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1.
Int J Mol Sci ; 22(15)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34360584

RESUMO

Trichostatin A (TSA) is a representative histone deacetylase (HDAC) inhibitor that modulates epigenetic gene expression by regulation of chromatin remodeling in cells. To investigate whether the regulation of chromatin de-condensation by TSA can affect the increase in the efficiency of Cas9 protein-gRNA ribonucleoprotein (RNP) indel formation from plant cells, genome editing efficiency using lettuce and tobacco protoplasts was examined after several concentrations of TSA treatments (0, 0.1, 1 and 10 µM). RNP delivery from protoplasts was conducted by conventional polyethylene glycol (PEG) transfection protocols. Interestingly, the indel frequency of the SOC1 gene from TSA treatments was about 3.3 to 3.8 times higher than DMSO treatment in lettuce protoplasts. The TSA-mediated increase of indel frequency of the SOC1 gene in lettuce protoplasts occurred in a concentration-dependent manner, although there was not much difference. Similar to lettuce, TSA also increased the indel frequency by 1.5 to 1.8 times in a concentration-dependent manner during PDS genome editing using tobacco protoplasts. The MNase test clearly showed that chromatin accessibility with TSA treatments was higher than that of DMSO treatment. Additionally, TSA treatment significantly increased the level of histone H3 and H4 acetylation from lettuce protoplasts. The qRT-PCR analysis showed that expression of cell division-related genes (LsCYCD1-1, LsCYCD3-2, LsCYCD6-1, and LsCYCU4-1) was increased by TSA treatment. These findings could contribute to increasing the efficiency of CRISPR/Cas9-mediated genome editing. Furthermore, this could be applied for the development of useful genome-edited crops using the CRISPR/Cas9 system with plant protoplasts.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Ácidos Hidroxâmicos/farmacologia , Alface/metabolismo , Proteínas de Plantas/metabolismo , Protoplastos/metabolismo , Tabaco/metabolismo , Divisão Celular , Genoma de Planta , Alface/efeitos dos fármacos , Alface/genética , Alface/crescimento & desenvolvimento , Células Vegetais , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Inibidores da Síntese de Proteínas/farmacologia , Protoplastos/efeitos dos fármacos , Tabaco/efeitos dos fármacos , Tabaco/genética , Tabaco/crescimento & desenvolvimento
2.
Aging (Albany NY) ; 13(13): 17489-17498, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34232916

RESUMO

BACKGROUND AND PURPOSE: Obesity is becoming a major global health issue and is mainly induced by the accumulation of adipose tissues mediated by adipogenesis, which is reported to be regulated by peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT enhancer-binding protein α (C/EBPα). Trichostatin A (TSA) is a novel histone deacetylase inhibitor (HDACI) that was recently reported to exert multiple pharmacological functions. The present study will investigate the inhibitory effect of TSA on adipogenesis, as well as the underlying mechanism. METHODS: The adipogenesis of 3T3-L1 cells was induced by stimulation with a differentiation cocktail (DMI) medium for 8 days. MTT assay was used to measure the cell viability and Oil Red O staining was used to evaluate the adipogenesis of 3T3-L1 cells. The total level of triglyceride and released glycerol were detected to evaluate the lipolysis during 3T3-L1 adipogenesis. The expression levels of Leptin, fatty acid-binding protein 4 (FABP4), and sterol regulatory element-binding protein (SREBP1C) were determined by qRT-PCR. qRT-PCR assay was utilized to detect the expression levels of PPARγ and C/EBPα in 3T3-L1 cells. A high-fat diet (HFD) was used to construct an obese mice model, followed by the treatment with TSA. HE staining was conducted to evaluate the pathological state of adipose tissues. Body weights and the weights of adipose tissues were recorded to evaluate the anti-obesity property of TSA. RESULTS: Firstly, the promoted lipid accumulation induced by DMI incubation was significantly reversed by the treatment with TSA in a dose-dependent manner. The elevated expression levels of Leptin, FABP4, SREBP1C, PPARγ, and C/EBPα induced by the stimulation with DMI incubation were dramatically inhibited by the introduction of TSA, accompanied by the upregulation of phosphorylated AMP-activated protein kinase (p-AMPK). Secondly, the inhibitory effect of TSA against the expression level of PPARγ and lipid accumulation was greatly abolished by an AMPK inhibitor. Lastly, the increased body weights and visceral adipocyte tissue weight, as well as the enlarged size of adipocytes induced by HFD were pronouncedly reversed by the administration of TSA. CONCLUSION: TSA inhibited adipogenesis in 3T3-L1 preadipocytes by activating the AMPK pathway.


Assuntos
Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Fármacos Antiobesidade/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Células 3T3-L1 , Tecido Adiposo/patologia , Animais , Peso Corporal/efeitos dos fármacos , Dieta Hiperlipídica , Proteínas de Ligação a Ácido Graxo/genética , Glicerol/metabolismo , Leptina/metabolismo , Lipólise/efeitos dos fármacos , Masculino , Camundongos , Camundongos Obesos , Obesidade/genética , Triglicerídeos/metabolismo
3.
Medicine (Baltimore) ; 100(23): e26135, 2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34114996

RESUMO

ABSTRACT: Pancreatic cancer (PC) is a malignant tumor which ranks fourth in cancer-related death. However, the specificity and sensitivity of traditional biomarkers such as carbohydrate antigen 19-9 no longer meet the clinical requirements.Tools as ONCOMINE and Gene Expression Profiling Interactive Analysis (GEPIA) were used to analyze the differential expression of matrix metalloproteinases (MMPs) in PC and adjacent tissues. For further analysis, we adopted database for annotation, visualization and integrated discovery (DAVID 6.8), transcriptional regulatory relationships unraveled by sentence-based text (TRRUST) and other tools. We also identified drugs targeted the selected MMPs.Eight MMPs (MMP1, MMP2, MMP7, MMP9, MMP11, MMP12, MMP14, and MMP28) were differentially expressed in PC and adjacent tissue. MMP1 (P = .0189), MMP7 (P = .000216), MMP11 (P = .0209), MMP14 (P = .00611) were correlated with the pathological stages of PC. Patients with higher expression of MMP1 (P = .0011), MMP2 (P = .011), MMP7 (P = .0081), MMP9 (P = .046), MMP11 (P = .0019), MMP12 (P = .0011), MMP14 (P = .0011), and MMP28 (P = 6.3e-06) showed poor prognosis. Ten transcription factors were associated with the up-regulation of selected MMPs. Marimastat (DB00786) was found to target selected MMPs.Our research revealed that selected MMPs played an important role in the early diagnosis and prognosis of PC.


Assuntos
Perfilação da Expressão Gênica/métodos , Ácidos Hidroxâmicos/farmacologia , Metaloproteinases da Matriz , Neoplasias Pancreáticas , Biomarcadores/metabolismo , Biologia Computacional , Regulação Neoplásica da Expressão Gênica , Humanos , Inibidores de Metaloproteinases de Matriz/farmacologia , Metaloproteinases da Matriz/classificação , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Metaloendopeptidases , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/metabolismo , Prognóstico
4.
Eur J Med Chem ; 222: 113569, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34111829

RESUMO

Novel 5-pyridinyl-1,2,4-triazoles were designed as dual inhibitors of histone deacetylase 2 (HDAC2) and focal adhesion kinase (FAK). Compounds 5d, 6a, 7c, and 11c were determined as potential inhibitors of both HDAC2 (IC50 = 0.09-1.40 µM) and FAK (IC50 = 12.59-36.11 nM); 6a revealed the highest activity with IC50 values of 0.09 µM and 12.59 nM for HDAC2 and FAK, respectively. Compound 6a was superior to reference drugs vorinostat and valproic acid in its ability to inhibit growth/proliferation of A-498 and Caki-1 renal cancer cells. Further investigation proved that 6a strongly arrests the cell cycle at the G2/M phase and triggers apoptosis in both A-498 and Caki-1 cells. Moreover, the enhanced Akt activity that is observed upon chronic application of HDAC inhibitors was effectively suppressed by the dual HDAC2/FAK inhibitor. Finally, the high potency and selectivity of 6a towards HDAC2 and FAK proteins were rationalized by molecular docking. Taken together, these findings highlight the potential of 6a as a promising dual-acting HDAC2/FAK inhibitor that could benefit from further optimization.


Assuntos
Antineoplásicos/farmacologia , Quinase 1 de Adesão Focal/antagonistas & inibidores , Histona Desacetilase 2/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Triazóis/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Benzamidas/química , Benzamidas/farmacologia , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Quinase 1 de Adesão Focal/metabolismo , Histona Desacetilase 2/metabolismo , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Humanos , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Triazóis/química , Células Tumorais Cultivadas
5.
Eur J Protistol ; 80: 125821, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34144311

RESUMO

Trypanosoma cruzi is a protozoan of great medical interest since it is the causative agent of Chagas disease, an endemic condition in Latin America. This parasite undergoes epigenetic events, such as phosphorylation, methylation and acetylation, which play a role in several cellular processes including replication, transcription and gene expression. Histone deacetylases (HDAC) are involved in chromatin compaction and post-translational modifications of cytoplasmic proteins, such as tubulin. Tubastatin A (TST) is a specific HDAC6 inhibitor that affects cell growth and promotes structural modifications in cancer cells and parasites. In the present study, we demonstrated that T. cruzi epimastigote cell proliferation and viability are reduced after 72 h of TST treatment. The results obtained through different microscopy methodologies suggest that this inhibitor impairs the polymerization dynamics of cytoskeleton microtubules, generating protozoa displaying atypical morphology and cellular patterns that include polynucleated parasites. Furthermore, the microtubules of treated protozoa were more intensely acetylated, especially at the anterior portion of the cell body. A cell cycle analysis demonstrated an increase in the number of trypanosomatids in the G2/M phase. Together, our results suggest that TST should be explored as a tool to study trypanosomatid cell biology, including microtubule cytoskeleton dynamics, and as an antiparasitic drug.


Assuntos
Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Citoesqueleto/metabolismo , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Trypanosoma cruzi/citologia , Trypanosoma cruzi/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos
6.
Int J Mol Sci ; 22(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071322

RESUMO

Rubinstein-Taybi syndrome (RSTS) is a rare neurodevelopmental disorder caused by mutations in CREBBP or EP300 genes encoding CBP/p300 lysine acetyltransferases. We investigated the efficacy of the histone deacetylase inhibitor (HDACi) Trichostatin A (TSA) in ameliorating morphological abnormalities of iPSC-derived young neurons from P149 and P34 CREBBP-mutated patients and hypoexcitability of mature neurons from P149. Neural progenitors from both patients' iPSC lines were cultured one week with TSA 20 nM and, only P149, for 6 weeks with TSA 0.2 nM, in parallel to neural progenitors from controls. Immunofluorescence of MAP2/TUJ1 positive cells using the Skeletonize Image J plugin evidenced that TSA partially rescued reduced nuclear area, and decreased branch length and abnormal end points number of both 45 days patients' neurons, but did not influence the diminished percentage of their neurons with respect to controls. Patch clamp recordings of TSA-treated post-mitotic P149 neurons showed complete/partial rescue of sodium/potassium currents and significant enhancement of neuron excitability compared to untreated replicas. Correction of abnormalities of P149 young neurons was also affected by valproic acid 1 mM for 72 h, with some variation, with respect to TSA, on the morphological parameter. These findings hold promise for development of an epigenetic therapy to attenuate RSTS patients cognitive impairment.


Assuntos
Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Adolescente , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Criança , Proteína p300 Associada a E1A/genética , Eletroencefalografia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Imageamento por Ressonância Magnética , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Mutação , Neurônios/metabolismo , Neurônios/fisiologia , Técnicas de Patch-Clamp , Síndrome de Rubinstein-Taybi/diagnóstico por imagem , Síndrome de Rubinstein-Taybi/genética , Síndrome de Rubinstein-Taybi/fisiopatologia
7.
Histochem Cell Biol ; 155(6): 637-653, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33974136

RESUMO

Histone deacetylase (HDAC) inhibitors have a potential therapeutic role for non-small cell lung cancer (NSCLC). However, more preclinical studies of HDAC inhibitors in NSCLC and normal lung epithelial cells are required to evaluate their antitumor activities and mechanisms. The bicellular tight junction molecule claudin-2 (CLDN-2) is highly expressed in lung adenocarcinoma tissues and increase the proliferation of adenocarcinoma cells. Downregulation of the tricellular tight junction molecule angulin-1/LSR induces malignancy via EGF-dependent CLDN-2 and TGF-ß-dependent cellular metabolism in human lung adenocarcinoma cells. In the present study, to investigate the detailed mechanisms of the antitumor activities of HDAC inhibitors in lung adenocarcinoma, human lung adenocarcinoma A549 cells and normal lung epithelial cells were treated with the HDAC inibitors Trichostatin A (TSA) and Quisinostat (JNJ-2648158) with or without TGF-ß. Both HDAC inhibitors increased anguin-1/LSR, decrease CLDN-2, promoted G1 arrest and prevented the migration of A549 cells. Furthermore, TSA but not Quisinostat with or without TGF-ß induced cellular metabolism indicated as the mitochondrial respiration measured using the oxygen consumption rate. In normal human lung epithelial cells, treatment with TSA and Quisinostat increased expression of LSR and CLDN-2 and decreased that of CLDN-1 with or without TGF-ß in 2D culture. Quisinostat but not TSA with TGF-ß increased CLDN-7 expression in 2D culture. Both HDAC inhibitors prevented disruption of the epithelial barrier measured as the permeability of FD-4 induced by TGF-ß in 2.5D culture. TSA and Quisinostat have potential for use in therapy for lung adenocarcinoma via changes in the expression of angulin-1/LSR and CLDN-2.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Proteínas de Junções Íntimas/antagonistas & inibidores , Antineoplásicos/química , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores de Histona Desacetilases/química , Humanos , Ácidos Hidroxâmicos/química , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas de Junções Íntimas/metabolismo
8.
Eur J Pharmacol ; 904: 174176, 2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34004213

RESUMO

Periprosthetic osteolysis (PPO) and subsequent aseptic loosening are major long-term complications after total joint arthroplasty and have become the first causes for further revision surgery. Since PPO is primarily caused by excessive bone resorption stimulated by released wear particles, osteoclast-targeted therapy is considered to be of great potential for PPO prevention and treatment. Accumulating evidences indicated that inhibition of histone deacetylases (HDACs) may represent a novel approach to suppress osteoclast differentiation. However, different inhibitors of HDACs were shown to exhibit distinct safety profiles and efficacy in inhibiting osteoclastogenesis. Quisinostat (Qst) is a hydroxamate-based histone deacetylase inhibitor, and exerts potent anti-cancer activity. However, its effect on osteoclastogenesis and its therapeutic potential in preventing PPO are still unclear. In this study, we found that Qst suppressed RANKL-induced production of TRAP-positive mature osteoclasts, expression of osteoclast-specific genes, formation of F-actin rings, and bone resorption activity at a nanomolar concentration as low as 2 nM in vitro. Furthermore, we found that as low as 30 µg/kg of Qst was sufficient to exert preventive effect on titanium particle-induced osteolysis in the murine calvarial osteolysis model. Mechanistically, we found that Qst suppressed osteoclastogenesis by interfering with NF-κB and c-Fos/NFATc1 pathways. Thus, our study revealed that Qst may serve as a potential therapeutic agent for prevention and treatment of PPO and other osteoclast-mediated diseases.


Assuntos
Reabsorção Óssea/tratamento farmacológico , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Osteogênese/efeitos dos fármacos , Osteólise/tratamento farmacológico , Actinas/metabolismo , Animais , Reabsorção Óssea/induzido quimicamente , Células Cultivadas , Modelos Animais de Doenças , Macrófagos/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/efeitos dos fármacos , Osteogênese/genética , Osteólise/induzido quimicamente , Próteses e Implantes/efeitos adversos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Crânio/diagnóstico por imagem , Crânio/efeitos dos fármacos , Crânio/patologia , Titânio/efeitos adversos
9.
J Med Chem ; 64(11): 7468-7482, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34043359

RESUMO

Nowadays, simultaneous inhibition of multiple targets through drug combination is an important anticancer strategy owing to the complex mechanism behind tumorigenesis. Recent studies have demonstrated that the inhibition of histone deacetylases (HDACs) will lead to compensated activation of a notorious cancer-related drug target, signal transducer and activator of transcription 3 (STAT3), in breast cancer through a cascade, which probably limits the anti-proliferation effect of HDAC inhibitors in solid tumors. By incorporating the pharmacophore of the HDAC inhibitor SAHA (vorinostat) into the STAT3 inhibitor pterostilbene, a series of potent pterostilbene hydroxamic acid derivatives with dual-target inhibition activity were synthesized. An excellent hydroxamate derivate, compound 14, inhibited STAT3 (KD = 33 nM) and HDAC (IC50 = 23.15 nM) with robust potency in vitro. Compound 14 also showed potent anti-proliferation ability in vivo and in vitro. Our study provides the first STAT3 and HDAC dual-target inhibitor for further exploration.


Assuntos
Antineoplásicos/química , Inibidores de Histona Desacetilases/química , Histona Desacetilases/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Sítios de Ligação , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Meia-Vida , Inibidores de Histona Desacetilases/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/química , Humanos , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/metabolismo , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Simulação de Acoplamento Molecular , Ratos , Ratos Sprague-Dawley , Fator de Transcrição STAT3/antagonistas & inibidores , Estilbenos/química , Estilbenos/metabolismo , Relação Estrutura-Atividade , Vorinostat/química , Vorinostat/metabolismo
10.
Biomed Res Int ; 2021: 5089371, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33959656

RESUMO

Triple-negative breast cancer (TNBC) is the most lethal subtype of breast cancer due to its lack of treatment options. Patients with TNBC frequently develop resistance to chemotherapy. As epigenetic-based antineoplastic drugs, histone deacetylase inhibitors (HDACis) have achieved particular efficacy in lymphoma but are less efficacious in solid tumors, and the resistance mechanism remains poorly understood. In this study, the GSE129944 microarray dataset from the Gene Expression Omnibus database was downloaded, and fold changes at the transcriptome level of a TNBC line (MDA-MB-231) after treatment with belinostat were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were used to identify the critical biological processes. Construction and analysis of the protein-protein interaction (PPI) network were performed to screen candidate genes related to cancer prognosis. A total of 465 DEGs were identified, including 240 downregulated and 225 upregulated genes. The cytokine-cytokine receptor pathway was identified as being significantly changed. Furthermore, the expression of CXCL1 was implicated as a favorable factor in the overall survival of breast cancer patients. With in vitro approaches, we also showed that belinostat could induce the expression of CXCL1 in another 2 TNBC cell lines (BT-549 and HCC-1937). We speculate that belinostat-induced CXCL1 expression could be one of the results of the stress clone evolution of cells after HDACi treatment. These findings provide new insights into clone evolution during HDACi treatment, which might guide us to a novel perspective that various mutation-targeted treatments should be implemented during the whole treatment cycle.


Assuntos
Quimiocina CXCL1/genética , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Sulfonamidas/farmacologia , Neoplasias de Mama Triplo Negativas , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Evolução Molecular , Feminino , Humanos , Prognóstico , Mapas de Interação de Proteínas/efeitos dos fármacos , Mapas de Interação de Proteínas/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , Neoplasias de Mama Triplo Negativas/diagnóstico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo
11.
Nat Commun ; 12(1): 2508, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33947865

RESUMO

Plant somatic cells can be reprogrammed into totipotent embryonic cells that are able to form differentiated embryos in a process called somatic embryogenesis (SE), by hormone treatment or through overexpression of certain transcription factor genes, such as BABY BOOM (BBM). Here we show that overexpression of the AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED 15 (AHL15) gene induces formation of somatic embryos on Arabidopsis thaliana seedlings in the absence of hormone treatment. During zygotic embryogenesis, AHL15 expression starts early in embryo development, and AH15 and other AHL genes are required for proper embryo patterning and development beyond the globular stage. Moreover, AHL15 and several of its homologs are upregulated and required for SE induction upon hormone treatment, and they are required for efficient BBM-induced SE as downstream targets of BBM. A significant number of plants derived from AHL15 overexpression-induced somatic embryos are polyploid. Polyploidisation occurs by endomitosis specifically during the initiation of SE, and is caused by strong heterochromatin decondensation induced by AHL15 overexpression.


Assuntos
Motivos AT-Hook , Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriologia , Arabidopsis/genética , Montagem e Desmontagem da Cromatina/genética , Regulação da Expressão Gênica de Plantas/genética , Técnicas de Embriogênese Somática de Plantas , Proteínas de Arabidopsis/genética , Segregação de Cromossomos/genética , Duplicação Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Resposta ao Choque Térmico/genética , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Poliploidia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima
12.
Leuk Res ; 106: 106575, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33878513

RESUMO

BACKGROUND: The metastasis and recurrence of Non-Hodgkin's lymphoma (NHL) is a major cause of morbidity and mortality. Recent work suggests that drugs capable of targeting epigenetic regulatory mechanisms may be well suited to the treatment of such disease progression. METHODS: This study was thus designed to evaluate the ability of the novel histone deacetylase (HDAC) inhibitor CUDC-101 to synergize with gemcitabine in order to kill human HUT78 and Pfeiffer NHL cells. To that end, we analyzed the viability of these NHL cells via CCK-8 assay, while the incidence of apoptosis among treated cells was evaluated via Annexin V-FITC/PI staining and by the Western blotting-mediated evaluation of proteins associate with apoptosis and related signaling pathways. RESULTS: We found that CUDC-101 and gemcitabine interacted synergistically to reduce NHL cell viability and to induce the apoptotic death of these cells via the EGFR/ PI3K/Akt and Erk pathways, which were regulated by HDAC signaling pathways. CONCLUSION: Together, our results highlight the anti-cancer properties of CUDC-101 alone or in combination with gemcitabine as an approach to inducing the apoptotic death of lymphoma cells in vitro, while also offering insight into the underlying molecular mechanisms governing this activity.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Quinazolinas/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Sinergismo Farmacológico , Humanos , Transdução de Sinais/efeitos dos fármacos
13.
Toxins (Basel) ; 13(5)2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33922825

RESUMO

Bites from elapid snakes typically result in neurotoxic symptoms in snakebite victims. Neurotoxins are, therefore, often the focus of research relating to understanding the pathogenesis of elapid bites. However, recent evidence suggests that some elapid snake venoms contain anticoagulant toxins which may help neurotoxic components spread more rapidly. This study examines the effects of venom from the West African black-necked spitting cobra (Naja nigricollis) on blood coagulation and identifies potential coagulopathic toxins. An integrated RPLC-MS methodology, coupled with nanofractionation, was first used to separate venom components, followed by MS, proteomics and coagulopathic bioassays. Coagulation assays were performed on both crude and nanofractionated N. nigricollis venom toxins as well as PLA2s and 3FTx purified from the venom. Assays were then repeated with the addition of either the phospholipase A2 inhibitor varespladib or the snake venom metalloproteinase inhibitor marimastat to assess whether either toxin inhibitor is capable of neutralizing coagulopathic venom activity. Subsequent proteomic analysis was performed on nanofractionated bioactive venom toxins using tryptic digestion followed by nanoLC-MS/MS measurements, which were then identified using Swiss-Prot and species-specific database searches. Varespladib, but not marimastat, was found to significantly reduce the anticoagulant activity of N. nigricollis venom and MS and proteomics analyses confirmed that the anticoagulant venom components mostly consisted of PLA2 proteins. We, therefore, conclude that PLA2s are the most likely candidates responsible for anticoagulant effects stimulated by N. nigricollis venom.


Assuntos
Acetatos/farmacologia , Anticoagulantes/toxicidade , Venenos Elapídicos/toxicidade , Indóis/farmacologia , Fosfolipases A2/farmacologia , Animais , Coagulação Sanguínea/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Venenos Elapídicos/antagonistas & inibidores , Cromatografia Gasosa-Espectrometria de Massas , Ácidos Hidroxâmicos/farmacologia , Naja , Proteômica
14.
Int J Mol Sci ; 22(6)2021 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-33809617

RESUMO

This study aimed to identify alternative anti-inflammatory compounds that modulate the activity of a relevant transcription factor, CCAAT/enhancer binding protein delta (C/EBPδ). C/EBPδ is a master regulator of inflammatory responses in macrophages (Mϕ) and is mainly regulated at the level of CEBPD gene transcription initiation. To screen for CEBPD-modulating compounds, we generated a THP-1-derived reporter cell line stably expressing secreted alkaline phosphatase (SEAP) under control of the defined CEBPD promoter (CEBPD::SEAP). A high-throughput screening of LOPAC®1280 and ENZO®774 libraries on LPS- and IFN-γ-activated THP-1 reporter Mϕ identified four epigenetically active hits: two bromodomain and extraterminal domain (BET) inhibitors, I-BET151 and Ro 11-1464, as well as two histone deacetylase (HDAC) inhibitors, SAHA and TSA. All four hits markedly and reproducibly upregulated SEAP secretion and CEBPD::SEAP mRNA expression, confirming screening assay reliability. Whereas BET inhibitors also upregulated the mRNA expression of the endogenous CEBPD, HDAC inhibitors completely abolished it. All hits displayed anti-inflammatory activity through the suppression of IL-6 and CCL2 gene expression. However, I-BET151 and HDAC inhibitors simultaneously upregulated the mRNA expression of pro-inflammatory IL-1ß. The modulation of CEBPD gene expression shown in this study contributes to our understanding of inflammatory responses in Mϕ and may offer an approach to therapy for inflammation-driven disorders.


Assuntos
Anti-Inflamatórios/farmacologia , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Genes Reporter , Ensaios de Triagem em Larga Escala , Inibidores de Histona Desacetilases/farmacologia , Macrófagos/metabolismo , Fosfatase Alcalina/metabolismo , Azepinas/farmacologia , Proteína delta de Ligação ao Facilitador CCAAT/antagonistas & inibidores , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Medições Luminescentes , Macrófagos/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células THP-1 , Tiofenos/farmacologia , Vorinostat/farmacologia
15.
Food Chem ; 356: 129696, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-33838605

RESUMO

This study aimed to assess the effects of acetylation levels on actomyosin disassociation and phosphorylation of lamb during incubation at 4 °C. Samples of whole proteins from lamb longissimus thoracis muscles were prepared and assigned into three treatments (high, middle and low acetylation groups). The results showed that deacetylation of myosin heavy chain and actin was inhibited by lysine deacetylase inhibitor trichostatin A and nicotinamide in this study. Phosphorylation levels of myosin heavy chain and actin were inhibited by their acetylation during incubation in vitro. Actomyosin disassociation degree in high acetylation group was significantly lower than that in middle and low acetylation groups (P < 0.05). The ATPase activity in high acetylation group was significantly higher than that in middle and low acetylation groups (P < 0.05). In conclusion, acetylation of myosin heavy chain and actin inhibited actomyosin dissociation by inhibiting their phosphorylation at 4 °C in vitro.


Assuntos
Actomiosina/metabolismo , Músculos/metabolismo , Acetilação/efeitos dos fármacos , Actinas/antagonistas & inibidores , Actinas/metabolismo , Actomiosina/antagonistas & inibidores , Animais , Sítios de Ligação , Temperatura Baixa , Ácidos Hidroxâmicos/farmacologia , Simulação de Dinâmica Molecular , Cadeias Pesadas de Miosina/antagonistas & inibidores , Cadeias Pesadas de Miosina/metabolismo , Niacinamida/farmacologia , Fosforilação , Ovinos
16.
Aging (Albany NY) ; 13(7): 9820-9837, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33744850

RESUMO

The aim of this study was to determine the effect of HDAC6 inhibition using the selective inhibitor Tubastatin A (TubA) on the regulation of tert-butyl hydroperoxide (TBHP)-treated chondrocytes and a mouse OA model. Using conventional molecular biology methods, our results showed that the level of HDAC6 increases both in the cartilage of osteoarthritis (OA) mice and TBHP-treated chondrocytes in vitro. TubA treatment effectively inhibits the expression of HDAC6, attenuates oxidative stress, reduces the level of apoptotic proteins to maintain chondrocyte survival, and suppresses the extracellular matrix (ECM) degradation. In addition, our results also revealed that HDAC6 inhibition by TubA activates autophagy in chondrocytes, whereas the protective effects of TubA were abolished by autophagy inhibitor intervention. Subsequently, the positive effects of HDAC6 inhibition by TubA were also found in a mouse OA model. Therefore, our study provide evidence that HDAC6 inhibition prevents OA development, and HDAC6 could be applied as a potential therapeutic target for OA management.


Assuntos
Autofagia/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Desacetilase 6 de Histona/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Osteoartrite/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Condrócitos/metabolismo , Modelos Animais de Doenças , Inibidores de Histona Desacetilases/uso terapêutico , Ácidos Hidroxâmicos/uso terapêutico , Indóis/uso terapêutico , Camundongos , Osteoartrite/metabolismo
17.
Mol Med Rep ; 23(5)2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33760120

RESUMO

Heart transplantation is widely used for the treatment of several heart diseases. Regulatory B cells (Breg cells) serve a critical role in immune tolerance. However, the role of Breg cells in immune tolerance in the context of allogeneic heart transplantation remains poorly understood. The present study aimed to explore the effect of histone deacetylase (HDAC) inhibitor trichostatin A (TSA)­regulated Breg on the regulation of immune tolerance in heart transplantation. By constructing anallogeneic heart transplantation mouse model, and performing flow cytometry, reverse transcription­quantitative PCR, western blotting and carboxyfluorescein succinimidyl esterstaining assays, TSA­regulated Breg cells and their effects on immune tolerance in heart transplantation were evaluated. The results demonstrated that TSA increased the frequency of CD19+CD5+CD1dhigh Breg cells both in vitro and in vivo. Moreover, TSA treatment increased the frequency of IL­10 and TGF­ß­producing CD19+CD5+CD1dhigh Breg cells, and IL­10 and TGF­ß levels in vitro and in vivo. TSA administration significantly prolonged the survival rate in a heart transplant experiment model. In addition, the IL­10 inhibitor ammonium trichloro(dioxoethylene­o,o')tellurate partially reduced the survival rate and the percentages of CD19+CD5+CD1dhigh Breg cells in mice receiving heart allografts. In contrast, anti­CD20 treatment significantly decreased the survival rate in these mice. Collectively, the present findings suggested that TSA may induce immune tolerance following heart transplantation by regulating CD19+CD5+CD1dhigh Breg cells. These results provide a theoretical basis for the prevention of immunological rejection in cardiac transplantation.


Assuntos
Linfócitos B Reguladores/imunologia , Transplante de Coração , Ácidos Hidroxâmicos/farmacologia , Tolerância Imunológica/imunologia , Animais , Antígenos CD19/imunologia , Antígenos CD1d/imunologia , Linfócitos B Reguladores/efeitos dos fármacos , Antígenos CD5/imunologia , Contagem de Células , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/imunologia , Modelos Animais de Doenças , Citometria de Fluxo , Inibidores de Histona Desacetilases/farmacologia , Humanos , Tolerância Imunológica/efeitos dos fármacos , Camundongos , Substâncias Protetoras/farmacologia
18.
Int J Mol Sci ; 22(4)2021 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-33669725

RESUMO

Trichostatin A ([R-(E,E)]-7-[4-(dimethylamino) phenyl]-N-hydroxy- 4,6-dimethyl- 7-oxo-2,4-heptadienamide, TSA) affects chromatin state through its potent histone deacetylase inhibitory activity. Interfering with the removal of acetyl groups from lysine residues in histones is one of many epigenetic regulatory processes that control gene expression. Histone deacetylase inhibition drives cells toward the differentiation stage, favoring the activation of specific genes. In this paper, we investigated the effects of TSA on H3 and H4 lysine acetylome and methylome profiling in mice embryonic stem cells (ES14), treated with trichostatin A (TSA) by using a new, untargeted approach, consisting of trypsin-limited proteolysis experiments coupled with MALDI-MS and LC-MS/MS analyses. The method was firstly set up on standard chicken core histones to probe the optimized conditions in terms of enzyme:substrate (E:S) ratio and time of proteolysis and, then, applied to investigate the global variations of the acetylation and methylation state of lysine residues of H3 and H4 histone in the embryonic stem cells (ES14) stimulated by TSA and addressed to differentiation. The proposed strategy was found in its simplicity to be extremely effective in achieving the identification and relative quantification of some of the most significant epigenetic modifications, such as acetylation and lysine methylation. Therefore, we believe that it can be used with equal success in wider studies concerning the characterization of all epigenetic modifications.


Assuntos
Células-Tronco Embrionárias/metabolismo , Histonas/metabolismo , Ácidos Hidroxâmicos/farmacologia , Lisina/metabolismo , Acetilação/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Dimetil Sulfóxido/farmacologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/química , Metilação/efeitos dos fármacos , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Peptídeos/química , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteólise/efeitos dos fármacos
19.
Nucleic Acids Res ; 49(7): 3781-3795, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33721015

RESUMO

Hydroxamate-based lysine deacetylase inhibitors (KDACis) are approved for clinical use against certain cancers. However, intrinsic and acquired resistance presents a major problem. Treatment of cells with hydroxamates such as trichostatin A (TSA) leads to rapid preferential acetylation of histone H3 already trimethylated on lysine 4 (H3K4me3), although the importance of this H3K4me3-directed acetylation in the biological consequences of KDACi treatment is not known. We address this utilizing Dictyostelium discoideum strains lacking H3K4me3 due to disruption of the gene encoding the Set1 methyltransferase or mutations in endogenous H3 genes. Loss of H3K4me3 confers resistance to TSA-induced developmental inhibition and delays accumulation of H3K9Ac and H3K14Ac. H3K4me3-directed H3Ac is mediated by Sgf29, a subunit of the SAGA acetyltransferase complex that interacts with H3K4me3 via a tandem tudor domain (TTD). We identify an Sgf29 orthologue in Dictyostelium with a TTD that specifically recognizes the H3K4me3 modification. Disruption of the gene encoding Sgf29 delays accumulation of H3K9Ac and abrogates H3K4me3-directed H3Ac. Either loss or overexpression of Sgf29 confers developmental resistance to TSA. Our results demonstrate that rapid acetylation of H3K4me3 histones regulates developmental sensitivity to TSA. Levels of H3K4me3 or Sgf29 will provide useful biomarkers for sensitivity to this class of chemotherapeutic drug.


Assuntos
Dictyostelium , Resistência a Medicamentos , Inibidores de Histona Desacetilases/farmacologia , Histonas/metabolismo , Ácidos Hidroxâmicos/farmacologia , Acetilação , Dictyostelium/efeitos dos fármacos , Dictyostelium/metabolismo
20.
Int J Mol Sci ; 22(3)2021 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-33573215

RESUMO

This study was conducted to explore whether trichostatin A-assisted epigenomic modulation (TSA-EM) can affect the expression of not only recombinant human α1,2-fucosyltransferase (rhα1,2-FT) and α-galactosidase A (rhα-Gal A) immune system enzymes but also Galα1→3Gal epitopes in ex vivo proliferating adult cutaneous fibroblast cells (ACFCs) derived from hFUT2×hGLA bi-transgenic pigs that had been produced for the needs of future xenotransplantation efforts. The ACFC lines were treated with 50 nM TSA for 24 h and then the expression profiles of rhα1,2-FT and rhα-Gal A enzymes were analyzed by Western blot and immunofluorescence. The expression profiles of the Galα1→3Gal epitope were determined by lectin blotting and lectin fluorescence. The ACFCs derived from non-transgenic (nTG) pigs were served as the negative (TSA-) and positive (TSA+) control groups. For both hFUT2×hGLA and nTG samples, the expression levels of α1,2-FT and α-Gal A proteins in TSA+ cells were more than twofold higher in comparison to TSA- cells. Moreover, a much lower expression of the Galα1→3Gal epitopes was shown in TSA- hFUT2×hGLA cells as compared to the TSA- nTG group. Interestingly, the levels of Galα1→3Gal expression in TSA-treated hFUT2×hGLA and nTG ACFCs were significantly higher than those noticed for their TSA-untreated counterparts. Summing up, ex vivo protection of effectively selected bi-transgenic ACFC lines, in which TSA-dependent epigenetic transformation triggered the enhancements in reprogrammability and subsequent expression of hFUT2 and hGLA transgenes and their corresponding transcripts, allows for cryopreservation of nuclear donor cells, nuclear-transferred female gametes, and resultant porcine cloned embryos. The latter can be used as a cryogenically conserved genetic resource of biological materials suitable for generation of bi-transgenic cloned offspring in pigs that is targeted at biomedical research in the field of cell/tissue xenotransplantation.


Assuntos
Epigênese Genética/efeitos dos fármacos , Epitopos/metabolismo , Rejeição de Enxerto/prevenção & controle , Ácidos Hidroxâmicos/farmacologia , Transplante Heterólogo/efeitos adversos , Animais , Animais Geneticamente Modificados , Linhagem Celular , Clonagem de Organismos/métodos , Criopreservação , Embrião de Mamíferos , Epitopos/genética , Epitopos/imunologia , Fibroblastos , Fucosiltransferases/genética , Fucosiltransferases/imunologia , Fucosiltransferases/metabolismo , Técnicas de Inativação de Genes , Rejeição de Enxerto/imunologia , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Pele/citologia , Suínos , Transplante Heterólogo/métodos , alfa-Galactosidase/genética , alfa-Galactosidase/imunologia , alfa-Galactosidase/metabolismo
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