Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 24.736
Filtrar
2.
Nat Commun ; 11(1): 4678, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938916

RESUMO

Diabetic foot ulcers (DFUs) are a life-threatening disease that often result in lower limb amputations and a shortened lifespan. However, molecular mechanisms contributing to the pathogenesis of DFUs remain poorly understood. We use next-generation sequencing to generate a human dataset of pathogenic DFUs to compare to transcriptional profiles of human skin and oral acute wounds, oral as a model of "ideal" adult tissue repair due to accelerated closure without scarring. Here we identify major transcriptional networks deregulated in DFUs that result in decreased neutrophils and macrophages recruitment and overall poorly controlled inflammatory response. Transcription factors FOXM1 and STAT3, which function to activate and promote survival of immune cells, are inhibited in DFUs. Moreover, inhibition of FOXM1 in diabetic mouse models (STZ-induced and db/db) results in delayed wound healing and decreased neutrophil and macrophage recruitment in diabetic wounds in vivo. Our data underscore the role of a perturbed, ineffective inflammatory response as a major contributor to the pathogenesis of DFUs, which is facilitated by FOXM1-mediated deregulation of recruitment of neutrophils and macrophages, revealing a potential therapeutic strategy.


Assuntos
Pé Diabético/genética , Pé Diabético/imunologia , Proteína Forkhead Box M1/imunologia , Cicatrização/imunologia , Adulto , Idoso , Animais , Proliferação de Células , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/imunologia , Pé Diabético/patologia , Modelos Animais de Doenças , Feminino , Proteína Forkhead Box M1/antagonistas & inibidores , Proteína Forkhead Box M1/metabolismo , Humanos , Inflamação/genética , Inflamação/imunologia , Masculino , Camundongos Endogâmicos , Pessoa de Meia-Idade , Mucosa Bucal/fisiologia , Piridinas/farmacologia , Tiofenos/farmacologia , Transcriptoma/fisiologia , Cicatrização/genética
3.
Anticancer Res ; 40(9): 5107-5114, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32878799

RESUMO

BACKGROUND/AIM: Epithelial-mesenchymal transition (EMT) via Sonic Hedgehog (Shh) signaling may be one of the mechanisms of progression of castration-resistant prostate cancer (CRPC). In this study, we investigated the possible therapeutic effect of vismodegib, a new Shh inhibitor, in a mouse CRPC model. MATERIALS AND METHODS: We determined cell proliferation, apoptosis and the expression of EMT-related genes for three prostate cancer cell lines; androgen-dependent LNCaP and independent C4-2B and PC-3 in the presence of vismodegib in vitro. Fifty mg/kg of vismodegib were orally administered into mice bearing C4-2B and PC-3 tumors, respectively every other week for 3 weeks. RESULTS: Vismodegib significantly inhibited cell proliferation and induced cell apoptosis in all cell lines in vitro (p<0.05). Vismodegib significantly inhibited EMT in CRPC cells and tumor growth in C4-2B-bearing mice compared to controls in vivo (p<0.05). Higher expression of caspase-3 and lower expression of vimentin in PC-3 and C4-2B tumors were induced by vismodegib in immunohistochemical analysis. CONCLUSION: Vismodegib inhibited cell proliferation via apoptosis and also suppressed EMT, showing anti-tumor effects in mice. Further mechanistic studies are needed to investigate the feasibility of vismodegib for CRPC treatment.


Assuntos
Anilidas/farmacologia , Antineoplásicos/farmacologia , Proteínas Hedgehog/metabolismo , Neoplasias de Próstata Resistentes à Castração/metabolismo , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Imunofenotipagem , Masculino , Camundongos , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
4.
PLoS Negl Trop Dis ; 14(9): e0008547, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898186

RESUMO

Envenomation by snakes is a major neglected human disease. Hospitalization and use of animal-derived antivenom are the primary therapeutic supports currently available. There is consensus that additional, not expensive, treatments that can be delivered even long after the snake bite are needed. We recently showed that the drug dubbed NUCC-390 shortens the time of recovery from the neuroparalysis caused by traumatic or toxic degeneration of peripheral motor neurons. These syndromes are characterized by the activation of a pro-regenerative molecular axis, consisting of the CXCR4 receptor expressed at the damaged site in neuronal axons and by the release of its ligand CXCL12α, produced by surrounding Schwann cells. This intercellular signaling axis promotes axonal growth and functional recovery from paralysis. NUCC-390 is an agonist of CXCR4 acting similarly to CXCL12α. Here, we have tested its efficacy in a murine model of neuroparalytic envenoming by a Papuan Taipan (Oxyuranus scutellatus) where a degeneration of the motor axon terminals caused by the presynaptic PLA2 toxin Taipoxin, contained in the venom, occurs. Using imaging of the neuromuscular junction and electrophysiological analysis, we found that NUCC-390 administration after injection of either the purified neuroparalytic Taipoxin or the whole Taipan venom, significantly accelerates the recovery from paralysis. These results indicate that NUCC-390, which is non-toxic in mice, should be considered for trials in humans to test its efficacy in accelerating the recovery from the peripheral neuroparalysis induced by Taipans. NUCC-390 should be tested as well in the envenomation by other snakes that cause neuroparalytic syndromes in humans. NUCC-390 could become an additional treatment, common to many snake envenomings, that can be delivered after the bite to reduce death by respiratory deficits and to shorten and improve functional recovery.


Assuntos
Venenos Elapídicos/toxicidade , Indazóis/farmacologia , Junção Neuromuscular/efeitos dos fármacos , Paralisia/terapia , Piperidinas/farmacologia , Piridinas/farmacologia , Receptores CXCR4/agonistas , Potenciais de Ação/efeitos dos fármacos , Animais , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Motores/efeitos dos fármacos
5.
Pestic Biochem Physiol ; 170: 104684, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32980064

RESUMO

As important chemical pesticides, protoporphyrinogen oxidase (PPO, EC 1.3.3.4) herbicides play a vital role in weed management. Herein, in a search for novel PPO herbicides, a series of phenoxypyridine-2-pyrrolidinone derivatives were synthesized and their herbicidal activities were tested. To confirm the structures of the newly synthesized compounds, a colorless single crystal of compound 9d was obtained and crystallographic data collected. PPO inhibition experiments showed that most compounds have PPO inhibitory effects. The half-maximal inhibitory concentration (IC50) of compound 9d and oxyfluorfen were 0.041 mg/L and 0.043 mg/L, respectively, which showed compound 9d was the most potent compound. Compound 9d reduced the Chlorophyll a (Chl a) and Chlorophyll b (Chl b) contents of Abutilon theophrasti (A. theophrasti), to 0.306 and 0.217 mg/g, respectively. Crop selectivity experiments and field trial indicated that compound 9d can potentially be used to develop post-emergence herbicides for weed control in rice, cotton, and peanut. Molecular docking studies showed that both oxyfluorfen and compound 9d can enter the PPO cavity to occupy the active site and compete with the porphyrin to block the chlorophyll synthesis process, affect photosynthesis, and eventually cause weed death. Compound 9d was found to be a promising lead compound for novel herbicide development.


Assuntos
Clorofila A , Herbicidas/farmacologia , Inibidores Enzimáticos/farmacologia , Simulação de Acoplamento Molecular , Protoporfirinogênio Oxidase , Piridinas/farmacologia , Pirrolidinonas , Relação Estrutura-Atividade
6.
Nat Commun ; 11(1): 4205, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826891

RESUMO

Triple negative breast cancer (TNBC) is a deadly form of breast cancer due to the development of resistance to chemotherapy affecting over 30% of patients. New therapeutics and companion biomarkers are urgently needed. Recognizing the elevated expression of glucose transporter 1 (GLUT1, encoded by SLC2A1) and associated metabolic dependencies in TNBC, we investigated the vulnerability of TNBC cell lines and patient-derived samples to GLUT1 inhibition. We report that genetic or pharmacological inhibition of GLUT1 with BAY-876 impairs the growth of a subset of TNBC cells displaying high glycolytic and lower oxidative phosphorylation (OXPHOS) rates. Pathway enrichment analysis of gene expression data suggests that the functionality of the E2F pathway may reflect to some extent OXPHOS activity. Furthermore, the protein levels of retinoblastoma tumor suppressor (RB1) strongly correlate with the degree of sensitivity to GLUT1 inhibition in TNBC, where RB1-negative cells are insensitive to GLUT1 inhibition. Collectively, our results highlight a strong and targetable RB1-GLUT1 metabolic axis in TNBC and warrant clinical evaluation of GLUT1 inhibition in TNBC patients stratified according to RB1 protein expression levels.


Assuntos
Transportador de Glucose Tipo 1/antagonistas & inibidores , Transportador de Glucose Tipo 1/metabolismo , Proteínas de Ligação a Retinoblastoma/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais , Neoplasias da Mama/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Transportador de Glucose Tipo 1/genética , Humanos , Camundongos , Fosforilação Oxidativa , Proteômica , Pirazóis/farmacologia , Piridinas/farmacologia , Quinolinas , RNA Mensageiro/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Ubiquitina-Proteína Ligases/genética
7.
Proc Natl Acad Sci U S A ; 117(33): 20127-20138, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747535

RESUMO

Medulloblastoma is the most common malignant brain tumor in children. Here we describe a medulloblastoma model using Induced pluripotent stem (iPS) cell-derived human neuroepithelial stem (NES) cells generated from a Gorlin syndrome patient carrying a germline mutation in the sonic hedgehog (SHH) receptor PTCH1. We found that Gorlin NES cells formed tumors in mouse cerebellum mimicking human medulloblastoma. Retransplantation of tumor-isolated NES (tNES) cells resulted in accelerated tumor formation, cells with reduced growth factor dependency, enhanced neurosphere formation in vitro, and increased sensitivity to Vismodegib. Using our model, we identified LGALS1 to be a GLI target gene that is up-regulated in both Gorlin tNES cells and SHH-subgroup of medulloblastoma patients. Taken together, we demonstrate that NES cells derived from Gorlin patients can be used as a resource to model medulloblastoma initiation and progression and to identify putative targets.


Assuntos
Proteínas Hedgehog/metabolismo , Meduloblastoma/genética , Células-Tronco Neurais/fisiologia , Anilidas/farmacologia , Animais , Síndrome do Nevo Basocelular/genética , Síndrome do Nevo Basocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Galectina 1/genética , Galectina 1/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Hedgehog/genética , Humanos , Camundongos , Neoplasias Experimentais , Receptor Patched-1/genética , Piridinas/farmacologia
8.
PLoS One ; 15(8): e0237442, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790767

RESUMO

BACKGROUND: Prostate cancer (PCa) is the most commonly diagnosed cancer and the second leading cause of cancer-related deaths among adult males globally. The poor prognosis of PCa is largely due to late diagnosis of the disease when it has already progressed to an advanced stage marked by androgen-independence, thus necessitating new strategies for early detection and treatment. We construe that these direly needed advances are limited by our poor understanding of early events in the progression of PCa and that would thus represent ideal targets for early intervention. To begin to fill this void, we interrogated molecular "oncophenotypes" that embody the transition of PCa from an androgen-dependent (AD) to-independent (AI) state. METHODS: To accomplish this aim, we used our previously established AD and AI murine PCa cell lines, PLum-AD and PLum-AI, respectively, which recapitulate primary and progressive PCa morphologically and molecularly. We statistically surveyed global gene expressions in these cell lines by microarray analysis. Differential profiles were functionally interrogated by pathways, gene set enrichment and topological gene network analyses. RESULTS: Gene expression analysis of PLum-AD and PLum-AI transcriptomes (n = 3 each), revealed 723 differentially expressed genes (392 upregulated and 331 downregulated) in PLum-AI compared to PLum-AD cells. Gene set analysis demonstrated enrichment of biological functions and pathways in PLum-AI cells that are central to tumor aggressiveness including cell migration and invasion facilitated by epithelial-to-mesenchymal transition (EMT). Further analysis demonstrated that the p38 mitogen-activated protein kinase (MAPK) was predicted to be significantly activated in the PLum-AI cells, whereas gene sets previously associated with favorable response to the p38 inhibitor SB203580 were attenuated (i.e., inversely enriched) in the PLum-AI cells, suggesting that these aggressive cells may be therapeutically vulnerable to p38 inhibition. Gene set and gene-network analysis also alluded to activation of other signaling networks particularly those associated with enhanced EMT, inflammation and immune function/response including, but not limited to Tnf, IL-6, Mmp 2, Ctgf, and Ptges. Accordingly, we chose SB203580 and IL-6 to validate their effect on PLum-AD and PLum-AI. Some of the common genes identified in the gene-network analysis were validated at the molecular and functional level. Additionally, the vulnerability to SB203580 and the effect of IL-6 were also validated on the stem/progenitor cell population using the sphere formation assay. CONCLUSIONS: In summary, our study highlights pathways associated with an augmented malignant phenotype in AI cells and presents new high-potential targets to constrain the aggressive malignancy seen in the castration-resistant PCa.


Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Imidazóis/farmacologia , Interleucina-6/farmacologia , Neoplasias da Próstata/patologia , Piridinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Androgênios/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Progressão da Doença , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Redes Reguladoras de Genes/genética , Imidazóis/uso terapêutico , Interleucina-6/uso terapêutico , Masculino , Camundongos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Piridinas/uso terapêutico , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
9.
Nat Commun ; 11(1): 4051, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792548

RESUMO

The RNA genome of the human immunodeficiency virus (HIV) is reverse-transcribed into DNA and integrated into the host genome, resulting in latent infections that are difficult to clear. Here we show an approach to eradicate HIV infections by selective elimination of host cells harboring replication-competent HIV (SECH), which includes viral reactivation, induction of cell death, inhibition of autophagy and the blocking of new infections. Viral reactivation triggers cell death specifically in HIV-1-infected T cells, which is promoted by agents that induce apoptosis and inhibit autophagy. SECH treatments can clear HIV-1 in >50% mice reconstituted with a human immune system, as demonstrated by the lack of viral rebound after withdrawal of treatments, and by adoptive transfer of treated lymphocytes into uninfected humanized mice. Moreover, SECH clears HIV-1 in blood samples from HIV-1-infected patients. Our results suggest a strategy to eradicate HIV infections by selectively eliminating host cells capable of producing HIV.


Assuntos
Infecções por HIV/prevenção & controle , HIV-1/patogenicidade , Animais , Antígenos CD34/metabolismo , Apoptose/efeitos dos fármacos , Autofagia/fisiologia , Azepinas/farmacologia , Linfócitos T CD4-Positivos/metabolismo , Humanos , Camundongos , Organofosfatos/farmacologia , Piperazinas/farmacologia , Piridinas/farmacologia , Pirimidinonas/farmacologia , RNA Viral/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Triazóis/farmacologia
10.
Nat Commun ; 11(1): 4249, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32843618

RESUMO

Aberrant cell cycle machinery and loss of the CDKN2A tumor suppressor locus make CDK4/6 a potential target in pancreatic ductal adenocarcinoma (PDAC). However, a vast majority of PDAC cases do not harbor a durable response to monotherapy of CDK4/6 inhibitor. Utilizing remote loading to co-encapsulate CDK4/6 inhibitor palbociclib (PAL) and an autophagy inhibitor hydroxychloroquine (HCQ), we demonstrate a ratiometrically designed mesoporous silica nanoformulation with synergistic efficacy in subcutaneous and orthotopic PDAC mouse models. The synergism is attributed to the effective intratumoral buildup of PAL/HCQ, which otherwise exhibit distinctly different circulatory and biodistribution profile. PAL/HCQ co-delivery nanoparticles lead to the most effective shrinkage of PDAC compared to various controls, including free drug mixture. Immunohistochemistry reveals that PAL/HCQ co-delivery nanoparticles trigger anti-apoptotic pathway after repetitive intravenous administrations in mice. When combined with a Bcl inhibitor, the performance of co-delivery nanoparticles is further improved, leading to a long-lasting anti-PDAC effect in vivo.


Assuntos
Autofagia/efeitos dos fármacos , Carcinoma Ductal Pancreático/tratamento farmacológico , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Sistemas de Liberação de Medicamentos , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Sinergismo Farmacológico , Humanos , Hidroxicloroquina/administração & dosagem , Hidroxicloroquina/química , Hidroxicloroquina/farmacologia , Camundongos , Nanopartículas/administração & dosagem , Nanopartículas/química , Piperazinas/administração & dosagem , Piperazinas/química , Piperazinas/farmacologia , Piridinas/administração & dosagem , Piridinas/química , Piridinas/farmacologia , Dióxido de Silício/administração & dosagem , Dióxido de Silício/química , Dióxido de Silício/farmacologia , Resultado do Tratamento
11.
Proc Natl Acad Sci U S A ; 117(33): 19854-19865, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32759214

RESUMO

The blood-retina barrier and blood-brain barrier (BRB/BBB) are selective and semipermeable and are critical for supporting and protecting central nervous system (CNS)-resident cells. Endothelial cells (ECs) within the BRB/BBB are tightly coupled, express high levels of Claudin-5 (CLDN5), a junctional protein that stabilizes ECs, and are important for proper neuronal function. To identify novel CLDN5 regulators (and ultimately EC stabilizers), we generated a CLDN5-P2A-GFP stable cell line from human pluripotent stem cells (hPSCs), directed their differentiation to ECs (CLDN5-GFP hPSC-ECs), and performed flow cytometry-based chemogenomic library screening to measure GFP expression as a surrogate reporter of barrier integrity. Using this approach, we identified 62 unique compounds that activated CLDN5-GFP. Among them were TGF-ß pathway inhibitors, including RepSox. When applied to hPSC-ECs, primary brain ECs, and retinal ECs, RepSox strongly elevated barrier resistance (transendothelial electrical resistance), reduced paracellular permeability (fluorescein isothiocyanate-dextran), and prevented vascular endothelial growth factor A (VEGFA)-induced barrier breakdown in vitro. RepSox also altered vascular patterning in the mouse retina during development when delivered exogenously. To determine the mechanism of action of RepSox, we performed kinome-, transcriptome-, and proteome-profiling and discovered that RepSox inhibited TGF-ß, VEGFA, and inflammatory gene networks. In addition, RepSox not only activated vascular-stabilizing and barrier-establishing Notch and Wnt pathways, but also induced expression of important tight junctions and transporters. Taken together, our data suggest that inhibiting multiple pathways by selected individual small molecules, such as RepSox, may be an effective strategy for the development of better BRB/BBB models and novel EC barrier-inducing therapeutics.


Assuntos
Células Endoteliais/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Barreira Hematorretiniana/efeitos dos fármacos , Barreira Hematorretiniana/metabolismo , Diferenciação Celular , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Claudina-5/genética , Claudina-5/metabolismo , Avaliação Pré-Clínica de Medicamentos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Edição de Genes , Genoma , Humanos , Camundongos , Camundongos Knockout , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Pirazóis/farmacologia , Piridinas/farmacologia , Junções Íntimas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
12.
Gene ; 760: 144989, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32717307

RESUMO

Kinesin 14 family member KIFC1 is a mitotic kinesin which contains a C-terminal motor domain and plays a vital role for clustering the amplified centrosomes. Overexpression of KIFC1 in prostate cancer (PCa) cells showed resistance to docetaxel (DTX). The present study revealed that small KIFC1 inhibitor AZ82 suppresed the transcription and translation of KIFC1 significantly in PCa cells. AZ82 inhibited the KIFC1 expression both in the cytoplasm and nucleus of PCa cells. Inhibition of KIFC1 by AZ82 caused multipolar mitosis in PCa cells via de-clustering the amplified centrosomes and decreased the rate of cancer cell growth and proliferation. Moreover, depletion of KIFC1 reduced cells entering the cell cycle and caused PCa cells death through apoptosis by increasing the expression of Bax and Cytochrome C. Thereby, KIFC1 silencing and inhibition decreased the PCa cells survival by inducing multipolar mitosis as well as apoptosis, suggesting inhibition of KIFC1 using AZ82 might be a strategy to treat PCa by controlling the cancer cell proliferation.


Assuntos
Alanina/análogos & derivados , Centrossomo/efeitos dos fármacos , Cinesina/antagonistas & inibidores , Neoplasias da Próstata/tratamento farmacológico , Piridinas/farmacologia , Alanina/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Centrossomo/metabolismo , Dineínas/metabolismo , Humanos , Cinesina/genética , Cinesina/metabolismo , Masculino , Mitose/efeitos dos fármacos , Miosinas/metabolismo , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia
14.
PLoS One ; 15(7): e0236175, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32697798

RESUMO

Adenoviruses cause upper respiratory infections, conjunctivitis, keratitis, and gastrointestinal illness. These can be fatal in immunocompromised individuals. Adenoviruses have also been engineered into viral vectors to deliver therapeutic genes or induce immunity as vaccine carriers. The success of ocular gene therapy is driven partly by the immunologic and biochemical influences of the intraocular environment. We have shown that versican and hyaluronan modulate adenoviral vector transgene expression through CD44 signaling. Herein we explored the role of these pathways on virus replication and viral protein expression of wild type adenovirus. We report that the addition of vitreous humor (which contains both versican and hyaluronan) increases viral hexon protein levels. Vitreous humor also increased wild type adenovirus DNA replication in vitro. Metalloproteinase and γ-secretase inhibitors, which inhibit CD44 proteolytic activation, blocked adenoviral replication in vitro. Similarly, protein kinase C and RhoA kinase inhibitors, both proteins associated with CD44 mediated pathways, also inhibited wild type adenoviral replication in vitro. Application of metalloproteinase and γ-secretase inhibitors to human conjunctival explants sharply decreased adenoviral vector gene expression. Our results demonstrate that pharmacologic delivery of these inhibitors is easily achievable. The inhibition of these enzymes should be explored as potential therapies of wild type adenoviral infections.


Assuntos
Infecções por Adenoviridae/tratamento farmacológico , Adenoviridae/efeitos dos fármacos , Antivirais/farmacologia , Vetores Genéticos/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Adenoviridae/fisiologia , Infecções por Adenoviridae/virologia , Administração Oftálmica , Amidas/farmacologia , Amidas/uso terapêutico , Antivirais/uso terapêutico , Túnica Conjuntiva/metabolismo , DNA Viral/genética , DNA Viral/isolamento & purificação , Diaminas/farmacologia , Diaminas/uso terapêutico , Dipeptídeos/farmacologia , Dipeptídeos/uso terapêutico , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/fisiologia , Células HeLa , Humanos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Indóis/farmacologia , Indóis/uso terapêutico , Maleimidas/farmacologia , Maleimidas/uso terapêutico , Metaloproteases/antagonistas & inibidores , Metaloproteases/metabolismo , Permeabilidade , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Proteólise/efeitos dos fármacos , Piridinas/farmacologia , Piridinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Tiazóis/farmacologia , Tiazóis/uso terapêutico , Versicanas/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Corpo Vítreo/metabolismo , Quinases Associadas a rho/metabolismo
15.
Life Sci ; 257: 118073, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32663574

RESUMO

AIMS: The preservation of pancreatic beta-cell function is crucial for the treatment of type 2 diabetes. Inhibition of class I histone deacetylase (HDAC) has been proved to protect beta-cells from palmitate- or cytokine-induced apoptosis and increase insulin secretion. However, the underlying molecular mechanism is unclear. MAIN METHODS: Rat islets were isolated for insulin secretion, real-time PCR, RNA- sequencing, ChIP-PCR, and oxygen consumption rate analysis after treated with the HDAC1 and HDAC3 inhibitor MS-275. KEY FINDINGS: MS-275 pretreatment significantly potentiated insulin secretion from rat islets. RNA-sequencing revealed that multiple signaling pathways were involved in MS-275-regulated islet function. Cacna1g and Adcy1 in calcium and cAMP signaling pathways were up-regulated in MS-275-treated islets, which was validated by real-time PCR. The expressions of the two genes displayed a similar increase in islets isolated from mice treated with MS-275. Knockdown of HDAC1 elevated Cacna1g and Adcy1 expressions in islets. ChIP-sequencing analysis showed that the pan-HDAC inhibitor sodium butyrate increased H3K27 acetylation level in the upstream region of Adcy1 and the promoter region of Cacna1g. ChIP-PCR revealed a similar result in MS-275-treated rat islets. However, MS-275 had minor effect on glucose-induced oxygen consumption rate in rat islets. Unlike glucose, MS-275 did not alter the expressions of glucose-sensitive genes such as Glut2 and Gck, but elevated intracellular Ca2+ concentration in beta-cells. SIGNIFICANCE: Our findings support the notion that MS-275-potentiated insulin secretion is involved in calcium and cAMP signaling-mediated gene expressions independent of glucose oxidation. Therefore, HDAC inhibition may serve as a therapeutic strategy for type 2 diabetes.


Assuntos
Benzamidas/farmacologia , Glucose/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Secreção de Insulina/efeitos dos fármacos , Piridinas/farmacologia , Animais , Cálcio/metabolismo , AMP Cíclico/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/fisiopatologia , Técnicas de Silenciamento de Genes , Histona Desacetilase 1/antagonistas & inibidores , Histona Desacetilases/efeitos dos fármacos , Células Secretoras de Insulina , Masculino , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
16.
Proc Natl Acad Sci U S A ; 117(30): 17808-17819, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32661168

RESUMO

p53 is the most frequently mutated, well-studied tumor-suppressor gene, yet the molecular basis of the switch from p53-induced cell-cycle arrest to apoptosis remains poorly understood. Using a combination of transcriptomics and functional genomics, we unexpectedly identified a nodal role for the caspase-8 paralog and only human pseudo-caspase, FLIP(L), in regulating this switch. Moreover, we identify FLIP(L) as a direct p53 transcriptional target gene that is rapidly up-regulated in response to Nutlin-3A, an MDM2 inhibitor that potently activates p53. Genetically or pharmacologically inhibiting expression of FLIP(L) using siRNA or entinostat (a clinically relevant class-I HDAC inhibitor) efficiently promoted apoptosis in colorectal cancer cells in response to Nutlin-3A, which otherwise predominantly induced cell-cycle arrest. Enhanced apoptosis was also observed when entinostat was combined with clinically relevant, p53-activating chemotherapy in vitro, and this translated into enhanced in vivo efficacy. Mechanistically, FLIP(L) inhibited p53-induced apoptosis by blocking activation of caspase-8 by the TRAIL-R2/DR5 death receptor; notably, this activation was not dependent on receptor engagement by its ligand, TRAIL. In the absence of caspase-8, another of its paralogs, caspase-10 (also transcriptionally up-regulated by p53), induced apoptosis in Nutlin-3A-treated, FLIP(L)-depleted cells, albeit to a lesser extent than in caspase-8-proficient cells. FLIP(L) depletion also modulated transcription of canonical p53 target genes, suppressing p53-induced expression of the cell-cycle regulator p21 and enhancing p53-induced up-regulation of proapoptotic PUMA. Thus, even in the absence of caspase-8/10, FLIP(L) silencing promoted p53-induced apoptosis by enhancing PUMA expression. Thus, we report unexpected, therapeutically relevant roles for FLIP(L) in determining cell fate following p53 activation.


Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Benzamidas/farmacologia , Caspase 8/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Sinergismo Farmacológico , Regulação da Expressão Gênica , Humanos , Imidazóis/metabolismo , Modelos Biológicos , Piperazinas/metabolismo , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Piridinas/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteína Supressora de Tumor p53/genética
17.
Proc Natl Acad Sci U S A ; 117(29): 17389-17398, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32632005

RESUMO

Wolfram syndrome is a rare multisystem disease characterized by childhood-onset diabetes mellitus and progressive neurodegeneration. Most cases are attributed to pathogenic variants in a single gene, Wolfram syndrome 1 (WFS1). There currently is no disease-modifying treatment for Wolfram syndrome, as the molecular consequences of the loss of WFS1 remain elusive. Because diabetes mellitus is the first diagnosed symptom of Wolfram syndrome, we aimed to further examine the functions of WFS1 in pancreatic ß cells in the context of hyperglycemia. Knockout (KO) of WFS1 in rat insulinoma (INS1) cells impaired calcium homeostasis and protein kinase B/Akt signaling and, subsequently, decreased cell viability and glucose-stimulated insulin secretion. Targeting calcium homeostasis with reexpression of WFS1, overexpression of WFS1's interacting partner neuronal calcium sensor-1 (NCS1), or treatment with calpain inhibitor and ibudilast reversed deficits observed in WFS1-KO cells. Collectively, our findings provide insight into the disease mechanism of Wolfram syndrome and highlight new targets and drug candidates to facilitate the development of a treatment for this disorder and similar diseases.


Assuntos
Antineoplásicos/farmacologia , Proteínas de Ligação a Calmodulina/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Piridinas/farmacologia , Síndrome de Wolfram/tratamento farmacológico , Animais , Cálcio/metabolismo , Proteínas de Ligação a Calmodulina/genética , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Glicoproteínas , Homeostase , Hiperglicemia , Secreção de Insulina , Proteínas de Membrana/genética , Receptor de Insulina , Transcriptoma , Síndrome de Wolfram/genética
18.
Am J Physiol Gastrointest Liver Physiol ; 319(2): G227-G237, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32597706

RESUMO

Antibiotic treatment is a standard therapy for Clostridioides difficile infection, but dysbiosis of the gut microbiota due to antibiotic exposure is also a major risk factor for the disease. Following an initial episode of C. difficile infection, a relentless cycle of recurrence can occur, where persistent treatment-related dysbiosis predisposes the patient to subsequent relapse. This study uses a longitudinal study design to compare the effects of a narrow-spectrum (ridinilazole) or broad-spectrum antibiotic (vancomycin) on intestinal bile acid profiles and their associations with gut bacteria over the course of C. difficile infection treatment. At the end of treatment (day 10), subjects receiving vancomycin showed a nearly 100-fold increase in the ratio of conjugated to secondary bile acids in their stool compared with baseline, whereas subjects receiving ridinilazole maintained this ratio near baseline levels. Correlation analysis detected significant positive associations between secondary bile acids and several Bacteroidales and Clostridiales families. These families were depleted in the vancomycin group but preserved at near-baseline abundance in the ridinilazole group. Enterobacteriaceae, which expanded to a greater extent in the vancomycin group, correlated negatively and positively with secondary and conjugated primary bile acids, respectively. Bile acid ratios at the end of treatment were significantly different between those who recurred and those who did not. These results indicate that a narrow-spectrum antibiotic maintains an intestinal bile acid profile associated with a lowered risk of recurrence.NEW & NOTEWORTHY This is the first study to demonstrate in humans the relationships between Clostridioides difficile antibiotic treatment choice and bile acid metabolism both during therapy and after treatment cessation. The results show a microbiota- and metabolome-preserving property of a novel narrow-spectrum agent that correlates with the agent's favorable sustained clinical response rates compared with broad-spectrum antibiotic treatment.


Assuntos
Antibacterianos/farmacologia , Benzimidazóis/farmacologia , Ácidos e Sais Biliares/química , Clostridiales/efeitos dos fármacos , Microbioma Gastrointestinal/efeitos dos fármacos , Piridinas/farmacologia , Ácidos e Sais Biliares/metabolismo , Fezes/química , Microbioma Gastrointestinal/fisiologia , Humanos
20.
J Headache Pain ; 21(1): 71, 2020 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-32522164

RESUMO

Migraine is a leading cause of disability worldwide, but it is still underdiagnosed and undertreated. Research on the pathophysiology of this neurological disease led to the discovery that calcitonin gene-related peptide (CGRP) is a key neuropeptide involved in pain signaling during a migraine attack. CGRP-mediated neuronal sensitization and glutamate-based second- and third-order neuronal signaling may be an important component involved in migraine pain. The activation of several serotonergic receptor subtypes can block the release of CGRP, other neuropeptides, and neurotransmitters, and can relieve the symptoms of migraine. Triptans were the first therapeutics developed for the treatment of migraine, working through serotonin 5-HT1B/1D receptors. The discovery that the serotonin 1F (5-HT1F) receptor was expressed in the human trigeminal ganglion suggested that this receptor subtype may have a role in the treatment of migraine. The 5-HT1F receptor is found on terminals and cell bodies of trigeminal ganglion neurons and can modulate the release of CGRP from these nerves. Unlike 5-HT1B receptors, the activation of 5-HT1F receptors does not cause vasoconstriction.The potency of different serotonergic agonists towards 5-HT1F was correlated in an animal model of migraine (dural plasma protein extravasation model) leading to the development of lasmiditan. Lasmiditan is a newly approved acute treatment for migraine in the United States and is a lipophilic, highly selective 5-HT1F agonist that can cross the blood-brain barrier and act at peripheral nervous system (PNS) and central nervous system (CNS) sites.Lasmiditan activation of CNS-located 5-HT1F receptors (e.g., in the trigeminal nucleus caudalis) could potentially block the release of CGRP and the neurotransmitter glutamate, thus preventing and possibly reversing the development of central sensitization. Activation of 5-HT1F receptors in the thalamus can block secondary central sensitization of this region, which is associated with progression of migraine and extracephalic cutaneous allodynia. The 5-HT1F receptors are also elements of descending pain modulation, presenting another site where lasmiditan may alleviate migraine. There is emerging evidence that mitochondrial dysfunction might be implicated in the pathophysiology of migraine, and that 5-HT1F receptors can promote mitochondrial biogenesis. While the exact mechanism is unknown, evidence suggests that lasmiditan can alleviate migraine through 5-HT1F agonist activity that leads to inhibition of neuropeptide and neurotransmitter release and inhibition of PNS trigeminovascular and CNS pain signaling pathways.


Assuntos
Benzamidas/farmacologia , Transtornos de Enxaqueca/fisiopatologia , Piperidinas/farmacologia , Piridinas/farmacologia , Receptores de Serotonina , Agonistas do Receptor de Serotonina/farmacologia , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Humanos , Neurônios/metabolismo , Gânglio Trigeminal/metabolismo , Gânglio Trigeminal/fisiopatologia , Triptaminas , Vasoconstrição/efeitos dos fármacos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA