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1.
Int J Mol Sci ; 22(17)2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34502316

RESUMO

Farrerol (FA) is a flavanone isolated from the Chinese herbal medicine "Man-shan-hong" (Rhododendron dauricum L.). In the present study, FA decreased the viability of SKOV3 cells in a dose- and time-dependent manner, and it induced G2/M cell cycle arrest and cell apoptosis. Cell cycle distribution analysis via flow cytometry showed that FA decreased G1 populations and increased G2/M populations in SKOV3 cells. Additionally, Western blotting confirmed an increase in the expression level of proteins involved in the cell cycle, e.g., CDK and cyclins. FA-induced apoptosis in SKOV3 cells was also investigated using a TUNEL assay, and increased expression levels of proapoptotic factors, including Caspase-3 and poly ADP ribose polymerase (PARP), through the Extracellular signal-regulated kinase (ERK)/MAPK pathway were investigated. Proinflammatory cytokines (e.g., IL-6, TNF-α, and IL-1) have been identified as a driver of the pathological mechanisms underlying involuntary weight loss and impaired physical function, i.e., cachexia, during cancer; in the present study, we showed that farrerol attenuates TNF-α-induced lipolysis and increases adipogenic differentiation in 3T3-L1 cells. Thus, farrerol could potentially be used as an anticancer agent or anticachetic drug.


Assuntos
Cromonas/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Lipólise/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Fator de Necrose Tumoral alfa/farmacologia , Apoptose , Ciclo Celular , Proliferação de Células , Feminino , Humanos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Células Tumorais Cultivadas
2.
Int J Mol Sci ; 22(16)2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34445612

RESUMO

Prostate cancer is a common cause of death worldwide. Here, we isolated cancer stem cells (CSCs) from four adenocarcinomas of the prostate (Gleason scores from 3 + 3 up to 4 + 5). CSCs were characterized by the expression of the stem cell markers TWIST, the epithelial cell adhesion molecule (EPCAM), the transcription factors SNAI1 (SNAIL) and SNAI2 (SLUG) and cancer markers such as CD44 and prominin-1 (CD133). All investigated CSC populations contained a fraction highly positive for aldehyde dehydrogenase (ALDH) function and displayed robust expressions of programmed cell death 1 (PD-1) ligands. Furthermore, we investigated immunotherapeutic approaches but had no success even with the clinically used PD-1 inhibitor pembrolizumab. In addition, we studied another death-inducing pathway via interferon gamma signaling and detected high-level upregulations of human leukocyte antigen A (HLA-A) and beta 2-microglobulin (B2M) with only moderate killing efficacy. To examine further killing mechanisms in prostate cancer stem cells (PCSCs), we analyzed NF-κB signaling. Surprisingly, two patient-specific populations of PCSCs were found: one with canonical NF-κB signaling and another one with blunted NF-κB activation, which can be efficiently killed by tumor necrosis factor (TNF). Thus, culturing of PCSCs and analysis of respective NF-κB induction potency after surgery might be a powerful tool for optimizing patient-specific treatment options, such as the use of TNF-inducing chemotherapeutics and/or NF-κB inhibitors.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Matadoras Naturais/patologia , NF-kappa B/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias da Próstata/patologia , Fator de Necrose Tumoral alfa/farmacologia , Antineoplásicos Imunológicos/farmacologia , Apoptose , Proliferação de Células , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Masculino , NF-kappa B/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Células Tumorais Cultivadas
3.
Molecules ; 26(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34361701

RESUMO

Arthritis is a chronic inflammatory disease accompanied by pathological reactions such as swelling, redness, fever, and pain in various joint areas. The drugs currently available to treat arthritis are associated with diverse side-effects. Therefore, there is a need for safer and more effective treatments to alleviate the inflammation of arthritis with fewer side-effects. In this study, a new sterol, Δ8(14)-ergostenol, was discovered, and its glycosides were synthesized and found to be more efficient in terms of synthesis or anti-inflammatory activity than either spinasterol or 5,6-dihydroergosterol is. Among these synthetic glycosides, galactosyl ergostenol inhibited the expression of inflammatory mediators in TNF-α-stimulated FLS and TNF-α-induced MMPs and collagen type II A1 degradation in human chondrocytes. These results suggest the new galactosyl ergostenol as a treatment candidate for arthritis.


Assuntos
Anti-Inflamatórios/farmacologia , Condrócitos/efeitos dos fármacos , Ergosterol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glicosídeos/farmacologia , Sinoviócitos/efeitos dos fármacos , Anti-Inflamatórios/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Ergosterol/química , Glicosídeos/síntese química , Humanos , Inflamação/prevenção & controle , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Modelos Biológicos , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Transdução de Sinais , Sinoviócitos/citologia , Sinoviócitos/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/farmacologia
4.
Int J Mol Sci ; 22(16)2021 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-34445450

RESUMO

Tumor necrosis factor alpha (TNFα) has been shown to impair the intestinal barrier, inducing and maintaining inflammatory states of the intestine. The aim of the current study was to analyze functional, molecular and regulatory effects of TNFα in a newly established non-transformed jejunal enterocyte model, namely IPEC-J2 monolayers. Incubation with 1000 U/mL TNFα induced a marked decrease in transepithelial electrical resistance (TEER), and an increase in permeability for the paracellular flux marker [3H]-D-mannitol compared to controls. Immunoblots revealed a significant decrease in tight junction (TJ) proteins occludin, claudin-1 and claudin-3. Moreover, a dose-dependent increase in the TNF receptor (TNFR)-1 was detected, explaining the exponential nature of pro-inflammatory effects, while TNFR-2 remained unchanged. Recovery experiments revealed reversible effects after the removal of the cytokine, excluding apoptosis as a reason for the observed changes. Furthermore, TNFα signaling could be inhibited by the specific myosin light chain kinase (MLCK) blocker ML-7. Results of confocal laser scanning immunofluorescence microscopy were in accordance with all quantitative changes. This study explains the self-enhancing effects of TNFα mediated by MLCK, leading to a differential regulation of TJ proteins resulting in barrier impairment in the intestinal epithelium.


Assuntos
Mucosa Intestinal/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Proteínas de Junções Íntimas/genética , Junções Íntimas , Fator de Necrose Tumoral alfa/metabolismo , Animais , Linhagem Celular , Claudina-1/genética , Claudina-3/genética , Regulação da Expressão Gênica , Mucosa Intestinal/fisiologia , Jejuno/metabolismo , Jejuno/fisiologia , Manitol/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Ocludina/genética , Permeabilidade , Transdução de Sinais , Sus scrofa/metabolismo , Sus scrofa/fisiologia , Fator de Necrose Tumoral alfa/farmacologia
5.
FASEB J ; 35(9): e21795, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34403508

RESUMO

Intervertebral disc degeneration is an irreversible process associated with accumulation of senescent nucleus pulposus (NP) cells. This study investigates the hypothesis that Tumor necrosis factor-α (TNF-α)-treated senescent NP cells propagate senescence of neighboring healthy cells via a paracrine effect that involves p-Stat3 signaling and the cytokine interleukin-6 (IL-6). NP cells isolated from bovine caudal intervertebral disc (IVD) were treated with TNF-α to induce senescence which was confirmed by demonstrating upregulation of senescence-associated ß-galactosidase and p16. This was correlated with downregulation of NP-associated markers, Aggrecan, Col2A1, and Sox9. Direct contact and non-contact co-culture of healthy and senescent cells showed that TNF-α-treated cells increased the senescence in healthy cells via a paracrine effect. The senescent cells have a secretory phenotype as indicated by increased gene and protein levels of IL-6. Phosphorylated Signal Transducer and Activator of Transcription 3 (pStat3) levels were also high in treated cells and appeared to upregulate IL-6 as inhibition of Stat3 phosphorylation by StatticV downregulated IL-6 mRNA expression in cells and protein levels in the culture media. All trans retinoic acid, an IL-6 inhibitor, also decreased the secretion of IL-6 and reduced the paracrine effect of senescent cells on healthy cells. Decreased pStat3 levels and inhibition of IL-6 secretion did not fully restore NP gene expression of Col2A1 but importantly, appeared to cause senescent cells to undergo apoptosis and cell death. This study demonstrated the paracrine effect of senescent NP cells which involves Stat3 and IL-6 and may explain why senescent NP cells accumulate in IVD with age. The role of pSTAT3 and IL-6 in mediating NP senescence requires further study as it may be a novel strategy for modulating the senescent-inducing effects of TNF-α.


Assuntos
Senescência Celular/efeitos dos fármacos , Núcleo Pulposo/citologia , Núcleo Pulposo/efeitos dos fármacos , Comunicação Parácrina/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Animais , Apoptose/efeitos dos fármacos , Bovinos , Morte Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-6/antagonistas & inibidores , Interleucina-6/metabolismo , Núcleo Pulposo/metabolismo , Fosforilação , Fator de Transcrição STAT3/metabolismo
6.
Molecules ; 26(15)2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34361563

RESUMO

The consumption of plant-based food is important for health promotion, especially regarding the prevention and management of chronic diseases such as diabetes. We investigated the effects of a lemon extract (LE), containing ≥20.0% total flavanones and ≥1.0% total hydroxycinnamic acids, on insulin signaling in murine 3T3-L1 adipocytes treated with TNF-α, which was used to mimic in vitro the insulin resistance condition that characterizes diabetes mellitus. Our results showed LE increased PPARγ, GLUT4 and DGAT-1 levels, demonstrating the potential of this lemon extract in the management of insulin resistance conditions associated with TNF-α pathway activation. LE treatment further decreased the release of interleukin 6 (IL-6) and restored triglyceride synthesis, which is the main feature of a healthy adipocyte.


Assuntos
Adipócitos/metabolismo , Citrus/química , Resistência à Insulina , Compostos Fitoquímicos , Extratos Vegetais , Fator de Necrose Tumoral alfa/efeitos adversos , Células 3T3-L1 , Animais , Camundongos , Compostos Fitoquímicos/química , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Fator de Necrose Tumoral alfa/farmacologia
7.
SLAS Discov ; 26(9): 1079-1090, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34269109

RESUMO

The recent renascence of phenotypic drug discovery (PDD) is catalyzed by its ability to identify first-in-class drugs and deliver results when the exact molecular mechanism is partially obscure. Acute respiratory distress syndrome (ARDS) is a severe, life-threatening condition with a high mortality rate that has increased in frequency due to the COVID-19 pandemic. Despite decades of laboratory and clinical study, no efficient pharmacological therapy for ARDS has been found. An increase in endothelial permeability is the primary event in ARDS onset, causing the development of pulmonary edema that leads to respiratory failure. Currently, the detailed molecular mechanisms regulating endothelial permeability are poorly understood. Therefore, the use of the PDD approach in the search for efficient ARDS treatment can be more productive than classic target-based drug discovery (TDD), but its use requires a new cell-based assay compatible with high-throughput (HTS) and high-content (HCS) screening. Here we report the development of a new plate-based image cytometry method to measure endothelial barrier function. The incorporation of image cytometry in combination with digital image analysis substantially decreases assay variability and increases the signal window. This new method simultaneously allows for rapid measurement of cell monolayer permeability and cytological analysis. The time-course of permeability increase in human pulmonary artery endothelial cells (HPAECs) in response to the thrombin and tumor necrosis factor α treatment correlates with previously published data obtained by transendothelial resistance (TER) measurements. Furthermore, the proposed image cytometry method can be easily adapted for HTS/HCS applications.


Assuntos
COVID-19/diagnóstico por imagem , Ensaios de Triagem em Larga Escala/métodos , Citometria por Imagem/métodos , Síndrome do Desconforto Respiratório/diagnóstico por imagem , COVID-19/diagnóstico , COVID-19/virologia , Permeabilidade da Membrana Celular/genética , Descoberta de Drogas , Células Endoteliais/ultraestrutura , Células Endoteliais/virologia , Humanos , Processamento de Imagem Assistida por Computador , Pandemias/prevenção & controle , Fenótipo , Artéria Pulmonar/diagnóstico por imagem , Artéria Pulmonar/patologia , Artéria Pulmonar/virologia , Edema Pulmonar/diagnóstico , Edema Pulmonar/diagnóstico por imagem , Edema Pulmonar/virologia , Síndrome do Desconforto Respiratório/diagnóstico , Síndrome do Desconforto Respiratório/virologia , Insuficiência Respiratória/diagnóstico , Insuficiência Respiratória/diagnóstico por imagem , Insuficiência Respiratória/virologia , SARS-CoV-2/patogenicidade , Trombina/farmacologia , Fator de Necrose Tumoral alfa/farmacologia
8.
Exp Cell Res ; 406(1): 112738, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34270981

RESUMO

We determined the role of time in adipose-derived stem/stromal cell (ASC) response to a model inflammatory environment. ASCs and other mesenchymal stem/stromal cells exhibit immune plasticity. We evaluated the persistence of pro- and anti-inflammatory phenotypes for ASCs exposed to a sustained or pulse inflammatory stimulus. Using qPCR, flow cytometry, and immunocytochemistry, we monitored the temporal expression and up-regulation patterns of a pro-inflammatory gene (caspase 1), a pleiotropic gene/protein (interleukin 6, IL-6), and an anti-inflammatory gene/protein (indoleamine 2, 3-dioxygenase, IDO1) after exposing ASCs to the cytokines tumor necrosis factor-α and interferon-γ. In response to sustained cytokine stimulation, we discovered that time played a role in the balance of pro- and anti-inflammatory ASC phenotypes. IL-6 was present at all time points for both cytokine-stimulated and non-stimulated conditions, whereas IDO1 was heterogeneously up-regulated in stimulated conditions at later time points. After a pulse stimulus, ASC immunoresponse remained consistent for 96-168 h. As a final measure of immune plasticity, we cultured cytokine-stimulated ASCs with blood-derived macrophages to observe macrophage polarization. While the presence of ASCs altered macrophage phenotype, there was no dependency on the length of ASC cytokine exposure time.


Assuntos
Caspase 1/genética , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Interferon gama/farmacologia , Interleucina-6/genética , Células-Tronco Mesenquimais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Tecido Adiposo/citologia , Tecido Adiposo/imunologia , Caspase 1/imunologia , Diferenciação Celular/efeitos dos fármacos , Técnicas de Cocultura , Regulação da Expressão Gênica , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Interleucina-6/imunologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/imunologia , Cultura Primária de Células , Transdução de Sinais , Fatores de Tempo
9.
Cell Mol Life Sci ; 78(17-18): 6069-6086, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34283248

RESUMO

TNF-α-induced NF-κB pathway is an essential component of innate and adaptive immune pathway, and it is tightly regulated by various post-translational modifications including ubiquitination. Oscillations in NF-κB activation and temporal gene expression are emerging as critical determinants of inflammatory response, however, the regulators of unique outcomes in different patho-physiological conditions are not well understood. Tripartite Motif-containing proteins (TRIMs) are RING domain-containing E3 ligases involved in the regulation of cellular homeostasis, metabolism, cell death, inflammation, and host defence. Emerging reports suggest that TRIMs are recruited at different steps of TNF-α-induced NF-κB pathway and modulate via their E3 ligase activity. TRIMs show synergy and antagonism in the regulation of the NF-κB pathway and also regulate it in a feedback manner. TRIMs also regulate pattern recognition receptors (PRRs) mediated inflammatory pathways and may have evolved to directly regulate a specific arm of immune signalling. The review emphasizes TRIM-mediated ubiquitination and modulation of TNF-α-regulated temporal and NF-κB signaling and its possible impact on unique transcriptional and functional outcomes.


Assuntos
NF-kappa B/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imunidade Inata , Inflamação/metabolismo , Inflamação/patologia , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacos
10.
Int J Mol Sci ; 22(12)2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34207810

RESUMO

Excess lipid droplets are frequently observed in arterial endothelial cells at sites of advanced atherosclerotic plaques. Here, the role of tumor necrosis factor alpha (TNFα) in modulating the low-density lipoprotein (LDL) content in confluent primary human aortic endothelial cells (pHAECs) was investigated. TNFα promoted an up to 2 folds increase in cellular cholesterol, which was resistant to ACAT inhibition. The cholesterol increase was associated with increased 125I-LDL surface binding. Using the non-hydrolysable label, Dil, TNFα could induce a massive increase in Dil-LDL by over 200 folds. The elevated intracellular Dil-LDL was blocked with excess unlabeled LDL and PCSK9, but not oxidized LDL (oxLDL), or apolipoprotein (apoE) depletion. Moreover, the TNFα-induced increase of LDL-derived lipids was elevated through lysosome inhibition. Using specific LDLR antibody, the Dil-LDL accumulation was reduced by over 99%. The effects of TNFα included an LDLR cell surface increase of 138%, and very large increases in ICAM-1 total and surface proteins, respectively. In contrast, that of scavenger receptor B1 (SR-B1) was reduced. Additionally, LDLR antibody bound rapidly in TNFα-treated cells by about 30 folds, inducing a migrating shift in the LDLR protein. The effect of TNFα on Dil-LDL accumulation was inhibited by the antioxidant tetramethythiourea (TMTU) dose-dependently, but not by inhibitors against NF-κB, stress kinases, ASK1, JNK, p38, or apoptosis caspases. Grown on Transwell inserts, TNFα did not enhance apical to basolateral LDL cholesterol or Dil release. It is concluded that TNFα promotes LDLR functions through combined increase at the cell surface and SR-B1 downregulation.


Assuntos
Artérias/metabolismo , LDL-Colesterol/metabolismo , Regulação para Baixo/efeitos dos fármacos , Células Endoteliais/metabolismo , Receptores de LDL/biossíntese , Receptores Depuradores Classe B/biossíntese , Fator de Necrose Tumoral alfa/farmacologia , Artérias/patologia , Células Endoteliais/patologia , Humanos , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Fator de Necrose Tumoral alfa/metabolismo
11.
Int J Mol Sci ; 22(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208434

RESUMO

Skullcapflavone II (SFII), a flavonoid derived from Scutellaria baicalensis, has been reported to have anti-inflammatory properties. However, its therapeutic potential for skin inflammatory diseases and its mechanism are unknown. Therefore, this study aimed to investigate the effect of SFII on TNF-α/IFN-γ-induced atopic dermatitis (AD)-associated cytokines, such as thymus- and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC). Co-stimulation with TNF-α/IFN-γ in HaCaT cells is a well-established model for induction of pro-inflammatory cytokines. We treated cells with SFII prior to TNF-α/IFN-γ-stimulation and confirmed that it significantly inhibited TARC and MDC expression at the mRNA and protein levels. Additionally, SFII also inhibited the expression of cathepsin S (CTSS), which is associated with itching in patients with AD. Using specific inhibitors, we demonstrated that STAT1, NF-κB, and p38 MAPK mediate TNF-α/IFN-γ-induced TARC and MDC, as well as CTSS expression. Finally, we confirmed that SFII significantly suppressed TNF-α/IFN-γ-induced phosphorylation of STAT1, NF-κB, and p38 MAPK. Taken together, our study indicates that SFII inhibits TNF-α/IFN-γ-induced TARC, MDC, and CTSS expression by regulating STAT1, NF-κB, and p38 MAPK signaling pathways.


Assuntos
Catepsinas/biossíntese , Quimiocina CCL17/biossíntese , Quimiocina CCL22/biossíntese , Flavonoides/farmacologia , Interferon gama/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Catepsinas/genética , Sobrevivência Celular/efeitos dos fármacos , Quimiocina CCL17/genética , Quimiocina CCL22/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células HaCaT , Humanos , Queratinócitos/metabolismo , NF-kappa B/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
12.
Int J Mol Sci ; 22(14)2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34299302

RESUMO

Short-chain fatty acid (SCFA) acetate, a byproduct of dietary fiber metabolism by gut bacteria, has multiple immunomodulatory functions. The anti-inflammatory role of acetate is well documented; however, its effect on monocyte chemoattractant protein-1 (MCP-1) production is unknown. Similarly, the comparative effect of SCFA on MCP-1 expression in monocytes and macrophages remains unclear. We investigated whether acetate modulates TNFα-mediated MCP-1/CCL2 production in monocytes/macrophages and, if so, by which mechanism(s). Monocytic cells were exposed to acetate with/without TNFα for 24 h, and MCP-1 expression was measured. Monocytes treated with acetate in combination with TNFα resulted in significantly greater MCP-1 production compared to TNFα treatment alone, indicating a synergistic effect. On the contrary, treatment with acetate in combination with TNFα suppressed MCP-1 production in macrophages. The synergistic upregulation of MCP-1 was mediated through the activation of long-chain fatty acyl-CoA synthetase 1 (ACSL1). However, the inhibition of other bioactive lipid enzymes [carnitine palmitoyltransferase I (CPT I) or serine palmitoyltransferase (SPT)] did not affect this synergy. Moreover, MCP-1 expression was significantly reduced by the inhibition of p38 MAPK, ERK1/2, and NF-κB signaling. The inhibition of ACSL1 attenuated the acetate/TNFα-mediated phosphorylation of p38 MAPK, ERK1/2, and NF-κB. Increased NF-κB/AP-1 activity, resulting from acetate/TNFα co-stimulation, was decreased by ACSL1 inhibition. In conclusion, this study demonstrates the proinflammatory effects of acetate on TNF-α-mediated MCP-1 production via the ACSL1/MAPK/NF-κB axis in monocytic cells, while a paradoxical effect was observed in THP-1-derived macrophages.


Assuntos
Acetatos/farmacologia , Quimiocina CCL2/biossíntese , Ácidos Graxos Voláteis/farmacologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Acetatos/administração & dosagem , Quimiocina CCL2/genética , Coenzima A Ligases/antagonistas & inibidores , Coenzima A Ligases/metabolismo , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Ácidos Graxos Voláteis/administração & dosagem , Humanos , Sistema de Sinalização das MAP Quinases , Modelos Biológicos , Monócitos/imunologia , NF-kappa B/metabolismo , Obesidade/etiologia , Obesidade/metabolismo , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células THP-1 , Triazenos/farmacologia , Fator de Necrose Tumoral alfa/administração & dosagem , Fator de Necrose Tumoral alfa/farmacologia
13.
Int J Mol Sci ; 22(14)2021 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-34299030

RESUMO

Neutrophil-derived microvesicles (NDMVs) have the potential to exert anti-inflammatory effects. Our study aimed to explore the effects of NDMVs on proinflammatory cytokines expressed by tumor necrosis factor α (TNFα)-stimulated fibroblast-like synoviocytes (FLS). FLS were isolated from the synovium of knee osteoarthritis (OA) patients undergoing surgery. NDMVs, isolated from TNFα-stimulated healthy neutrophils, were characterized by electron microscopy and nanoparticle tracking analysis. MTT and scratch wound healing assays were used to measure FLS viability and migration after treatment with NDMVs, while internalization of fluorescently labeled NDMVs was appraised by flow cytometry and confocal microscopy. Levels of proinflammatory cytokines in supernatants were quantified by the Bio-Plex system. Incubation of FLS with NDMVs at a vesicle/cell ratio of 100 resulted in a time-dependent uptake, with 35% of synoviocytes containing microvesicles over a 6-24 h time period, with no significant change in cell viability. TNFα stimulated the cytokine expression in FLS, and NDMVs down-regulated TNFα-induced expression of IL-5, IL-6, IL-8, MCP-1, IFNγ and MIP-1ß. However, this down-regulation was selective, as NDMVs had no significant effects on TNFα-stimulated expression of IL-2 or IL-4. NDMVs were internalized by FLS to inhibit TNFα-stimulated broad-spectrum proinflammatory cytokine secretion. NDMVs, therefore, may exhibit an anti-inflammatory role in the regulation of the FLS function.


Assuntos
Micropartículas Derivadas de Células/metabolismo , Fibroblastos/metabolismo , Mediadores da Inflamação/metabolismo , Neutrófilos/metabolismo , Osteoartrite do Joelho/metabolismo , Sinoviócitos/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Micropartículas Derivadas de Células/patologia , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Fibroblastos/patologia , Humanos , Neutrófilos/patologia , Osteoartrite do Joelho/tratamento farmacológico , Osteoartrite do Joelho/imunologia , Osteoartrite do Joelho/patologia , Sinoviócitos/efeitos dos fármacos , Sinoviócitos/imunologia , Sinoviócitos/patologia
14.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201751

RESUMO

Cylindromatosis (CYLD) is a deubiquitinase (DUB) enzyme that was initially characterized as a tumor suppressor of adnexal skin tumors in patients with CYLD syndrome. Later, it was also shown that the expression of functionally inactive mutated forms of CYLD promoted tumor development and progression of non-melanoma skin cancer (NMSC). However, the ability of wild-type CYLD to inhibit skin tumorigenesis in vivo in immunocompetent mice has not been proved. Herein, we generated transgenic mice that express the wild type form of CYLD under the control of the keratin 5 (K5) promoter (K5-CYLDwt mice) and analyzed the skin properties of these transgenic mice by WB and immunohistochemistry, studied the survival and proliferating characteristics of primary keratinocytes, and performed chemical skin carcinogenesis experiments. As a result, we found a reduced activation of the nuclear factor kappa B (NF-κB) pathway in the skin of K5-CYLDwt mice in response to tumor necrosis factor-α (TNF-α); accordingly, when subjected to insults, K5-CYLDwt keratinocytes are prone to apoptosis and are protected from excessive hyperproliferation. Skin carcinogenesis assays showed inhibition of tumor development in K5-CYLDwt mice. As a mechanism of this tumor suppressor activity, we found that a moderate increase in CYLD expression levels reduced NF-κB activation, which favored the differentiation of tumor epidermal cells and inhibited its proliferation; moreover, it decreased tumor angiogenesis and inflammation. Altogether, our results suggest that increased levels of CYLD may be useful for anti-skin cancer therapy.


Assuntos
Carcinoma de Células Escamosas/patologia , Enzima Desubiquitinante CYLD/genética , Neoplasias Cutâneas/patologia , Animais , Carcinoma de Células Escamosas/irrigação sanguínea , Carcinoma de Células Escamosas/genética , Diferenciação Celular/genética , Proliferação de Células/genética , Células Cultivadas , Enzima Desubiquitinante CYLD/metabolismo , Genes Supressores de Tumor , Imunocompetência , Queratinócitos/efeitos dos fármacos , Queratinócitos/patologia , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , NF-kappa B/metabolismo , Neovascularização Patológica/genética , Ésteres de Forbol/toxicidade , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Cutâneas/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
15.
Int J Mol Sci ; 22(14)2021 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-34299094

RESUMO

The root bark of Cudrania tricuspidata has been reported to have anti-sclerotic, anti-inflammatory, antioxidant, neuroprotective, hepatoprotective, and cytotoxic activities. In the present study, the effect of 16 compounds from C. tricuspidata on tumor necrosis factor-α+interferon-γ-treated HaCaT cells were investigated. Among these 16 compounds, 11 decreased IL-6 production and 15 decreased IL-8 production. The six most effective compounds, namely, steppogenin (2), cudraflavone C (6), macluraxanthone B (12), 1,6,7-trihydroxy-2-(1,1-dimethyl-2-propenyl)-3- methoxyxanthone (13), cudraflavanone B (4), and cudratricusxanthone L (14), were selected for further experiments. These six compounds decreased the expression levels of chemokines, such as regulated on activation, normal T cell expressed and secreted (RANTES) and thymus and activation-regulated chemokine (TARC), and downregulated the protein expression levels of intercellular adhesion molecule-1. Compounds 2, 6, 12, 4, and 14 inhibited nuclear factor-kappa B p65 translocation to the nucleus; however, compound 13 showed no significant effects. In addition, extracellular signal regulatory kinase-1/2 phosphorylation was only inhibited by compound 14, whereas p38 phosphorylation was inhibited by compounds 13 and 4. Taken together, the compounds from C. tricuspidata showed potential to be further developed as therapeutic agents to suppress inflammation in skin cells.


Assuntos
Anti-Inflamatórios/farmacologia , Inflamação/tratamento farmacológico , Queratinócitos/efeitos dos fármacos , Moraceae/química , Compostos Fitoquímicos/química , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/farmacologia , Quimiocinas/metabolismo , Citocinas/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/patologia , Interferon gama/metabolismo , Queratinócitos/metabolismo , NF-kappa B/metabolismo , Fosforilação , Compostos Fitoquímicos/classificação , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologia
16.
Cell Death Dis ; 12(7): 649, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34172716

RESUMO

Endothelial-mesenchymal transition (EndMT) is an important source of cancer-associated fibroblasts (CAFs), which facilitates tumour progression. PDAC is characterised by abundant CAFs and tumour necrosis factor-α (TNF-α). Here, we show that TNF-α strongly induces human endothelial cells to undergo EndMT. Interestingly, TNF-α strongly downregulates the expression of the endothelial receptor TIE1, and reciprocally TIE1 overexpression partially prevents TNF-α-induced EndMT, suggesting that TNF-α acts, at least partially, through TIE1 regulation in this process. We also show that TNF-α-induced EndMT is reversible. Furthermore, TNF-α treatment of orthotopic mice resulted in an important increase in the stroma, including CAFs. Finally, secretome analysis identified TNFSF12, as a regulator that is also present in PDAC patients. With the aim of restoring normal angiogenesis and better access to drugs, our results support the development of therapies targeting CAFs or inducing the EndMT reversion process in PDAC.


Assuntos
Fibroblastos Associados a Câncer/efeitos dos fármacos , Carcinoma Ductal Pancreático/patologia , Células Endoteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias Pancreáticas/patologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Células Cultivadas , Citocina TWEAK/genética , Citocina TWEAK/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Masculino , Camundongos Transgênicos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Receptor de TIE-1/genética , Receptor de TIE-1/metabolismo , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo
18.
Nat Commun ; 12(1): 3958, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34172753

RESUMO

Astrocytes play important roles in neurological disorders such as stroke, injury, and neurodegeneration. Most knowledge on astrocyte biology is based on studies of mouse models and the similarities and differences between human and mouse astrocytes are insufficiently characterized, presenting a barrier in translational research. Based on analyses of acutely purified astrocytes, serum-free cultures of primary astrocytes, and xenografted chimeric mice, we find extensive conservation in astrocytic gene expression between human and mouse samples. However, the genes involved in defense response and metabolism show species-specific differences. Human astrocytes exhibit greater susceptibility to oxidative stress than mouse astrocytes, due to differences in mitochondrial physiology and detoxification pathways. In addition, we find that mouse but not human astrocytes activate a molecular program for neural repair under hypoxia, whereas human but not mouse astrocytes activate the antigen presentation pathway under inflammatory conditions. Here, we show species-dependent properties of astrocytes, which can be informative for improving translation from mouse models to humans.


Assuntos
Astrócitos/fisiologia , Animais , Apresentação do Antígeno , Astrócitos/efeitos dos fármacos , Células Cultivadas , Expressão Gênica/efeitos dos fármacos , Humanos , Inativação Metabólica , Inflamação , Camundongos , Mitocôndrias/metabolismo , Doenças do Sistema Nervoso/tratamento farmacológico , Doenças do Sistema Nervoso/patologia , Estresse Oxidativo , Poli I-C/farmacologia , Poli I-C/uso terapêutico , Especificidade da Espécie , Transcriptoma/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Fator de Necrose Tumoral alfa/uso terapêutico
19.
Methods Mol Biol ; 2277: 125-132, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080148

RESUMO

Mitochondrial dysfunction is regarded as a key factor involved in the pathogenesis of septic disorders, leading to a decline in energy supply. The influence of short- and medium-chain fatty acids (SCFA/MCFA) on mitochondrial respiration under inflammatory conditions has thus far not been investigated. In the following protocol we describe the assessment of mitochondrial respiration using high-resolution respirometry under inflammatory and baseline conditions. For this approach, human endothelial cells and monocytes were pretreated with TNF-α to mimic inflammation followed by incubation with SCFA/MCFA and then subjected to high-resolution respirometry. Mitochondrial DNA content was assessed by PCR .


Assuntos
Ácidos Graxos/farmacologia , Inflamação/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , DNA Mitocondrial/genética , Ácidos Graxos/química , Ácidos Graxos Voláteis/química , Ácidos Graxos Voláteis/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Inflamação/patologia , Mitocôndrias/patologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Monócitos/patologia , Fator de Necrose Tumoral alfa/farmacologia
20.
Int J Mol Sci ; 22(9)2021 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-34063048

RESUMO

Pazopanib is a multikinase inhibitor with anti-tumor activity. As of now, the anti-obesity effect and mode of action of pazopanib are unknown. In this study, we investigated the effects of pazopanib on lipid accumulation, lipolysis, and expression of inflammatory cyclooxygenase (COX)-2 in differentiating and differentiated 3T3-L1 cells, a murine preadipocyte. Of note, pazopanib at 10 µM markedly decreased lipid accumulation and triglyceride (TG) content during 3T3-L1 preadipocyte differentiation with no cytotoxicity. Furthermore, pazopanib inhibited not only expression of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), and perilipin A but also phosphorylation of signal transducer and activator of transcription (STAT)-3 during 3T3-L1 preadipocyte differentiation. In addition, pazopanib treatment increased phosphorylation of cAMP-activated protein kinase (AMPK) and its downstream effector ACC during 3T3-L1 preadipocyte differentiation. However, in differentiated 3T3-L1 adipocytes, pazopanib treatment did not stimulate glycerol release and hormone-sensitive lipase (HSL) phosphorylation, hallmarks of lipolysis. Moreover, pazopanib could inhibit tumor necrosis factor (TNF)-α-induced expression of COX-2 in both 3T3-L1 preadipocytes and differentiated cells. In summary, this is the first report that pazopanib has strong anti-adipogenic and anti-inflammatory effects in 3T3-L1 cells, which are mediated through regulation of the expression and phosphorylation of C/EBP-α, PPAR-γ, STAT-3, ACC, perilipin A, AMPK, and COX-2.


Assuntos
Adipócitos/citologia , Adipócitos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Indazóis/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Sulfonamidas/farmacologia , Células 3T3-L1 , Adenilato Quinase/metabolismo , Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ácido Graxo Sintases/metabolismo , Glicerol/metabolismo , Leptina/metabolismo , Camundongos , PPAR gama/metabolismo , Perilipina-1/metabolismo , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Resistina/metabolismo , Fator de Transcrição STAT3/metabolismo , Esterol Esterase/metabolismo , Triglicerídeos/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
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