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1.
Molecules ; 24(10)2019 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-31130671

RESUMO

The increasing prevalence of drug resistant and/or high-risk cancers indicate further drug discovery research is required to improve patient outcome. This study outlines a simplified approach to identify lead compounds from natural products against several cancer cell lines, and provides the basis to better understand structure activity relationship of the natural product cephalotaxine. Using high-throughput screening, a natural product library containing fractions and pure compounds was interrogated for proliferation inhibition in acute lymphoblastic leukemia cellular models (SUP-B15 and KOPN-8). Initial hits were verified in control and counter screens, and those with EC50 values ranging from nanomolar to low micromolar were further characterized via mass spectrometry, NMR, and cytotoxicity measurements. Most of the active compounds were alkaloid natural products including cephalotaxine and homoharringtonine, which were validated as protein synthesis inhibitors with significant potency against several cancer cell lines. A generated BODIPY-cephalotaxine probe provides insight into the mode of action of cephalotaxine and further rationale for its weaker potency when compared to homoharringtonine. The steroidal natural products (ecdysone and muristerone A) also showed modest biological activity and protein synthesis inhibition. Altogether, these findings demonstrate that natural products continue to provide insight into structure and function of molecules with therapeutic potential against drug resistant cancer cell models.


Assuntos
Produtos Biológicos/farmacologia , Citostáticos/química , Citostáticos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Produtos Biológicos/química , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Descoberta de Drogas , Mepesuccinato de Omacetaxina/química , Mepesuccinato de Omacetaxina/farmacologia , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Relação Estrutura-Atividade
2.
J Nat Prod ; 82(5): 1301-1311, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31084028

RESUMO

Natural products continue to provide a platform to study biological systems. A bioguided study of cancer cell models led us to a new member of the jatrophane natural products from Jatropha gossypiifolia, which was independently identified and characterized as jatrogossone A (1). Purification and structure elucidation was performed by column chromatography and high-performance liquid chromatography-mass spectrometry and NMR techniques, and the structure was confirmed via X-ray crystallography. The unique molecular scaffold of jatrogossone A prompted an evaluation of its mode of action. Cytotoxicity assays demonstrated that jatrogossone A displays selective antiproliferative activity against cancer cell models in the low micromolar range with a therapeutic window. Jatrogossone A (1) affects mitochondrial membrane potential (ΔΨm) in a time- and dose-dependent manner. This natural product induces radical oxygen species (ROS) selectively in cancer cellular models, with minimal ROS induction in noncancerous cells. Compound 1 induces ROS in the mitochondria, as determined by colocalization studies, and it induces mitophagy. It promotes also in vitro cell death by causing cell arrest at the G2/M stage, caspase (3/7) activation, and PARP-1 cleavage. The combined findings provide a potential mechanism by which 1 relies on upregulation of mitochondrial ROS to potentiate cytotoxic effects through intracellular signaling.

3.
Eur J Med Chem ; 164: 391-398, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30611980

RESUMO

Although pediatric leukemia is generally treatable, certain leukemic subtypes face poor prognosis in the clinic suggesting new selective therapeutic agents are needed. Thus, to identify selective apoptosis inducers, a small-molecule library screening approach was conducted using an isogenic leukemic murine p185+ B-ALL cell line pair (BCR-ABL-WT and the BAX/BAK deficient BCR-ABL-DKO). Gratifyingly, the investigation revealed several compounds featuring substituted aromatic five-membered-ring heterocycles with significant activity against murine and human leukemic cellular models. The identified compounds represent potentially novel antileukemic molecular scaffolds exemplified by compounds 1, 2 and 7, which demonstrated EC50 values in the nanomolar and low micromolar range against various leukemia subtypes (SUP-B15, KOPN-8, NALM-06, UoC-B1 cellular models) and pro-apoptotic properties in solid tumor cell models (MDA-MB-231, SUM149) with ample therapeutic index in normal cells. Herein, we highlight compounds 1, 2 and 7 which promote cell death mediated by caspase 3/7 induction. Our study establishes a strategic platform for the development of potent and selective anti-leukemic agents.


Assuntos
Antineoplásicos/farmacologia , Compostos Heterocíclicos/uso terapêutico , Leucemia/tratamento farmacológico , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Caspases/genética , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos/métodos , Indução Enzimática/efeitos dos fármacos , Compostos Heterocíclicos/química , Humanos , Camundongos , Bibliotecas de Moléculas Pequenas/uso terapêutico , Índice Terapêutico
4.
Eur J Med Chem ; 146: 501-510, 2018 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-29407975

RESUMO

Acute lymphoblastic leukemia (ALL) is the most common type of leukemia in children. Up to a quarter of ALL patients relapse and face poor prognosis. To identify new compound leads, we conducted a phenotypic screen using terrestrial natural product (NP) fractions against immortalized ALL cellular models. We identified vitexin, a flavonoid, as a promising hit with biological activity (EC50 = 30 µM) in pre-B cell ALL models with no toxicity against normal human tissue (BJ cells) at the tested concentrations. To develop more potent compounds against ALL and elucidate its potential mode of action, a vitexin-inspired compound library was synthesized. Thus, we developed an improved and scalable protocol for the direct synthesis of 4-quinolone core heterocycles containing an N-sulfonamide using a one-pot condensation reaction protocol. The newly generated compounds represent a novel molecular scaffold against ALL as exemplified by compounds 13 and 15, which demonstrated EC50 values in the low micromolar range (0.3-10 µM) with little to no toxicity in normal cellular models. Computational studies support the hypothesis that these compounds are potential CDK inhibitors. The compounds induced apoptosis, caused cell arrest at G0/G1 and G2/M, and induced ROS in cancer cells.


Assuntos
Apigenina/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Apigenina/síntese química , Apigenina/química , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Relação Estrutura-Atividade
5.
F1000Res ; 6: 1136, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28928948

RESUMO

The first challenge in the 2014 competition launched by the Teach-Discover-Treat (TDT) initiative asked for the development of a tutorial for ligand-based virtual screening, based on data from a primary phenotypic high-throughput screen (HTS) against malaria. The resulting Workflows were applied to select compounds from a commercial database, and a subset of those were purchased and tested experimentally for anti-malaria activity. Here, we present the two most successful Workflows, both using machine-learning approaches, and report the results for the 114 compounds tested in the follow-up screen. Excluding the two known anti-malarials quinidine and amodiaquine and 31 compounds already present in the primary HTS, a high hit rate of 57% was found.

6.
Res Q Exerc Sport ; 88(3): 269-281, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28644729

RESUMO

PURPOSE: Prenatal exercise is a health behavior that is receiving growing attention amid concern that women in Western societies are gaining excess weight during pregnancy and contributing to future obesity in both the mother and child. In this article, we draw on insights from the fields of social epidemiology and social theory of the body to examine existing prenatal exercise interventions and to propose a multidimensional framework intended to guide future theorizing and intervention design. METHOD: A scoping review of existing prenatal exercise programs and interventions focused on controlling gestational weight gain was conducted. Articles published prior to January 2017 were obtained from PubMed and CINAHL, and relevant articles were identified (n = 62) using specified inclusion and exclusion criteria. Identified articles were further analyzed to classify the level(s) of the socioecological model targeted in the intervention or program. RESULTS: The majority of existing interventions target intrapersonal factors during pregnancy and do not attend to the role that cumulative exposure of social and structural disadvantage over the lifetime-not just during the prenatal period-plays in shaping health outcomes. In response, a multidimensional framework is proposed that includes key concepts that facilitate a life-course perspective, as well as attention to the integration of biological and social factors as they relate to health and health-related behaviors. CONCLUSION: Efforts to promote prenatal exercise and to improve maternal and infant health should attend to how systemic inequality impacts women's health.


Assuntos
Exercício Físico , Comportamentos Relacionados com a Saúde , Gravidez/psicologia , Cuidado Pré-Natal/métodos , Cultura , Feminino , Promoção da Saúde , Humanos , Obesidade/prevenção & controle , Fatores Socioeconômicos , Ganho de Peso/fisiologia
7.
Am J Public Health ; 106(5): 796-9, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26985618

RESUMO

Nature-based physical activity programming (e.g., countryside walks, hiking, horseback riding) has been found to be an effective way to help improve the health of people with mental illness. Exercise referral initiatives, whereby health practitioners prescribe exercise in an attempt to prevent or treat chronic illnesses, have helped make such nature-based activities accessible to this population in the United Kingdom and Australia; however, there is a dearth of research related to the most prominent exercise referral program in the United States: Exercise is Medicine. Taking into account the barriers to physical activity faced by people with mental illness, we explore how nature-based programming for this population might be mobilized in the United States through the growing Exercise is Medicine initiative.


Assuntos
Exercício Físico/psicologia , Promoção da Saúde/organização & administração , Nível de Saúde , Transtornos Mentais/terapia , Meio Selvagem , Doença Crônica , Comportamento Cooperativo , Humanos , Relações Interprofissionais , Parques Recreativos , Qualidade de Vida , Índice de Gravidade de Doença , Estados Unidos
8.
BMC Physiol ; 11: 16, 2011 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-22059553

RESUMO

BACKGROUND: This work tests the hypothesis that increased levels of vascular endothelial growth factor (VEGF) observed during bladder inflammation modulates nerve plasticity. METHODS: Chronic inflammation was induced by intravesical instillations of Bacillus Calmette-Guérin (BCG) into the urinary bladder and the density of nerves expressing the transient receptor potential vanilloid subfamily 1 (TRPV1) or pan-neuronal marker PGP9.5 was used to quantify alterations in peripheral nerve plasticity. Some mice were treated with B20, a VEGF neutralizing antibody to reduce the participation of VEGF. Additional mice were treated systemically with antibodies engineered to specifically block the binding of VEGF to NRP1 (anti-NRP1B) and NRP2 (NRP2B), or the binding of semaphorins to NRP1 (anti-NRP1 A) to diminish activity of axon guidance molecules such as neuropilins (NRPs) and semaphorins (SEMAs). To confirm that VEGF is capable of inducing inflammation and neuronal plasticity, another group of mice was instilled with recombinant VEGF165 or VEGF121 into the urinary bladder. RESULTS: The major finding of this work was that chronic BCG instillation resulted in inflammation and an overwhelming increase in both PGP9.5 and TRPV1 immunoreactivity, primarily in the sub-urothelium of the urinary bladder. Treatment of mice with anti-VEGF neutralizing antibody (B20) abolished the effect of BCG on inflammation and nerve density.NRP1A and NRP1B antibodies, known to reduce BCG-induced inflammation, failed to block BCG-induced increase in nerve fibers. However, the NRP2B antibody dramatically potentiated the effects of BCG in increasing PGP9.5-, TRPV1-, substance P (SP)-, and calcitonin gene-related peptide (CGRP)-immunoreactivity (IR). Finally, instillation of VEGF121 or VEGF165 into the mouse bladder recapitulated the effects of BCG and resulted in a significant inflammation and increase in nerve density. CONCLUSIONS: For the first time, evidence is being presented supporting that chronic BCG instillation into the mouse bladder promotes a significant increase in peripheral nerve density that was mimicked by VEGF instillation. Effects of BCG were abolished by pre-treatment with neutralizing VEGF antibody. The present results implicate the VEGF pathway as a key modulator of inflammation and nerve plasticity, introduces a new animal model for investigation of VEGF-induced nerve plasticity, and suggests putative mechanisms underlying this phenomenon.


Assuntos
Vacina BCG/farmacologia , Inflamação/metabolismo , Plasticidade Neuronal/efeitos dos fármacos , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Vacina BCG/imunologia , Calcitonina/imunologia , Calcitonina/metabolismo , Feminino , Inflamação/induzido quimicamente , Inflamação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/imunologia , Neuropilina-1/imunologia , Neuropilina-1/metabolismo , Neuropilina-2/imunologia , Neuropilina-2/metabolismo , Neuropilinas/efeitos dos fármacos , Neuropilinas/imunologia , Neuropilinas/metabolismo , Precursores de Proteínas/imunologia , Precursores de Proteínas/metabolismo , Proteínas Recombinantes/farmacologia , Semaforinas/imunologia , Semaforinas/metabolismo , Transdução de Sinais , Substância P/imunologia , Substância P/metabolismo , Canais de Cátion TRPV/imunologia , Canais de Cátion TRPV/metabolismo , Ubiquitina Tiolesterase/imunologia , Ubiquitina Tiolesterase/metabolismo , Bexiga Urinária/imunologia , Bexiga Urinária/patologia , Urotélio/efeitos dos fármacos , Urotélio/imunologia , Urotélio/metabolismo
9.
Am J Physiol Renal Physiol ; 299(6): F1245-56, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20861073

RESUMO

Recent findings indicate that VEGF receptors and coreceptors (neuropilins; NRP) are expressed on nonendothelial cells in human bladder urothelium, in one human bladder cancer cell line (J82), and in the mouse bladder urothelium. In addition, VEGFR1, VEGFR2, NRP1, and NRP2 expressions were upregulated in animal models of chronic bladder inflammation induced by four weekly instillations of protease-activated receptors (PAR)-activating peptides or bacillus Calmette-Guérin (BCG) into the mouse bladder. Here, we used four weekly instillations of BCG as a model for chronic bladder inflammation to further investigate whether VEGF receptors and NRPs play a role in the migration of inflammatory cells and inflammation-induced lymphangiogenesis and angiogenesis. For this purpose, we used neutralizing antibodies that were engineered to specifically block the binding of VEGF to NRP (anti-NRP1(B)) and the binding of semaphorins to NRP (anti-NRP1(A)). C57BL/6 mice received intraperitoneal injections of PBS, anti-NRP1(A)- or anti-NRP1(B)-neutralizing antibodies and then were challenged chronically with intravesical PBS or BCG. At the end of chronic challenge period, a fluorescent internalizable tracer, scVEGF/Cy5.5, was administered to all mice and near-infrared fluorescence images were obtained in vivo and in real time. BCG increased the overall accumulation of scVEGF/Cy5.5 in the urinary bladder urothelium and inflammatory cells. In addition, BCG increased the density of blood and lymphatic vessels concomitantly with an upregulation of NRP2 expression in lymphatic vessels. Treatment of the mice with NRP1-neutralizing antibodies dramatically reduced scVEGF/Cy5.5 uptake, polymorphonuclear (myeloperoxidase-positive cells) and dendritic cell (CD11c-positive cells) infiltration, and decreased the overall density of BCG-induced blood and lymphatic vessels. These results implicate NRPs as critical in vivo regulators of the vascular and inflammatory responses to the intravesical administration of BCG.


Assuntos
Cistite/fisiopatologia , Neuropilina-1/fisiologia , Neuropilinas/fisiologia , Receptores de Fatores de Crescimento do Endotélio Vascular/fisiologia , Transdução de Sinais/fisiologia , Animais , Vacina BCG , Movimento Celular/imunologia , Cistite/induzido quimicamente , Feminino , Humanos , Linfangiogênese/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/fisiopatologia , Neuropilina-1/imunologia , Bexiga Urinária/irrigação sanguínea , Bexiga Urinária/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese
10.
Bioorg Med Chem Lett ; 20(1): 149-52, 2010 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19963377

RESUMO

A series of halo-nitrobenzamide were synthesized and evaluated for their ability to block proliferation of Trypanosoma brucei brucei. A number of these compounds had significant activity against the parasite, particularly 2-chloro-N-(4-chlorophenyl)-5-nitrobenzamide 17 which exhibited low micromolar inhibitory potency against T. brucei and selectivity towards both malaria and mammalian cells.


Assuntos
Benzamidas/química , Tripanossomicidas/química , Benzamidas/síntese química , Benzamidas/farmacologia , Descoberta de Drogas , Humanos , Testes de Sensibilidade Parasitária , Tripanossomicidas/síntese química , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase Africana/tratamento farmacológico
11.
Cancer Res ; 69(22): 8797-806, 2009 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-19887618

RESUMO

Tumor-microenvironment interactions are increasingly recognized to influence tumor progression. To understand the competitive dynamics of tumor cells in diverse microenvironments, we experimentally parameterized a hybrid discrete-continuum mathematical model with phenotypic trait data from a set of related mammary cell lines with normal, transformed, or tumorigenic properties. Surprisingly, in a resource-rich microenvironment, with few limitations on proliferation or migration, transformed (but not tumorigenic) cells were most successful and outcompeted other cell types in heterogeneous tumor simulations. Conversely, constrained microenvironments with limitations on space and/or growth factors gave a selective advantage to phenotypes derived from tumorigenic cell lines. Analysis of the relative performance of each phenotype in constrained versus unconstrained microenvironments revealed that, although all cell types grew more slowly in resource-constrained microenvironments, the most aggressive cells were least affected by microenvironmental constraints. A game theory model testing the relationship between microenvironment resource availability and competitive cellular dynamics supports the concept that microenvironmental independence is an advantageous cellular trait in resource-limited microenvironments.


Assuntos
Neoplasias da Mama/patologia , Modelos Teóricos , Adesão Celular/fisiologia , Comunicação Celular/fisiologia , Linhagem Celular Tumoral , Progressão da Doença , Matriz Extracelular/metabolismo , Feminino , Teoria do Jogo , Humanos
12.
Am J Physiol Renal Physiol ; 295(6): F1613-23, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18815217

RESUMO

Interstitial cystitis (IC) is a chronic and painful bladder syndrome of unknown cause with no reliable biological marker or effective therapy. Vascular endothelial growth factor (VEGF), which plays a key role in bladder inflammation, is closely associated with the vascular alterations observed in patients with IC. However, our recent findings of VEGF receptors (VEGF-Rs) and VEGF coreceptors on nonendothelial cells in human and mouse urothelium suggest that additional VEGF targets and functions are possible in IC bladders. We report here that VEGF-Rs and coreceptors (neuropilins; NRP) are strongly expressed in both the human bladder urothelium and in the human bladder cancer cell line (J82) and that the expression of NRP2 and VEGF-R1 is significantly downregulated in IC compared with control subjects. In addition, treatment of J82 cells with bacillus Calmette-Guérin (BCG), a novel treatment strategy for IC, upregulates the messages for NRPs and VEGF-Rs. Furthermore, intravesical instillation of an internalizable VEGF fluorescent tracer (scVEGF/Cy5.5) into mouse urinary bladders results in a marked ligand accumulation in the urothelium and bladder parenchyma, indicating that urothelial VEGF-Rs are functionally active and capable of ligand interaction and internalization. Our results suggest that the VEGF pathway is altered in IC, that urinary VEGF may gain access to the bladder wall via these receptors, and that BCG treatment may replenish the missing VEGF-Rs/NRP receptors. Together, these results suggest that levels of NRPs, VEGF-Rs, and VEGF are new putative markers for the diagnosis of IC and that modulating these receptors can be exploited as therapeutic strategies.


Assuntos
Cistite Intersticial/fisiopatologia , Neuropilinas/fisiologia , Receptores de Fatores de Crescimento do Endotélio Vascular/fisiologia , Animais , Linhagem Celular , Cistite Intersticial/genética , Feminino , Humanos , Inflamação/fisiopatologia , Masculino , Camundongos , Reação em Cadeia da Polimerase , Bexiga Urinária/fisiopatologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
13.
Am J Physiol Renal Physiol ; 295(1): F60-72, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18463314

RESUMO

Recent evidence supports a role for vascular endothelium growth factor (VEGF) signaling in bladder inflammation. However, it is not clear what bladder cells are targeted by VEGF. Therefore, we determined the nature of cells responding to VEGF in normal and inflamed bladders by tagging such cells in vivo with a targeted fluorescent tracer, scVEGF/Cy, an engineered single-chain VEGF labeled with Cy5.5 dye, which identifies cells with accessible and functionally active VEGF receptors. Inflammation was induced by intravesical instillation of PAR-activating peptides or BCG. In vivo NIRF imaging with intravenously injected scVEGF/Cy revealed accumulation of the tracer in the control mouse bladder and established that inflammation increased the steady-state levels of tracer uptake. Ex vivo colocalization of Cy5.5 dye revealed that in normal and at a higher level in inflamed bladder, accumulation of scVEGF/Cy occurs in both urothelial and ganglial cells, expressing VEGF receptors VEGFR-1 and VEGFR-2, as well as VEGF coreceptors neuropilins (NRP) NRP1 and NRP2. PCR results indicate that the messages for VEGF-Rs and NRPs are present in the bladder mucosa and ChIP/QPCR analysis indicated that inflammation induced upregulation of genes encoding VEGFRs and NRPs. Our results strongly suggest new and blossoming VEGF-driven processes in bladder urothelial cells and ganglia in the course of inflammation. We expect that molecular imaging of the VEGF pathway in the urinary tract by receptor-mediated cell tagging in vivo will be useful for clinical diagnosis and therapeutic monitoring, and will help to accelerate the development of bladder-targeting drugs and treatments.


Assuntos
Cistite/metabolismo , Neuropilinas/biossíntese , Receptores de Fatores de Crescimento do Endotélio Vascular/biossíntese , Bexiga Urinária/metabolismo , Adulto , Idoso , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Neurônios/metabolismo , Neuropilina-1/biossíntese , Neuropilina-2/biossíntese , Espectroscopia de Luz Próxima ao Infravermelho , Bexiga Urinária/citologia , Urotélio/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese
14.
BMC Cancer ; 7: 219, 2007 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-18047671

RESUMO

BACKGROUND: The lymphatics form a second circulatory system that drains the extracellular fluid and proteins from the tumor microenvironment, and provides an exclusive environment in which immune cells interact and respond to foreign antigen. Both cancer and inflammation are known to induce lymphangiogenesis. However, little is known about bladder lymphatic vessels and their involvement in cancer formation and progression. METHODS: A double transgenic mouse model was generated by crossing a bladder cancer-induced transgenic, in which SV40 large T antigen was under the control of uroplakin II promoter, with another transgenic mouse harboring a lacZ reporter gene under the control of an NF-kappaB-responsive promoter (kappaB-lacZ) exhibiting constitutive activity of beta-galactosidase in lymphatic endothelial cells. In this new mouse model (SV40-lacZ), we examined the lymphatic vessel density (LVD) and function (LVF) during bladder cancer progression. LVD was performed in bladder whole mounts and cross-sections by fluorescent immunohistochemistry (IHC) using LYVE-1 antibody. LVF was assessed by real-time in vivo imaging techniques using a contrast agent (biotin-BSA-Gd-DTPA-Cy5.5; Gd-Cy5.5) suitable for both magnetic resonance imaging (MRI) and near infrared fluorescence (NIRF). In addition, IHC of Cy5.5 was used for time-course analysis of co-localization of Gd-Cy5.5 with LYVE-1-positive lymphatics and CD31-positive blood vessels. RESULTS: SV40-lacZ mice develop bladder cancer and permitted visualization of lymphatics. A significant increase in LVD was found concomitantly with bladder cancer progression. Double labeling of the bladder cross-sections with LYVE-1 and Ki-67 antibodies indicated cancer-induced lymphangiogenesis. MRI detected mouse bladder cancer, as early as 4 months, and permitted to follow tumor sizes during cancer progression. Using Gd-Cy5.5 as a contrast agent for MRI-guided lymphangiography, we determined a possible reduction of lymphatic flow within the tumoral area. In addition, NIRF studies of Gd-Cy5.5 confirmed its temporal distribution between CD31-positive blood vessels and LYVE-1 positive lymphatic vessels. CONCLUSION: SV40-lacZ mice permit the visualization of lymphatics during bladder cancer progression. Gd-Cy5.5, as a double contrast agent for NIRF and MRI, permits to quantify delivery, transport rates, and volumes of macromolecular fluid flow through the interstitial-lymphatic continuum. Our results open the path for the study of lymphatic activity in vivo and in real time, and support the role of lymphangiogenesis during bladder cancer progression.


Assuntos
Biomarcadores Tumorais/análise , Carcinoma in Situ/diagnóstico , Carcinoma in Situ/patologia , Vasos Linfáticos/diagnóstico por imagem , Vasos Linfáticos/patologia , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/patologia , Animais , Carcinoma in Situ/imunologia , Modelos Animais de Doenças , Glicoproteínas/análise , Glicoproteínas/imunologia , Imuno-Histoquímica , Linfangiogênese , Vasos Linfáticos/metabolismo , Linfografia/métodos , Imagem por Ressonância Magnética , Proteínas de Membrana/análise , Proteínas de Membrana/imunologia , Proteínas de Membrana Transportadoras , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Espectroscopia de Luz Próxima ao Infravermelho , Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/imunologia , Uroplaquina II
15.
BMC Immunol ; 8: 17, 2007 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-17705868

RESUMO

BACKGROUND: All four PARs are present in the urinary bladder, and their expression is altered during inflammation. In order to search for therapeutic targets other than the receptors themselves, we set forth to determine TFs downstream of PAR activation in the C57BL/6 urinary bladders. METHODS: For this purpose, we used a protein/DNA combo array containing 345 different TF consensus sequences. Next, the TF selected was validated by EMSA and IHC. As mast cells seem to play a fundamental role in bladder inflammation, we determined whether c-kit receptor deficient (Kit w/Kit w-v) mice have an abrogated response to PAR stimulation. Finally, TFEB antibody was used for CHIP/Q-PCR assay and revealed up-regulation of genes known to be downstream of TFEB. RESULTS: TFEB, a member of the MiTF family of basic helix-loop-helix leucine zipper, was the only TF commonly up-regulated by all PAR-APs. IHC results confirm a correlation between inflammation and TFEB expression in C57BL/6 mice. In contrast, Kit w/Kit w-v mice did not exhibit inflammation in response to PAR activation. EMSA results confirmed the increased TFEB binding activity in C57BL/6 but not in Kit w/Kit w-v mice. CONCLUSION: This is the first report describing the increased expression of TFEB in bladder inflammation in response to PAR activation. As TFEB belongs to a family of TFs essential for mast cell survival, our findings suggest that this molecule may influence the participation of mast cells in PAR-mediated inflammation and that targeting TFEB/MiTF activity may be a novel approach for the treatment of bladder inflammatory disorders.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Cistite/metabolismo , Redes Reguladoras de Genes , Inflamação/metabolismo , Receptores Ativados por Proteinase/metabolismo , Bexiga Urinária/metabolismo , Animais , Feminino , Mastócitos/citologia , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL/metabolismo , Camundongos Mutantes , Membrana Mucosa/citologia , Membrana Mucosa/metabolismo , Receptores Ativados por Proteinase/isolamento & purificação
16.
Comp Med ; 57(3): 255-66, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17605340

RESUMO

FVB/NJ mice frequently are used as transgenic hosts, but the suitability of this genetic background for transgenic and congenic models of systemic autoimmunity have not been reported. In this study, FVB/NJ mice were evaluated for the presence of serum autoantibodies and autoimmune kidney pathology. Previously unreported albuminuria was observed in aged female FVB/NJ mice; however, serum autoantibody testing, light microscopic evaluation of differentially stained renal sections, and evaluation of renal sections for immunoglobulin deposits revealed that the albuminuria was not of autoimmune etiology. Anecdotally, multiple characteristics of the FVB/NJ strain, including albuminuria, cholesterolemia, mild podocyte foot process effacement in aged female FVB/NJ kidneys and predisposition to enhanced Th2 immune responses, is reminiscent of human minimal change nephrotic syndrome (MCNS). We propose that mapping of genetic polymorphisms that are responsible for these traits in FVB/NJ mice may lead to increased understanding of mild nephrotic syndromes including MCNS and other proteinurias.


Assuntos
Albuminúria/metabolismo , Autoimunidade/fisiologia , Modelos Animais de Doenças , Animais , Anticorpos Antinucleares/sangue , Colesterol/sangue , Creatinina/urina , Feminino , Imunofluorescência , Membrana Basal Glomerular/metabolismo , Membrana Basal Glomerular/ultraestrutura , Imunoglobulina G/metabolismo , Rim/metabolismo , Rim/patologia , Rim/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Nefrose Lipoide/imunologia , Podócitos/ultraestrutura , Organismos Livres de Patógenos Específicos
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