Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 6.231
Filtrar
1.
PLoS One ; 15(1): e0227030, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31910224

RESUMO

Endothelial activation and alteration during dengue virus (DENV) infection are multifactorial events; however, the role of extracellular vesicles (EVs) in these phenomena is not known. In the present study, we characterized the EVs released by DENV-2 infected U937 macrophage cell line and evaluated the changes in the physiology and integrity of the EA.hy926 endothelial cells exposed to them. U937 macrophages were infected, supernatants were collected, and EVs were purified and characterized. Then, polarized endothelial EA.hy926 cells were exposed to the EVs for 24 h, and the transendothelial electrical resistance (TEER), monolayer permeability, and the expression of tight junction and adhesion proteins and cytokines were evaluated. The isolated EVs from infected macrophages corresponded to exosomes and apoptotic bodies, which contained the viral NS3 protein and different miRs, among other products. Exposure of EA.hy926 cells to EVs induced an increase in TEER, as well as changes in the expression of VE-cadherin and ICAM in addition leads to an increase in TNF-α, IP-10, IL-10, RANTES, and MCP-1 secretion. These results suggest that the EVs of infected macrophages transport proteins and miR that induce early changes in the physiology of the endothelium, leading to its activation and eliciting a defense program against damage during first stages of the disease, even in the absence of the virus.


Assuntos
Vírus da Dengue , Células Endoteliais/metabolismo , Vesículas Extracelulares/virologia , Macrófagos/ultraestrutura , Antígenos CD/metabolismo , Caderinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Citocinas/metabolismo , Dengue/imunologia , Vírus da Dengue/imunologia , Células Endoteliais/imunologia , Vesículas Extracelulares/fisiologia , Humanos , Macrófagos/virologia , Permeabilidade , Células U937
2.
Med Chem ; 16(1): 63-68, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-30734682

RESUMO

BACKGROUND: A convenient approach to modulation of the inflammation has an influence on the production of inflammatory mediators - icosanoids, generated in arachidonic acid (AA) metabolism. The common therapeutic activity of non-steroidal anti-inflammatory drugs (NSAID), such as aspirin, includes inhibition of two crucial enzymes of AA metabolism - cyclooxygenase- 1 and -2 (COX-1/2), with certain risk for gastrointestinal and renal intolerance. Ever since the enrolment of COX-2, particularly overabundance of its main products prostaglandin E2 (PGE2) and thromboxane A2 (TXA2) in numerous pathological processes was recognized, it became a significant therapeutic target. OBJECTIVE: The aim of this study was to examine the effects of synthesized organo-fluorine compounds on PGE2 and TXA2 production in the inflammation process. METHODS: Trifluoromethyl compounds were synthesized from N-benzyl trifluoromethyl aldimine, commercially available 2-methyl or 2-phenyl α-bromo esters (ß-lactams trans-1 and trans-2 and trifluoromethyl ß-amino ester, respectively) and methyl 2-isocyanoacetate (2-imidazoline trans-4). The reactions proceeded with high geometric selectivity, furnishing the desired products in good yields. The influence of newly synthesized compounds on PGE2 and TXA2 production in human leukemic U937 macrophages on both enzyme activity and gene expression levels was observed. RESULTS: Among the tested trifluoromethyl compounds, methyl trans-1-benzyl-5-(trifluoromethyl)- 4,5-dihydro-1H-imidazole-4-carboxylate (trans-4) can be distinguished as the most powerful antiinflammatory agent, probably due to its trifluoromethyl-imidazoline moiety. CONCLUSION: Some further structural modifications in tested compounds and particularly in the synthesis of different trifluoromethyl imidazolines could contribute to the development of new COX-2 inhibitors and potent anti-inflammatory agents.


Assuntos
Dinoprostona/biossíntese , Hidrocarbonetos Fluorados/farmacologia , Macrófagos/efeitos dos fármacos , Tromboxano A2/biossíntese , Relação Dose-Resposta a Droga , Humanos , Hidrocarbonetos Fluorados/síntese química , Hidrocarbonetos Fluorados/química , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Macrófagos/metabolismo , Estrutura Molecular , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Células U937
3.
J Biochem Mol Toxicol ; 34(1): e22412, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31714645

RESUMO

Acute myeloid leukemia (AML) is a cancer of hematopoietic stem cells with a rapid progression. The progression of AML can be regulated by estrogenic signals. Our present data showed that an industrial endocrine-disrupting chemical, bisphenol A (BPA), can promote the proliferation of AML cells and decrease their sensitivity to daunorubicin and cytarabine treatment. Among the tested cytokines, BPA treatment can decrease the expression of interleukin-4 (IL-4) while increasing the expression of IL-6. Overexpression of IL-4 or neutralization antibody of IL-6 (anti-IL-6) can attenuate BPA-induced proliferation of AML cells and reverse BPA-suppressed chemosensitivity. Furthermore, activation of nuclear factor kappa B is essential for BPA-induced upregulation of IL-6 in AML cells. As to IL-4, BPA can increase the expression of NFAT1 to inhibit its transcription. Collectively, our data showed that BPA can trigger the malignancy of AML cells via regulation of IL-4 and IL-6.


Assuntos
Anticorpos Neutralizantes/imunologia , Compostos Benzidrílicos/farmacologia , Estrogênios não Esteroides/farmacologia , Interleucina-4/metabolismo , Interleucina-6/metabolismo , Leucemia Mieloide Aguda/patologia , Fenóis/farmacologia , Proliferação de Células/efeitos dos fármacos , Células HL-60 , Humanos , Interleucina-4/imunologia , Interleucina-6/imunologia , Leucemia Mieloide Aguda/metabolismo , Fatores de Transcrição NFATC/metabolismo , Células U937
4.
Life Sci ; 242: 117228, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31881227

RESUMO

AIMS: Berberine (BBR) is reported to induce apoptosis and inhibit migration of leukemic cells, but the underlying pharmacological mechanisms have not been fully revealed. This study aims to investigate the possible mechanisms from the perspective of autophagy. MAIN METHODS: P-53-null leukemic cell lines Jurkat and U937 were used for the in vitro study. MDC staining was used for observation of autophagy in leukemic cells, and Western blot analysis was for determination of the expression levels of autophagy-associated proteins. Apoptosis of the leukemic cells was detected by flow cytometry. Cellular location of MDM2 was observed with immunofluorescence staining. Ubiquitination of MDM2 was assessed by immunoprecipitation. Male 6-8-week-old NOD/SCID mice were used for evaluating the effect of BBR on chemotherapy sensitivity in vivo. KEY FINDINGS: BBR induced autophagy in p53-null leukemic cells, which was inhibited by autophagy inhibitors 3-methyladenine. 3-methyladenine also inhibited BBR-induced apoptosis in leukemic cells. In addition, BBR not only decreased MDM2 mRNA expression, but also enhanced MDM2 self-ubiquitination in leukemic cells. Forced overexpression of MDM2 reversed the effect of BBR on autophagy and apoptosis. Furthermore, BBR promoted doxorubicin-induced autophagy and cell death in the leukemic cells and overexpression of MDM2 suppressed these effects. In vivo, BBR combined with doxorubicin achieved better therapeutic effect than doxorubicin alone. SIGNIFICANCE: MDM2 inhibits autophagy and apoptosis in leukemic cells in a p53-independent manner. BBR induces autophagy in p53-null leukemic cells through downregulating MDM2 expression at both transcriptional and post-transcriptional levels, which may contribute to the anti-cancer effect of BBR in leukemia.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Berberina/farmacologia , Células Jurkat/efeitos dos fármacos , Leucemia Experimental/tratamento farmacológico , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Células U937/efeitos dos fármacos , Animais , Western Blotting , Citometria de Fluxo , Imunofluorescência , Humanos , Células Jurkat/metabolismo , Leucemia Experimental/metabolismo , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Reação em Cadeia da Polimerase em Tempo Real , Células U937/metabolismo , Ubiquitinação
5.
Life Sci ; 242: 117229, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31887298

RESUMO

AIMS: Neutrophil elastase (NE) is a critical proteolytic enzyme that is involved in cancer. We previously reported high NE expression in peripheral blood neutrophils from acute promyelocytic leukemia (APL) patients. The present study aimed to elucidate the specific role and mechanisms of NE in APL development. MATERIALS AND METHODS: NE expression was detected in APL bone marrow samples and analyzed in the BloodSpot database. CCK-8 assay and flow cytometry were used to assess cell proliferation and cell cycle distribution, respectively. The expression levels of proliferation and differentiation markers were measured by Western blotting and quantitative real-time PCR. The co-expression and interaction of NE and p200 cut-like homeobox 1 (CUX1) were evaluated by indirect immunofluorescence, co-immunoprecipitation, and in situ proximity ligation assay. KEY FINDINGS: NE was highly expressed in APL bone marrow and blood neutrophils. NE overexpression promoted the proliferation and inhibited the differentiation of NB4 cells, whereas NE downregulation achieved the opposite results in U937 cells. Mechanistically, NE interacted with and effectively hydrolyzed the tumor suppressor p200 CUX1. Rescue experiments revealed that p200 CUX1 upregulation reversed the functional influence of NE on APL cells. SIGNIFICANCE: NE-mediated proteolysis of the tumor suppressor p200 CUX1 promotes APL progression. NE/p200 CUX1 axis is a novel and promising therapeutic target for APL treatment.


Assuntos
Proteínas de Homeodomínio/metabolismo , Leucemia Promielocítica Aguda/enzimologia , Elastase de Leucócito/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Adolescente , Adulto , Western Blotting , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Feminino , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Células HL-60 , Proteínas de Homeodomínio/fisiologia , Humanos , Imunoprecipitação , Leucemia Promielocítica Aguda/metabolismo , Elastase de Leucócito/fisiologia , Masculino , Proteólise , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sincalida/metabolismo , Fatores de Transcrição/fisiologia , Células U937
6.
Mol Pharmacol ; 97(3): 212-225, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31871304

RESUMO

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor family, playing pivotal roles in regulating glucose and lipid metabolism as well as inflammation. While characterizing potential PPARγ ligand activity of natural compounds in macrophages, we investigated their influence on the expression of adipophilin [perilipin 2 (PLIN2)], a well-known PPARγ target. To confirm that a compound regulates PLIN2 expression via PPARγ, we performed experiments using the widely used PPARγ antagonist 2-chloro-5-nitro-N-phenylbenzamide (GW9662). Surprisingly, instead of blocking upregulation of PLIN2 expression in THP-1 macrophages, expression was concentration-dependently induced by GW9662 at concentrations and under conditions commonly used. We found that this unexpected upregulation occurs in many human and murine macrophage cell models and also primary cells. Profiling expression of PPAR target genes showed upregulation of several genes involved in lipid uptake, transport, and storage as well as fatty acid synthesis by GW9662. In line with this and with upregulation of PLIN2 protein, GW9662 elevated lipogenesis and increased triglyceride levels. Finally, we identified PPARδ as a mediator of the substantial unexpected effects of GW9662. Our findings show that: 1) the PPARγ antagonist GW9662 unexpectedly activates PPARδ-mediated signaling in macrophages, 2) GW9662 significantly affects lipid metabolism in macrophages, 3) careful validation of experimental conditions and results is required for experiments involving GW9662, and 4) published studies in a context comparable to this work may have reported erroneous results if PPARγ independence was demonstrated using GW9662 only. In light of our findings, certain existing studies might require reinterpretation regarding the role of PPARγ SIGNIFICANCE STATEMENT: Peroxisome proliferator-activated receptors (PPARs) are targets for the treatment of various diseases, as they are key regulators of inflammation as well as lipid and glucose metabolism. Hence, reliable tools to characterize the molecular effects of PPARs are indispensable. We describe profound and unexpected off-target effects of the PPARγ antagonist 2-chloro-5-nitro-N-phenylbenzamide (GW9662) involving PPARδ and in turn affecting macrophage lipid metabolism. Our results question certain existing studies using GW9662 and make better experimental design of future studies necessary.


Assuntos
Anilidas/farmacologia , Lipogênese/fisiologia , PPAR delta/metabolismo , PPAR gama/metabolismo , Perilipina-2/biossíntese , Triglicerídeos/metabolismo , Animais , Células Cultivadas , Feminino , Expressão Gênica , Humanos , Lipogênese/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , PPAR delta/antagonistas & inibidores , PPAR gama/antagonistas & inibidores , Perilipina-2/genética , Células RAW 264.7 , Células U937
7.
Life Sci ; 243: 117234, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31887299

RESUMO

PURPOSE: In acute myeloid leukemia (AML), complete remission can be achieved in parts of patients using cytarabine/anthracycline combination-based chemotherapy, however, drug resistance-related recurrence is still a common cause of treatment failure, leading to high mortality among patients. In our research, we revealed the molecular mechanisms that were sufficient to improve sensitivity of AML cells to the anthracycline daunorubicin (DNR). METHODS: We evaluated the effects of autophagy and apoptosis induced by DNR using two AML cell lines HL60 and U937.Western blot was preformed to analyze the apoptotic pathway protein expression and flow cytometric analysis was used to detect the level of apoptosis in AML cells. The levels of autophagy-related proteins were detected by western blotting and autophagic vesicles were observed by electron microscopy. RESULTS: DNR effectively induced autophagy in two AML cell lines HL60 and U937 confirming by upregulation of LC3-II lipidation, formation of autophagosomes. Inhibition of autophagy by pharmacologic inhibitor HCQ promoted apoptosis induced by DNR, suggesting that autophagy played a vital role in pro-survival in AML. Furthermore, ULK1 inhibition by a highly selective kinase inhibitor SBI-0206965 and shRNA enhanced cytotoxicity of DNR against AML cells. Independent of mTOR -ULK1 signaling pathway, activation of autophagy of DNR was proved to be mediated by AMPK (pThr172)/ULK1 pathway. CONCLUSIONS: These results revealed that pro-survival autophagy induced by ULK1 activation was one of the potential mechanisms of AML resistance to DNR. Targeting ULK1 selectively could be a promising therapeutic strategy to enhance sensitivity of DNR for AML therapy.


Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/antagonistas & inibidores , Daunorrubicina/uso terapêutico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Antibióticos Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Daunorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/farmacologia , Células HL-60 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/patologia , Fosforilação , Células U937
8.
Biochim Biophys Acta Gene Regul Mech ; 1863(1): 194475, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31870784

RESUMO

Targeting the apoptosis machinery is a promising therapeutic approach in myeloid malignancies. BCL2L1 is a well-known glucocorticoid-responsive gene and a key apoptosis regulator that, when over-expressed, can contribute to tumor development, progression and therapeutic resistance. Moreover, synthetic glucocorticoids, like dexamethasone, are frequently used in the treatment of hematopoietic diseases due to its pro-apoptotic properties. We report here that the trithorax protein ASH2L, considered one of the core subunits of H3K4-specific MLL/SET methyltransferase complexes, contributes to anti-apoptotic BCL-XL over-expression and cell survival in patient-derived myeloid leukemia cells. We find that the unliganded glucocorticoid receptor (uGR) and ASH2L interact in a common protein complex through a chromatin looping determined by uGR and ASH2L binding to BCL2L1 specific +58 HRE and promoter region, respectively. Upon addition of dexamethasone, GR and ASH2L recruitment is reduced, BCL-XL expression diminishes and apoptosis is induced consequently. Overall, our findings indicate that uGR and ASH2L may act as key regulatory players of BCL- XL upregulation in AML cells.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Glucocorticoides/farmacologia , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/metabolismo , Receptores de Glucocorticoides/metabolismo , Fatores de Transcrição/metabolismo , Proteína bcl-X/genética , Apoptose , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/metabolismo , Regiões Promotoras Genéticas , Elementos de Resposta , Células U937 , Proteína bcl-X/metabolismo
9.
J Enzyme Inhib Med Chem ; 35(1): 1-20, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31619080

RESUMO

Inflammatory bowel disease (IBD) is a chronic immuno-inflammation in gastrointestinal tract. We have evaluated the activity of the compounds to inhibit the adhesion of monocytes to colon epithelial cells is triggered by a pro-inflammatory cytokine, tumour necrosis factor (TNF)-α. The in vitro activity of the compounds, 13b (an ureido-derivative), 14c, 14j, 14k, 14n (thioureido-), 18c and 18d (sulfonamido-), was in correlation with in vivo anti-colitis activity revealed as significant recovery in body- and colon-weights and colon myeloperoxidase level, a biochemical marker of inflammation reflecting neutrophil infiltration. In vivo, TNBS-induced changes in the expression of inflammatory cytokines (TNF-α, IL-6, IL-1ß, IL-10, and TGF-ß), NLRP3 inflammasome components (NLRP-3, Caspase-1, and IL-18), and epithelial junction molecules (E-cadherin, claudin2/3, and ZO-1) were blocked and recovered by oral administration of the compounds (1 mg/kg). Compound 14n which showed the best efficacy can be a promising lead for orally available therapeutics for pathology of IBD.


Assuntos
Anti-Inflamatórios/farmacologia , Doenças Inflamatórias Intestinais/tratamento farmacológico , Piridinas/farmacologia , Animais , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/química , Células Cultivadas , Colite/induzido quimicamente , Colite/tratamento farmacológico , Citocinas/antagonistas & inibidores , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Feminino , Células HT29 , Humanos , Doenças Inflamatórias Intestinais/patologia , Estrutura Molecular , Piridinas/síntese química , Piridinas/química , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Ácido Trinitrobenzenossulfônico , Células U937
10.
mSphere ; 4(5)2019 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-31578246

RESUMO

Gene diversification is a common mechanism pathogens use to alter surface structures to aid in immune avoidance. Neisseria gonorrhoeae uses a gene conversion-based diversification system to alter the primary sequence of the gene encoding the major subunit of the pilus, pilE Antigenic variation occurs when one of the nonexpressed 19 silent copies donates part of its DNA sequence to pilE We have developed a method using Pacific Biosciences (PacBio) amplicon sequencing and custom software to determine pilin antigenic variation frequencies. The program analyzes 37 variable regions across the strain FA1090 1-81-S2 pilE gene and can be modified to determine sequence variation from other starting pilE sequences or other diversity generation systems. Using this method, we measured pilin antigenic variation frequencies for various derivatives of strain FA1090 and showed we can also analyze pilin antigenic variation frequencies during macrophage infection.IMPORTANCE Diversity generation systems are used by many unicellular organism to provide subpopulations of cell with different properties that are available when needed. We have developed a method using the PacBio DNA sequencing technology and a custom computer program to analyze the pilin antigenic variation system of the organism that is the sole cause of the sexually transmitted infection, gonorrhea.


Assuntos
Variação Antigênica , Proteínas de Fímbrias/genética , Neisseria gonorrhoeae/genética , Análise de Sequência de DNA/métodos , Proteínas de Fímbrias/imunologia , Gonorreia/microbiologia , Humanos , Software , Células U937
11.
Nat Commun ; 10(1): 4408, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31562318

RESUMO

Intestinal epithelial cells (IEC) have important functions in nutrient absorption, barrier integrity, regeneration, pathogen-sensing, and mucus secretion. Goblet cells are a specialized cell type of IEC that secrete Trefoil factor 3 (TFF3) to regulate mucus viscosity and wound healing, but whether TFF3-responsiveness requires a receptor is unclear. Here, we show that leucine rich repeat receptor and nogo-interacting protein 2 (LINGO2) is essential for TFF3-mediated functions. LINGO2 immunoprecipitates with TFF3, co-localizes with TFF3 on the cell membrane of IEC, and allows TFF3 to block apoptosis. We further show that TFF3-LINGO2 interactions disrupt EGFR-LINGO2 complexes resulting in enhanced EGFR signaling. Excessive basal EGFR activation in Lingo2 deficient mice increases disease severity during colitis and augments immunity against helminth infection. Conversely, TFF3 deficiency reduces helminth immunity. Thus, TFF3-LINGO2 interactions de-repress inhibitory LINGO2-EGFR complexes, allowing TFF3 to drive wound healing and immunity.


Assuntos
Colite/imunologia , Receptores ErbB/imunologia , Helmintíase/imunologia , Mucosa Intestinal/imunologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/imunologia , Fator Trefoil-3/imunologia , Animais , Linhagem Celular Tumoral , Colite/induzido quimicamente , Colite/metabolismo , Sulfato de Dextrana , Receptores ErbB/genética , Receptores ErbB/metabolismo , Células Caliciformes/imunologia , Células Caliciformes/metabolismo , Células Caliciformes/parasitologia , Células HEK293 , Helmintíase/metabolismo , Helmintíase/parasitologia , Helmintos/imunologia , Helmintos/fisiologia , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/parasitologia , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Organofosfonatos , Fator Trefoil-3/genética , Fator Trefoil-3/metabolismo , Células U937
12.
J Toxicol Sci ; 44(9): 585-600, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31474740

RESUMO

Amino acid derivative reactivity assay (ADRA) has previously been developed as an alternative method to direct peptide reactivity assay (DPRA) to evaluate key event 1 in skin sensitization mechanisms. However, when using alternative methods for skin sensitization, integrated approaches to testing and assessment (IATA) that combine the results of multiple tests evaluating different key events are generally required. To verify whether ADRA can be used in IATA, we replaced DPRA with ADRA in five IATA methods combining DPRA, KeratinoSens, and h-CLAT: (i) the "2 out of 3" approach, (ii) the "3 out of 3" approach, (iii) sequential testing strategy (STS), (iv) integrated testing strategy by scoring approach (ITS-SA), and (v) the "ITS by two methods approach" (ITS-2MA). The prediction accuracy of the "2 out of 3" approach using ADRA (1 mM) and ADRA (0.5 mg/mL) was 90.0% and 91.1%, respectively, for human data, and was very similar to that obtained using DPRA (91.1%). The "3 out of 3" approach also showed good predictability (83.2%) using either ADRA (1 mM) or ADRA (0.5 mg/mL) compared to DPRA. Regarding the accuracy of the prediction of sensitization intensity for the human data by the third classification, prediction accuracy using ADRA was almost the same as STS, ITS-SA, or ITS-2MA using DPRA. As a result, this study showed that ADRA can be used as a test method for key event 1 in the evaluation of skin sensitization by combining multiple alternative methods.


Assuntos
Aminoácidos/imunologia , Alternativas aos Testes com Animais/métodos , Imunização/métodos , Pele/imunologia , Linhagem Celular , Humanos , Células U937
13.
Cell Host Microbe ; 26(4): 551-563.e6, 2019 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-31540829

RESUMO

During infection, Legionella pneumophila translocates over 300 effector proteins into the host cytosol, allowing the pathogen to establish an endoplasmic reticulum (ER)-like Legionella-containing vacuole (LCV) that supports bacterial replication. Here, we perform a genome-wide CRISPR-Cas9 screen and secondary targeted screens in U937 human monocyte/macrophage-like cells to systematically identify host factors that regulate killing by L. pneumophila. The screens reveal known host factors hijacked by L. pneumophila, as well as genes spanning diverse trafficking and signaling pathways previously not linked to L. pneumophila pathogenesis. We further characterize C1orf43 and KIAA1109 as regulators of phagocytosis and show that RAB10 and its chaperone RABIF are required for optimal L. pneumophila replication and ER recruitment to the LCV. Finally, we show that Rab10 protein is recruited to the LCV and ubiquitinated by the effectors SidC/SdcA. Collectively, our results provide a wealth of previously undescribed insights into L. pneumophila pathogenesis and mammalian cell function.


Assuntos
Legionella pneumophila/patogenicidade , Doença dos Legionários/patologia , Fagocitose/imunologia , Proteínas/genética , Vacúolos/microbiologia , Animais , Proteínas de Bactérias/metabolismo , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Retículo Endoplasmático/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Células HeLa , Humanos , Legionella pneumophila/genética , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Células RAW 264.7 , Células U937 , Fatores de Virulência/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
14.
Nature ; 574(7776): 57-62, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31534221

RESUMO

The causative agent of plague, Yersinia pestis, uses a type III secretion system to selectively destroy immune cells in humans, thus enabling Y. pestis to reproduce in the bloodstream and be transmitted to new hosts through fleabites. The host factors that are responsible for the selective destruction of immune cells by plague bacteria are unknown. Here we show that LcrV, the needle cap protein of the Y. pestis type III secretion system, binds to the N-formylpeptide receptor (FPR1) on human immune cells to promote the translocation of bacterial effectors. Plague infection in mice is characterized by high mortality; however, Fpr1-deficient mice have increased survival and antibody responses that are protective against plague. We identified FPR1R190W as a candidate resistance allele in humans that protects neutrophils from destruction by the Y. pestis type III secretion system. Thus, FPR1 is a plague receptor on immune cells in both humans and mice, and its absence or mutation provides protection against Y. pestis. Furthermore, plague selection of FPR1 alleles appears to have shaped human immune responses towards other infectious diseases and malignant neoplasms.


Assuntos
Macrófagos/metabolismo , Neutrófilos/metabolismo , Peste/microbiologia , Receptores de Formil Peptídeo/metabolismo , Yersinia pestis/metabolismo , Alelos , Animais , Antígenos de Bactérias/metabolismo , Aderência Bacteriana , Sistemas CRISPR-Cas , Quimiotaxia/imunologia , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/citologia , Neutrófilos/imunologia , Neutrófilos/microbiologia , Peste/imunologia , Peste/prevenção & controle , Polimorfismo de Nucleotídeo Único/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Receptores de Formil Peptídeo/antagonistas & inibidores , Receptores de Formil Peptídeo/deficiência , Receptores de Formil Peptídeo/genética , Sistemas de Secreção Tipo III/efeitos dos fármacos , Células U937 , Yersinia pestis/química , Yersinia pestis/imunologia , Yersinia pestis/patogenicidade
15.
J Biomed Sci ; 26(1): 63, 2019 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-31470848

RESUMO

BACKGROUND: Chemotherapy is the main treatment for acute myeloid leukemia (AML), but the cure rates for AML patients remain low, and the notorious adverse effects of chemotherapeutic drugs drastically reduce the life quality of patients. Penfluridol, a long-acting oral antipsychotic drug, has an outstanding safety record and exerts oncostatic effects on various solid tumors. Until now, the effect of penfluridol on AML remains unknown. METHODS: AML cell lines harboring wild-type (WT) Fms-like tyrosine kinase 3 (FLT3) and internal tandem duplication (ITD)-mutated FLT3 were used to evaluate the cytotoxic effects of penfluridol by an MTS assay. A flow cytometric analysis and immunofluorescence staining were employed to determine the cell-death phenotype, cell cycle profile, and reactive oxygen species (ROS) and acidic vesicular organelle (AVO) formation. Western blotting and chemical inhibitors were used to explore the underlying mechanisms involved in penfluridol-mediated cell death. RESULTS: We observed that penfluridol concentration-dependently suppressed the cell viability of AML cells with FLT3-WT (HL-60 and U937) and FLT3-ITD (MV4-11). We found that penfluridol treatment not only induced apoptosis as evidenced by increases of nuclear fragmentation, the sub-G1 populations, poly (ADP ribose) polymerase (PARP) cleavage, and caspase-3 activation, but also triggered autophagic responses, such as the light chain 3 (LC3) turnover and AVO formation. Interestingly, blocking autophagy by the pharmacological inhibitors, 3-methyladenine and chloroquine, dramatically enhanced penfluridol-induced apoptosis, indicating the cytoprotective role of autophagy in penfluridol-treated AML cells. Mechanistically, penfluridol-induced apoptosis occurred through activating protein phosphatase 2A (PP2A) to suppress Akt and mitogen-activated protein kinase (MAPK) activities. Moreover, penfluridol's augmentation of intracellular ROS levels was critical for the penfluridol-induced autophagic response. In the clinic, we observed that patients with AML expressing high PP2A had favorable prognoses. CONCLUSIONS: These findings provide a rationale for penfluridol being used as a PP2A activator for AML treatment, and the combination of penfluridol with an autophagy inhibitor may be a novel strategy for AML harboring FLT3-WT and FLT3-ITD.


Assuntos
Antipsicóticos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Penfluridol/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Tirosina Quinase 3 Semelhante a fms/metabolismo , Linhagem Celular Tumoral , Citoproteção/efeitos dos fármacos , Células HL-60 , Humanos , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Transdução de Sinais , Células U937
16.
Colloids Surf B Biointerfaces ; 181: 959-962, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31382346

RESUMO

The therapeutic effect of indomethacin, a water-insoluble non-steroidal anti-inflammatory drug, requires its efficient transport through cellular membranes and accumulation inside the target cells. The application of dendritic polymers has been proposed for the improvement of the drug's solubility and intracellular delivery. In this study we evaluated the anti-inflammatory potential of novel, highly-biocompatible 4-carbomethoxypyrrolidone-coated PAMAM dendrimers loaded with indomethacin. Our results indicate that complexation with dendrimers do not hamper the inhibitory action of indomethacin towards cyclooxygenases. Drug-dendrimer formulations exhibited improved anti-inflammatory activity in in vitro-cultured cellular models, showing enhanced inhibition of prostaglandin secretion and significantly decreased expression of NF-κB marker genes compared to free drug.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Dendrímeros/química , Indometacina/farmacologia , Pirrolidinonas/química , Anti-Inflamatórios não Esteroides/química , Humanos , Indometacina/química , NF-kappa B/metabolismo , Prostaglandinas/metabolismo , Células U937
17.
Anticancer Res ; 39(8): 4533-4537, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31366556

RESUMO

BACKGROUND/AIM: Serum-derived macrophage activating factor, serum-MAF, is known to increase the phagocytic activity of macrophages by enhancing the engulfment efficiency. To elucidate the mechanisms underlying phagocytic activation, morphological changes were observed and analyzed. MATERIALS AND METHODS: Morphological changes in macrophages were observed and quantitatively analyzed using scanning electron microscope (SEM) and confocal microscope. RESULTS: SEM and confocal microscopy images revealed frill-like structures and active actin accumulations, respectively, in serum-MAF treated macrophages. Actin accumulation was induced within 5 min following serum-MAF treatment. CONCLUSION: Serum-MAF induced a rapid rearrangement of cytoskeletal actin and enhanced phagocytic activity. Findings of the current study may contribute to the development of techniques that facilitate activation of the human immune system, which in turn may be beneficial for cancer immunotherapy.


Assuntos
Actinas/química , Macrófagos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-maf/farmacologia , Actinas/ultraestrutura , Humanos , Imunoterapia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Microscopia Confocal , Microscopia Eletrônica de Varredura , Proteínas Proto-Oncogênicas c-maf/genética , Células U937 , Proteína de Ligação a Vitamina D/química , Proteína de Ligação a Vitamina D/metabolismo
18.
Mol Biol (Mosk) ; 53(4): 638-647, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31397437

RESUMO

The ubiquitin-proteasome system (UPS) performs proteolysis of most intracellular proteins. The key components of the UPS are the proteasomes, multi-subunit protein complexes, playing an important role in cellular adaptation to various types of stress. We analyzed the dynamics of the proteasome activity, the content of proteasome subunits, and the expression levels of genes encoding catalytic subunits of proteasomes in the human histiocytic lymphoma U937 cell line immediately, 2, 4, 6, 9, 24, and 48 h after a heat shock (HS). The initial decrease (up to 62%) in the proteasome activity in cellular lysates was revealed, then 10 h after HS the activity began to recover. The amount of proteasomal α-subunits in the cells decreased 2 h after HS, and was restored to 24-48 h after HS. Fluctuations in the levels of mRNAs encoding proteasome catalytic subunits with the maximum expression 2 h after HS and a gradual decrease to 48 h after HS were observed. The average estimated number of mRNA copies per cell ranged from 10 for weakly to 150 for highly expressed proteasome genes. Thus, the recovery efficiency of UPS functionality after HS, which reflects the important role of proteasomes in maintaining cell homeostasis, was evaluated.


Assuntos
Resposta ao Choque Térmico , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Subunidades Proteicas/genética , Proteólise , Células U937 , Ubiquitina/metabolismo
19.
Molecules ; 24(17)2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31461974

RESUMO

Within non-communicable diseases, chronic inflammatory conditions represent one of the biggest challenges for modern medicine. Traditional Chinese Medicine (TCM) has been practiced over centuries and has accumulated tremendous empirical knowledge on the treatment of such diseases. Huangqi Jianzhong Tang (HQJZT) is a famous TCM herbal formula composed of Radix Astragali, Ramulus Cinnamomi, Radix et Rhizoma Glycyrrhizae Praeparata cum Melle, Radix Paeoniae Alba, Rhizoma Zingiberis Recens, Fructus Jujubae and Saccharum Granorum (maltose), which has been used for the treatment of various chronic inflammatory gastrointestinal diseases. However, there is insufficient knowledge about its active constituents and the mechanisms responsible for its effects. The present study aimed at identifying constituents contributing to the bioactivity of HQJZT by combining in vitro cytokine production assays and LC-MS metabolomics techniques. From the HQJZT decoction as well as from its single herbal components, extracts of different polarities were prepared. Phytochemical composition of the extracts was analyzed by means of UPLC-QTOF-MS/MS. The inhibitory effects of the extracts on TNF-α, IL-1ß and IFN-γ production were studied in U937 cells. Phytochemical and pharmacological bioactivity data were correlated by orthogonal projection to latent structures discriminant analysis (OPLS-DA) in order to identify those HQJZT constituents which may be relevant for the observed pharmacological activities. The investigations resulted in the identification of 16 HQJZT constituents, which are likely to contribute to the activities observed in U937 cells. Seven of them, namely calycosin, formononetin, astragaloside I, liquiritigenin, 18ß-glycyrrhetinic acid, paeoniflorin and albiflorin were unambiguously identified. The predicted results were verified by testing these compounds in the same pharmacological assays as for the extracts. In conclusion, the anti-inflammatory activity of HQJZT could be substantiated by in vitro pharmacological screening, and the predicted activities of the OPLS-DA hits could be partially verified. Moreover, the benefits and limitations of MVDA for prediction pharmacologically active compounds contributing to the activity of a TCM mixture could be detected.


Assuntos
Anti-Inflamatórios/química , Citocinas/metabolismo , Medicamentos de Ervas Chinesas/química , Lipopolissacarídeos/efeitos adversos , Metabolômica/métodos , Anti-Inflamatórios/farmacologia , Cromatografia Líquida , Citocinas/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Medicamentos de Ervas Chinesas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interferon gama/metabolismo , Interleucina-1beta/metabolismo , Espectrometria de Massas em Tandem , Fator de Necrose Tumoral alfa/metabolismo , Células U937
20.
J Exp Clin Cancer Res ; 38(1): 308, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31307525

RESUMO

BACKGROUND: At present, it is generally believed that leukemia stem cells are the source of AML, so the killing of leukemia stem cells has become important. Previous studies have suggested that HHT combined with ATO can synergistically kill U937 cells, and HHT has also demonstrated the ability to kill leukemia stem cells. We evaluated whether HHT combined with ATO can systematically kill leukemia stem cells (LSCs) and explored the synergistic effect and molecular mechanism. METHODS: CCK-8 was used to detect cell viability. The changes of cell cycle (PI staining), apoptosis (Annexin V/PI) and surface markers (CD34, CD38, CD96, CD45) were detected by flow cytometry. The cells of CD34+ primary leukemia and CD38- KG-1, and TF-1 were separated by flow cytometry. High-throughput mRNA sequencing was used to analysis mRNA level changes after the application of the two drugs. Western blot was used to verify the changes of pathway protein expression. NRG mice were used as the receptor of xenograft model. Histological H&E staining assess the invaded ability of leukemia cells, and laser scanning confocal microscopy evaluated the molecule markers change. RESULTS: HHT and ATO synergistically killed KG-1 (CD34+/CD96+/CD38+/-) and Kasumi-1 (CD34+/CD38-) cells. Their combination had a stronger effect of inducing apoptosis and blocking the cell cycle than HHT or ATO administrator alone, meanwhile significantly reducing the numbers of LSCs. Further, CD34+CD38- cells in KG-1, KG-1a, TF-1, and primary leukemia cells were more sensitive to HHT and ATO. High-throughput mRNA sequencing suggested that HHT alone could significantly upregulate molecules related to the Notch, P53, and NF-κB signaling pathways. When combined with ATO, HHT further upregulated P53, whereas HHT-induced NF-κB pathway activation was significantly suppressed. Western blot analysis verified the change of protein expression in the above pathways and further demonstrated that GSI, could eliminate these effects. In vivo, HHT combined with ATO significantly reduced the LSC burden, and weakened the expression of LSC markers. CONCLUSIONS: This is the first evidence that HHT combined with arsenic can synergistically kill LSCs in vitro and in vivo, along with identification of the underlying mechanism, highlighting a potentially effective treatment strategy.


Assuntos
Antineoplásicos/administração & dosagem , Trióxido de Arsênio/administração & dosagem , Mepesuccinato de Omacetaxina/administração & dosagem , Leucemia Mieloide Aguda/tratamento farmacológico , Células-Tronco Neoplásicas/metabolismo , Animais , Antineoplásicos/farmacologia , Trióxido de Arsênio/farmacologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Mepesuccinato de Omacetaxina/farmacologia , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Células-Tronco Neoplásicas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Células U937 , Ensaios Antitumorais Modelo de Xenoenxerto
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA