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1.
Hepatology ; 78(1): 26-44, 2023 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-36107019

RESUMEN

BACKGROUND AND AIM: Drug-induced liver injury (DILI) is a common disorder that involves both direct liver cell toxicity and immune activation. The bile acid receptor, G-protein-coupled bile acid receptor 1 (GPBAR1; Takeda G-protein-coupled receptor 5 [TGR5]), and cysteinyl leukotriene receptor (CYSLTR) 1 are G-protein-coupled receptors activated by bile acids and leukotrienes, exerting opposite effects on cell-to-cell adhesion, inflammation, and immune cell activation. To investigate whether GPBAR1 and CYSLTR1 mutually interact in the development of DILI, we developed an orally active small molecule, CHIN117, that functions as a GPBAR1 agonist and CYSLTR1 antagonist. APPROACH AND RESULTS: RNA-sequencing analysis of liver explants showed that acetaminophen (APAP) intoxication positively modulates the leukotriene pathway, CYSLTR1, 5-lipoxygenase, and 5-lipoxygenase activating protein, whereas GPBAR1 gene expression was unchanged. In mice, acute liver injury induced by orally dosing APAP (500 mg/kg) was severely exacerbated by Gpbar1 gene ablation and attenuated by anti-Cysltr1 small interfering RNA pretreatment. Therapeutic dosing of wild-type mice with CHIN117 reversed the liver damage caused by APAP and modulated up to 1300 genes, including 38 chemokines and receptors, that were not shared by dosing mice with a selective GPBAR1 agonist or CYSLTR1 antagonist. Coexpression of the two receptors was detected in liver sinusoidal endothelial cells (LSECs), monocytes, and Kupffer cells, whereas combinatorial modulation of CYSLTR1 and GPBAR1 potently reversed LSEC/monocyte interactions. CHIN117 reversed liver damage and liver fibrosis in mice administered CCl 4 . CONCLUSIONS: By genetic and pharmacological approaches, we demonstrated that GPBAR1 and CYSLTR1 mutually interact in the development of DILI. A combinatorial approach designed to activate GPBAR1 while inhibiting CYSLTR1 reverses liver injury in models of DILI.


Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas , Hepatopatías , Ratones , Animales , Ácidos y Sales Biliares/metabolismo , Araquidonato 5-Lipooxigenasa/metabolismo , Células Endoteliales/metabolismo , Acetaminofén/toxicidad , Receptores Acoplados a Proteínas G/metabolismo , Hepatopatías/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Leucotrienos/metabolismo , Proteínas de Unión al GTP/metabolismo
2.
Biochem Biophys Res Commun ; 681: 36-40, 2023 Nov 12.
Artículo en Inglés | MEDLINE | ID: mdl-37748257

RESUMEN

Piezo1 and Piezo2 are mechanoreceptors involved in sensing both internal and external mechanical forces converting them in electrical signals to the brain. Piezo1 is mainly expressed in the endothelial system and in epidermis sensing shear stress and light touch. The internal traction forces generated by Myosin Light Chain Kinase (MYLK) activate Piezo1, regulating cell contraction. We observed Oenothera biennis cell culture hydro-soluble extract (ObHEx) activated MYLK regulating cell contraction ability. The aim of this work was to test the hypothesis that ObHEx activates Piezo1 through MYLK pathway using CHO cell overexpressing Piezo1, HUVEC and SHSY5Y cells endogenously expressing high levels of Piezo1. Results showed that ObHEx extracts were able to activate Piezo1 and the effect is due to Liriodendrin and Salvadoraside, the two most abundant lignans produced by the cell culture. The effect is lost in presence of MYLK specific inhibitors confirming the key role of this pathway and providing indication about the mechanism of action in Piezo1 activation by lignans. In summary, these results confirmed the connection between Piezo1 and MYLK, opening the possibility of using lignans-containing natural extracts to activate Piezo1.

3.
FASEB J ; 36(1): e22060, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34862975

RESUMEN

Farnesoid-x-receptor (FXR) agonists, currently trialed in patients with non-alcoholic steatosis (NAFLD), worsen the pro-atherogenic lipid profile and might require a comedication with statin. Here we report that mice feed a high fat/high cholesterol diet (HFD) are protected from developing a pro-atherogenic lipid profile because their ability to dispose cholesterol through bile acids. This protective mechanism is mediated by suppression of FXR signaling in the liver by muricholic acids (MCAs) generated in mice from chenodeoxycholic acid (CDCA). In contrast to CDCA, MCAs are FXR antagonists and promote a CYP7A1-dependent increase of bile acids synthesis. In mice feed a HFD, the treatment with obeticholic acid, a clinical stage FXR agonist, failed to improve the liver histopathology while reduced Cyp7a1 and Cyp8b1 genes expression and bile acids synthesis and excretion. In contrast, treating mice with atorvastatin mitigated liver and vascular injury caused by the HFD while increased the bile acids synthesis and excretion. Atorvastatin increased the percentage of 7α-dehydroxylase expressing bacteria in the intestine promoting the formation of deoxycholic acid and litocholic acid, two GPBAR1 agonists, along with the expression of GPBAR1-regulated genes in the white adipose tissue and colon. In conclusion, present results highlight the central role of bile acids in regulating lipid and cholesterol metabolism in response to atorvastatin and provide explanations for limited efficacy of FXR agonists in the treatment of NAFLD.


Asunto(s)
Atorvastatina/farmacología , Hígado Graso/tratamiento farmacológico , Hígado/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Enfermedades Vasculares/tratamiento farmacológico , Animales , Bacterias/metabolismo , Ácidos y Sales Biliares/metabolismo , Colesterol 7-alfa-Hidroxilasa/metabolismo , Colesterol en la Dieta/efectos adversos , Colesterol en la Dieta/farmacología , Hígado Graso/inducido químicamente , Hígado Graso/metabolismo , Hígado Graso/microbiología , Microbioma Gastrointestinal/efectos de los fármacos , Masculino , Ratones , Esteroide 12-alfa-Hidroxilasa/metabolismo , Enfermedades Vasculares/inducido químicamente , Enfermedades Vasculares/metabolismo , Enfermedades Vasculares/microbiología
4.
Bioorg Chem ; 138: 106620, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37229937

RESUMEN

In recent years, thiadiazolopyrimidine derivatives have been acknowledged for their striking poly-pharmacological framework, thus representing an interesting scaffold for the development of new therapeutic candidates. This paper examines the synthesis and the interactome characterization of a novel bioactive thiadiazolopyrimidone (compound 1), endowed with cytotoxic activity on HeLa cancer cells. In detail, starting from a small set of synthesized thiadiazolopyrimidones, a multi-disciplinary strategy has been carried out on the most bioactive one to disclose its potential biological targets by functional proteomics, using a label-free mass spectrometry based platform coupling Drug Affinity Responsive Target Stability and targeted Limited Proteolysis-Multiple Reaction Monitoring. The identification of Annexin A6 (ANXA6) as compound 1 most reliable cellular partner paved the way to deepen the protein-ligand interaction through bio-orthogonal approaches and to prove compound 1 action on migration and invasion processes governed by ANXA6 modulation. The identification of compund 1 as the first ANXA6 protein modulator represents a relevant tool to further explore the biological role of ANXA6 in cancer, as well as to develop novel anticancer candidates.


Asunto(s)
Antineoplásicos , Neoplasias , Humanos , Proteómica , Antineoplásicos/farmacología , Proteolisis
5.
Int J Mol Sci ; 24(11)2023 May 24.
Artículo en Inglés | MEDLINE | ID: mdl-37298148

RESUMEN

The impact of innovative technologies on the target discovery has been employed here to characterize the interactome of STIRUR 41, a promising 3-fluoro-phenyl-5-pyrazolyl-urea derivative endowed with anti-cancer activity, on neuroblastoma-related cells. A drug affinity responsive target stability-based proteomic platform has been optimized to elucidate the molecular mechanism at the basis of STIRUR 41 action, together with immunoblotting analysis and in silico molecular docking. Ubiquitin Specific Protease 7 (USP-7), one of the deubiquitinating enzymes which protect substrate proteins from proteasomal degradation, has been identified as the most affine STIRUR 41 target. As further demonstrated by in vitro and in-cell assays, STIRUR 41 was able to inhibit both the enzymatic activity of USP-7 and its expression levels in neuroblastoma-related cells, thus laying an encouraging base for the blockade of USP-7 downstream signaling.


Asunto(s)
Neuroblastoma , Urea , Humanos , Simulación del Acoplamiento Molecular , Proteómica , Immunoblotting
6.
Int J Mol Sci ; 24(2)2023 Jan 06.
Artículo en Inglés | MEDLINE | ID: mdl-36674610

RESUMEN

Fabry disease is a lysosomal storage disease caused by mutations in the GLA gene that encodes alpha-galactosidase (AGAL). The disease causes abnormal globotriaosylceramide (Gb3) storage in the lysosomes. Variants responsible for the genotypic spectrum of Fabry disease include mutations that abolish enzymatic activity and those that cause protein instability. The latter can be successfully treated with small molecules that either bind and stabilize AGAL or indirectly improve its cellular activity. This paper describes the first attempt to reposition curcumin, a nutraceutical, to treat Fabry disease. We tested the efficacy of curcumin in a cell model and found an improvement in AGAL activity for 80% of the tested mutant genotypes (four out of five tested). The fold-increase was dependent on the mutant and ranged from 1.4 to 2.2. We produced evidence that supports a co-chaperone role for curcumin when administered with AGAL pharmacological chaperones (1-deoxygalactonojirimycin and galactose). The combined treatment with curcumin and either pharmacological chaperone was beneficial for four out of five tested mutants and showed fold-increases ranging from 1.1 to 2.3 for DGJ and from 1.1 to 2.8 for galactose. Finally, we tested a long-term treatment on one mutant (L300F) and detected an improvement in Gb3 clearance and lysosomal markers (LAMP-1 and GAA). Altogether, our findings confirmed the necessity of personalized therapies for Fabry patients and paved the way to further studies and trials of treatments for Fabry disease.


Asunto(s)
Curcumina , Enfermedad de Fabry , Humanos , Enfermedad de Fabry/tratamiento farmacológico , Enfermedad de Fabry/genética , alfa-Galactosidasa/metabolismo , Curcumina/farmacología , Curcumina/uso terapéutico , Curcumina/metabolismo , Galactosa/metabolismo , Mutación , Lisosomas/metabolismo , 1-Desoxinojirimicina/farmacología , 1-Desoxinojirimicina/uso terapéutico
7.
Int J Mol Sci ; 24(13)2023 Jun 29.
Artículo en Inglés | MEDLINE | ID: mdl-37446018

RESUMEN

Sex is a biological variable that can reflect clinical outcomes in terms of quality of life, therapy effectiveness, responsiveness and/or toxicity. Sphingosine-1-phosphate (S1P) is a lipidic mediator whose activity can be influenced by sex. To evaluate whether the S1P axis underlies sex 'instructions' in the lung during physiological and oncological lung conditions, sphingosine and S1P were quantified in the blood of healthy (H) volunteers, lung adenocarcinoma (ADK) and squamous cell carcinoma (SCC) patients of both sexes. S1P receptors and their metabolic enzymes were evaluated in the tissues. Circulating levels of S1P were similar among H female and male subjects and female SCC patients. Instead, male and female ADK patients had lower circulating S1P levels. S1P receptor 3 (S1PR3) was physiologically expressed in the lung, but it was overexpressed in male SCC, and female and male ADK, but not in female SCC patients, who showed a significantly reduced ceramide synthase 1 (CERS1) mRNA and an overexpression of the ceramidase (ASAH1) precursor in lung tumor tissues, compared to male SCC and both male and female ADK patients. These findings highlighted sex differences in S1P rheostat in pathological conditions, but not in physiological conditions, identifying S1P as a prognostic mediator depending on lung cancer histotype.


Asunto(s)
Neoplasias Pulmonares , Esfingosina , Humanos , Masculino , Femenino , Esfingosina/metabolismo , Ceramidasas/metabolismo , Caracteres Sexuales , Calidad de Vida , Lisofosfolípidos/metabolismo , Pulmón/metabolismo , Neoplasias Pulmonares/metabolismo
8.
FASEB J ; 35(1): e21271, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33368684

RESUMEN

Autophagy is a highly conserved catabolic process activated by fasting and caloric restriction. FXR, a receptor for primary bile acids, reverses the activity of cAMP-response element binding protein (CREB) on autophagy-related genes (Atg)s and terminates autophagy in the fed state. GPBAR1, a receptor for secondary bile acids, exerts its genomic effects via cAMP-CREB pathway. By genetic and pharmacological approaches, we have obtained evidence that GPBAR1 functions as a positive modulator of autophagy in liver and white adipose tissue (WAT) in fasting. Mechanistically, we found that Gpbar1-/- mice lack the expression of Cyp2c70 a gene essential for generation of muricholic acids which are FXR antagonists, and have an FXR-biased bile acid pool. Because FXR represses autophagy, Gpbar1-/- mice show a defective regulation of autophagy in fasting. BAR501, a selective GPBAR1 agonist, induces autophagy in fed mice. Defective regulation of autophagy in Gpbar1-/- could be reversed by FXR antagonism, while repression of autophagy by feeding was partially abrogated by FXR gene ablation, and FXR activation repressed Atgs in the fast state. BAR501 reversed the negative regulatory effects of feeding and FXR agonism on autophagy and promoted the recruitment of CREB to a CRE on the LC3 promoter. In mice exposed to chronic high caloric intake, GPBAR1 agonism ameliorated insulin sensitivity and induced Atgs expression in the liver and WAT. In summary, GPBAR1 is required for positive regulation of autophagy in fasting and its ligands reverse the repressive effects exerted on liver and WAT autophagy flow by FXR in fed.


Asunto(s)
Tejido Adiposo Blanco/metabolismo , Autofagia/efectos de los fármacos , Ácidos Cólicos/farmacología , Hígado/metabolismo , Receptores Citoplasmáticos y Nucleares , Receptores Acoplados a Proteínas G , Animales , Autofagia/genética , Ratones , Ratones Noqueados , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo
9.
Int J Mol Sci ; 23(15)2022 Jul 25.
Artículo en Inglés | MEDLINE | ID: mdl-35897773

RESUMEN

In the last few years, several efforts have been made to identify original strategies against glioblastoma multiforme (GBM): this requires a more detailed investigation of the molecular mechanism of GBM so that novel targets can be identified for new possible therapeutic agents. Here, using a combined biochemical and proteomic approach, we evaluated the ability of a blood-brain barrier-permeable 2,3-benzodiazepin-4-one, called 1g, to interfere with the activity and the expression of brain glycogen phosphorylase (PYGB) on U87MG cell line in parallel with the capability of this compound to inhibit the cell growth and cycle. Thus, our results highlighted PYGB as a potential therapeutic target in GBM prompting 1g as a capable anticancer drug thanks to its ability to negatively modulate the uptake and metabolism of glucose, the so-called "Warburg effect", whose increase is considered a common feature of cancer cells in respect of their normal counterparts.


Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Encéfalo/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Glucógeno Fosforilasa/metabolismo , Humanos , Proteómica
10.
Int J Mol Sci ; 23(9)2022 May 04.
Artículo en Inglés | MEDLINE | ID: mdl-35563496

RESUMEN

Fabry disease is caused by a deficiency of lysosomal alpha galactosidase and has a very large genotypic and phenotypic spectrum. Some patients who carry hypomorphic mutations can benefit from oral therapy with a pharmacological chaperone. The drug requires a very precise regimen because it is a reversible inhibitor of alpha-galactosidase. We looked for molecules that can potentiate this pharmacological chaperone, among drugs that have already been approved for other diseases. We tested candidate molecules in fibroblasts derived from a patient carrying a large deletion in the gene GLA, which were stably transfected with a plasmid expressing hypomorphic mutants. In our cell model, three drugs were able to potentiate the action of the pharmacological chaperone. We focused our attention on one of them, acetylsalicylic acid. We expect that acetylsalicylic acid can be used in synergy with the Fabry disease pharmacological chaperone and prolong its stabilizing effect on alpha-galactosidase.


Asunto(s)
Enfermedad de Fabry , alfa-Galactosidasa , 1-Desoxinojirimicina/farmacología , 1-Desoxinojirimicina/uso terapéutico , Aspirina/farmacología , Aspirina/uso terapéutico , Reposicionamiento de Medicamentos , Enfermedad de Fabry/tratamiento farmacológico , Enfermedad de Fabry/genética , Humanos , Lisosomas , Chaperonas Moleculares/genética , Mutación , alfa-Galactosidasa/genética , alfa-Galactosidasa/uso terapéutico
11.
Int J Mol Sci ; 23(23)2022 Dec 02.
Artículo en Inglés | MEDLINE | ID: mdl-36499490

RESUMEN

Accumulation of senescent dermal fibroblasts drives skin aging. The reactivation of proliferation is one strategy to modulate cell senescence. Recently, we reported the exact chemical composition of the hydrophilic extract of Oenothera biennis cell cultures (ObHEx) and we showed its skin anti-aging properties. The aim of this work is to assess its biological effect specifically on cell senescence. ObHEx action has been evaluated on normal human dermal fibroblasts subjected to stress-induced premature senescence (SIPS) through an ultra-deep proteomic analysis, leading to the most global senescence-associated proteome so far. Mass spectrometry data show that the treatment with ObHEx re-establishes levels of crucial mitotic proteins, strongly downregulated in senescent cells. To validate our proteomics findings, we proved that ObHEx can, in part, restore the activity of 'senescence-associated-ß-galactosidase', the most common hallmark of senescent cells. Furthermore, to assess if the upregulation of mitotic protein levels translates into a cell cycle re-entry, FACS experiments have been carried out, demonstrating a small but significative reactivation of senescent cell proliferation by ObHEx. In conclusion, the deep senescence-associated global proteome profiling published here provides a panel of hundreds of proteins deregulated by SIPS that can be used by the community to further understand senescence and the effect of new potential modulators. Moreover, proteomics analysis pointed to a specific promitotic effect of ObHEx on senescent cells. Thus, we suggest ObHEx as a powerful adjuvant against senescence associated with skin aging.


Asunto(s)
Oenothera biennis , Proteómica , Humanos , Fibroblastos/metabolismo , Senescencia Celular , Piel , Células Cultivadas
12.
Molecules ; 27(3)2022 Feb 07.
Artículo en Inglés | MEDLINE | ID: mdl-35164377

RESUMEN

Plant extracts have shown beneficial properties in terms of skin repair, promoting wound healing through a plethora of mechanisms. In particular, the poly-/oligosaccharidic aqueous extract of Triticum vulgare (TVE), as well as TVE-based products, shows interesting biological assets, hastening wound repair. Indeed, TVE acts in the treatment of tissue regeneration mainly on decubitus and venous leg ulcers. Moreover, on scratched monolayers, TVE prompts HaCat cell migration, correctly modulating the expression of metalloproteases toward a physiological matrix remodeling. Here, using the same HaCat-based in vitro scratch model, the TVE effect has been investigated thanks to an LFQ proteomic analysis of HaCat secretomes and immunoblotting. Indeed, the unbiased TVE effect on secreted proteins has not yet been fully understood, and it could be helpful to obtain a comprehensive picture of its bio-pharmacological profile. It has emerged that TVE treatment induces significant up-regulation of several proteins in the secretome (153 to be exact) whereas only a few were down-regulated (72 to be exact). Interestingly, many of the up-regulated proteins are implicated in promoting wound-healing-related processes, such as modulating cell-cell interaction and communication, cell proliferation and differentiation, and prompting cell adhesion and migration.


Asunto(s)
Queratinocitos/metabolismo , Extractos Vegetales/farmacología , Proteoma/metabolismo , Proteómica/métodos , Triticum/química , Cicatrización de Heridas , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Humanos , Queratinocitos/efectos de los fármacos , Proteoma/análisis
13.
J Cell Physiol ; 236(7): 4926-4943, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33284486

RESUMEN

Mesoglycan is a drug based on a mixture of glycosaminoglycans mainly used for the treatment of blood vessel diseases acting as antithrombotic and profibrinolytic drugs. Besides the numerous clinical studies, there is no information about its function on the fibrinolytic cascade. Here, we have elucidated the mechanism of action by which mesoglycan induces the activation of plasmin from endothelial cells. Surprisingly, by a proteomic analysis, we found that, following mesoglycan treatment, these cells show a notable amount of annexin A2 (ANXA2) at the plasma membrane. This protein has been widely associated with fibrinolysis and appears able to move to the membrane when phosphorylated. In our model, this translocation has proven to enhance cell migration, invasion, and angiogenesis. Furthermore, the interaction of mesoglycan with syndecan 4 (SDC4), a coreceptor belonging to the class of heparan sulfate proteoglycans, represents the upstream event of the ANXA2 behavior. Indeed, the activation of SDC4 triggers the motility of endothelial cells culminating in angiogenesis. Interestingly, mesoglycan can induce the release of plasmin in endothelial cell supernatants only in the presence of ANXA2. This evaluation suggests that mesoglycan triggers the formation of a chain mechanism starting from the activation of SDC4, and the related cascade of events, including src complex and PKCα activation, promoting the phosphorylation of ANXA2 and its translocation to plasma membrane. This indicates a connection among mesoglycan, SDC4-(PKCα-src), and ANXA2 which, in turn, links the tissue plasminogen activator bringing it closer to plasminogen. This latter is so cleaved to release the plasmin and degrade fibrin sleeves.


Asunto(s)
Fibrinolisina/metabolismo , Fibrinólisis/fisiología , Fibrinolíticos/farmacología , Glicosaminoglicanos/farmacología , Activador de Tejido Plasminógeno/metabolismo , Anexina A2/genética , Anexina A2/metabolismo , Línea Celular , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Células Endoteliales/metabolismo , Fibrinólisis/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Neovascularización Fisiológica/efectos de los fármacos , Proteína Quinasa C-alfa/metabolismo , Proteómica , Interferencia de ARN , ARN Interferente Pequeño/genética , Sindecano-4/genética , Sindecano-4/metabolismo
14.
Cell Physiol Biochem ; 55(2): 222-234, 2021 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-33914445

RESUMEN

BACKGROUND/AIMS: Sphingosine-1-phosphate (S1P) is a membrane-derived bioactive phospholipid involved in many lung physiological and pathological processes. Higher levels of S1P have been registered in a broad range of respiratory diseases, including inflammatory disorders and cancer. The aim of our study was to understand the role of S1P in healthy versus tumor cells after Toll-Like Receptors (TLRs) activation, well-known modulators of sphingolipid metabolism. METHODS: Lung adenocarcinoma cells and non-pathological human fibroblasts were stimulated with unmethylated Cytosine phosphate Guanosine (CpG), the TLR9 ligand, and S1P-dependent TNF-α release was evaluated by means of ELISA. Immunofluorescence and LC-MS/MS analysis were performed to evaluate/quantify S1P generation following TLR9 activation. RESULTS: We found that S1P was involved in TLR9-induced TNF-α release in that the inhibition of both ceramidase and sphingosine kinase I/II (SPHK I/II) significantly reduced the levels of TNF-α after TLR9 triggering in lung adenocarcinoma cells. These results were not observed in healthy fibroblasts, implying that this pathway was mainly involved in pathological conditions. Moreover, the activation of TLR4 by means of LPS did not have similar effects as in the case of CpG-stimulated TLR9. Importantly, the activation of TLR9 induced S1P generation and allowed it to interact on the outside membrane receptor S1P1 and S1P3 via the efflux through its membrane transporter SPNS2. Indeed, both the blockade of S1P3 and the transporter SPNS2 significantly reduced the activity of S1P on TNF-α release from lung adenocarcinoma cells. CONCLUSION: Our study identifies a novel inflammatory pathway in that TLR9 increases the pro-inflammatory cytokine release, such as TNF-α, via the induction of a ceramide/S1P imbalance in favor of S1P, adding a novel puzzle piece in TLR9-orchestrated inflammatory pathway and shedding more light on the role of the higher levels of S1P during inflammatory conditions.


Asunto(s)
Adenocarcinoma del Pulmón/metabolismo , Neoplasias Pulmonares/metabolismo , Lisofosfolípidos/metabolismo , Esfingosina/análogos & derivados , Receptor Toll-Like 9/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Células A549 , Western Blotting , Técnica del Anticuerpo Fluorescente , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Pulmón/inmunología , Pulmón/metabolismo , Esfingosina/metabolismo , Espectrometría de Masas en Tándem
15.
Cell Physiol Biochem ; 55(5): 539-552, 2021 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-34473432

RESUMEN

BACKGROUND/AIMS: The pleiotropic lipid mediator sphingosine-1-phosphate (S1P) exerts a multitude of effects on respiratory cell physiology and pathology through five S1P receptors (S1PR1-5). Epidemiological studies proved high levels of circulating S1P in non-small cell lung cancer (NSCLC) patients. Studies in literature suggest that high levels of S1P support carcinogenesis but the exact mechanism is still elusive. The aim of this study was to understand the mechanism/s underlying S1P-mediated lung tumor cell proliferation. METHODS: We used human samples of NSCLC, a mouse model of first-hand smoking and of Benzo(a)pyrene (BaP)-induced tumor-bearing mice and A549 lung adenocarcinoma cells. RESULTS: We found that the expression of S1PR3 was also into the nucleus of lung cells in vitro, data that were confirmed in lung tissues of NSCLC patients, smoking and tumor bearing BaP-exposed mice. The intranuclear, but not the membrane, localization of S1PR3 was associated to S1P-mediated proliferation of lung adenocarcinoma cells. Indeed, the inhibition of the membrane S1PR3 did not alter tumor cell proliferation after Toll Like Receptor (TLR) 9 activation. Instead, according to the nuclear localization of sphingosine kinase (SPHK) II, the inhibition of the kinase completely blocked the endogenous S1P-induced tumor cell proliferation. CONCLUSION: These results prove that the nuclear S1PR3/SPHK II axis is involved in lung tumor cell proliferation, highlighting a novel molecular mechanism which could provide differential therapeutic approaches especially in non-responsive lung cancer patients.


Asunto(s)
Adenocarcinoma del Pulmón/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Transducción de Señal , Receptores de Esfingosina-1-Fosfato/metabolismo , Células A549 , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Animales , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Proteínas de Neoplasias/genética , Receptores de Esfingosina-1-Fosfato/genética
16.
Bioorg Chem ; 115: 105168, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34284173

RESUMEN

In recent years, 5-pyrazolyl-ureas have mostly been known for their attractive poly-pharmacological outline and, in particular, ethyl 1-(2-hydroxypentyl)-5-(3-(3-(trifluoromethyl) phenyl) ureido)-1H-pyrazole-4-carboxylate (named GeGe-3) has emerged as a capable anti-angiogenic compound. This paper examines its interactome by functional proteomics using a label-free mass spectrometry based platform, coupling Drug Affinity Responsive Target Stability and targeted Limited Proteolysis-Multiple Reaction Monitoring. Calreticulin has been recognized as the GeGe-3 principal target and this evidence has been supported by immunoblotting and in silico molecular docking. Furthermore, cell studies have shown that GeGe-3 lowers cell calcium mobilization, cytoskeleton organization and focal adhesion kinase expression, thus linking its biological potential to calreticulin binding and, ultimately, shedding light on the reasonable action mechanism of this molecule as an anti-angiogenic factor.


Asunto(s)
Inhibidores de la Angiogénesis/química , Proteoma/metabolismo , Proteómica/métodos , Pirazoles/química , Urea/química , Inhibidores de la Angiogénesis/metabolismo , Inhibidores de la Angiogénesis/farmacología , Sitios de Unión , Calcio/metabolismo , Calreticulina/química , Calreticulina/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Quinasa 1 de Adhesión Focal/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ionomicina/farmacología , Simulación del Acoplamiento Molecular , Proteoma/efectos de los fármacos
17.
FASEB J ; 33(2): 2809-2822, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30303744

RESUMEN

Nonalcoholic steatohepatitis (NASH) is associated with an increased risk of developing cardiovascular complications and mortality, suggesting that treatment of NASH might benefit from combined approaches that target the liver and the cardiovascular components of NASH. Using genetic and pharmacologic approaches, we show that G protein-coupled bile acid-activated receptor 1 (GPBAR1) agonism reverses liver and vascular damage in mouse models of NASH. NASH is associated with accelerated vascular inflammation representing an independent risk factor for development of cardiovascular diseases and cardiovascular-related mortality. GPBAR1, also known as TGR5, is a G protein-coupled receptor for secondary bile acids that reduces inflammation and promotes energy expenditure. Using genetic and pharmacologic approaches, we investigated whether GPBAR1 agonism by 6ß-ethyl-3α,7ß-dihydroxy-5ß-cholan-24-ol (BAR501) reverses liver and vascular damage induced by exposure to a diet enriched in fat and fructose (HFD-F). Treating HFD-F mice with BAR501 reversed liver injury and promoted the browning of white adipose tissue in a Gpbar1-dependent manner. Feeding HFD-F resulted in vascular damage, as shown by the increased aorta intima-media thickness and increased expression of inflammatory genes (IL-6,TNF-α, iNOS, and F4/80) and adhesion molecules (VCAM, intercellular adhesion molecule-1, and endothelial selectin) in the aorta, while reducing the expression of genes involved in NO and hydrogen sulfide generation, severely altering vasomotor activities of aortic rings in an ex vivo assay. BAR501 reversed this pattern in a Gpbar1-dependent manner, highlighting a potential role for GPBAR1 agonism in treating the liver and vascular component of NASH.-Carino, A., Marchianò, S., Biagioli, M., Bucci, M., Vellecco, V., Brancaleone, V., Fiorucci, C., Zampella, A., Monti, M. C., Distrutti, E., Fiorucci, S. Agonism for the bile acid receptor GPBAR1 reverses liver and vascular damage in a mouse model of steatohepatitis.


Asunto(s)
Colestanoles/farmacología , Modelos Animales de Enfermedad , Inflamación/prevención & control , Hepatopatías/prevención & control , Enfermedad del Hígado Graso no Alcohólico/fisiopatología , Receptores Acoplados a Proteínas G/agonistas , Enfermedades Vasculares/prevención & control , Animales , Dieta Alta en Grasa/efectos adversos , Inflamación/etiología , Inflamación/metabolismo , Inflamación/patología , Hepatopatías/etiología , Hepatopatías/metabolismo , Hepatopatías/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/etiología , Receptores Acoplados a Proteínas G/fisiología , Enfermedades Vasculares/etiología , Enfermedades Vasculares/metabolismo , Enfermedades Vasculares/patología
18.
J Nat Prod ; 83(5): 1495-1504, 2020 05 22.
Artículo en Inglés | MEDLINE | ID: mdl-32275146

RESUMEN

A wide range of prescreening tests for antimicrobial activity of 59 bacterial isolates from sediments of Ria Formosa Lagoon (Algarve, Portugal) disclosed Vibrio spartinae 3.6 as the most active antibacterial producing strain. This bacterial strain, which has not previously been submitted for chemical profiling, was subjected to de novo whole genome sequencing, which aided in the discovery and elucidation of a prodigiosin biosynthetic gene cluster that was predicted by the bioinformatic tool KEGG BlastKoala. Comparative genomics led to the identification of a new membrane di-iron oxygenase-like enzyme, annotated as Vspart_02107, which is likely to be involved in the biosynthesis of cycloprodigiosin and analogues. The combined genomics-metabolomics profiling of the strain led to the isolation and identification of one new branched-chain prodigiosin (5) and to the detection of two new cyclic forms. Furthermore, the evaluation of the minimum inhibitory concentrations disclosed the major prodigiosin as very effective against multi-drug-resistant pathogens including Stenotrophomonas maltophilia, a clinical isolate of Listeria monocytogenes, as well as some human pathogens reported by the World Health Organization as prioritized targets.


Asunto(s)
Antibacterianos/biosíntesis , Indoles/química , Pirroles/química , Vibrio/genética , Vibrio/metabolismo , Antibacterianos/química , Bacterias/efectos de los fármacos , Biología Computacional , Ciclización , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Genómica , Listeria monocytogenes/efectos de los fármacos , Metabolómica , Pruebas de Sensibilidad Microbiana , Espectrometría de Masa por Ionización de Electrospray , Stenotrophomonas maltophilia/efectos de los fármacos
19.
Mar Drugs ; 18(5)2020 May 20.
Artículo en Inglés | MEDLINE | ID: mdl-32443698

RESUMEN

Rhamnolipids (RLs) are surface-active molecules mainly produced by Pseudomonas spp. Antarctica is one of the less explored places on Earth and bioprospecting for novel RL producer strains represents a promising strategy for the discovery of novel structures. In the present study, 34 cultivable bacteria isolated from Edmonson Point Lake, Ross Sea, Antarctica were subjected to preliminary screening for the biosurfactant activity. The positive strains were identified by 16S rRNA gene sequencing and the produced RLs were characterized by liquid chromatography coupled to high resolution mass spectrometry (LC-HRESIMS) and liquid chromatography coupled with tandem spectrometry (LC-MS/MS), resulting in a new mixture of 17 different RL congeners, with six previously undescribed RLs. We explored the influence of the carbon source on the RL composition using 12 different raw materials, such as monosaccharides, polysaccharides and petroleum industry derivatives, reporting for the first time the production of RLs using, as sole carbon source, anthracene and benzene. Moreover, we investigated the antimicrobial potential of the RL mixture, towards a panel of both Gram-positive and Gram-negative pathogens, reporting very interesting results towards Listeria monocytogenes with a minimum inhibitory concentration (MIC) value of 3.13 µg/mL. Finally, we report for the first time the antimicrobial activity of RLs towards three strains of the emerging multidrug resistant Stenotrophomonas maltophilia with MIC values of 12.5 µg/ml.


Asunto(s)
Antibacterianos/farmacología , Decanoatos/farmacología , Pseudomonas , Ramnosa/análogos & derivados , Tensoactivos/química , Animales , Regiones Antárticas , Decanoatos/química , Humanos , Listeria monocytogenes/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Ramnosa/química , Ramnosa/farmacología , Stenotrophomonas maltophilia/efectos de los fármacos
20.
Molecules ; 25(12)2020 Jun 23.
Artículo en Inglés | MEDLINE | ID: mdl-32585851

RESUMEN

Pterostilbene, the 3,5-dimethoxy derivative of resveratrol, is a well-known polyphenolic compound, mainly found in blueberries, grapevines, and Pterocarpus marsupium heartwood, which has recently attracted a great deal of attention due to its wide bio-pharmacological profile. Moreover, pterostilbene is more lipophilic than resveratrol, with a consequently better bioavailability and a more interesting therapeutic potential. In this work, a chemoproteomic approach, based on affinity chromatography, was applied on pterostilbene in the attempt to identify the biological targets responsible for its bioactivity. On this basis, syntaxins, a group of proteins involved in the formation of SNARE complexes mediating vesicles exocytosis, were selected among the most interesting pterostilbene interactors. In vitro and in cell assays gave evidence of the pterostilbene ability to reduce insulin secretion on glucose-stimulated pancreatic beta cells, opening the way to potential applications of pterostilbene as a supplement in the care of insulin-dependent metabolic disorders.


Asunto(s)
Quimioinformática , Secreción de Insulina/efectos de los fármacos , Proteómica , Estilbenos/farmacología , Cromatografía de Afinidad , Células HeLa , Humanos , Espectrometría de Masas , Estilbenos/química
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