of URB597. Interestingly, LPS induced transcription of both SphK1 and SphK2, and the selective inhibitors of SphK1 (SLP7111228) and SphK2 (SLM6031434) strongly reduced LPS-induced TNFα and IL-1ß production. Thus, the two SphKs were pro-inflammatory in BV2 cells in a non-redundant manner. Most importantly, the inhibition of FAAH by URB597, as well as the activation of CB2 by JWH133, prevented LPS-stimulated transcription of SphK1 and SphK2. These results present SphK1 and SphK2 at the intersection of pro-inflammatory LPS and anti-inflammatory eCB signaling, and suggest the further development of inhibitors of FAAH or SphKs for the treatment of neuroinflammatory diseases.
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Endocannabinoides , Factor de Necrosis Tumoral alfa , Ratones , Animales , Factor de Necrosis Tumoral alfa/farmacología , Endocannabinoides/farmacología , Lipopolisacáridos/farmacología , Microglía , Esfingosina/farmacología , Antiinflamatorios/farmacologíaRESUMEN
Sphingosine 1-phosphate (S1P) is one of the lipid mediators involved in diverse physiological functions. S1P circulates in blood and lymph bound to carrier proteins. Three S1P carrier proteins have been reported, albumin, apolipoprotein M (ApoM) and apolipoprotein A4 (ApoA4). The carrier-bound S1P exerts its functions via specific S1P receptors (S1PR1-5) on target cells. Previous studies showed several differences in physiological functions between albumin-bound S1P and ApoM-bound S1P. However, molecular mechanisms underlying the carrier-dependent differences have not been clarified. In addition, ApoA4 is a recently identified S1P carrier protein, and its functional differences from albumin and ApoM have not been addressed. Here, we compared the three carrier proteins in the processes of S1P degradation, release from S1P-producing cells and receptor activation. ApoM retained S1P more stable than albumin and ApoA4 in the cell culture medium when compared in the equimolar amounts. ApoM facilitated theS1P release from endothelial cells most efficiently. Furthermore, ApoM-bound S1P showed a tendency to induce prolonged activation of Akt via S1PR1 and S1PR3. These results suggest that the carrier-dependent functional differences of S1P are partly ascribed to the differences in the S1P stability, S1P-releasing efficiency and signaling duration.
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Lisofosfolípidos , Proteínas Proto-Oncogénicas c-akt , Humanos , Apolipoproteínas M/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Lisofosfolípidos/farmacología , Esfingosina/farmacología , Proteínas Portadoras/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Albúminas/metabolismoRESUMEN
Sphingosine-1-phosphate (S1P) is emerging as a crucial sphingolipid modulating neuroinflammation and cognition. S1P levels in the brain have been found to be decreased in cognitive impairment. S1P lyase (S1PL) is the key enzyme in metabolizing S1P and has been implicated in neuroinflammation. This study evaluated the effect of S1PL inhibition on cognition in type 2 diabetic mice. Fingolimod (0.5 mg/kg and 1 mg/kg) rescued cognition in high-fat diet and streptozotocin-induced diabetic mice, as evident in the Y maze and passive avoidance test. We further evaluated the effect of fingolimod on the activation of microglia in the pre-frontal cortex (PFC) and hippocampus of diabetic mice. Our study revealed that fingolimod inhibited S1PL and promoted anti-inflammatory microglia in both PFC and hippocampus of diabetic mice as it increased Ym-1 and arginase-1. The levels of p53 and apoptotic proteins (Bax and caspase-3) were elevated in the PFC and hippocampus of type 2 diabetic mice which fingolimod reversed. The underlying mechanism promoting anti-inflammatory microglial phenotype was also explored in this study. TIGAR, TP53-associated glycolysis and apoptosis regulator, is known to foster anti-inflammatory microglia and was found to be downregulated in the brain of type 2 diabetic mice. S1PL inhibition decreased the levels of p53 and promoted TIGAR, thereby increasing anti-inflammatory microglial phenotype and inhibiting apoptosis in the brain of diabetic mice. Our study reveals that S1PL inhibition could be beneficial in mitigating cognitive deficits in diabetic mice.
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Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Ratones , Animales , Esfingosina/farmacología , Esfingosina/metabolismo , Clorhidrato de Fingolimod/metabolismo , Clorhidrato de Fingolimod/farmacología , Microglía , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/farmacología , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/metabolismo , Enfermedades Neuroinflamatorias , Cognición , Diabetes Mellitus Tipo 2/metabolismo , Fosfatos/metabolismo , Fosfatos/farmacología , Monoéster Fosfórico Hidrolasas/metabolismo , Monoéster Fosfórico Hidrolasas/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismoRESUMEN
Sphingosine kinase 1 (SK1) converts the pro-death lipid sphingosine to the pro-survival sphingosine-1-phosphate (S1P) and is upregulated in several cancers. DNA damaging agents, such as the chemotherapeutic doxorubicin (Dox), have been shown to degrade SK1 protein in cancer cells, a process dependent on wild-type p53. As mutations in p53 are very common across several types of cancer, we evaluated the effects of Dox on SK1 in p53 mutant cancer cells. In the p53 mutant breast cancer cell line MDA-MB-231, we show that Dox treatment significantly increases SK1 protein and S1P. Using MDA-MB-231 cells with CRISPR-mediated knockout of SK1 or the selective SK1 inhibitor PF-543, we implicated SK1 in both Dox-induced migration and in a newly uncovered proangiogenic program induced by Dox. Mechanistically, inhibition of SK1 suppressed the induction of the cytokine BMP4 and of the EMT transcription factor Snail in response to Dox. Interestingly, induction of BMP4 by SK1 increased Snail levels following Dox treatment by stabilizing Snail protein. Furthermore, we found that SK1 was required for Dox-induced p38 MAP kinase phosphorylation and that active p38 MAPK in turn upregulated BMP4 and Snail, positioning p38 downstream of SK1 and upstream of BMP4/Snail. Modulating production of S1P by inhibition of de novo sphingolipid synthesis or knockdown of the S1P-degrading enzyme S1P lyase identified S1P as the sphingolipid activator of p38 in this model. This work establishes a novel angiogenic pathway in response to a commonly utilized chemotherapeutic and highlights the potential of SK1 as a secondary drug target for patients with p53 mutant cancer.
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Neoplasias , Proteína p53 Supresora de Tumor , Humanos , Regulación hacia Arriba , Proteína p53 Supresora de Tumor/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Esfingolípidos , Doxorrubicina/farmacología , Esfingosina/farmacología , Esfingosina/metabolismo , Lisofosfolípidos/farmacologíaRESUMEN
Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that regulates fundamental cellular processes such as proliferation, migration, apoptosis, and differentiation through 5 cognate G protein-coupled receptors (S1P1-S1P5). We previously demonstrated that blockade of S1P2 signaling in S1P2-deficient mice attenuates high-fat diet-induced adipocyte hypertrophy and glucose intolerance and an S1P2-specific antagonist JTE-013 inhibits, whereas an S1P1/S1P3 dual antagonist (VPC23019) activates, adipogenic differentiation of preadipocytes. Based on those observations, this study examined whether an S1P1-specific agonist, SEW-2871, VPC23019, or their combination acts on obesity and glucose intolerance in leptin-deficient ob/ob mice. The oral administration of SEW-2871 or JTE-013 induced significant reductions in body/epididymal fat weight gains and epididymal/inguinal fat adipocyte sizes and improved glucose intolerance and adipocyte inflammation in ob/ob mice but not in their control C57BL/6J mice. Both SEW-2871 and JTE-013 decreased messenger RNA levels of tumor necrosis factor-α and CD11c, whereas they increased those of CD206 and adiponectin in the epididymal fats isolated from ob/ob mice with no changes in the levels of peroxisome proliferator activated receptor γ and its regulated genes. By contrast, VPC23019 did not cause any such alterations but counteracted with all those SEW-2871 actions in these mice. In conclusion, the S1P1 agonist SEW-2871 acted like the S1P2 antagonist JTE-013 to reduce body/epididymal fats and improve glucose tolerance in obese mice. Therefore, this study raises the possibility that endogenous S1P could promote obesity/type 2 diabetes through the S1P2, whereas exogenous S1P could act against them through the S1P1.
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Diabetes Mellitus Tipo 2 , Intolerancia a la Glucosa , Animales , Masculino , Ratones , Glucosa , Lisofosfolípidos/farmacología , Lisofosfolípidos/fisiología , Ratones Endogámicos C57BL , Ratones Obesos , Obesidad , Receptores de Lisoesfingolípidos/genética , Esfingosina/farmacología , Esfingosina/fisiología , Receptores de Esfingosina-1-FosfatoRESUMEN
Microglia, together with astrocytes and pericytes, cooperate to ensure blood-brain barrier (BBB) stability, modulating endothelial responses to inflammatory insults. Agonists of the sphingosine 1 phosphate (S1P) receptors, such as siponimod (BAF-312), are important pharmacological tools in multiple sclerosis and other inflammatory diseases. Modulation of S1P receptors may result in a reduced inflammatory response and increased BBB stability. An in vitro BBB model was reproduced using human-derived endothelial cells, astrocytes and microglia. Co-cultures were exposed to inflammatory cytokines (TNFα, 10 UI and IFNγ, 5 UI) in the presence of BAF-312 (100 nM), and the BBB properties and microglia role were evaluated. The drug facilitated microglial migration towards endothelial/astrocyte co-cultures, involving the activity of the metalloprotease 2 (MMP2). Microglia actively cooperated with astrocytes in the maintenance of endothelial barrier stability: in the triple co-culture, selective treatment of microglial cells with BAF-312 significantly prevented cytokines' effects on the endothelial barrier. In conclusion, BAF-312, modulating S1P receptors in microglia, may contribute to the reinforcement of the endothelial barrier at the BBB, suggesting an additional effect of the drug in the treatment of multiple sclerosis.
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Barrera Hematoencefálica , Esclerosis Múltiple , Azetidinas , Compuestos de Bencilo , Citocinas , Células Endoteliales , Humanos , Metaloproteinasa 2 de la Matriz , Microglía , Esclerosis Múltiple/tratamiento farmacológico , Fosfatos , Esfingosina/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Retinal pigment epithelium (RPE) cells, essential for preserving retina homeostasis, also contribute to the development of retina proliferative diseases, through their exacerbated migration, epithelial to mesenchymal transition (EMT) and inflammatory response. Uncovering the mechanisms inducing these changes is crucial for designing effective treatments for these pathologies. Sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) are bioactive sphingolipids that promote migration and inflammation in several cell types; we recently established that they stimulate the migration of retina Müller glial cells (Simón et al., 2015; Vera et al., 2021). We here analyzed whether S1P and C1P regulate migration, inflammation and EMT in RPE cells. We cultured two human RPE cell lines, ARPE-19 and D407 cells, and supplemented them with either 5 µM S1P or 10 µM C1P, or their vehicles, for 24 h. Analysis of cell migration by the scratch wound assay showed that S1P addition significantly enhanced migration in both cell lines. Pre-treatment with W146 and BML-241, antagonists for S1P receptor 1 (S1P1) and 3 (S1P3), respectively, blocked exogenous S1P-induced migration. Inhibiting sphingosine kinase 1 (SphK1), the enzyme involved in S1P synthesis, significantly reduced cell migration and exogenous S1P only partially restored it. Addition of C1P markedly stimulated cell migration. Whereas inhibiting C1P synthesis did not affect C1P-induced migration, inhibiting S1P synthesis strikingly decreased it; noteworthy, addition of C1P promoted the transcription of SphK1. These results suggest that S1P and C1P stimulate RPE cell migration and their effect requires S1P endogenous synthesis. Both S1P and C1P increase the transcription of pro-inflammatory cytokines IL-6 and IL-8, and of EMT marker α-smooth muscle actin (α-SMA) in ARPE-19 cells. Collectively, our results suggest new roles for S1P and C1P in the regulation of RPE cell migration and inflammation; since the deregulation of sphingolipid metabolism is involved in several proliferative retinopathies, targeting their metabolism might provide new tools for treating these pathologies.
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Actinas , Epitelio Pigmentado de la Retina , Humanos , Receptores de Esfingosina-1-Fosfato , Epitelio Pigmentado de la Retina/metabolismo , Transición Epitelial-Mesenquimal , Interleucina-6 , Interleucina-8 , Lisofosfolípidos/farmacología , Lisofosfolípidos/metabolismo , Esfingosina/farmacología , Esfingosina/metabolismo , Ceramidas/farmacología , Ceramidas/metabolismo , Inflamación/metabolismo , FosfatosRESUMEN
Sphingosine-1-phosphate (S1P) is involved in inflammatory signaling/s associated with the development of respiratory disorders, including cancer. However, the underlying mechanism/s are still elusive. The aim of this study was to investigate the role of S1P on circulating blood cells obtained from healthy volunteers and non-small cell lung cancer (NSCLC) patients. To pursue our goal, peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with S1P. We found that the administration of S1P did not induce healthy PBMCs to release pro-inflammatory cytokines. In sharp contrast, S1P significantly increased the levels of TNF-α and IL-6 from lung cancer-derived PBMCs. This effect was S1P receptor 3 (S1PR3)-dependent. The pharmacological blockade of ceramidase and sphingosine kinases (SPHKs), key enzymes for S1P synthesis, completely reduced the release of both TNF-α and IL-6 after S1P addition on lung cancer-derived PBMCs. Interestingly, S1P-induced IL-6, but not TNF-α, release from lung cancer-derived PBMCs was mTOR- and K-Ras-dependent, while NF-κB was not involved. These data identify S1P as a bioactive lipid mediator in a chronic inflammation-driven diseases such as NSCLC. In particular, the higher presence of S1P could orchestrate the cytokine milieu in NSCLC, highlighting S1P as a pro-tumor driver.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Neumonía , Carcinoma de Pulmón de Células no Pequeñas/patología , Citocinas , Humanos , Inflamación/patología , Interleucina-6 , Leucocitos Mononucleares , Neoplasias Pulmonares/patología , Lisofosfolípidos , Neumonía/patología , Esfingosina/análogos & derivados , Esfingosina/farmacología , Factor de Necrosis Tumoral alfaRESUMEN
BACKGROUND: Endoplasmic reticulum stress (ER stress) is involved in the development and progression of various forms of heart disease and may lead to myocardial apoptosis. Sphingosine-1-phosphate (S1P) possesses cardioprotective properties, including anti-apoptosis. However, little is known about the link between S1P and ER stress-induced myocardial apoptosis. This study investigated the regulatory role of S1P in ER stress-induced apoptosis in cardiomyocytes. METHODS: ER stress and myocardial apoptosis were induced by transverse aortic constriction (TAC) or tunicamycin in mice, which were then treated with 2-acetyl-5-tetrahydroxybutyl imidazole (THI) or S1P. AC16 cells were treated with tunicamycin or thapsigargin, or pretreated with S1P, sphingosine-1-phosphate receptor (S1PR) subtype antagonists, S1PR1 agonist, and PI3K and MEK inhibitors. Cardiac function, the level of S1P in plasma and heart, ER stress markers, cell viability, and apoptosis were detected. RESULTS: S1P reduced the expression of ER stress-related molecules and ER stress-induced myocardial apoptosis in mice subjected to TAC or an injection of tunicamycin. Furthermore, in AC16 cells exposed to thapsigargin or tunicamycin, S1P decreased the expression of ER stress-related molecules, promoting cell viability and survival. Nevertheless, the S1PR1 antagonist abrogated the protection of S1P. Subsequently, in TAC S1PR1 heterozygous (S1PR1+/-) mice, S1P had no effect on ER stress and apoptosis in cardiomyocytes. Notably, in vitro, the impact of anti-ER stress-induced myocardial apoptosis by the S1PR1 agonist was reversed by PI3K and MEK inhibitors. CONCLUSION: This study is the first to demonstrate that S1P relieves ER stress-induced myocardial apoptosis via S1PR1/AKT and S1PR1/ERK1/2, which are potential therapeutic targets for heart disease.
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Estrés del Retículo Endoplásmico , Cardiopatías , Animales , Imidazoles/farmacología , Lisofosfolípidos/farmacología , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/farmacología , Miocitos Cardíacos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Lisoesfingolípidos/genética , Receptores de Lisoesfingolípidos/metabolismo , Transducción de Señal , Esfingosina/análogos & derivados , Esfingosina/farmacología , Receptores de Esfingosina-1-Fosfato , Tapsigargina/farmacología , Tunicamicina/farmacologíaRESUMEN
The bioactive lysophospholipid sphingosine-1-phosphate (S1P) acts via five different subtypes of S1P receptors (S1PRs) - S1P1-5. S1P5 is predominantly expressed in nervous and immune systems, regulating the egress of natural killer cells from lymph nodes and playing a role in immune and neurodegenerative disorders, as well as carcinogenesis. Several S1PR therapeutic drugs have been developed to treat these diseases; however, they lack receptor subtype selectivity, which leads to side effects. In this article, we describe a 2.2 Å resolution room temperature crystal structure of the human S1P5 receptor in complex with a selective inverse agonist determined by serial femtosecond crystallography (SFX) at the Pohang Accelerator Laboratory X-Ray Free Electron Laser (PAL-XFEL) and analyze its structure-activity relationship data. The structure demonstrates a unique ligand-binding mode, involving an allosteric sub-pocket, which clarifies the receptor subtype selectivity and provides a template for structure-based drug design. Together with previously published S1PR structures in complex with antagonists and agonists, our structure with S1P5-inverse agonist sheds light on the activation mechanism and reveals structural determinants of the inverse agonism in the S1PR family.
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Receptores de Lisoesfingolípidos , Esfingosina , Humanos , Sistema Inmunológico , Lisofosfolípidos/farmacología , Esfingosina/análogos & derivados , Esfingosina/farmacologíaRESUMEN
Human cardiac fibroblasts (HCFs) play key roles in normal physiological functions and pathological processes in the heart. Our recent study has found that, in HCFs, sphingosine 1-phosphate (S1P) can upregulate the expression of cyclooxygenase-2 (COX-2) leading to prostaglandin E2 (PGE2) generation mediated by S1P receptors/PKCα/MAPKs cascade-dependent activation of NF-κB. Alternatively, G protein-coupled receptor- (GPCR-) mediated transactivation of receptor tyrosine kinases (RTKs) has been proved to induce inflammatory responses. However, whether GPCR-mediated transactivation of RTKs participated in the COX-2/PGE2 system induced by S1P is still unclear in HCFs. We hypothesize that GPCR-mediated transactivation of RTKs-dependent signaling cascade is involved in S1P-induced responses. This study is aimed at exploring the comprehensive mechanisms of S1P-promoted COX-2/PGE2 expression and apoptotic effects on HCFs. Here, we used pharmacological inhibitors and transfection with siRNA to evaluate whether matrix metalloprotease (MMP)2/9, heparin-binding- (HB-) epidermal growth factor (EGF), EGF receptor (EGFR), PI3K/Akt, MAPKs, and transcription factor AP-1 participated in the S1P-induced COX-2/PGE2 system determined by Western blotting, real-time polymerase chain reaction (RT-PCR), chromatin immunoprecipitation (ChIP), and promoter-reporter assays in HCFs. Our results showed that S1PR1/3 activated by S1P coupled to Gq- and Gi-mediated MMP9 activity to stimulate EGFR/PI3K/Akt/MAPKs/AP-1-dependent activity of transcription to upregulate COX-2 accompanied with PGE2 production, leading to stimulation of caspase-3 activity and apoptosis. Moreover, S1P-enhanced c-Jun bound to COX-2 promoters on its corresponding binding sites, which was attenuated by these inhibitors of protein kinases, determined by a ChIP assay. These results concluded that transactivation of MMP9/EGFR-mediated PI3K/Akt/MAPKs-dependent AP-1 activity was involved in the upregulation of the COX-2/PGE2 system induced by S1P, in turn leading to apoptosis in HCFs.
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Apoptosis/efectos de los fármacos , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Fibroblastos/metabolismo , Lisofosfolípidos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/metabolismo , Miocardio/citología , Esfingosina/análogos & derivados , Activación Transcripcional/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Ciclooxigenasa 2/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/genética , Metaloproteinasa 9 de la Matriz/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Esfingosina/farmacología , Factor de Transcripción AP-1/metabolismo , TransfecciónRESUMEN
Gastric cancer is one of the most common cancers with few effective treatments, a new treatment agent is desperately needed. C-2, a Jaspine B derivative, has shown anti-cancer efficacy in gastric cancer cells. The anti-cancer mechanism, however, remains unknown. As a result, we investigate the anti-cancer effect and the underlying mechanism of C-2 in gastric cancer cells. The results showed that C-2 selectively reduced the proliferation of gastric cancer cells when compared to normal epithelial gastric cells. Western blotting and flow cytometry further demonstrated that Caspase9 is involved in causing cell death. Meanwhile, C-2 triggered autophagy in gastric cancer cells, inhibition of which with LY294002 can enhance the anti-proliferative activity of C-2. Next, we found that C-2 triggered autophagy through activating JNK/ERK, and that inhibitors of these proteins exacerbated C-2 induced cell death. Mechanically, enhanced phosphorylation of JNK/ERK elevated Beclin-1 by disturbing Beclin-1/Bcl-xL or Beclin-1/Bcl-2 complexes, resulting in autophagy and up-regulation of p62. Finally, p62 binds Keap1 competitively to release Nrf2, boosting Nrf2 translocation from the cytoplasm to the nucleus and triggering expression of Nrf2 target genes, so enhancing survival. C-2 inhibited the growth of gastric cancer cells, while JNK/ERK dependent autophagy antagonized C-2 induced cell growth inhibition through p62/Keap1/Nrf2 pathway.