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1.
Elife ; 82019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31453809

RESUMO

Spinal cord patterning is orchestrated by multiple cell signalling pathways. Neural progenitors are maintained by Notch signalling, whereas ventral neural fates are specified by Hedgehog (Hh) signalling. However, how dynamic interactions between Notch and Hh signalling drive the precise pattern formation is still unknown. We applied the PHRESH (PHotoconvertible REporter of Signalling History) technique to analyse cell signalling dynamics in vivo during zebrafish spinal cord development. This approach reveals that Notch and Hh signalling display similar spatiotemporal kinetics throughout spinal cord patterning. Notch signalling functions upstream to control Hh response of neural progenitor cells. Using gain- and loss-of-function tools, we demonstrate that this regulation occurs not at the level of upstream regulators or primary cilia, but rather at the level of Gli transcription factors. Our results indicate that Notch signalling maintains Hh responsiveness of neural progenitors via a Gli-dependent mechanism in the spinal cord.


Assuntos
Padronização Corporal , Ouriços-Cacheiros/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Medula Espinal/embriologia , Proteína GLI1 em Dedos de Zinco/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento , Peixe-Zebra
2.
J Agric Food Chem ; 66(45): 11926-11934, 2018 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-30354116

RESUMO

Obesity is a risk factor for numerous metabolic disorders. In this study, we investigated the effects of the isothiocyanates sulforaphane (SA) and sulforaphene (SE) on adipogenesis in 3T3-L1 adipocytes. SE, a compound that is abundant in radish, inhibited adipogenesis by suppressing the adipogenic transcription factors peroxisome proliferator-activated receptor γ (PPARγ, 69.2 ± 2.4%, P < 0.05) and CCAAT/enhancer-binding protein α (C/EBPα, 36.1 ± 3.1%, P < 0.05), thereby reducing fat accumulation in 3T3-L1 adipocytes (45.6 ± 2.7%, P < 0.05); SA was less effective. SE exerted these activities through the activation of the Hedgehog (Hh) signaling pathway by restoring Smo ((2.1 ± 0.2)-fold, P < 0.05) and Gli1 ((2.8 ± 0.1)-fold, P < 0.05) expression, which was suppressed by adipogenic signals. These effects of SE were abrogated by treatment with the Hh inhibitor vismodegib. Thus, SE inhibits adipocyte differentiation via Hh signaling and may be an effective natural agent for preventing adipocyte hyperplasia and obesity.


Assuntos
Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Fármacos Antiobesidade/farmacologia , Ouriços-Cacheiros/metabolismo , Isotiocianatos/farmacologia , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Ouriços-Cacheiros/genética , Camundongos , PPAR gama/genética , PPAR gama/metabolismo , Transdução de Sinais/efeitos dos fármacos
3.
Elife ; 72018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-30014845

RESUMO

Understanding how patterning influences cell behaviors to generate three dimensional morphologies is a central goal of developmental biology. Additionally, comparing these regulatory mechanisms among morphologically diverse tissues allows for rigorous testing of evolutionary hypotheses. Zebrafish skin is endowed with a coat of precisely patterned bony scales. We use in-toto live imaging during scale development and manipulations of cell signaling activity to elucidate core features of scale patterning and morphogenesis. These analyses show that scale development requires the concerted activity of Wnt/ß-catenin, Ectodysplasin (Eda) and Fibroblast growth factor (Fgf) signaling. This regulatory module coordinates Hedgehog (HH) dependent collective cell migration during epidermal invagination, a cell behavior not previously implicated in skin appendage morphogenesis. Our analyses demonstrate the utility of zebrafish scale development as a tractable system in which to elucidate mechanisms of developmental patterning and morphogenesis, and suggest a single, ancient origin of skin appendage patterning mechanisms in vertebrates.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Pele/embriologia , Pesos e Medidas , Via de Sinalização Wnt , Peixe-Zebra/embriologia , beta Catenina/metabolismo , Animais , Padronização Corporal , Movimento Celular , Ectodisplasinas/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Ouriços-Cacheiros/metabolismo , Microscopia Intravital , Morfogênese
4.
J Cell Sci ; 131(15)2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29930086

RESUMO

Hedgehog (Hh) transduces signals by promoting cell surface accumulation and activation of the G-protein-coupled receptor (GPCR)-family protein Smoothened (Smo) in Drosophila, but the molecular mechanism underlying the regulation of Smo trafficking remains poorly understood. Here, we identified the Cul4-DDB1 E3 ubiquitin ligase complex as being essential for Smo ubiquitylation and cell surface clearance. We found that the C-terminal intracellular domain of Smo recruits Cul4-DDB1 through the ß subunit of trimeric G protein (Gß), and that Cul4-DDB1-Gß promotes the ubiquitylation of both Smo and its binding partner G-protein-coupled-receptor kinase 2 (Gprk2) and induces the internalization and degradation of Smo. Hh dissociates Cul4-DDB1 from Smo by recruiting the catalytic subunit of protein kinase A (PKA) to phosphorylate DDB1, which disrupts its interaction with Gß. Inactivation of the Cul4-DDB1 complex resulted in elevated Smo cell surface expression, whereas an excessive amount of Cul4-DDB1 blocked Smo accumulation and attenuated Hh pathway activation. Taken together, our study identifies an E3 ubiquitin ligase complex targeting Smo for ubiquitylation and provides new insight into how Hh signaling regulates Smo trafficking and cell surface expression.


Assuntos
Proteínas Culina/metabolismo , Proteínas de Drosophila/metabolismo , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , Animais , Proteínas Culina/genética , Proteínas de Drosophila/genética , Ouriços-Cacheiros/genética , Ouriços-Cacheiros/metabolismo , Ligação Proteica , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética
5.
J Immunol Res ; 2018: 7438608, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29675438

RESUMO

Fibroblast growth factor receptor-like-1 (FGFRL1) has been identified as the fifth fibroblast growth factor receptor. So far, little is known about its biological functions, particularly in cancer development. Here, for the first time, we demonstrated the roles of FGFRL1 in ovarian carcinoma (OC). An array and existing databases were used to investigate the expression profile of FGFRL1 and the relationship between FGFRL1 expression and clinicopathological parameters. FGFRL1 was significantly upregulated in OC patients, and high FGFRL1 expression was correlated with poor prognosis. In vitro cell proliferation, apoptosis and migration assays, and in vivo subcutaneous xenograft tumor models were used to determine the role of FGFRL1. Loss of function of FGFRL1 significantly influenced cell proliferation, apoptosis, and migration of OC cells in vitro and tumor growth in vivo. Chromatin immunoprecipitation PCR analysis and microarray hybridization were performed to uncover the mechanism. FGFRL1 expression could be induced by hypoxia through hypoxia-inducible factor 1α, which directly binds to the promoter elements of FGFRL1. FGFRL1 promoted tumor progression by crosstalk with Hedgehog (Hh) signaling. Taken together, FGFRL1 is a potential predictor and plays an important role in tumor growth and Hh signaling which could serve as potential therapeutic targets for the treatment of OC.


Assuntos
Carcinogênese , Neoplasias Ovarianas/metabolismo , Receptor Tipo 5 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Estudos de Coortes , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Ouriços-Cacheiros/metabolismo , Xenoenxertos , Humanos , Camundongos , Análise em Microsséries , Transplante de Neoplasias , Neoplasias Ovarianas/patologia , RNA Interferente Pequeno/genética , Receptor Tipo 5 de Fator de Crescimento de Fibroblastos/genética , Transdução de Sinais
6.
Pharm Res ; 35(1): 17, 2018 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-29305793

RESUMO

PURPOSE: The aim of this study was to determine whether co-administration of hedgehog (Hh) pathway inhibitor cyclopamine (CYP) and microtubule stabilizer docetaxel (DTX) as polymer-drug conjugates, methoxy poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylenecarbonate-graft-dodecanol-graft-cyclopamine) (P-CYP) and methoxy poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol-graft-docetaxel) (P-DTX) could synergistically inhibit orthotopic pancreatic tumor growth in NSG mice. METHODS: P-DTX and P-CYP were synthesized from mPEG-b-PCC through carbodiimide coupling reaction and characterized by 1H-NMR. The micelles were prepared by film hydration and particle size was measured by dynamic light scattering (DLS). Cytotoxicity, apoptosis and cell cycle analysis of P-DTX and P-CYP were evaluated in MIA PaCa-2 cells. In vivo efficacy of P-DTX and P-CYP were evaluated in NSG mice bearing MIA PaCa-2 cells derived orthotopic pancreatic tumor. RESULTS: P-CYP and P-DTX self-assembled into micelles of <90 nm and their combination therapy efficiently inhibited the proliferation of MIA PaCa-2 cells, induced apoptosis and cell cycle arrest at M-phase more efficiently than P-CYP and P-DTX monotherapies. Furthermore, the combination therapy of P-CYP and P-DTX significantly reduced Hh component expression compared to P-CYP alone as determined by Western blot analysis. Lastly, the combination therapy induced greater inhibition of orthotopic pancreatic tumor growth in NSG mice compared to their monotherapies. CONCLUSION: Combination of polymer conjugated anticancer drug (P-DTX) with polymer conjugated Hh inhibitor (P-CYP) enhanced pancreatic cancer cell killing, apoptosis as well as in vivo tumor growth inhibition with no obvious toxicities.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Polímeros/química , Taxoides/farmacologia , Alcaloides de Veratrum/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Docetaxel , Portadores de Fármacos , Liberação Controlada de Fármacos , Ouriços-Cacheiros/metabolismo , Humanos , Camundongos , Micelas , Microtúbulos/metabolismo , Metástase Neoplásica , Neoplasias Pancreáticas/patologia , Espectroscopia de Prótons por Ressonância Magnética , Taxoides/administração & dosagem , Taxoides/química , Alcaloides de Veratrum/administração & dosagem , Alcaloides de Veratrum/química
7.
Exp Neurol ; 303: 72-79, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29337143

RESUMO

BACKGROUND: Facial nerve paralysis is a significant cause of morbidity, affecting facial appearance, emotional expression, speech, oral competence, and vision. A more complete understanding of the complex cellular events required for successful nerve regeneration may reveal new therapeutic targets. The role of fibroblasts in regeneration, and the process by which the nerve reforms its three-dimensional structure after a transection injury, are not fully understood. The Hedgehog signaling pathway has been shown to mediate nerve sheath formation during development. We therefore sought to characterize the role of Hedgehog-responsive cells following transection of the facial nerve. METHODS: Two transgenic mouse lines with reporters for the downstream effector of Hedgehog signaling, Gli1, were used. The animals underwent a unilateral facial nerve transection injury, and the contralateral side served as a control. Facial nerves were analyzed via immunohistochemistry and immunofluorescence at predetermined time points as the facial nerve regenerated after the transection injury. RESULTS: There was a statistically significant increase in Gli1+ cells both at the site of injury and within the distal nerve segment over time. Gli1+ cells are fibroblasts within the nerve and appear to contribute to the reformation of the nerve sheath after injury. CONCLUSION: These findings describe a key signaling pathway by which fibroblasts participate in motor nerve regeneration. Fibroblasts that reside within the nerve respond to injury and may represent a novel therapeutic target in the context of facial nerve regeneration after transection injury.


Assuntos
Traumatismos do Nervo Facial/patologia , Fibroblastos/metabolismo , Ouriços-Cacheiros/metabolismo , Regeneração Nervosa/genética , Proteína GLI1 em Dedos de Zinco/metabolismo , Animais , Antígenos/metabolismo , Modelos Animais de Doenças , Fibronectinas/metabolismo , Citometria de Fluxo , Galactosídeos/genética , Galactosídeos/metabolismo , Regulação da Expressão Gênica/genética , Ouriços-Cacheiros/genética , Indóis/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/metabolismo , Neurônios/patologia , Neurônios/ultraestrutura , Proteoglicanas/metabolismo , Receptor de Fator de Crescimento Neural/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Transdução de Sinais/genética , Proteína GLI1 em Dedos de Zinco/genética
8.
Bull Environ Contam Toxicol ; 100(3): 361-368, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29333580

RESUMO

The residues of persistent organochlorinated pollutants (POPs), namely polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) (HCHs, CHLs, HCCPs, DDTs, and dicofol congeners) were investigated in the hair and muscle of road-killed Erinaceus roumanicus and E. concolor in Turkey. Mean residue levels were as follows: in hair, PCBs = 7.43 ± 4.88 ng/g and OCPs = 9.21 ± 1.27 ng/g; in muscle, PCBs = 30.73 ± 2.51 ng/g and OCPs = 145.04 ± 16.59 ng/g. There was no significant difference between species and sex, while there was significant difference between habitats and regions in terms of either total PCB and OCP levels, or POP levels (p < 0.05). Age was a determinative factor for the bio-accumulation of POPs. The contaminant levels were high in the species, sample areas, and habitats. The data also showed that tissues of hedgehogs are suitable for monitoring and evaluating the bioaccumulation of POP levels in Turkey.


Assuntos
Monitoramento Ambiental/métodos , Ouriços-Cacheiros/metabolismo , Hidrocarbonetos Clorados/análise , Resíduos de Praguicidas/análise , Bifenilos Policlorados/análise , Animais , Feminino , Cabelo/química , Masculino , Músculos/química , Especificidade da Espécie , Turquia
9.
JCI Insight ; 2(21)2017 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-29093271

RESUMO

Advanced basal cell carcinomas (BCCs) circumvent Smoothened (SMO) inhibition by activating GLI transcription factors to sustain the high levels of Hedgehog (HH) signaling required for their survival. Unfortunately, there is a lack of efficacious therapies. We performed a gene expression-based drug repositioning screen in silico and identified the FDA-approved histone deacetylase (HDAC) inhibitor, vorinostat, as a top therapeutic candidate. We show that vorinostat only inhibits proliferation of BCC cells in vitro and BCC allografts in vivo at high dose, limiting its usefulness as a monotherapy. We leveraged this in silico approach to identify drug combinations that increase the therapeutic window of vorinostat and identified atypical PKC Ɩ/ʎ (aPKC) as a HDAC costimulator of HH signaling. We found that aPKC promotes GLI1-HDAC1 association in vitro, linking two positive feedback loops. Combination targeting of HDAC1 and aPKC robustly inhibited GLI1, lowering drug doses needed in vitro, in vivo, and ex vivo in patient-derived BCC explants. We identified a bioavailable and selective small-molecule aPKC inhibitor, bringing the pharmacological blockade of aPKC and HDAC1 into the realm of clinical possibility. Our findings provide a compelling rationale and candidate drugs for combined targeting of HDAC1 and aPKC in HH-dependent cancers.


Assuntos
Carcinoma Basocelular/tratamento farmacológico , Histona Desacetilase 1/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Isoenzimas/efeitos dos fármacos , Proteína Quinase C/efeitos dos fármacos , Neoplasias Cutâneas/tratamento farmacológico , Aloenxertos , Animais , Carcinoma Basocelular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Biologia Computacional , Combinação de Medicamentos , Descoberta de Drogas , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ouriços-Cacheiros/genética , Ouriços-Cacheiros/metabolismo , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Inibidores de Histona Desacetilases/química , Isoenzimas/metabolismo , Camundongos , Camundongos Knockout , Proteína Quinase C/metabolismo , Transdução de Sinais , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/genética , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo
10.
PLoS Negl Trop Dis ; 11(2): e0005137, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28231240

RESUMO

Schistosomiasis affects approximately 240 million people in the world. Schistosoma mansoni eggs in the liver induce periportal fibrosis and hepatic failure driven by monocyte recruitment and macrophage activation, resulting in robust Th2 response. Here, we suggested a possible involvement of Galectin-3 (Gal-3), histone deacetylases (HDACs), and Hedgehog (Hh) signaling with macrophage activation during Th1/Th2 immune responses, fibrogranuloma reaction, and tissue repair during schistosomiasis. Gal-3 is highly expressed by liver macrophages (Kupffer cells) around Schistosoma eggs. HDACs and Hh regulate macrophage polarization and hepatic stellate cell activation during schistosomiasis-associated fibrogenesis. Previously, we demonstrated an abnormal extracellular matrix distribution in the liver that correlated with atypical monocyte-macrophage differentiation in S. mansoni-infected, Gal-3-deficient (Lgals3-/-) mice. New findings explored in this review focus on the chronic phase, when wild-type (Lgals3+/+) and Lgals3-/- mice were analyzed 90 days after cercariae infection. In Lgals3-/- infected mice, there was significant inflammatory infiltration with myeloid cells associated with egg destruction (hematoxylin and eosin staining), phagocytes (specifically Kupffer cells), numerically reduced and diffuse matrix extracellular deposition in fibrotic areas (Gomori trichrome staining), and severe disorganization of collagen fibers surrounding the S. mansoni eggs (reticulin staining). Granuloma-derived stromal cells (GR cells) of Lgals3-/- infected mice expressed lower levels of alpha smooth muscle actin (α-SMA) and eotaxin and higher levels of IL-4 than Lgals3+/+ mice (real-time PCR). The relevant participation of macrophages in these events led us to suggest distinct mechanisms of activation that culminate in defective fibrosis in the liver of Lgals3-/- infected mice. These aspects were discussed in this review, as well as the possible interference between Gal-3, HDACs, and Hh signaling during progressive liver fibrosis in S. mansoni-infected mice. Further studies focused on macrophage roles could elucidate these questions and clear the potential utility of these molecules as antifibrotic targets.


Assuntos
Galectina 3/metabolismo , Ouriços-Cacheiros/metabolismo , Histona Desacetilases/metabolismo , Cirrose Hepática/metabolismo , Esquistossomose/complicações , Animais , Galectina 3/genética , Ouriços-Cacheiros/genética , Histona Desacetilases/genética , Humanos , Cirrose Hepática/etiologia , Cirrose Hepática/genética , Esquistossomose/parasitologia , Esquistossomose Japônica/parasitologia , Transdução de Sinais
11.
J Biomol Struct Dyn ; 35(8): 1654-1671, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27421773

RESUMO

Nicotinic acetylcholine receptors (nAChRs) are neuromuscular proteins responsible for muscle contraction upon binding with chemical stimulant acetylcholine (ACh). The α-neurotoxins of snake mimic the structure of ACh and attacks nAChRs, which block the flow of ACh and leads to numbness and paralysis. The toxin-binding site of alpha subunit in the nAChRs is highly conserved throughout chordate lineages with few exceptions in resistance organisms. In this study, we have analyzed the sequence and structures of toxin-binding/resistant nAChRs and their interaction stability with toxins through molecular docking and molecular dynamics simulation (MDS). We have reported the potential glycosylation residues within the toxin-binding cleft adding sugar moieties through N-linked glycosylation in resistant organisms. Residue variations at key positions alter the secondary structure of binding cleft, which might interfere with toxin binding and it could be one of the possible explanations for the resistance to snake venoms. Analysis of nAChR-α-neurotoxin complexes has confirmed the key interacting residues. In addition, drastic variation in the binding stability of Mongoose nAChR-α-Bungarotoxin (α-BTX) and human nAChR-α-BTX complexes were found at specific phase of MDS. Our findings suggest that specific mutations in the binding site of toxin are potentially preventing the formation of stable complex of receptor-toxin, which might lead to mechanism of resistance. This in silico study on the binding cleft of nAChR and the findings of interacting residues will assist in designing potential inhibitors as therapeutic targets.


Assuntos
Bungarotoxinas/química , Neurotoxinas/química , Receptores Nicotínicos/química , Mordeduras de Serpentes/prevenção & controle , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bungarotoxinas/metabolismo , Colubridae/fisiologia , Cristalografia por Raios X , Ouriços-Cacheiros/metabolismo , Herpestidae/metabolismo , Humanos , Camundongos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Naja haje/fisiologia , Neurotoxinas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Receptores Nicotínicos/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Musaranhos/metabolismo , Mordeduras de Serpentes/metabolismo , Termodinâmica
12.
Elife ; 52016 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-27705744

RESUMO

Cholesterol is necessary for the function of many G-protein coupled receptors (GPCRs). We find that cholesterol is not just necessary but also sufficient to activate signaling by the Hedgehog (Hh) pathway, a prominent cell-cell communication system in development. Cholesterol influences Hh signaling by directly activating Smoothened (SMO), an orphan GPCR that transmits the Hh signal across the membrane in all animals. Unlike many GPCRs, which are regulated by cholesterol through their heptahelical transmembrane domains, SMO is activated by cholesterol through its extracellular cysteine-rich domain (CRD). Residues shown to mediate cholesterol binding to the CRD in a recent structural analysis also dictate SMO activation, both in response to cholesterol and to native Hh ligands. Our results show that cholesterol can initiate signaling from the cell surface by engaging the extracellular domain of a GPCR and suggest that SMO activity may be regulated by local changes in cholesterol abundance or accessibility.


Assuntos
Colesterol/metabolismo , Ouriços-Cacheiros/metabolismo , Transdução de Sinais , Receptor Smoothened/agonistas , Animais , Linhagem Celular , Células Epiteliais/fisiologia , Fibroblastos/fisiologia , Humanos , Camundongos
13.
Artigo em Inglês | MEDLINE | ID: mdl-27544300

RESUMO

The African hedgehog, Atelerix albiventris, is a spiny mammal that has become popular as an exotic pet in many countries. To elucidate the ability of hedgehogs to metabolize xenobiotics, the animals were exposed to polycyclic aromatic hydrocarbon, pyrene. The in vivo exposure study indicated that pyrene was biotransformed to glucuronide and sulfate conjugates, such as pyrene-1-glucuronide, pyrene-1-sulfate, and pyrenediol-sulfate, and excreted in the urine. Pyrene-1-glucuronide was the main metabolite, and limited sulfate conjugate excretion was observed. The main products excreted in feces were 1-hydroxypyrene and pyrene. Based on the results of the in vivo exposure study, in vitro enzymatic kinetic experiments were performed using various substrates and compared to rats and pigs. The enzyme efficiencies of cytochrome P450 (CYP)-mediated ethoxyresorufin O-deethylase activity and warfarin 4'-, 6-, and 8-hydroxylation activity in hedgehogs were lower than those of rats. Furthermore, UDP-glucuronosyltransferase activity in hedgehogs also had a lower Km value than that in pigs. Interestingly, the enzyme efficiencies of sulfation activity toward 1-hydroxypyrene and ß-estradiol in hedgehogs were significantly lower than those in pigs. These observations suggested that phenol and estrogen sulfotransferases may have limited roles in xenobiotic metabolism in hedgehogs.


Assuntos
Ouriços-Cacheiros/metabolismo , Fígado/enzimologia , Desintoxicação Metabólica Fase II , Desentoxicação Metabólica Fase I , Pirenos/metabolismo , Animais , Fezes/química , Glucuronídeos/metabolismo , Ouriços-Cacheiros/urina , Cinética , Masculino , Pirenos/toxicidade , Pirenos/urina , Ratos , Eliminação Renal , Especificidade da Espécie , Especificidade por Substrato , Sulfatos/metabolismo , Sus scrofa
14.
Nat Prod Commun ; 11(6): 747-8, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27534107

RESUMO

Two new 13, 14/14, 15-disecopregnane-type skeleton C21 steroidal aglycones, neocynapanogenin G (1) and neocynapanogenin H (2), were isolated from the hydrolyzed extract of the CHCl3 soluble extract of the roots of Cynanchun paniculatum. Their structures were determined on the basis of chemical evidence and extensive spectroscopic methods, including 1D and 2D NMR spectroscopy. Compound 1 displayed signifidant inhibition of the Hedgehog signaling pathway in vitro.


Assuntos
Cynanchum/química , Medicamentos de Ervas Chinesas/química , Iridoides/química , Raízes de Plantas/química , Esteroides/química , Animais , Linhagem Celular , Ouriços-Cacheiros/genética , Ouriços-Cacheiros/metabolismo , Humanos , Estrutura Molecular , Transdução de Sinais/efeitos dos fármacos
15.
Int Immunopharmacol ; 34: 129-138, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26945831

RESUMO

The present study aimed to investigate the effect of palmatine (Pal) in a rabbit osteoarthritis (OA) model in vivo and rabbit interleukin-1ß (IL-1ß)-stimulated chondrocytes in vitro. Appropriate concentrations of Pal were identified by the MTT assay and used to preincubate IL-1ß-induced chondrocytes, as well as an activator or inhibitor of Wnt and Hedgehog signaling pathways. Matrix metalloproteinase (MMP)-1, 3, and 13; tissue inhibitor of metalloproteinase (TIMP)-1; collagenase II; aggrecan; and the related molecules of the Wnt/ß-catenin and Hedgehog signaling pathways were investigated. Protein expression was detected by Western blot analysis and messenger RNA (mRNA) expression was examined by PCR analysis. Pal (0.3 mL, 100 mg/L) was injected into rabbit knee joints and histological examination, immunohistochemistry, and Mankin scoring of the articular cartilage were performed. Pal (10-100 mg/L) had no effect on chondrocyte viability, decreased the expression of the MMPs, and increased the synthesis of TIMP-1whereas collagenase II and aggrecan were inhibited by IL-1ß. When the activator (Licl) and inhibitor (DKK-1) of the Wnt/ß-catenin signaling pathway as well as the inhibitor (cyclopamine) of the Hedgehog signaling pathway were added, the Wnt/ß-catenin signaling pathway was less inhibited by Pal, and a similar inhibitory effect of cyclopamine on the Hedgehog signaling pathway was evident. Additionally, Pal enhanced the effect of cyclopamine. The histological examination, immunohistochemistry and Mankin scoring also demonstrated the protective effect of Pal, and the inhibition of the Wnt and Hedgehog signaling pathways by Pal. Pal may be useful in the treatment of OA, in which its effect is likely mediated via the Wnt/ß-catenin and Hedgehog signaling pathways.


Assuntos
Alcaloides de Berberina/uso terapêutico , Condrócitos/efeitos dos fármacos , Ouriços-Cacheiros/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Osteoartrite/tratamento farmacológico , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Células Cultivadas , Condrócitos/fisiologia , Sinergismo Farmacológico , Humanos , Interleucina-1beta/imunologia , Cloreto de Lítio/farmacologia , Metaloproteinases da Matriz/metabolismo , Coelhos , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Veratrum/imunologia , Alcaloides de Veratrum/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos
16.
BMC Cancer ; 16: 150, 2016 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-26911235

RESUMO

BACKGROUND: Aberrant Hedgehog (Hh) signaling is associated with the development of many cancers including prostate cancer, gastrointestinal cancer, lung cancer, pancreatic cancer, ovarian cancer, and basal cell carcinoma. The Hh signaling pathway has been one of the most intensely investigated targets for cancer therapy, and a number of compounds inhibiting Hh signaling are being tested clinically for treating many cancers. Lung cancer causes more deaths than the next three most common cancers (colon, breast, and prostate) combined. Cyclopamine was the first compound found to inhibit Hh signaling and has been invaluable for understanding the function of Hh signaling in development and cancer. To find novel strategies for combating lung cancer, we decided to characterize the effect of cyclopamine tartrate (CycT), an improved analogue of cyclopamine, on lung cancer cells and its mechanism of action. METHODS: The effect of CycT on oxygen consumption and proliferation of non-small-cell lung cancer (NSCLC) cell lines was quantified by using an Oxygraph system and live cell counting, respectively. Apoptosis was detected by using Annexin V and Propidium Iodide staining. CycT's impact on ROS generation, mitochondrial membrane potential, and mitochondrial morphology in NSCLC cells was monitored by using fluorometry and fluorescent microscopy. Western blotting and fluorescent microscopy were used to detect the levels and localization of Hh signaling targets, mitochondrial fission protein Drp1, and heme-related proteins in various NSCLC cells. RESULTS: Our findings identified a novel function of CycT, as well as another Hh inhibitor SANT1, to disrupt mitochondrial function and aerobic respiration. Our results showed that CycT, like glutamine depletion, caused a substantial decrease in oxygen consumption in a number of NSCLC cell lines, suppressed NSCLC cell proliferation, and induced apoptosis. Further, we found that CycT increased ROS generation, mitochondrial membrane hyperpolarization, and mitochondrial fragmentation, thereby disrupting mitochondrial function in NSCLC cells. CONCLUSIONS: Together, our work demonstrates that CycT, and likely other Hh signaling inhibitors, can interrupt NSCLC cell function by promoting mitochondrial fission and fragmentation, mitochondrial membrane hyperpolarization, and ROS generation, thereby diminishing mitochondrial respiration, suppressing cell proliferation, and causing apoptosis. Our work provides novel mechanistic insights into the action of Hh inhibitors in cancer cells.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Ouriços-Cacheiros/metabolismo , Neoplasias Pulmonares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tartaratos/farmacocinética , Alcaloides de Veratrum/farmacologia , Animais , Apoptose , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Respiração Celular/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
17.
Artigo em Inglês | MEDLINE | ID: mdl-26393434

RESUMO

Bats exhibit higher paracellular absorption of glucose-sized molecules than non-flying mammals, a phenomenon that may be driven by higher permeability of the intestinal tight junctions. The various claudins, occludin, and other proteins making up the tight junctions are thought to determine their permeability properties. Here we show that absorption of the paracellular probe l-arabinose is higher in a bat (Eptesicus fuscus) than in a vole (Microtus pennsylvanicus) or a hedgehog (Atelerix albiventris). Furthermore, histological measurements demonstrated that hedgehogs have many more enterocytes in their intestines, suggesting that bats cannot have higher absorption of arabinose simply by having more tight junctions. We therefore investigated the mRNA levels of several claudins and occludin, because these proteins may affect permeability of tight junctions to macronutrients. To assess the expression levels of claudins per tight junction, we normalized the mRNA levels of the claudins to the constitutively expressed tight junction protein ZO-1, and combined these with measurements previously made in a bat and a rodent to determine if there were among-species differences. Although expression ratios of several genes varied among species, there was not a consistent difference between bats and non-flyers in the expression ratio of any particular gene. Protein expression patterns may differ from mRNA expression patterns, and might better explain differences among species in arabinose absorption.


Assuntos
Claudinas/genética , Regulação da Expressão Gênica , Absorção Intestinal , Mucosa Intestinal/metabolismo , Intestinos/citologia , Animais , Arvicolinae/genética , Arvicolinae/metabolismo , Quirópteros/genética , Quirópteros/metabolismo , Ouriços-Cacheiros/genética , Ouriços-Cacheiros/metabolismo , Especificidade da Espécie
18.
Elife ; 42015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26371509

RESUMO

Alterations in Hedgehog (Hh) signaling lead to birth defects and cancers including medulloblastoma, the most common pediatric brain tumor. Although inhibitors targeting the membrane protein Smoothened suppress Hh signaling, acquired drug resistance and tumor relapse call for additional therapeutic targets. Here we show that phosphodiesterase 4D (PDE4D) acts downstream of Neuropilins to control Hh transduction and medulloblastoma growth. PDE4D interacts directly with Neuropilins, positive regulators of Hh pathway. The Neuropilin ligand Semaphorin3 enhances this interaction, promoting PDE4D translocation to the plasma membrane and cAMP degradation. The consequent inhibition of protein kinase A (PKA) enhances Hh transduction. In the developing cerebellum, genetic removal of Neuropilins reduces Hh signaling activity and suppresses proliferation of granule neuron precursors. In mouse medulloblastoma allografts, PDE4D inhibitors suppress Hh transduction and inhibit tumor growth. Our findings reveal a new regulatory mechanism of Hh transduction, and highlight PDE4D as a promising target to treat Hh-related tumors.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Ouriços-Cacheiros/metabolismo , Meduloblastoma/patologia , Neuropilina-1/metabolismo , Neuropilina-2/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Proliferação de Células , Humanos , Camundongos , Camundongos Knockout
19.
Mech Dev ; 137: 11-22, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25936631

RESUMO

We report that the morphogen Hedgehog (Hh) is an axonal chemoattractant in the midline of Drosophila melanogaster embryos. Hh is present in the ventral nerve cord during axonal guidance and overexpression of hh in the midline causes ectopic midline crossing of FasII-positive axonal tracts. In addition, we show that Hh influences axonal guidance via a non-canonical signalling pathway dependent on Ptc. Our results reveal that the Hh pathway cooperates with the Netrin/Frazzled pathway to guide axons through the midline in invertebrates.


Assuntos
Axônios/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Ouriços-Cacheiros/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/fisiologia , Animais , Axônios/fisiologia , Drosophila melanogaster/fisiologia , Fatores de Crescimento Neural/metabolismo , Receptores de Netrina , Neurogênese/fisiologia
20.
Angew Chem Int Ed Engl ; 54(19): 5596-602, 2015 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-25736574

RESUMO

Biology-oriented synthesis employs the structural information encoded in complex natural products to guide the synthesis of compound collections enriched in bioactivity. The trans-hydrindane dehydro-δ-lactone motif defines the characteristic scaffold of the steroid-like withanolides, a plant-derived natural product class with a diverse pattern of bioactivity. A withanolide-inspired compound collection was synthesized by making use of three key intermediates that contain this characteristic framework derivatized with different reactive functional groups. Biological evaluation of the compound collection in cell-based assays that monitored biological signal-transduction processes revealed a novel class of Hedgehog signaling inhibitors that target the protein Smoothened.


Assuntos
Produtos Biológicos/farmacologia , Ouriços-Cacheiros/metabolismo , Transdução de Sinais/efeitos dos fármacos , Vitanolídeos/farmacologia , Animais , Produtos Biológicos/síntese química , Produtos Biológicos/química , Relação Dose-Resposta a Droga , Conformação Molecular , Relação Estrutura-Atividade , Vitanolídeos/síntese química , Vitanolídeos/química
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