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1.
Cell Prolif ; 54(3): e12997, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33511708

RESUMO

OBJECTIVES: Stromal cell-derived factor-1 (SDF-1) actively directs endogenous cell homing. Exendin-4 (EX-4) promotes stem cell osteogenic differentiation. Studies revealed that EX-4 strengthened SDF-1-mediated stem cell migration. However, the effects of SDF-1 and EX-4 on periodontal ligament stem cells (PDLSCs) and bone regeneration have not been investigated. In this study, we aimed to evaluate the effects of SDF-1/EX-4 cotherapy on PDLSCs in vitro and periodontal bone regeneration in vivo. METHODS: Cell-counting kit-8 (CCK8), transwell assay, qRT-PCR and western blot were used to determine the effects and mechanism of SDF-1/EX-4 cotherapy on PDLSCs in vitro. A rat periodontal bone defect model was developed to evaluate the effects of topical application of SDF-1 and systemic injection of EX-4 on endogenous cell recruitment, osteoclastogenesis and bone regeneration in vivo. RESULTS: SDF-1/EX-4 cotherapy had additive effects on PDLSC proliferation, migration, alkaline phosphatase (ALP) activity, mineral deposition and osteogenesis-related gene expression compared to SDF-1 or EX-4 in vitro. Pretreatment with ERK inhibitor U0126 blocked SDF-1/EX-4 cotherapy induced ERK signal activation and PDLSC proliferation. SDF-1/EX-4 cotherapy significantly promoted new bone formation, recruited more CXCR4+ cells and CD90+ /CD34- stromal cells to the defects, enhanced early-stage osteoclastogenesis and osteogenesis-related markers expression in regenerated bone compared to control, SDF-1 or EX-4 in vivo. CONCLUSIONS: SDF-1/EX-4 cotherapy synergistically regulated PDLSC activities, promoted periodontal bone formation, thereby providing a new strategy for periodontal bone regeneration.


Assuntos
Regeneração Óssea/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Quimiocina CXCL12/metabolismo , Exenatida/farmacologia , Ligamento Periodontal/citologia , Células Estromais/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Exenatida/metabolismo , Humanos , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células Estromais/metabolismo
2.
Life Sci ; 269: 119001, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33421527

RESUMO

AIMS: Osteoarthritis (OA) is a common joint disease and the main cause of disability. We sought to determine the effective concentration of emodin on chondrocytes and to identify the dosage of emodin that induces a comparable therapeutic effect with the COX-2 inhibitor drug, celecoxib that is currently used to treat OA. MATERIAL AND METHODS: In vitro experiments induced inflammation of chondrocytes by IL-1ß, and an osteoarthritis model was established in vivo by cutting rat anterior cruciate ligament. Western Blot, Real-time PCR, HE staining, Safranin O-green staining and immunohistochemistry were performed to detect MMP-3, MMP-13, ADAMTS-4, iNOS and COL2A1 on the chondrocytes or the tibial plateau. The cytokine activity and content in serum of six groups of rats were measured by kit. RESULTS: It was found that the surface layer of the cartilage was thicker and smoother after the administration of emodin. Tissue expression of MMP-3, MMP-13, ADAMTS-4 and iNOS were significantly (p < 0.05) decreased in chondrocytes and cartilage treated with different doses of emodin, and the content of COL2A1 was reversed. Emodin also significantly decreased the blood levels of COX-2 and PGE2. The effective emodin in vitro was 5 µmol/L, whereas emodin at 80 mg/kg was equivalent to celecoxib in vivo. CONCLUSION: Emodin reduces the expression of cartilage matrix degradation biomarkers, thereby reducing the degradation of cartilage matrix and protecting the knee joint cartilage. Emodin at 5 µmol/L shows the best concentration to treat chondrocytes, and the protective effect of emodin at 80 mg/kg is comparable to that of celecoxib.


Assuntos
Cartilagem Articular/patologia , Emodina/farmacologia , Matriz Extracelular/metabolismo , Articulação do Joelho/patologia , Substâncias Protetoras/farmacologia , Proteína ADAMTS4/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Condrócitos/patologia , Ciclo-Oxigenase 2/sangue , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Emodina/administração & dosagem , Matriz Extracelular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Metaloproteinases da Matriz/metabolismo , Óxido Nítrico/sangue , Óxido Nítrico Sintase Tipo II/sangue , Óxido Nítrico Sintase Tipo II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Células Estromais/patologia
3.
Ecotoxicol Environ Saf ; 207: 111511, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33254391

RESUMO

Decidualization, which endows the endometrium competency to adopt developing embryo and maintain appropriate milieu for following growth, is a pivotal process for human pregnancy. The delicate collaboration between ovarian steroid hormones estrogen and progesterone governs the process of decidualization and subsequent establishment of embryo implantation. Mycotoxin zearalenone (ZEA) is well known as endocrine disruptor due to its potent estrogenic activity. In this study, we investigated effects of ZEA on decidualization of human endometrial stromal cells. Results indicated that ZEA exhibited its inhibitory action through nuclear translocation of ERα. ZEA exposure led to dampened progress of decidualization, which could be attenuated by estrogen receptor antagonist. Notably, resveratrol (RSV) administration restored impaired decidualization process by induction of anti-oxidative gene glutathione peroxidase 3 (GPX3). This study provides novel insights into the mechanism underlying adverse effects of ZEA in human decidual stromal cells and suggests RSV a potential therapeutic candidate to alleviate ZEA-induced cytotoxicity during decidualization.


Assuntos
Disruptores Endócrinos/toxicidade , Estrogênios não Esteroides/toxicidade , Substâncias Protetoras/farmacologia , Resveratrol/farmacologia , Zearalenona/toxicidade , Células Cultivadas , Decídua/efeitos dos fármacos , Implantação do Embrião/efeitos dos fármacos , Endométrio/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Receptor alfa de Estrogênio , Estrogênios/farmacologia , Feminino , Humanos , Gravidez , Progesterona/farmacologia , Células Estromais/efeitos dos fármacos
4.
Life Sci ; 261: 118371, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32882267

RESUMO

AIMS: Metformin is a clinical drug administered to patients to treat type 2 diabetes mellitus that was found to be associated with a lower risk of occurrence of cancer and cancer-related death. The present study investigated the effects of metformin on human adipose-derived stromal cells (ADSC) - breast cancer cell line interactions. MAIN METHODS: ADSCs grown from lipoaspirates were tested for growth-stimulating and migration-controlling activity on breast cancer cell lines after pretreatment with metformin. Furthermore, secreted proteins of ADSCs, phosphorylation of intracellular proteins and the effect of metformin on adipocytic differentiation of ADSCs were assayed. KEY FINDINGS: Compared to breast cancer cell lines (4.0 ± 3.5% reduction of proliferation), 2 mM metformin significantly inhibited the proliferation of ADSC lines (19.2 ± 8.4% reduction of proliferation). This effect on ADSCs seems to be mediated by altered phosphorylation of GSK-3, CREB and PRAS40. Furthermore, treatment with metformin abolished the induction of differentiation of three ADSC lines to adipocytes. 1 and 2 mM metformin significantly impaired the migration of breast cancer cell lines MDA-MB-231 and MDA-MB-436 in scratch assays. SIGNIFICANCE: Metformin showed low direct inhibitory effects on breast cancer cell lines at physiological concentrations but exerted a significant retardation of the growth and the adipocytic differentiation of ADSCs. Thus, the anticancer activity of metformin in breast cancer at physiological drug concentrations seems to be mediated by an indirect mechanism that lowers the supportive activity of ADSCs.


Assuntos
Tecido Adiposo/patologia , Neoplasias da Mama/patologia , Metformina/farmacologia , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Proteínas de Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Células Estromais/patologia
5.
PLoS One ; 15(8): e0236839, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32780746

RESUMO

The majority of chronic myeloid leukemia (CML) cases are caused by a chromosomal translocation linking the breakpoint cluster region (BCR) gene to the Abelson murine leukemia viral oncogene-1 (ABL1), creating the mutant fusion protein BCR-ABL1. Downstream of BCR-ABL1 is growth factor receptor-bound protein-2 (GRB2), an intracellular adapter protein that binds to BCR-ABL1 via its src-homology-2 (SH2) domain. This binding constitutively activates growth pathways, downregulates apoptosis, and leads to an over proliferation of immature and dysfunctional myeloid cells. Utilizing novel synthetic methods, we developed four furo-quinoxaline compounds as GRB2 SH2 domain antagonists with the goal of disrupting this leukemogenic signaling. One of the four antagonists, NHD2-15, showed a significant reduction in proliferation of K562 cells, a human BCR-ABL1+ leukemic cell line. To elucidate the mode of action of these compounds, various biophysical, in vitro, and in vivo assays were performed. Surface plasmon resonance (SPR) assays indicated that NHD2-15 antagonized GRB2, binding with a KD value of 119 ± 2 µM. Cellulose nitrate (CN) assays indicated that the compound selectively bound the SH2 domain of GRB2. Western blot assays suggested the antagonist downregulated proteins involved in leukemic transformation. Finally, NHD2-15 was nontoxic to primary cells and adult zebrafish, indicating that it may be an effective clinical treatment for CML.


Assuntos
Proliferação de Células/efeitos dos fármacos , Proteína Adaptadora GRB2/antagonistas & inibidores , Quinoxalinas/farmacologia , Animais , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/metabolismo , Proteína Adaptadora GRB2/química , Proteína Adaptadora GRB2/metabolismo , Humanos , Células K562 , Rim/citologia , Cinética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Ligação Proteica , Quinoxalinas/química , Quinoxalinas/metabolismo , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Ressonância de Plasmônio de Superfície , Peixe-Zebra , Domínios de Homologia de src
6.
J Virol ; 94(18)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32641474

RESUMO

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that can cause severe clinical disease in allograft recipients and infants infected in utero Virus-neutralizing antibodies defined in vitro have been proposed to confer protection against HCMV infection, and the virion envelope glycoprotein B (gB) serves as a major target of neutralizing antibodies. The viral fusion protein gB is nonfusogenic on its own and requires glycoproteins H (gH) and L (gL) for membrane fusion, which is in contrast to requirements of related class III fusion proteins, including vesicular stomatitis virus glycoprotein G (VSV-G) or baculovirus gp64. To explore requirements for gB's fusion activity, we generated a set of chimeras composed of gB and VSV-G or gp64, respectively. These gB chimeras were intrinsically fusion active and led to the formation of multinucleated cell syncytia when expressed in the absence of other viral proteins. Utilizing a panel of virus-neutralizing gB-specific monoclonal antibodies (MAbs), we could demonstrate that syncytium formation of the fusogenic gB/VSV-G chimera can be significantly inhibited by only a subset of neutralizing MAbs which target antigenic domain 5 (AD-5) of gB. This observation argues for differential modes of action of neutralizing anti-gB MAbs and suggests that blocking the membrane fusion function of gB could be one mechanism of antibody-mediated virus neutralization. In addition, our data have important implications for the further understanding of the conformation of gB that promotes membrane fusion as well as the identification of structures in AD-5 that could be targeted by antibodies to block this early step in HCMV infection.IMPORTANCE HCMV is a major global health concern, and antiviral chemotherapy remains problematic due to toxicity of available compounds and the emergence of drug-resistant viruses. Thus, an HCMV vaccine represents a priority for both governmental and pharmaceutical research programs. A major obstacle for the development of a vaccine is a lack of knowledge of the nature and specificities of protective immune responses that should be induced by such a vaccine. Glycoprotein B of HCMV is an important target for neutralizing antibodies and, hence, is often included as a component of intervention strategies. By generation of fusion-active gB chimeras, we were able to identify target structures of neutralizing antibodies that potently block gB-induced membrane fusion. This experimental system provides an approach to screen for antibodies that interfere with gB's fusogenic activity. In summary, our data will likely contribute to both rational vaccine design and the development of antibody-based therapies against HCMV.


Assuntos
Anticorpos Neutralizantes/farmacologia , Citomegalovirus/genética , Proteínas Mutantes Quiméricas/genética , Proteínas do Envelope Viral/genética , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Anticorpos Antivirais/farmacologia , Sítios de Ligação , Fusão Celular , Linhagem Celular , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/virologia , Expressão Gênica , Células Gigantes/efeitos dos fármacos , Células Gigantes/metabolismo , Células Gigantes/ultraestrutura , Células Gigantes/virologia , Células HEK293 , Humanos , Camundongos , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/metabolismo , Cultura Primária de Células , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Células Estromais/virologia , Vesiculovirus/genética , Vesiculovirus/metabolismo , Proteínas do Envelope Viral/metabolismo
7.
Leukemia ; 34(11): 2981-2991, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32409689

RESUMO

FLT3-ITD mutations occur in 20-30% of AML patients and are associated with aggressive disease. Patients with relapsed FLT3-mutated disease respond well to 2nd generation FLT3 TKIs but inevitably relapse within a short timeframe. In this setting, until overt relapse occurs, the bone marrow microenvironment facilitates leukemia cell survival despite continued on-target inhibition. We demonstrate that human bone marrow derived conditioned medium (CM) protects FLT3-ITD+ AML cells from the 2nd generation FLT3 TKI quizartinib and activates STAT3 and STAT5 in leukemia cells. Extrinsic activation of STAT5 by CM is the primary mediator of leukemia cell resistance to FLT3 inhibition. Combination treatment with quizartinib and dasatinib abolishes STAT5 activation and significantly reduces the IC50 of quizartinib in FLT3-ITD+ AML cells cultured in CM. We demonstrate that CM protects FLT3-ITD+ AML cells from the inhibitory effects of quizartinib on glycolysis and that this is partially reversed by treating cells with the combination of quizartinib and dasatinib. Using a doxycycline-inducible STAT5 knockdown in the FLT3-ITD+ MOLM-13 cell line, we show that dasatinib-mediated suppression of leukemia cell glycolytic activity is STAT5-independent and provide a preclinical rationale for combination treatment with quizartinib and dasatinib in FLT3-ITD+ AML.


Assuntos
Benzotiazóis/farmacologia , Dasatinibe/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos/genética , Metabolismo Energético , Duplicação Gênica , Técnicas de Silenciamento de Genes , Glicólise , Humanos , Fosforilação , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Tirosina Quinase 3 Semelhante a fms/genética
8.
Breast Cancer Res ; 22(1): 41, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32370801

RESUMO

BACKGROUND: In utero endocrine disruption is linked to increased risk of breast cancer later in life. Despite numerous studies establishing this linkage, the long-term molecular changes that predispose mammary cells to carcinogenic transformation are unknown. Herein, we investigated how endocrine disrupting compounds (EDCs) drive changes within the stroma that can contribute to breast cancer susceptibility. METHODS: We utilized bisphenol A (BPA) as a model of estrogenic endocrine disruption to analyze the long-term consequences in the stroma. Deregulated genes were identified by RNA-seq transcriptional profiling of adult primary fibroblasts, isolated from female mice exposed to in utero BPA. Collagen staining, collagen imaging techniques, and permeability assays were used to characterize changes to the extracellular matrix. Finally, gland stiffness tests were performed on exposed and control mammary glands. RESULTS: We identified significant transcriptional deregulation of adult fibroblasts exposed to in utero BPA. Deregulated genes were associated with cancer pathways and specifically extracellular matrix composition. Multiple collagen genes were more highly expressed in the BPA-exposed fibroblasts resulting in increased collagen deposition in the adult mammary gland. This transcriptional reprogramming of BPA-exposed fibroblasts generates a less permeable extracellular matrix and a stiffer mammary gland. These phenotypes were only observed in adult 12-week-old, but not 4-week-old, mice. Additionally, diethylstilbestrol, known to increase breast cancer risk in humans, also increases gland stiffness similar to BPA, while bisphenol S does not. CONCLUSIONS: As breast stiffness, extracellular matrix density, and collagen deposition have been directly linked to breast cancer risk, these data mechanistically connect EDC exposures to molecular alterations associated with increased disease susceptibility. These alterations develop over time and thus contribute to cancer risk in adulthood.


Assuntos
Disruptores Endócrinos/toxicidade , Matriz Extracelular/patologia , Glândulas Mamárias Animais/patologia , Efeitos Tardios da Exposição Pré-Natal/patologia , Células Estromais/patologia , Animais , Compostos Benzidrílicos/toxicidade , Estrogênios não Esteroides/farmacologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/imunologia , Feminino , Fibroblastos/imunologia , Fibroblastos/patologia , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/imunologia , Glândulas Mamárias Animais/metabolismo , Camundongos , Fenóis/toxicidade , Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Células Estromais/efeitos dos fármacos , Células Estromais/imunologia , Transcriptoma
9.
Nat Commun ; 11(1): 2054, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32345968

RESUMO

Classical dendritic cells (cDCs) are rare sentinel cells specialized in the regulation of adaptive immunity. Modeling cDC development is crucial to study cDCs and harness their therapeutic potential. Here we address whether cDCs could differentiate in response to trophic cues delivered by mesenchymal components of the hematopoietic niche. We find that mesenchymal stromal cells engineered to express membrane-bound FLT3L and stem cell factor (SCF) together with CXCL12 induce the specification of human cDCs from CD34+ hematopoietic stem and progenitor cells (HSPCs). Engraftment of engineered mesenchymal stromal cells (eMSCs) together with CD34+ HSPCs creates an in vivo synthetic niche in the dermis of immunodeficient mice driving the differentiation of cDCs and CD123+AXL+CD327+ pre/AS-DCs. cDC2s generated in vivo display higher levels of resemblance with human blood cDCs unattained by in vitro-generated subsets. Altogether, eMSCs provide a unique platform recapitulating the full spectrum of cDC subsets enabling their functional characterization in vivo.


Assuntos
Células Dendríticas/citologia , Nicho de Células-Tronco , Animais , Biomarcadores/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Quimiocina CXCL12/farmacologia , Análise por Conglomerados , Colágeno/farmacologia , Células Dendríticas/efeitos dos fármacos , Combinação de Medicamentos , Humanos , Laminina/farmacologia , Proteínas de Membrana/metabolismo , Camundongos , Organoides/efeitos dos fármacos , Organoides/metabolismo , Proteoglicanas/farmacologia , Nicho de Células-Tronco/efeitos dos fármacos , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo
10.
Nat Commun ; 11(1): 1435, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32188843

RESUMO

Regeneration of corneal stroma has always been a challenge due to its sophisticated structure and keratocyte-fibroblast transformation. In this study, we fabricate grid poly (ε-caprolactone)-poly (ethylene glycol) microfibrous scaffold and infuse the scaffold with gelatin methacrylate (GelMA) hydrogel to obtain a 3 D fiber hydrogel construct; the fiber spacing is adjusted to fabricate optimal construct that simulates the stromal structure with properties most similar to the native cornea. The topological structure (3 D fiber hydrogel, 3 D GelMA hydrogel, and 2 D culture dish) and chemical factors (serum, ascorbic acid, insulin, and ß-FGF) are examined to study their effects on the differentiation of limbal stromal stem cells to keratocytes or fibroblasts and the phenotype maintenance, in vitro and in vivo tissue regeneration. The results demonstrate that fiber hydrogel and serum-free media synergize to provide an optimal environment for the maintenance of keratocyte phenotype and the regeneration of damaged corneal stroma.


Assuntos
Substância Própria/fisiologia , Gelatina/farmacologia , Hidrogéis/farmacologia , Metacrilatos/farmacologia , Poliésteres/farmacologia , Polietilenoglicóis/farmacologia , Regeneração , Animais , Substância Própria/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Limbo da Córnea/citologia , Masculino , Ratos Sprague-Dawley , Regeneração/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Estresse Mecânico , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Suínos , Tecidos Suporte/química , Vimentina/metabolismo
11.
PLoS One ; 15(3): e0219275, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32163417

RESUMO

Pathogenic bacteria often damage tissues by secreting toxins that form pores in cell membranes, and the most common pore-forming toxins are cholesterol-dependent cytolysins. During bacterial infections, glutamine becomes a conditionally essential amino acid, and glutamine is an important nutrient for immune cells. However, the role of glutamine in protecting tissue cells against pore-forming toxins is unclear. Here we tested the hypothesis that glutamine supports the protection of tissue cells against the damage caused by cholesterol-dependent cytolysins. Stromal and epithelial cells were sensitive to damage by the cholesterol-dependent cytolysins, pyolysin and streptolysin O, as determined by leakage of potassium and lactate dehydrogenase from cells, and reduced cell viability. However, glutamine deprivation increased the leakage of lactate dehydrogenase and reduced the viability of cells challenged with cholesterol-dependent cytolysins. Without glutamine, stromal cells challenged with pyolysin leaked lactate dehydrogenase (control vs. pyolysin, 2.6 ± 0.6 vs. 34.4 ± 4.5 AU, n = 12), which was more than three-fold the leakage from cells supplied with 2 mM glutamine (control vs. pyolysin, 2.2 ± 0.3 vs. 9.4 ± 1.0 AU). Glutamine cytoprotection did not depend on glutaminolysis, replenishing the Krebs cycle via succinate, changes in cellular cholesterol, or regulators of cell metabolism (AMPK and mTOR). In conclusion, although the mechanism remains elusive, we found that glutamine supports the protection of tissue cells against the damage caused by cholesterol-dependent cytolysins from pathogenic bacteria.


Assuntos
Colesterol/metabolismo , Citoproteção/efeitos dos fármacos , Citotoxinas/toxicidade , Glutamina/farmacologia , Animais , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Bovinos , Células HeLa , Proteínas Hemolisinas/toxicidade , Humanos , L-Lactato Desidrogenase/metabolismo , Estreptolisinas/toxicidade , Células Estromais/efeitos dos fármacos
12.
Proc Natl Acad Sci U S A ; 117(10): 5532-5541, 2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32079724

RESUMO

The role of stromal fibroblasts in chronic inflammation is unfolding. In rheumatoid arthritis, leukocyte-derived cytokines TNF and IL-17A work together, activating fibroblasts to become a dominant source of the hallmark cytokine IL-6. However, IL-17A alone has minimal effect on fibroblasts. To identify key mediators of the synergistic response to TNF and IL-17A in human synovial fibroblasts, we performed time series, dose-response, and gene-silencing transcriptomics experiments. Here we show that in combination with TNF, IL-17A selectively induces a specific set of genes mediated by factors including cut-like homeobox 1 (CUX1) and IκBζ (NFKBIZ). In the promoters of CXCL1, CXCL2, and CXCL3, we found a putative CUX1-NF-κB binding motif not found elsewhere in the genome. CUX1 and NF-κB p65 mediate transcription of these genes independent of LIFR, STAT3, STAT4, and ELF3. Transcription of NFKBIZ, encoding the atypical IκB factor IκBζ, is IL-17A dose-dependent, and IκBζ only mediates the transcriptional response to TNF and IL-17A, but not to TNF alone. In fibroblasts, IL-17A response depends on CUX1 and IκBζ to engage the NF-κB complex to produce chemoattractants for neutrophil and monocyte recruitment.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Artrite Reumatoide/metabolismo , Fibroblastos/metabolismo , Proteínas de Homeodomínio/metabolismo , Inflamação/metabolismo , Interleucina-17/fisiologia , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Artrite Reumatoide/genética , Células Cultivadas , Quimiocina CXCL1/genética , Quimiocina CXCL2/genética , Quimiocinas CXC/genética , Fatores Quimiotáticos/genética , Fibroblastos/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Humanos , Inflamação/genética , Interleucina-17/farmacologia , Interleucina-6/genética , Metaloproteinase 3 da Matriz/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/fisiologia , RNA Interferente Pequeno/genética , Proteínas Repressoras/genética , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Líquido Sinovial , Fator de Transcrição RelA/metabolismo , Fatores de Transcrição/genética , Transcriptoma/efeitos da radiação , Fator de Necrose Tumoral alfa/farmacologia
13.
J Steroid Biochem Mol Biol ; 200: 105640, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32087250

RESUMO

Bisphenol A(BPA) is one of the most widespread endocrine disruptors in the environment and is associated with reproductive diseases. In this study, we focused on the correlation between environmentally relevant levels of BPA exposure and histone modification during endometrial stromal cells decidualization. BPA exposure changed the morphology of decidualized endometrial stromal cells, with inhibition of mixed-lineage leukemia 1(MLL1) and induction of enhancer of zeste homolog2 (EZH2) during in vitro decidualization. The expression of HOXA10, PRL and IGFBP-1 was down-regulated upon BPA treatment. Furthermore, chromatin immunoprecipitation quantitative PCR(ChIP-qPCR) was performed to evaluate the recruitment of histone-3, lysine-4 trimethylation (H3K4me3) and histone-3, lysine-27 trimethylation (H3K27me3) at the gene promoters. The decreased H3K4me3 and the increased H3K27me3 at HOXA10, PRL and IGFBP-1 promoter regions were consistent with the expression of MLL1 and EZH2 respectively. The effect of BPA on MLL1 and EZH2 could be abrogated by ICI 182,780. Our study provides the first indication that environmentally relevant levels of BPA exposure can regulate the expression of decidualization-related genes by affecting histone modification, impairing endometrial decidualization.


Assuntos
Compostos Benzidrílicos/farmacologia , Disruptores Endócrinos/farmacologia , Fenóis/farmacologia , Células Estromais/efeitos dos fármacos , Adulto , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Endométrio/citologia , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Epigênese Genética , Feminino , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Pessoa de Meia-Idade , Proteína de Leucina Linfoide-Mieloide/metabolismo , Regiões Promotoras Genéticas , Células Estromais/metabolismo , Células Estromais/fisiologia
14.
Int J Med Sci ; 17(2): 224-233, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32038106

RESUMO

Background: Adenomyosis is a quite common gynecological disorder and above 30% of patients have typical secondary and progressive dysmenorrhea. Current treatments still have many disadvantages and thereby the novel treatment aiming to relieve dysmenorrhea still needs to be further investigated. Mifepristone is a wonderful drug because it is effective, safe and cheap in many diseases including adenomyosis. In this study, we aim to investigate if mifepristone could be used in the treatment of adenomyosis-associated dysmenorrhea. Methods: Human primary endometrial epithelial and stromal cells from adenomyosis patients were isolated and treated with mifepristone. RNA-sequencing was then performed to detect the gene changes of pain-related inflammatory mediators. Meanwhile, the effect of mifepristone on the infiltration and degranulation of mast cells were investigated in adenomyosis lesions. Additionally, the role of mifepristone on the density of nerve fibers was also studied in the ectopic endometrium. At last, to evaluate the therapeutic efficacy of mifepristone on dysmenorrhea of adenomyosis, twenty participants were included and the visual analog scale (VAS) score was assessed and compared before and after treatment with mifepristone. Results: We demonstrated that mifepristone reduced the secretion of IL-6 and TNF-α from endometrial epithelial and stromal cells, restricted the infiltration and degranulation of mast cells in eutopic and ectopic endometrium and decreased the density of nerve fibers by inhibiting the migration capacity of nerve cells in adenomyosis. Meanwhile, we found that mifepristone could significantly relieve dysmenorrhea of adenomyosis. Conclusion: The findings demonstrated that mifepristone could be applied in the treatment of dysmenorrhea for the adenomyosis patients.


Assuntos
Adenomiose/complicações , Dismenorreia/tratamento farmacológico , Dismenorreia/etiologia , Mifepristona/uso terapêutico , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Inflamação/metabolismo , Interleucina-6/metabolismo , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
15.
Eur J Pharmacol ; 873: 172985, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32017934

RESUMO

Prostate smooth muscle contraction and prostate enlargement contribute to lower urinary tract symptoms suggestive of benign prostatic hyperplasia. Recent evidence demonstrated that inhibitors for polo-like kinases (PLKs) inhibit smooth muscle contraction of human prostate tissues. However, their additive effects to α1-blockers, and effects on prostate growth are unknown. Here, we examined effects of a novel and highly selective PLK1 inhibitor, onvansertib on prostate smooth muscle contraction alone and in combination with α1-blockers, and on proliferation and viability of prostate stromal cells (WPMY-1). Prostate tissues were obtained from radical prostatectomy. Contractions were studied in an organ bath. Proliferation and viability were assessed by plate colony, EdU, and CCK-8 assay. Electric field stimulation (EFS)-induced contractions of human prostate tissues were inhibited to 34% by 100 nM and 1 µM onvansertib at 32 Hz, and to 48% and 47% by the α1-blockers tamsulosin and silodosin. Combination of onvansertib with tamsulosin or silodosin further reduced EFS-induced contractions in comparison to α1-blockers alone (59% and 61% respectively), and to onvansertib alone (68% for both). Noradrenaline-, phenylephrine-, methoxamine-, endothelin-1-, and ATP-induced contractions were inhibited by onvansertib (100 nM) to similar extent. Viability and proliferation of WPMY-1 cells were reduced in a concentration- and time-dependent manner (24-72 h, 10-100 nM). Onvansertib inhibits neurogenic, adrenergic, and endothelin-1- and ATP-induced contractions of human prostate smooth muscle, and proliferation of stromal cells. Contractions are reduced not more than 50% by α1-blockers. Combination of α1-blockers with onvansertib provides additive inhibition of prostate contractions.


Assuntos
Antagonistas de Receptores Adrenérgicos alfa 2/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Músculo Liso/efeitos dos fármacos , Piperazinas/farmacologia , Próstata/citologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Pirazóis/farmacologia , Quinazolinas/farmacologia , Células Estromais/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Sinergismo Farmacológico , Humanos , Técnicas In Vitro , Masculino , Contração Muscular/efeitos dos fármacos , Próstata/efeitos dos fármacos , Ensaio Tumoral de Célula-Tronco
16.
Nutrients ; 12(2)2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32019160

RESUMO

Vitamin D status has been implicated in obesity and adipose tissue inflammation. In the present study, we explored the effects of dietary vitamin D supplementation on adipose tissue inflammation and immune cell population, and the effects of in vitro 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) treatment on pro-inflammatory cytokine production by stromal vascular cells (SVCs) and adipocytes in lean and high-fat diet-induced obese mice. The results show that epididymal fat Mcp-1 and Rantes mRNA levels, which were higher in obese mice compared with lean mice, were significantly down-regulated by vitamin D supplementation. While obese mice had higher numbers of macrophages and natural killer (NK) cells within adipose tissue, these remained unaltered by vitamin D supplementation. In accordance with these in vivo findings, the in vitro 1,25(OH)2D3 treatment decreased IL-6, MCP-1, and IL-1ß production by SVCs from obese mice, but not by adipocytes. In addition, 1,25(OH)2D3 treatment significantly decreased Tlr2 expression and increased mRNA levels of Iκba and Dusp1 in SVCs. These findings suggest that vitamin D supplementation attenuates inflammatory response in adipose tissue, especially in SVCs, possibly through inhibiting NF-κB and MAPK signaling pathways in SVCs but not by the inhibition of macrophage infiltration.


Assuntos
Adipócitos/efeitos dos fármacos , Calcitriol/farmacologia , Obesidade/imunologia , Células Estromais/efeitos dos fármacos , Vitaminas/farmacologia , Adipócitos/imunologia , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/imunologia , Animais , Citocinas/efeitos dos fármacos , Citocinas/imunologia , Dieta Hiperlipídica , Modelos Animais de Doenças , Inflamação , Camundongos , Camundongos Obesos , Obesidade/terapia , Células Estromais/imunologia
17.
Int J Mol Sci ; 21(2)2020 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-31936485

RESUMO

Understanding of adipogenesis is important to find remedies for obesity and related disorders. In addition, it is also critical in bone disorders because there is a reciprocal relationship between adipogenesis and osteogenesis in bone micro-environment. Oxysterols are pro-osteogenic and anti-adipogenic molecules via hedgehog activation in pluripotent bone marrow stomal cells. However, no study has evaluated the role of specific oxysterols in C3H10T1/2 cells, which are a good cell model for studying osteogenesis and adipogenesis in bone-marrows. Thus, we investigated the effects of specific oxysterols on adipogenesis and expression of adipogenic transcripts in C3H10T1/2 cells. Treatment of cells with DMITro significantly induced mRNA expression of Pparγ. This induction was significantly inhibited by 25-HC. The expression of C/cepα, Fabp4 and Lpl was also inhibited by 25-HC. To determine the mechanism by which 25-HC inhibits adipogenesis, the effects of the hedgehog signalling pathway inhibitor, cyclopamine and CUR61414, were evaluated. Treatment of C3H10T1/2 cells with DMITro + cyclopamine or DMITro + CUR61414 for 96h did not modulate adipocyte differentiation; cyclopamine and CUR61414 did not reverse the inhibitory effects of 25-HC, suggesting that the canonical hedgehog signalling may not play a role in the anti-adipogenic effects of 25-HC in C3H10T1/2 cells. In addition, LXR agonist did not inhibit adipogenesis, but 25-HC strongly inhibits adipogenesis of C3H10T1/2 cells. Our observations showed that 25-HC was the most potent oxysterol in inhibiting adipogenesis and the expression of key adipogenic transcripts in C3H10T1/2 cells among the tested oxysterols, suggesting its potential application in providing an intervention in osteoporosis and obesity. We also report that the inhibitory effects of 25-HC on adipogenic differentiation in C3H10T1/2 cells are not mediated by hedgehog signaling and LXR.


Assuntos
Adipogenia/efeitos dos fármacos , Hidroxicolesteróis/farmacologia , Células-Tronco Pluripotentes/citologia , Adipogenia/genética , Animais , Benzoatos/farmacologia , Benzilaminas/farmacologia , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Receptores X do Fígado/agonistas , Receptores X do Fígado/metabolismo , Camundongos , Oxisteróis/farmacologia , Células-Tronco Pluripotentes/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Fatores de Tempo
18.
J Biol Chem ; 295(8): 2248-2258, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-31937587

RESUMO

The regulation mechanisms involved in matrix metalloproteinase (MMP) expression and the motility of human endometrial and decidual stromal cells (ESCs and DSCs, respectively) during decidualization remain unclear. DSCs show significant increased cell motility and expression of FOS-like 1 (FOSL1) and MMP1, MMP2, and MMP9 compared with ESCs, whereas lack of decidualization inducers leads to a rapid decrease in FOSL1 and MMP1 and MMP9 expression in DSCs in vitro Therefore, we hypothesized that a link exists between decidualization inducers and FOSL1 in up-regulation of motility during decidualization. Based on the response of ESCs/DSCs to different decidualization systems in vitro, we found that progesterone (P4) alone had no significant effect and that 17ß-estradiol (E2) significantly increased cell motility and FOSL1 and MMP1 and MMP9 expression at the mRNA and protein levels, whereas 8-bromo-cAMP significantly decreased cell motility and FOSL1 and MMP9 expression in the presence of P4. In addition, we showed that E2 triggered phosphorylation of estrogen receptor 1 (ESR1), which could directly bind to the promoter of FOSL1 in ESCs/DSCs. Additionally, we also revealed silencing of ESR1 expression by siRNA abrogated E2-induced FOSL1 expression at the transcript and protein levels. Moreover, silencing of FOSL1 expression by siRNA was able to block E2-induced MMP1 and MMP9 expression and cell motility in ESCs/DSCs. Taken together, our data suggest that, in addition to its enhancement of secretory function, the change in MMP expression and cell motility is another component of the decidualization of ESCs/DSCs, including estrogen-dependent MMP1 and MMP9 expression mediated by E2-ESR1-FOSL1 signaling.


Assuntos
Movimento Celular , Decídua/citologia , Endométrio/citologia , Estrogênios/farmacologia , Metaloproteinases da Matriz/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Adulto , Movimento Celular/efeitos dos fármacos , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Pessoa de Meia-Idade , Prolactina/metabolismo , Células Estromais/citologia , Células Estromais/efeitos dos fármacos , Células Estromais/enzimologia , Adulto Jovem
19.
Med Sci Monit ; 26: e920670, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31929497

RESUMO

BACKGROUND Intrauterine adhesion (IUA) is a common reproductive system disease in women, characterized by endometrial stromal cell proliferation, increasing fibroblasts and increasing extracellular matrix secretion. The purpose of this study was to investigate the effect of mitomycin C on reducing endometrial fibrosis for IUA. MATERIAL AND METHODS Firstly, a rat IUA model was constructed by intrauterine mechanical injury. The endometrial stromal cells and fibroblasts were isolated and treated with mitomycin C. After that, Cell Counting Kit-8 (CCK-8) assay was used to investigate the endometrial stromal cell viability. Furthermore, cell cycle and apoptosis assays of endometrial stromal cells and fibroblasts were performed, respectively. Finally, the cell viability of human endometrial cells or human uterus adhesion fibroblasts treated with mitomycin C was determined using CCK-8 assay with or without estradiol. RESULTS Endometrial stromal cells were isolated from a rat IUA model. Cell cycle assay results showed that mitomycin C inhibited cell viability and promoted G1 cell cycle arrest and apoptosis in rat IUA endometrial stromal cells. Fibroblasts were also isolated from the rat IUA model. We found that mitomycin C inhibited the synthesis and secretion of collagen type I by western blotting analysis. Furthermore, mitomycin C promoted G1 cell cycle arrest and apoptosis in IUA rat uterine fibroblasts. We found that estradiol decreased the inhibitory effects of cell viability of human endometrial cells and human uterus adhesion fibroblasts by mitomycin C. CONCLUSIONS Our findings revealed that mitomycin C could reduce endometrial fibrosis for intrauterine adhesion.


Assuntos
Endométrio/patologia , Mitomicina/uso terapêutico , Aderências Teciduais/tratamento farmacológico , Útero/patologia , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Estradiol/farmacologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Mitomicina/farmacologia , Ratos Sprague-Dawley , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo
20.
Nat Rev Cancer ; 20(3): 174-186, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31980749

RESUMO

Cancer-associated fibroblasts (CAFs) are a key component of the tumour microenvironment with diverse functions, including matrix deposition and remodelling, extensive reciprocal signalling interactions with cancer cells and crosstalk with infiltrating leukocytes. As such, they are a potential target for optimizing therapeutic strategies against cancer. However, many challenges are present in ongoing attempts to modulate CAFs for therapeutic benefit. These include limitations in our understanding of the origin of CAFs and heterogeneity in CAF function, with it being desirable to retain some antitumorigenic functions. On the basis of a meeting of experts in the field of CAF biology, we summarize in this Consensus Statement our current knowledge and present a framework for advancing our understanding of this critical cell type within the tumour microenvironment.


Assuntos
Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Neoplasias/etiologia , Neoplasias/patologia , Microambiente Tumoral , Animais , Biomarcadores , Fibroblastos Associados a Câncer/efeitos dos fármacos , Plasticidade Celular , Ensaios Clínicos como Assunto , Suscetibilidade a Doenças , Humanos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Transdução de Sinais , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Células Estromais/patologia , Resultado do Tratamento
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