RESUMO
Matrix metalloproteinases (MMP) are a family of proteolytic enzymes, the expression of which in a key step of tumor progression has recently been better defined. The overexpression of one or more MMPs is thus common among malignant tumors. It may characterize tumor progression and help predict its response to chemotherapy. Consequently, the development of a device for measuring MMP activities is an important challenge for diagnosis and prognosis. In this study, we describe an innovative supramolecular peptide/surface assembly for screening MMP activities. This sensor was used to discriminate various MMP activities and to distinguish between invasive and noninvasive cancerous cell suspensions. Our results confirm the proof-of-concept of a powerful tool for the determination of the tumor aggressiveness and a technical building block for future development of MMP lab-on-chip devices.
Assuntos
Corantes Fluorescentes/química , Metaloproteinases da Matriz/metabolismo , Neoplasias/diagnóstico , Peptídeos/química , Sequência de Aminoácidos , Linhagem Celular Tumoral , Células HeLa , Humanos , Cinética , Dispositivos Lab-On-A-Chip , Microscopia de Fluorescência , Neoplasias/enzimologia , Neoplasias/patologia , Peptídeos/metabolismo , Índice de Gravidade de Doença , Espectrometria de Fluorescência , Propriedades de Superfície , beta-Ciclodextrinas/químicaRESUMO
It has been widely reported that the tear film, which is crucially important as a protective barrier of the eye, undergoes biochemical changes as a result of a wide range of ocular pathology. This tends to suggest the possibility of early detection of ocular diseases on the basis of biochemical analysis of tears. However, studies of tears by conventional methods of biomolecular and biochemical analysis are often limited by methodological difficulties. Moreover, such analysis could not be applied in the clinic, where structural and morphological analyses by, mainly, slit-lamp biomicroscopy remains the recommended method. In this study, we assessed, for the first time, the potential of FTIR spectroscopy combined with advanced chemometric processing of spectral data for analysis of raw tears for diagnosis purposes. We first optimized sampling and spectral acquisition (tears collection method, tear sample volume, and preservation of the samples) for accurate spectral measurement. On the basis of the results, we focused our study on the possibility of discriminating tears from normal individuals from those of patients with different ocular pathologies, and showed that the most discriminating spectral range is that corresponding to variations of CH2 and CH3 of lipid aliphatic chains. We also report more subtle discrimination of tears from patients with keratoconus and those from patients with non-specific inflammatory ocular diseases, on the basis of variations in spectral ranges attributed notably to lipid and carbohydrate vibrations. Finally, we also succeeded in distinguishing tears from patients with early-stage and late-stage keratoconus on the basis of spectral features attributed to protein structure. Therefore, this study strongly suggests that FTIR spectral analysis of tears could be developed as a valuable and cost-saving tool for biochemical-based detection of ocular diseases, potentially before the appearance of the first morphological signs of diseases. Combined with supervised modelling methods and with use of a spectral data base acquired for representative patients, such a spectral approach could be a useful addition to current methods of clinical analysis for improvement of patient care.
Assuntos
Oftalmopatias/diagnóstico , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Lágrimas/química , Adulto , Idoso , Feminino , Humanos , Lipídeos/química , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
A triethyleneglycol (TEG) chain, a linear peptide, and a cyclic peptide labeled with 7-methoxycoumarin-3-carboxylic acid (MC) and 7-diethylaminocoumarin-3-carboxylic acid (DAC) were used to thoroughly study Förster resonance energy transfer (FRET) in inclusion complexes. (1) H NMR evidence was given for the formation of a 1:1 inclusion complex between ß-cyclodextrin (ß-CD) and the fluorophore moieties of model compounds. The binding constant was 20 times higher for DAC than for MC derivatives. Molecular modeling provided additional information. The UV/Vis absorption and fluorescence properties were studied and the energy transfer process was quantified. Fluorescence quenching was particularly strong for the peptide derivatives. The presence of ß-CDs reduced the FRET efficiency slightly. Dye-labeled peptide derivatives can thus be used to form inclusion complexes with ß-CDs and retain most of their FRET properties. This paves the way for their subsequent use in analytical devices that are designed to measure the activity of matrix metalloproteinases.
Assuntos
Etilenoglicol/química , Corantes Fluorescentes/química , Peptídeos/química , beta-Ciclodextrinas/química , Cumarínicos/química , Transferência Ressonante de Energia de Fluorescência , Modelos MolecularesRESUMO
The PI3K/Akt-signaling pathway, associated with cancer development and disease progression, is recognized to be an anti-tumor drug target that could present important therapeutic benefit. However, no targeted Akt medicines have been commercialized yet, reflecting that drug selection procedures requires significant improvement from early research to clinical trials. Thus, new methods permitting both the evaluation of cytotoxic and proliferation inhibition effect on cancer cells but also to provide a global fingerprint of the drug action mechanism of new Akt inhibitor candidates are of major interest. Because it can detect very subtle molecular changes and could provide a global fingerprint of drug effects on cells, Fourier-transform infrared (FTIR) spectroscopy appears to be a promising method to develop new time- and cost-saving tools for chemical library screening improvements. In this study, we combine FTIR spectroscopy, advanced chemometrics analysis and cross-validation by standard biological assays to establish a basis of a mid-throughput methodology for rapid and automated assessment of cell response to Akt inhibitors and quantitative evaluation of their anti-proliferative effects. Our results shows that our methodology is able (1) to detect cell response to an Akt inhibitor exposure even for very low doses, (2) to provide biochemical information of interest about its effects on the cell metabolism, lipidome, and proteome, (3) to predict accurately resulting cell proliferation inhibition rate. Thus, further based on a large spectral data base, our methodology could contribute to facilitate preliminary screening of chemical libraries and improving the selection procedure of drug candidates in laboratory routine.
Assuntos
Ensaios de Seleção de Medicamentos Antitumorais/métodos , Leucemia/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Automação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Ensaios de Triagem em Larga Escala/métodos , Humanos , Leucemia/tratamento farmacológico , Leucemia/fisiopatologia , Modelos Biológicos , Inibidores de Proteínas Quinases/análiseRESUMO
Characterization of matrix metalloprotease (MMP) activities is of increasing interest for cancer prognosis or treatment follow-up. Indeed, MMP-1, -2 and -9 are widely involved in the growth of many tumors and progression steps such as angiogenesis, invasion, and metastasis. Fluorogenic peptide MMP substrates were previously synthesized with the aim of detecting MMP activities. One of their drawbacks is their limited solubility in biological media. Grafting them onto a solid support represented a novel way to yield efficient analysis devices whilst at the same time decreasing the quantities of peptides used. Novel peptide arrays were designed in order to detect MMP activities in biological fluids. Silicon plates were used as the solid support for the design of these novel tools. These were functionalized by organic self-assembled monolayers (SAMs) on which fluorogenic peptides were covalently coupled. SAM and peptide grafting on silicon plates were confirmed by epifluorescence, ellipsometry, and FT-IR analysis. Enzymatic assays were monitored by fluorescence spectrometry and showed that immobilized linear peptides were recognized and cleaved by MMPs.
Assuntos
Metaloproteinases da Matriz/metabolismo , Peptídeos/química , Microscopia de Fluorescência , Espectroscopia de Infravermelho com Transformada de Fourier , Especificidade por SubstratoRESUMO
Following our search for antimalarial compounds, novel series of ferrocenyl-substituted pyrrolo[1,2-a]quinoxalines 1-2 were synthesized from ferrocene-carboxaldehyde and tested for their in vitro activity upon the erythrocytic development of Plasmodium falciparum strains with different chloroquine-resistance status. The ferrocenic pyrrolo[1,2-a]quinoxalines 1-2 were prepared in 6 or 9 steps through a Barton-Zard reaction. Promising pharmacological results against FcB1, K1 and F32 strains were obtained with ferrocenyl pyrrolo[1,2-a]quinoxalines 1j-l linked by a bis-(3-aminopropyl)piperazine linker substituted by a nitrobenzyl moiety.
Assuntos
Antimaláricos/farmacologia , Compostos Ferrosos/química , Plasmodium falciparum/efeitos dos fármacos , Pirróis/farmacologia , Quinoxalinas/farmacologia , Antimaláricos/síntese química , Antimaláricos/química , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Humanos , Metalocenos , Modelos Moleculares , Estrutura Molecular , Testes de Sensibilidade Parasitária , Pirróis/síntese química , Pirróis/química , Quinoxalinas/síntese química , Quinoxalinas/química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
PURPOSE: Angiogenesis plays a critical role in tumor growth. This phenomena is regulated by numerous mediators such as vascular endothelial growth factor (VEGF). CBO-P11, a cyclo-peptide, has proven to specifically bind to receptors of VEGF and may be used as targeting ligand for tumor angiogenesis. We herein report the design of novel nanoparticles conjugated to CBO-P11 in order to specifically target tumor site. METHODS: The conjugation of CBO-P11 on the surface of poly(vinylidene fluoride) (PVDF) nanoparticles was investigated using the copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition known as "click" reaction. CBO-P11 was modified with a near-infrared cyanine dye bearing an alkyne function, allowing both "click" coupling on azido-modified nanoparticles and fluorescence labelling. Each step of this nanodevice construction was judiciously performed in aqueous solution and successfully characterized. The cytotoxicity of nanoparticles was evaluated in human brain endothelial cell line and their affinity for VEGF receptors was determined via fluorescence-based uptake assays on porcine aortic endothelial cell line. RESULTS: Nanoparticles were found to be spherical, dense, monodisperse and stable. No cytotoxicity was observed after four days of incubation demonstrating the biocompatibility of nanoparticles. Fluorescence highlighted the specific interaction of these functionalized nanoparticles for VEGF receptors, suggesting that the targeting peptide bioactivity was retained. CONCLUSIONS: These results demonstrate the potential of these functionalized nanoparticles for targeting tumor angiogenesis and their possible use as multifunctional platform for cancer treatment if coupled with therapeutic agents.
Assuntos
Sistemas de Liberação de Medicamentos , Nanopartículas/química , Peptídeos/metabolismo , Polivinil/química , Receptores de Fatores de Crescimento do Endotélio Vascular/química , Animais , Linhagem Celular , Química Click , Fatores de Crescimento Endotelial/química , Humanos , Estrutura Molecular , Neovascularização Patológica/tratamento farmacológico , Peptídeos Cíclicos/química , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de Superfície , SuínosRESUMO
A novel series of isoindolo[2,1-a]quinoxaline and indolo[1,2-a]quinoxaline derivatives was synthesized and evaluated in vitro against various human cancer cell lines for antiproliferative activity. These new compounds displayed activity against leukemia and breast cancer cell lines in the 3- to 18-µM concentration range.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Quinoxalinas/síntese química , Quinoxalinas/farmacologia , Antineoplásicos/química , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Concentração Inibidora 50 , Leucemia/tratamento farmacológico , Modelos Moleculares , Estrutura Molecular , Quinoxalinas/química , Relação Estrutura-AtividadeRESUMO
PET (Positron Emission Tomography) allows imaging of the in vivo distribution of biochemical compounds labeled with a radioactive tracer, mainly 18F-FDG (2-deoxy-2-[18F] fluoro-D-glucose). 18F only allows a relatively poor spatial resolution (2-3 mm) which does not allow imaging of small tumors or specific small size tissues, e.g. vasculature. Unfortunately, angiogenesis is a key process in various physiologic and pathologic processes and is, for instance, involved in modern anticancer approaches. Thus ability to visualize angiogenesis could allow early diagnosis and help to monitor the response of cancer to specific chemotherapies. Therefore, indirect analytical techniques are required to assess the localization of fluorinated compounds at a micrometric scale. Multimodality imaging approaches could provide accurate information on the metabolic activity of the target tissue. In this article, PIGE method (Particle Induced Gamma-ray Emission) was used to determine fluorinated tracers by the nuclear reaction of 19F(p,p'γ)19F in tissues. The feasibility of this approach was assessed on polyfluorinated model glucose compounds and novel peptide-based tracer designed for angiogenesis imaging. Our results describe the first mapping of the biodistribution of fluorinated compounds in both vascularized normal tissue and tumor tissue.
RESUMO
Malignant gliomas are very aggressive tumors, highly angiogenic and invading heterogeneously the surrounding brain parenchyma, making their resection very difficult. To overcome the limits of current diagnostic imaging techniques used for gliomas, we proposed using FTIR imaging, with a spatial resolution from 6 to 10 µm, to provide molecular information for their histological examination, based on discrimination between normal and tumor vasculature. Differentiation between normal and tumor blood vessel spectra by hierarchical cluster analysis was performed on tissue sections obtained from xenografted brain tumors of Rag-gamma mice 28 days after intracranial implantation of glioma cells, as well as for human brain tumors obtained in clinics. Classical pathological examination and immunohistochemistry were performed in parallel to the FTIR spectral imaging of brain tissues. First on the animal model, classification of FTIR spectra of blood vessels could be performed using spectral intervals based on fatty acyl (3050-2800 cm(-1)) and carbohydrate (1180-950 cm(-1)) absorptions, with the formation of two clusters corresponding to healthy and tumor parts of the tissue sections. Further data treatments on these two spectral intervals provided interpretable information about the molecular contents involved in the differentiation between normal and tumor blood vessels, the latter presenting a higher level of fatty acyl chain unsaturation and an unexpected loss of absorption from osidic residues. This classification method was further successfully tested on human glioma tissue sections. These findings demonstrate that FTIR imaging could highlight discriminant molecular markers to distinguish between normal and tumor vasculature, and help to delimitate areas of corresponding tissue.
Assuntos
Neoplasias Encefálicas/patologia , Capilares/anatomia & histologia , Capilares/patologia , Diagnóstico por Imagem/métodos , Glioma/patologia , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Animais , Neoplasias Encefálicas/química , Neoplasias Encefálicas/classificação , Neoplasias Encefálicas/diagnóstico , Capilares/química , Carboidratos/análise , Linhagem Celular Tumoral , Análise por Conglomerados , Ácidos Graxos/análise , Glioma/química , Glioma/classificação , Glioma/diagnóstico , Humanos , Masculino , Camundongos , Camundongos Endogâmicos , Transplante de Neoplasias , Neovascularização Patológica , Transplante HeterólogoRESUMO
PURPOSE: Matrix metalloproteinases (MMP) are a family of proteolytic enzymes, the expression of which in a key step of tumor progression has been better defined recently. The studies highlighted the ongoing need for very specific inhibitors, substrates or release devices designed to be selective for one or at least very few MMPs. METHODS: This report deals with the design, synthesis and in vitro evaluation of linear and especially novel cyclic peptidic moieties, embodying MMP cleavable sequences designed to answer these questions. FRET (fluorescence resonance energy transfer) labelling via chromophore-modified amino-acids was used to give access to enzyme kinetics. RESULTS: Evaluation of these peptides showed that cyclisation gives rise to high specificity for certain MMP, suggesting that this approach could provide very specific MMP substrate. Moreover, cyclic structures present a very good plasma stability. CONCLUSIONS: These original derivatives could allow the design of MMP-controlled delivery devices, the specificity of which will be retained in complex biological media and in vivo.
Assuntos
Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Inibidores de Metaloproteinases de Matriz , Peptídeos/síntese química , Peptídeos/uso terapêutico , Sequência de Aminoácidos , Ciclização , Transferência Ressonante de Energia de Fluorescência , Humanos , Cinética , Dados de Sequência MolecularRESUMO
UNLABELLED: A significant antitumor effect was previously observed with radioimmunotherapy using anti-carcinoembryonic antigen (131)I-F6 monoclonal antibody in medullary thyroid cancer-bearing nude mice. Nevertheless, no complete response was observed. As seen with chemotherapy, drugs targeting the tumor microenvironment might improve radioimmunotherapy efficacy. This study evaluated the toxicity and efficacy of combining radioimmunotherapy with thalidomide or a cyclopeptidic vascular endothelial growth inhibitor (CBOP11) in mice grafted with the TT human medullary thyroid cancer cell line. METHODS: Six to 10 nude mice treated with 92.5 MBq of (131)I-F6 in association with 200 mg/kg/d of oral thalidomide during 20 d by force-feeding or 0.45 mg/kg/d of CBOP11 during 25 d using subcutaneous minipumps were compared with control mice receiving either treatment or naked F6 or nonspecific (131)I-734. Combined therapies included (131)I-F6 at day 0 followed by thalidomide between days 20 and 40, thalidomide between days 0 and 20 followed by (131)I-F6 at day 25, (131)I-F6 at day 0 and CBOP11 between days 0 and 25, CBOP11 between days 0 and 25 followed by (131)I-F6 at day 25, and (131)I-F6 at day 0 followed by CBOP11 between days 20 and 45. Animal weight, hematologic toxicity, tumor volume, and serum calcitonin were monitored for the following 3 mo. Improvement of (125)I-F6 tumor biodistribution by antiangiogenic drug was studied after pretreatment by thalidomide. Follow-up of the tumor after combined antiangiogenic and radioimmunotherapy therapies was performed by histology studies. RESULTS: Combined associations, as compared with radioimmunotherapy alone, increased leukopenia but not thrombocytopenia. Tumor volume-quadrupling time (TVQT) was 22.8 +/- 3.3 d in the control group, 29.9 +/- 3.6 d in the group treated with thalidomide, 34.6 +/- 4.4 d in the group treated with CBOP11, and 51.0 +/- 2.8 d after radioimmunotherapy alone. As compared with radioimmunotherapy, TVQT was significantly longer (P < 0.01) after thalidomide followed by radioimmunotherapy (69.83 +/- 3.9), CBOP11 followed by radioimmunotherapy (71.3 +/- 6.1), and CBOP11-radioimmunotherapy in concomitance (64.2 +/- 6.1). Nevertheless, TVQT was not increased after radioimmunotherapy followed by thalidomide (48.8 +/- 4) and radioimmunotherapy followed by CBOP11 (56.8 +/- 4.8). Surprisingly, pretreatment by CBOP11 or thalidomide sensitized larger tumors (>300 mm(3)) to radioimmunotherapy. Change in calcitonin levels confirmed morphologic tumor response. Tumor uptake 24 h after injection of (125)I-F6 was 4.5 +/- 0.6 percentage injected dose per gram (%ID/g) without pretreatment and 8.7 +/- 1.3 %ID/g with pretreatment by thalidomide. An increase of the antitumor effect observed using the antiangiogenic drug combined with radioimmunotherapy was correlated with a decrease of blood vessels shown by von Willebrand immunostaining. CONCLUSION: Pretreatment with antiangiogenic therapies improved radioimmunotherapy efficacy, with acceptable toxicity. Future investigations will be performed to understand how antiangiogenic agents sensitize large tumors to radioimmunotherapy.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Antígeno Carcinoembrionário/metabolismo , Regulação Neoplásica da Expressão Gênica , Radioimunoterapia , Neoplasias da Glândula Tireoide/terapia , Ensaios Antitumorais Modelo de Xenoenxerto , Inibidores da Angiogênese/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/uso terapêutico , Apoptose/efeitos dos fármacos , Antígeno Carcinoembrionário/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Terapia Combinada , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Fatores de Crescimento Endotelial/farmacocinética , Fatores de Crescimento Endotelial/farmacologia , Fatores de Crescimento Endotelial/uso terapêutico , Humanos , Radioisótopos do Iodo/química , Camundongos , Peptídeos Cíclicos/farmacocinética , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/uso terapêutico , Talidomida/farmacologia , Talidomida/uso terapêutico , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Distribuição Tecidual , Resultado do Tratamento , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Ortho-nitro- and ortho-alkoxy-oligo-meta-aniline units fold in solution through hydrogen bonds and aromatic stacking into compact structures that were characterized in the solid state as cylindrical beta-sheet like structures.
Assuntos
Compostos de Anilina/química , Cristalografia por Raios X , Ligação de Hidrogênio , Conformação MolecularRESUMO
Vascular basement membrane remodeling is involved in tumor angiogenesis to enable tumor invasion and growth. FT-IR spectral imaging was used to determine changes in tumor blood vessels to reveal protein secondary structure in Rag-gamma immuno-deficient mice sacrificed 14 and 21 days after subcutaneous glioma implantation. For the oldest blood capillaries (diameter >20 microns), tumor growth induced a decrease in triple-helix content (1638 cm(-1); -7.3%; P < 0.05) and an increase in beta turns (1666 and 1615 cm(-1); +4%; P < 0.01). These protein-structure alterations, mainly from type IV collagen, reflected the high angiogenic stress of growing tumors. We propose to use these molecular markers of vascular basement membrane protein alterations for gradation of solid tumors by FT-IR spectral imaging.
Assuntos
Colágeno Tipo IV/análise , Glioma/irrigação sanguínea , Proteínas de Membrana/análise , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Animais , Membrana Basal/química , Membrana Basal/metabolismo , Membrana Basal/patologia , Capilares/química , Capilares/metabolismo , Capilares/patologia , Colágeno Tipo IV/química , Colágeno Tipo IV/metabolismo , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Estrutura Secundária de Proteína , Ratos , Transplante HeterólogoRESUMO
Tumor development leading to cancer is a complex process involving several steps. Among them, angiogenesis, ie growth of new tumor induced blood vessels is one of the most important therapeutic targets in the search for anticancer agents. One point which remain to be addressed is the detection of tumor angiogenic areas, ie tumor angiogenesis imaging. After presenting the key points of tumor development which lead to neoangiogenesis, and providing an overview of the main therapeutic approaches in this field, this review focuses on the recent progress in angiogenesis imaging, namely the one dealing with matrix metalloproteases. These enzymes are indeed present to major phenomena of the tumor progression. The different imaging approaches are described, namely the ones using optical, radiochemical or magnetic resonance ones.
Assuntos
Diagnóstico por Imagem/tendências , Neoplasias/irrigação sanguínea , Neovascularização Patológica/diagnóstico , HumanosRESUMO
Thrombin inhibition protects against liver fibrosis. However, it is not known whether the thrombin profibrogenic effect is due to effects on blood coagulation or to signaling via protease-activated receptors (PARs). We took advantage of the lack of blood coagulation defects in PAR-1-knockout mice. Acute carbon tetrachloride (CCl(4)) toxicity was similar in wild-type (WT), PAR-1(-/-), and PAR-1(+/-) mice as judged by aminotransferase levels, area of liver necrosis, and liver peroxidation measured by Fourier-transformed infrared spectroscopy. Fifteen mice/group received CCl(4) or its solvent for 6 wk (300 microl/kg, 3 times a week). Fibrosis area was increased 10-fold by CCl(4) treatment in WT mice. PAR-1 deficiency protected against fibrosis, with 36% and 56% decrease in PAR-1(+/-) and PAR-1(-/-) mice, respectively (P < 0.001). Similar results were obtained for area of activated fibrogenic cells (64% and 79% decrease in PAR-1(+/-) and PAR-1(-/-) mice, respectively, P < 0.001). These findings were corroborated by measurements of type I collagen, matrix metalloproteinase-2, and PDGF-beta receptor mRNA levels. There was also a significant decrease in T lymphocyte infiltration in PAR-1-deficient mice. Altogether, these results suggest that thrombin profibrogenic effects are independent of effects on blood coagulation and are instead due to direct effects on fibrogenic cells and possibly on T lymphocytes.
Assuntos
Cirrose Hepática/prevenção & controle , Fígado/metabolismo , Receptor PAR-1/metabolismo , Trombina/metabolismo , Animais , Tetracloreto de Carbono , Hipóxia Celular , Quimiocina CCL2/metabolismo , Quimiotaxia de Leucócito , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Fator de Crescimento do Tecido Conjuntivo , Modelos Animais de Doenças , Genótipo , Proteínas Imediatamente Precoces/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peroxidação de Lipídeos , Fígado/enzimologia , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Masculino , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Necrose , RNA Mensageiro/metabolismo , Receptor PAR-1/deficiência , Receptor PAR-1/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Linfócitos T/metabolismo , Transaminases/sangueRESUMO
In this study, we report a rapid sonochemical synthesis of monodisperse nonaggregated Fe(3)O(4)@SiO(2) magnetic nanoparticles (NPs). We found that coprecipitation of Fe(II) and Fe(III) in aqueous solutions under the effect of power ultrasound yields smaller Fe(3)O(4) NPs with a narrow size distribution (4-8 nm) compared to the silent reaction. Moreover, the coating of Fe(3)O(4) NPs with silica using an alkaline hydrolysis of tetraethyl orthosilicate in ethanol-water mixture is accelerated many-fold in the presence of a 20 kHz ultrasonic field. The thickness of the silica shell can be easily controlled in the range of several nanometers during sonication. Mossbauer spectra revealed that nonsuperparamagnetic behavior of obtained core-shell NPs is mostly related to the dipole-dipole interactions of magnetic cores and not to the particle size effect. Core-shell Fe(3)O(4)@SiO(2) NPs prepared with sonochemistry exhibit a higher magnetization value than that for NPs obtained under silent conditions owing to better control of the deposited silica quantities as well as to the high speed of sonochemical coating, which prevents the magnetite from oxidizing.
Assuntos
Cristalização/métodos , Compostos Férricos/química , Magnetismo/métodos , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Dióxido de Silício/química , Sonicação , Compostos Férricos/efeitos da radiação , Substâncias Macromoleculares/química , Substâncias Macromoleculares/efeitos da radiação , Teste de Materiais , Conformação Molecular/efeitos da radiação , Nanoestruturas/efeitos da radiação , Nanotecnologia/métodos , Tamanho da Partícula , Dióxido de Silício/efeitos da radiação , Propriedades de Superfície/efeitos da radiaçãoRESUMO
Fourier-transform infrared (FT-IR) spectral imaging was used for analyzing biochemical changes in tumor cells. Metabolic parameters of human lung A549/8 adenocarcinoma and U87 glioma cells were compared under stress conditions in culture along with tumor progression after cell implantation onto the chick embryo chorio-allantoic membrane. In cell culture, glucose consumption and lactic acid release were higher in U87 cells. A549/8 cells were less sensitive to oxidative stress as observed through changes in fatty acyl chains. In vivo biochemical mapping of highly (U87) vs. poorly (A549/8) angiogenic tumors provided results comparable to culture models. Therefore, FT-IR imaging allows detecting subtle chemical changes in tumors, which might be useful for diagnosis.
Assuntos
Neoplasias/metabolismo , Neoplasias/patologia , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Glucose/farmacologia , Humanos , Transplante de Neoplasias , Neoplasias/irrigação sanguíneaRESUMO
Tumor ischemia participates in angiogenesis and cancer progression through cellular responses to hypoxia and nutrient deprivation. However, the contribution of amino acids limitation to this process remains poorly understood. Using serum-free cell culture conditions, we tested the impact of L-glutamine deprivation on metabolic and angiogenic responses in A549/8 carcinoma cells. In these cells, lowering glutamine concentration modified the cell cycle distribution and significantly induced apoptosis/necrosis. Although glutamine deprivation led to a HIF-independent increase in VEGF-A mRNA, the corresponding protein level remained low and correlated with the inhibition of protein synthesis and activation of the GCN2/eIF2alpha pathway. Limitation of glutamine availability also hampers hypoxia- and hypoglycemia-induced VEGF-A protein upregulation. Thus, glutamine deprivation may have no direct effect on VEGF-dependent angiogenesis, compared to hypoxia or to glucose deprivation, and may instead be detrimental to cancer progression by antagonizing ischemia-induced stresses.