RESUMO
This study explores the potential anticancer effects of lesbicoumestan from Lespedeza bicolor against human leukemia cancer cells. Flow cytometry and fluorescence microscopy were used to investigate antiproliferative effects. The degradation of mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) was evaluated using immunoprecipitation, Western blotting, and confocal microscopy. Apoptosis was investigated using three-dimensional (3D) Jurkat cell resistance models. Lesbicoumestan induced potent mitochondrial depolarization on the Jurkat cells via upregulated expression levels of mitochondrial reactive oxygen species. Furthermore, the underlying apoptotic mechanisms of lesbicoumestan through the MALT1/NF-κB pathway were comprehensively elucidated. The analysis showed that lesbicoumestan significantly induced MALT1 degradation, which led to the inhibition of the NF-κB pathway. In addition, molecular docking results illustrate how lesbicoumestan could effectively bind with MALT1 protease at the latter's active pocket. Similar to traditional 2D cultures, apoptosis was markedly induced upon lesbicoumestan treatment in 3D Jurkat cell resistance models. Our data support the hypothesis that lesbicoumestan is a novel inhibitor of MALT1, as it exhibited potent antiapoptotic effects in Jurkat cells.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/metabolismo , Apoptose/fisiologia , Caspases/metabolismo , Proliferação de Células/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Células Jurkat , Mitocôndrias/efeitos dos fármacos , Simulação de Acoplamento Molecular , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/química , Estresse Oxidativo/fisiologia , Esferoides CelularesRESUMO
Recent studies found that short-chain fatty acids (SCFAs), which are produced through bacterial fermentation in the gastrointestinal tract, have oncoprotective effects against cervical cancer. The most common SCFAs that are well known include acetic acid, butyric acid, and propionic acid, among which propionic acid (PA) has been reported to induce apoptosis in HeLa cells. However, the mechanism in which SCFAs suppress HeLa cell viability remain poorly understood. Our study aims to provide a more detailed look into the mechanism of PA in HeLa cells. Flow cytometry analysis revealed that PA induces reactive oxygen species (ROS), leading to the dysfunction of the mitochondrial membrane. Moreover, PA inhibits NF-κB and AKT/mTOR signaling pathways and induces LC3B protein levels, resulting in autophagy. PA also increased the sub-G1 cell population that is characteristic of cell death. Therefore, the results of this study propose that PA inhibits HeLa cell viability through a mechanism mediated by the induction of autophagy. The study also suggests a new approach for cervical cancer therapeutics.
Assuntos
Antineoplásicos/farmacologia , Propionatos/farmacologia , Neoplasias do Colo do Útero/patologia , Antineoplásicos/química , Autofagia/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Feminino , Células HeLa , Humanos , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , NF-kappa B/metabolismo , Propionatos/química , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias do Colo do Útero/metabolismoRESUMO
Vav1 is a Rho/Rac (Ras-related C3 botulinum toxin substrate) guanine nucleotide exchange factor expressed in hematopoietic and endothelial cells that are involved in a wide range of cellular functions. It is also stabilized under hypoxic conditions when it regulates the accumulation of the transcription factor HIF (Hypoxia Inducible Factor)-1α, which activates the transcription of target genes to orchestrate a cellular response to low oxygen. One of the genes induced by HIF-1α is GLUT (Glucose Transporter)-1, which is the major glucose transporter expressed in vessels that supply energy to the brain. Here, we identify a role for Vav1 in providing glucose to the brain. We found that Vav1 deficiency downregulates HIF-1α and GLUT-1 levels in endothelial cells, including blood-brain barrier cells. This downregulation of GLUT-1, in turn, reduced glucose uptake to endothelial cells both in vitro and in vivo, and reduced glucose levels in the brain. Furthermore, endothelial cell-specific Vav1 knock-out in mice, which caused glucose uptake deficiency, also led to a learning delay in fear conditioning experiments. Our results suggest that Vav1 promotes learning by activating HIF-1α and GLUT-1 and thereby distributing glucose to the brain. We further demonstrate the importance of glucose transport by endothelial cells in brain functioning and reveal a potential new axis for targeting GLUT-1 deficiency syndromes and other related brain diseases.
Assuntos
Glicemia/metabolismo , Encéfalo/metabolismo , Regulação da Expressão Gênica , Transportador de Glucose Tipo 1/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Proteínas Proto-Oncogênicas c-vav/genética , Animais , Feminino , Transportador de Glucose Tipo 1/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-vav/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , TransfecçãoRESUMO
Hypertension increases the pressure load on the heart and is associated with a poorly understood chronic systemic inflammatory state. Interleukin 33 (IL-33) binds to membrane-bound ST2 (ST2L) and has antihypertrophic and antifibrotic effects in the myocardium. In contrast, soluble ST2 appears to act as a decoy receptor for IL-33, blocking myocardial and vascular benefits, and is a prognostic biomarker in patients with cardiovascular diseases. Here we report that a highly local intramyocardial IL-33/ST2 conversation regulates the heart's response to pressure overload. Either endothelial-specific deletion of IL33 or cardiomyocyte-specific deletion of ST2 exacerbated cardiac hypertrophy with pressure overload. Furthermore, pressure overload induced systemic circulating IL-33 as well as systemic circulating IL-13 and TGF-beta1; this was abolished by endothelial-specific deletion of IL33 but not by cardiomyocyte-specific deletion of IL33. Our study reveals that endothelial cell secretion of IL-33 is crucial for translating myocardial pressure overload into a selective systemic inflammatory response.
Assuntos
Cardiomegalia/fisiopatologia , Hipertensão/fisiopatologia , Inflamação/fisiopatologia , Interleucinas/fisiologia , Miocárdio/patologia , Animais , Cardiomegalia/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Hipertensão/metabolismo , Hipertensão/patologia , Inflamação/metabolismo , Inflamação/patologia , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-33 , Interleucinas/metabolismo , Camundongos , Camundongos Knockout , Receptores de Interleucina/genética , Receptores de Interleucina/fisiologiaRESUMO
Similar to IL-1α and IL-33, IL-1 family member IL-37b translocates to the nucleus and is associated with suppression of innate and adaptive immunity. Here we demonstrate an extracellular function of the IL-37 precursor and a processed form. Recombinant IL-37 precursor reduced LPS-induced IL-6 by 50% (P < 0.001) in highly inflammatory human blood-derived M1 differentiated macrophages derived from selective subjects but not M2 macrophages. In contrast, a neutralizing monoclonal anti-IL-37 increased LPS-induced IL-6, TNFα and IL-1ß (P < 0.01). The suppression by IL-37 was consistently observed at low picomolar but not nanomolar concentrations. Whereas LPS induced a 12-fold increase in TNFα mRNA, IL-37 pretreatment decreased the expression to only 3-fold over background (P < 0.01). Mechanistically, LPS-induced p38 and pERK were reduced by IL-37. Recombinant IL-37 bound to the immobilized ligand binding α-chain of the IL-18 receptor as well as to the decoy receptor IL-1R8. In M1 macrophages, LPS increased the surface expression of IL-1R8. Compared with human blood monocytes, resting M1 cells express more surface IL-1R8 as well as total IL-1R8; there was a 16-fold increase in IL-1R8 mRNA levels when pretreated with IL-37. IL-37 reduced LPS-induced TNFα and IL-6 by 50-55% in mouse bone marrow-derived dendritic cells, but not in dendritic cells derived from IL-1R8-deficient mice. In mice subjected to systemic LPS-induced inflammation, pretreatment with IL-37 reduced circulating and organ cytokine levels. Thus, in addition to a nuclear function, IL-37 acts as an extracellular cytokine by binding to the IL-18 receptor but using the IL-1R8 for its anti-inflammatory properties.
Assuntos
Imunidade Inata , Inflamação/imunologia , Interleucina-1/química , Interleucina-1/metabolismo , Receptores de Interleucina-1/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Endotoxemia/metabolismo , Endotoxemia/patologia , Ativação Enzimática/efeitos dos fármacos , Espaço Extracelular/química , Citometria de Fluxo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Imobilizadas/metabolismo , Inflamação/patologia , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/metabolismo , Camundongos , Testes de Neutralização , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Interleucina-1/química , Proteínas Recombinantes/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Although it has been established that diabetes increases susceptibility to infections, the role of insulin (INS) in the immune response is unknown. Here, we investigated the immunological function of INS. Proinsulin dimer (pINSd) was a potent immune stimulus that induced inflammatory cytokines, but mature INS was unable to induce an immune response. An affinity-purified rabbit polyclonal antibody raised against mature IL-1α recognized IL-1α and pINS but failed to detect mature INS and IL-1ß. Analysis of the pINS sequence revealed the existence of an INS/IL-1α motif in the C-peptide of pINS. Surprisingly, the INS/IL-1α motif was recognized by monoclonal antibody raised against IL-1α. Deleting the INS/IL-1α motif in pINSd and IL-1α changed their activities. To investigate the pINSd receptor, the reconstitution of IL-1 receptor 1 (IL-1R1) in Wish cells restored pINSd activity that was reversed by an IL-1R antagonist. These data suggested that pINSd needs IL-1R1 for inflammatory cytokine induction. Mouse embryo fibroblast cells of IL-1R1-deficient mice further confirmed that pINSd promotes immune responses through IL-1R1.
Assuntos
Citocinas/biossíntese , Mediadores da Inflamação/metabolismo , Interleucina-1alfa/metabolismo , Proinsulina/metabolismo , Receptores de Interleucina-1/metabolismo , Animais , Células Cultivadas , Interleucina-1alfa/química , Camundongos , Proinsulina/químicaRESUMO
IL-37 is a fundamental inhibitor of innate immunity. Human IL-37 has a caspase-1 cleavage site and translocates to the nucleus upon LPS stimulation. Here, we investigated whether caspase-1 processing affects IL-37-mediated suppression of LPS-induced cytokines and the release from cells by analyzing a caspase-1 cleavage site mutant IL-37 (IL-37D20A). Nuclear translocation of IL-37D20A is significantly impaired compared with WT IL-37 in transfected cells. LPS-induced IL-6 was decreased in cells expressing WT IL-37 but not IL-37D20A. The function of IL-37 in transfected bone marrow-derived macrophages is nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome-dependent, because IL-37 transfection in apoptosis-associated speck-like protein containing a carboxyl-terminal caspase recruitment domain- and NLRP3-deficient cells does not reduce levels of IL-6 and IL-1ß upon LPS stimulation. IL-37-expressing macrophages release both precursor and mature IL-37, but only the externalization of mature IL-37 was dependent on ATP. Precursor and mature IL-37 was also secreted from human dendritic cells and peripheral blood mononuclear cells. To determine whether IL-37 is active in the extracellular compartment, we pretreated IL-37 transgenic mice with IL-37-neutralizing antibodies before LPS challenge. In IL-37-expressing mice, neutralizing IL-37 antibodies reversed the suppression of LPS-induced serum IL-6. In contrast, the addition of neutralizing antibody did not reverse suppression of LPS-induced IL-6 in mouse macrophages transfected with IL-37. Although caspase-1 is required for nuclear translocation of intracellular IL-37 and for secretion of mature IL-37, the release of the IL-37 precursor is independent of caspase-1 activation. IL-37 now emerges as a dual-function cytokine with intra- and extracellular properties for suppressing innate inflammation.
Assuntos
Anticorpos Neutralizantes/imunologia , Caspase 1/metabolismo , Núcleo Celular/metabolismo , Imunidade Inata/imunologia , Interleucina-1/metabolismo , Transporte Ativo do Núcleo Celular/imunologia , Animais , Western Blotting , Caspase 1/genética , Linhagem Celular , Escherichia coli , Imunofluorescência , Humanos , Interleucina-6/sangue , Lipopolissacarídeos , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Mutagênese Sítio-DirigidaRESUMO
Interleukin-33 (IL-33) receptors are composed of ST2 (also known as IL-1R4), a ligand binding chain, and IL-1 receptor accessory protein (IL-1RAcP, also known as IL-1R3), a signal transducing chain. IL-1R3 is a common receptor for IL-1α, and IL-1ß, IL-33, and three IL-36 isoforms. A549 human lung epithelial cells are highly sensitive to IL-1α and IL-1ß but not respond to IL-33. The lack of responsiveness to IL-33 is due to ST2 expression. ST2 was stably transfected into A549 cells to reconstitute its activity. RT-PCR and FACS analysis confirmed ST2 expression on the cell surface of A549/ST2 cells. Upon IL-33 stimulation, A549/ST2 cells induced IL-8 and IL-6 production in a dose dependent manner while A549/mock cells remained unresponsive. There was no difference in IL-1α and IL-1ß activity in A549/ST2 cells compared to A549/mock cells despite the fact that IL-33 shares IL-1R3 with IL-1α/ß. IL-33 activated inflammatory signaling molecules in a time- and dose-dependent manner. Anti-ST2 antibody and soluble recombinant ST2-Fc abolished IL-33-induced IL-6 and IL-8 production in A549/ST2 cells but the IL-1 receptor antagonist failed to block IL-33-induced cytokines. This result demonstrates for the first time the reconstitution of ST2 in A549 human lung epithelial cell line and verified its function in IL-33-mediated cytokine production and signal transduction.
Assuntos
Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Interleucina-33/metabolismo , Transdução de Sinais/fisiologia , Células A549 , Regulação da Expressão Gênica/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteína Antagonista do Receptor de Interleucina 1/genética , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Interleucina-1alfa/genética , Interleucina-1beta/genética , Interleucina-33/genética , Interleucina-6/sangue , Interleucina-6/genética , Interleucina-8/biossíntese , Interleucina-8/genéticaRESUMO
The rationale of α1-antitrypsin (AAT) augmentation therapy to treat progressive emphysema in AAT-deficient patients is based on inhibition of neutrophil elastase; however, the benefit of this treatment remains unclear. Here we show that clinical grade AAT (with elastase inhibitory activity) and a recombinant form of AAT (rAAT) without anti-elastase activity reduces lung inflammatory responses to LPS in elastase-deficient mice. WT and elastase-deficient mice treated with either native AAT or rAAT exhibited significant reductions in infiltrating neutrophils (23% and 68%), lavage fluid levels of TNF-α (70% and 80%), and the neutrophil chemokine KC (CXCL1) (64% and 90%), respectively. Lung parenchyma TNF-α, DNA damage-inducible transcript 3 and X-box binding protein-1 mRNA levels were reduced in both mouse strains treated with AAT; significantly lower levels of these genes, as well as IL-1ß gene expression, were observed in lungs of AAT-deficient patients treated with AAT therapy compared with untreated patients. In vitro, LPS-induced cytokines from WT and elastase-deficient mouse neutrophils, as well as neutrophils of healthy humans, were similarly reduced by AAT or rAAT; human neutrophils adhering to endothelial cells were decreased by 60-80% (P < 0.001) with either AAT or rAAT. In mouse pancreatic islet macrophages, LPS-induced surface expression of MHC II, Toll-like receptor-2 and -4 were markedly lower (80%, P < 0.001) when exposed to either AAT or rAAT. Consistently, in vivo and in vitro, rAAT reduced inflammatory responses at concentrations 40- to 100-fold lower than native plasma-derived AAT. These data provide evidence that the anti-inflammatory and immunomodulatory properties of AAT can be independent of elastase inhibition.
Assuntos
Anti-Inflamatórios/farmacologia , Fatores Imunológicos/farmacologia , Elastase de Leucócito/metabolismo , Enfisema Pulmonar/tratamento farmacológico , alfa 1-Antitripsina/farmacologia , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/imunologia , Adulto , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/deficiência , Lipopolissacarídeos/toxicidade , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Enfisema Pulmonar/genética , Enfisema Pulmonar/imunologia , Proteínas Recombinantes/farmacologia , Deficiência de alfa 1-Antitripsina/tratamento farmacológico , Deficiência de alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/imunologiaRESUMO
It is well known that activated microglia produce nitric oxide (NO), which has an important role in the pathophysiology of several neurodegenerative diseases such as Alzheimer's disease. In the course of searching for novel therapeutic agents from medicinal plants against neuroinflammatory diseases, the methanolic extract of Tetrapanax papyriferus was found to have significant NO inhibitory activity in lipopolysaccharide (LPS)-stimulated BV2 microglia cells. Nine oleanane-type triterpenes, including two new compounds, epipapyriogenin C-3-O-ß-d-glucopyranoside (6) and 11-O-butylpapyrioside LIIc (9), were isolated from the leaves and stems of Tetrapanax papyriferus. The structures of these compounds were elucidated with 1D- and 2D-NMR and MS data. Among these Δ(11,13) oleanane-type triterpenes, compound 3 showed significant NO inhibitory activity in BV-2 cells, reducing the LPS-induced expression of COX-2 and pro-inflammatory cytokines such as TNF-α and IL-6. Compounds 7 and 9 also showed NO inhibitory activities among the Δ(12) oleanane-type triterpene saponins. These results show that oleanane-type triterpenes isolated from T. papyriferus could be a potential natural resource of NO inhibitors used in the treatment of neurodegenerative disorders.
Assuntos
Anti-Inflamatórios/química , Óxido Nítrico/biossíntese , Ácido Oleanólico/química , Extratos Vegetais/química , Anti-Inflamatórios/administração & dosagem , Araliaceae/química , Ciclo-Oxigenase 2 , Citocinas/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Lipopolissacarídeos/toxicidade , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Doenças Neurodegenerativas/tratamento farmacológico , Óxido Nítrico/antagonistas & inibidores , Ácido Oleanólico/administração & dosagem , Ácido Oleanólico/isolamento & purificação , Extratos Vegetais/administração & dosagem , Folhas de Planta/química , Plantas Medicinais/químicaRESUMO
Interleukin-32 (IL-32) is a cytokine produced by T lymphocytes, natural killer (NK) cells, monocytes and epithelial cells. There are five splicing variants (α, ß, γ, δ, and ε) and IL-32γ is the most active isoform. We generated human IL-32γ transgenic (IL-32γ TG) mice, displaying a high level of IL-32γ expression in the pancreas. We investigated the effect of IL-32γ on streptozotocin (STZ)-induced type 1 diabetes model using IL-32γ TG mice. After a suboptimal diabetogenic dose of STZ administration, IL-32γ TG mice showed significantly increased blood glucose level comparing with that of wild type (WT) mice at day 5. Inflammatory cytokines levels such as, IL-6, TNFα, IFNγ and IL-1ß, in pancreas and liver lysates were accessed by a specific cytokine ELISA. The proinflammatory cytokines were significantly enhanced in the pancreas of IL-32γ TG mice comparing to that of WT mice whereas those cytokines levels in liver of IL-32γ TG and WT mice were not changed by STZ. These data indicate that the overexpression of IL-32γ contributes to initial islet ß-cells injury and inflammation in pancreas and aggravates STZ-induced type 1 diabetes.
Assuntos
Glicemia/análise , Diabetes Mellitus Experimental/patologia , Células Secretoras de Insulina/patologia , Interleucinas/biossíntese , Animais , Ensaio de Imunoadsorção Enzimática , Glucose/metabolismo , Humanos , Hiperglicemia/induzido quimicamente , Inflamação/imunologia , Inflamação/patologia , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/metabolismo , Interferon gama/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Interleucinas/genética , Fígado/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Isoformas de Proteínas/genética , Estreptozocina , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Atractylodes macrocephala Koidz (AMK) is a traditional herbal medicine used for thousands of years in East Asia to improve a variety of illnesses and conditions, including cancers. This study explored the effect of AMK extract on apoptosis and tumor-grafted mice using AGS human gastric adenocarcinoma cells. We investigated the compounds, target genes, and associated diseases of AMK using the Traditional Chinese Medical Systems Pharmacy (TCMSP) database platform. Cell viability assay, cell cycle and mitochondrial depolarization analysis, caspase activity assay, reactive oxygen species (ROS) assay, and wound healing and spheroid formation assay were used to investigate the anti-cancer effects of AMK extract on AGS cells. Also, in vivo studies were conducted using subcutaneous xenografts. AMK extract reduced the viability of AGS cells and increased the sub-G1 cell fraction and the mitochondrial membrane potential. Also, AMK extract increased the production of ROS. AMK extract induced the increased caspase activities and modulated the mitogen-activated protein kinases (MAPK). In addition, AMK extract effectively inhibited AGS cell migration and led to a notable reduction in the growth of AGS spheroids. Moreover, AMK extract hindered the growth of AGS xenograft tumors in NSG mice. Our results suggest that AMK has anti-cancer effects by promoting cell cycle arrest and inhibiting the proliferation of AGS cancer cells and a xenograft model through apoptosis. This study could provide a novel approach to treat gastric cancer.
Assuntos
Atractylodes , Neoplasias Gástricas , Humanos , Animais , Camundongos , Neoplasias Gástricas/tratamento farmacológico , Espécies Reativas de Oxigênio , Caspases , Extratos Vegetais/farmacologiaRESUMO
BACKGROUND: Colon cancer, a prominent contributor to global cancer-related deaths, prompts the need for innovative treatment strategies. Euphorbia resinifera O. Berg (E. resinifera) and Euphorbia officinarum subsp. echinus Hook. f. & Coss Vindt (E. echinus) and their bee-derived products have been integral to traditional Moroccan medicine due to their potential health benefits. These plants have historical use in addressing various health issues, including cancer. However, their effects against colon cancer remain unclear, and the specific mechanisms underlying their anti-cancer effects lack comprehensive investigation. METHODS: The study aimed to assess the potential anti-cancer effects of Euphorbia extract on colon cancer cell lines (DLD-1) through various techniques. The apoptosis, migration, and proliferation of DLD-1 cells were measured in DLD-1 cells. In addition, we conducted High-Performance Liquid Chromatography (HPLC) analysis to identify the profile of phenolic compounds present in the studied extracts. RESULTS: The extracts demonstrated inhibition of colon cancer cell migration. E. resinifera flower and E. echinus stem extracts show significant anti-migratory effects. Regarding anti-proliferative activity, E. resinifera flower extract hindered proliferation, whereas E. echinus flower extract exhibited dose-dependent inhibition. Apoptosis assays revealed E. resinifera flower extract inducing early-stage apoptosis and E. echinus flower extract promoting late-stage apoptosis. While apoptotic protein expression indicated, E. resinifera stem and propolis extracts had minimal impact on apoptosis. CONCLUSION: The findings provide evidence supporting the beneficial effects of E resinifera and E. echinus extracts on colon cancer and exerting anti-cancer properties.
Assuntos
Apoptose , Proliferação de Células , Neoplasias do Colo , Euphorbia , Extratos Vegetais , Euphorbia/química , Humanos , Neoplasias do Colo/tratamento farmacológico , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Linhagem Celular Tumoral , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Antineoplásicos Fitogênicos/farmacologia , MarrocosRESUMO
IL-1 family ligand does not possess a typical hydrophobic signal peptide and needs a processing enzyme for maturation. The maturation process of IL-33 (IL-1F11), a new member of the IL-1 family ligand, remains unclear. Precursor IL-33 ligand affinity column isolates neutrophil proteinase 3 (PR3) from human urinary proteins. PR3 is a known IL-1 family ligand-processing enzyme for IL-1ß (IL-1F2) and IL-18 (IL-1F4), including other inflammatory cytokines. We investigated PR3 in the maturation process of precursor IL-33 because we isolated urinary PR3 by using the precursor IL-33 ligand affinity column. PR3 converted inactive human and mouse precursor IL-33 proteins to biological active forms; however, the increase of PR3 incubation time abrogated IL-33 activities. Unlike caspase-1-cleaved precursor IL-18, PR3 cut precursor IL-33 and IL-18 at various sites and yielded multibands. The increased incubation period of PR3 abated mature IL-33 in a time-dependent manner. The result is consistent with the decreased bioactivity of IL-33 along with the increased PR3 incubation time. Six different human and mouse recombinant IL-33 proteins were expressed by the predicted consensus amino acid sequence of PR3 cleavage sites and tested for bioactivities. The human IL-33/p1 was highly active, but human IL-33/p2 and p3 proteins were inactive. Our results suggest the dual functions (activation/termination) of PR3 in IL-33 biological activity.
Assuntos
Interleucinas/metabolismo , Mieloblastina/metabolismo , Precursores de Proteínas/metabolismo , Animais , Caspase 1/genética , Caspase 1/metabolismo , Linhagem Celular , Humanos , Interleucina-18/genética , Interleucina-18/metabolismo , Interleucina-33 , Interleucinas/genética , Camundongos , Mieloblastina/genética , Precursores de Proteínas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
α1-Antitrypsin (AAT) is a member of the serine proteinase inhibitor family that impedes the enzymatic activity of serine proteinases, including human neutrophil elastase, cathepsin G and neutrophil proteinase 3. Here, we expressed recombinant AAT by fusing the intact AAT gene to the constant region of IgG1 to generate soluble recombinant AAT-Fc protein. The recombinant AAT-Fc protein was produced in Chinese hamster ovary (CHO) cells and purified using mini-protein A affinity chromatography. Recombinant AAT-Fc protein was tested for antiinflammatory function and AAT-Fc sufficiently suppressed tumor necrosis factor (TNF)-α-induced interleukin (IL)-6 in human peripheral blood mononuclear cells (PBMCs) and inhibited cytokine-induced TNFα by different cytokines in mouse macrophage Raw 264.7 cells. However, AAT-Fc failed to suppress lipopolysaccharide-induced cytokine production in both PBMCs and macrophages. In addition, our data showed that AAT-Fc blocks the development of hyperglycemia in a streptozotocin-induced mouse model of diabetes. Interestingly, we also found that plasma-derived AAT specifically inhibited the enzymatic activity of elastase but that AAT-Fc had no inhibitory effect on elastase activity.
Assuntos
Anti-Inflamatórios/farmacologia , Imunoglobulina G/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , alfa 1-Antitripsina/farmacologia , Animais , Anti-Inflamatórios/uso terapêutico , Glicemia/análise , Células CHO , Linhagem Celular , Células Cultivadas , Cricetinae , Cricetulus , Citocinas/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Feminino , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/farmacologia , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Imunoglobulina G/genética , Imunoglobulina G/uso terapêutico , Interleucina-6/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Elastase Pancreática/metabolismo , Proteínas Recombinantes de Fusão/uso terapêutico , Fator de Necrose Tumoral alfa/metabolismo , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/uso terapêuticoRESUMO
Inflammatory cytokines mediate inflammatory bowel diseases (IBDs) and cytokine blocking therapies often ameliorate the disease severity. IL-32 affects inflammation by increasing the production of IL-1, TNFα, and several chemokines. Here, we investigated the role of IL-32 in intestinal inflammation by generating a transgenic (TG) mouse expressing human IL-32γ (IL-32γ TG). Although IL-32γ TG mice are healthy, constitutive serum and colonic tissue levels of TNFα are elevated. Compared with wild-type (WT) mice, IL-32γ TG mice exhibited a modestly exacerbated acute inflammation early following the initiation of dextran sodium sulfate (DSS)-induced colitis. However, after 6 d, there was less colonic inflammation, reduced tissue loss, and improved survival rate compared with WT mice. Associated with attenuated tissue damage, colonic levels of TNFα and IL-6 were significantly reduced in the IL-32γ TG mice whereas IL-10 was elevated. Cultured colon explants from IL-32γ TG mice secreted higher levels of IL-10 compared with WT mice and lower levels of TNFα and IL-6. Constitutive levels of IL-32γ itself in colonic tissues were significantly lower following DSS colitis. Although the highest level of serum IL-32γ occurred on day 3 of colitis, IL-32 was below constitutive levels on day 9. The ability of IL-32γ to increase constitutive IL-10 likely reduces TNFα, IL-6, and IL-32 itself accounting for less inflammation. In humans with ulcerative colitis (UC), serum IL-32 is elevated and colonic biopsies contain IL-32 in inflamed tissues but not in uninvolved tissues. Thus IL-32γ emerges as an example of how innate inflammation worsens as well as protects intestinal integrity.
Assuntos
Colite/patologia , Inflamação/etiologia , Interleucinas/fisiologia , Animais , Colite/induzido quimicamente , Citocinas/sangue , Sulfato de Dextrana , Humanos , Interleucinas/sangue , Camundongos , Camundongos Transgênicos , Taxa de Sobrevida , Fatores de TempoRESUMO
IL-33/IL-1F11 is a new member of the IL-1 family ligand and provokes T helper-type immune responses. IL-33 is the ligand of ST2 and IL-1 receptor accessory protein (IL-1RAcP) that triggers nuclear factor-κ light chain enhancer of activated B cells (NF-κB) and MAPK signaling. We discovered a novel short splice variant of IL-33 that was termed spIL-33. The new spIL-33 lacks exon 3 containing a proposed caspase-1 cleavage site. We isolated spIL-33 cDNA from the Huh7 human hepatocarcinoma cell line and expressed the recombinant spIL-33 protein in Escherichia coli. The recombinant spIL-33 and pro-IL-33 were not cleaved by caspase-1, unlike IL-18 (IL-1F4). The recombinant spIL-33 was constitutively active, and spIL-33-induced inflammatory cytokine production was caspase-1-independent in HMC-1 and Raw 264.7 cells. The recombinant spIL-33 induced the phosphorylation of IL-1 receptor-associated kinase (IRAK1), NF-κB, p38 MAPK, p44/42 MAPK, and JNK in a time- and dose-dependent manner. Anti-ST2 monoclonal antibody specifically blocked the spIL-33-induced cytokine production. In this study, we identified and characterized a new IL-33 splice variant, which was a constitutively active IL-33 isoform. The existence of constitutively active spIL-33 suggests that the biological activity of IL-33 could be triggered by diverse stimulations during immune responses. Further investigation of the spIL-33 expression pattern may contribute to understanding the involvement of IL-33 in inflammatory disorders.
Assuntos
Processamento Alternativo/fisiologia , Interleucinas/biossíntese , Precursores de Proteínas/biossíntese , Animais , Sequência de Bases , Humanos , Inflamação/genética , Inflamação/metabolismo , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Interleucina-33 , Interleucinas/genética , Células Jurkat , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Fosforilação/fisiologia , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Precursores de Proteínas/genética , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Células U937RESUMO
OBJECTIVE: To evaluate the role of IL-32 in granulomatosis with polyangiitis (GPA) patients and the relationship between IL-32 and disease activity, as PR3 has the ability to bind and activate IL-32, which has been described as a novel cytokine that induces inflammatory cytokines. METHODS: We investigated the level of IL-32, PR3, TNF-α and IL-6 in GPA patients by using ELISA. Northern blot was used to analyse the level of IL-32 mRNA in leucocytes of GPA patients. The intracellular colocalization of IL-32 and PR3 in leucocytes was examined by IF staining. RESULTS: We observed that IL-32 and PR3 levels in GPA patients were increased significantly when compared with normal individuals and each was tightly associated (P < 0.001). Northern blot analysis revealed that the mRNA level of IL-32 was prominently elevated in leucocytes of GPA patients. The intracellular colocalization of IL-32 and PR3 in leucocytes from GPA patients vs normal individuals was verified by IF staining. CONCLUSION: IL-32 level was elevated in GPA patients but its level was changed by treatment response. IL-32 could be an index in GPA and play a role in the aetiology of GPA.
Assuntos
Granulomatose com Poliangiite/etiologia , Interleucinas/fisiologia , Adulto , Idoso , Northern Blotting , Ensaio de Imunoadsorção Enzimática , Feminino , Granulomatose com Poliangiite/metabolismo , Humanos , Interleucina-6/metabolismo , Interleucinas/metabolismo , Leucócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Mieloblastina/metabolismo , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Vasculite/etiologia , Vasculite/metabolismoRESUMO
Interferons (IFNs) are commonly grouped into type I and type II IFN. Type I IFNs are known as antiviral IFNs including IFN-α, IFN-ß, and IFN-ω whereas type II IFN is referred to immune IFN and IFN-γ is only member of the type II IFN. Type I IFNs are induced by virus invading however type II IFN is produced by mitogenic or antigenic stimuli. IFN-τ was first identified in ruminant ungulates as a pregnancy recognition hormone, trophoblastin. IFN-τ constitutes a new class of type I IFN, which possesses the common features of type I IFN, such as the ability to prevent viral infection and to limit cell proliferation. In addition, IFN-τ is unique in that it is induced by pregnancy unlike other type I IFNs. We cloned Bos taurus (B. T.) Coreanae IFN-τ from peripheral blood mononuclear cells. The amino acid sequence of B. T. Coreanae IFN-τ shares only 90.3% identity with that of Holstein dairy cow. Recombinant B. T. Coreanae and Holstein IFN-τ proteins were expressed in Escherichia coli and the antiviral activity of IFN-τ proteins were examined. Both recombinant proteins were active and protected human WISH and bovine MDBK cells from the cytopathic effect of vesicular stomatitis virus. The recombinant IFN-τ protein of B. T. Coreanae and Holstein properly induced the expression of antiviral genes including 2',5'-oligoadenylate synthetase (OAS) and Mx GTPase 1 (Mx-1).
Assuntos
Aminoácidos/química , Bovinos/metabolismo , Interferon Tipo I/química , Proteínas da Gravidez/química , 2',5'-Oligoadenilato Sintetase/metabolismo , Sequência de Aminoácidos , Animais , Antivirais/farmacologia , Sequência de Bases , Células Sanguíneas/efeitos dos fármacos , Células Sanguíneas/imunologia , Linhagem Celular , Clonagem Molecular , Efeito Citopatogênico Viral/efeitos dos fármacos , Cães , GTP Fosfo-Hidrolases/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Interferon Tipo I/farmacologia , Dados de Sequência Molecular , Proteínas da Gravidez/genética , Proteínas da Gravidez/metabolismo , Proteínas da Gravidez/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
Interleukin-32 (IL-32) is an inflammatory cytokine, and its activity is associated with various auto-inflammatory disorders as well as infectious pathogens such as Mycobacterium tuberculosis, and viral infections. However, the precise antiviral mechanism of IL-32 remains unclear. We assessed the IL-32 level in the sera of H1N1 influenza A patients and IL-32 level was significantly elevated. Next we examined the antiviral activity of recombinant IL-32γ (rIL-32γ) with WISH cells infected by vesicular stomatitis virus (VSV) but no antiviral activity was observed. Therefore we investigated the supernatant of rIL-32-treated THP-1 cells since this cell line effectively responded to rIL-32γ. The supernatant of rIL-32-treated THP-1 cell possessed an antiviral effect and in addition, an agonistic monoclonal antibody further enhanced a specific antiviral activity of rIL-32γ. The fractionation and mass spectrometer analysis of the THP-1 cell supernatant revealed that the antiviral activity of rIL-32γ is via a THP-1 cell-produced factor, transferrin, rather than the direct effects of rIL-32γ on epithelial cells. We also characterized a secreted soluble IL-32γ protein in serum of IL-32γ transgenic mouse (TG), but not in that of IL-32α TG. The present results suggest that IL-32γ expression and its genetic variation in individual could be an important aspect of viral infections.