RESUMO
BACKGROUND: Phagocytosis of apoptotic cells by macrophages induces a suppressor phenotype. Previous data from our group suggested that this occurs via Platelet-activating factor receptor (PAF-R)-mediated pathways. In the present study, we investigated the impact of apoptotic cell inoculation or induction by a chemotherapeutic agent (dacarbazine, DTIC) on tumour growth, microenvironmental parameters and survival, and the effect of treatment with a PAF-R antagonist (WEB2170). These studies were performed in murine tumours: Ehrlich Ascitis Tumour (EAT) and B16F10 melanoma. METHODS: Tumour growth was assessed by direct counting of EAT cells in the ascitis or by measuring the volume of the solid tumour. Parameters of the tumour microenvironment, such as the frequency of cells expressing cyclo-oxygenase-2 (COX-2), caspase-3 and galectin-3, and microvascular density, were determined by immunohistochemistry. Levels of vascular endothelium growth factor (VEGF) and prostaglandin E2 (PGE2) were determined by ELISA, and levels of nitric oxide (NO) by Griess reaction. PAF-R expression was analysed by immunohistochemistry and flow cytometry. RESULTS: Inoculation of apoptotic cells before EAT implantation stimulated tumour growth. This effect was reversed by in vivo pre-treatment with WEB2170. This treatment also reduced tumour growth and modified the microenvironment by reducing PGE2, VEGF and NO production. In B16F10 melanoma, WEB2170 alone or in association with DTIC significantly reduced tumour volume. Survival of the tumour-bearing mice was not affected by WEB2170 treatment but was significantly improved by the combination of DTIC with WEB2170. Tumour microenvironment elements were among the targets of the combination therapy since the relative frequency of COX-2 and galectin-3 positive cells and the microvascular density within the tumour mass were significantly reduced by treatment with WEB2170 or DTIC alone or in combination. Antibodies to PAF-R stained the cells from inside the tumour, but not the tumour cells grown in vitro. At the tissue level, a few cells (probably macrophages) stained positively with antibodies to PAF-R. CONCLUSIONS: We suggest that PAF-R-dependent pathways are activated during experimental tumour growth, modifying the microenvironment and the phenotype of the tumour macrophages in such a way as to favour tumour growth. Combination therapy with a PAF-R antagonist and a chemotherapeutic drug may represent a new and promising strategy for the treatment of some tumours.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma de Ehrlich/tratamento farmacológico , Melanoma Experimental/tratamento farmacológico , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Antineoplásicos Alquilantes/administração & dosagem , Apoptose/efeitos dos fármacos , Azepinas/administração & dosagem , Carcinoma de Ehrlich/irrigação sanguínea , Carcinoma de Ehrlich/metabolismo , Carcinoma de Ehrlich/patologia , Caspase 3/metabolismo , Proliferação de Células/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Dacarbazina/administração & dosagem , Dinoprostona/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Galectina 3/metabolismo , Imuno-Histoquímica , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neovascularização Patológica/metabolismo , Neovascularização Patológica/prevenção & controle , Óxido Nítrico/metabolismo , Fenótipo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Fatores de Tempo , Triazóis/administração & dosagem , Carga Tumoral , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Proteoglycans were accurately localized in mouse pubic symphyseal tissues using the cuprolinic blue method. Specific glycosaminoglycans degradative enzymes, together with chondroitin sulfate and decorin antibodies, allowed the identification of glycosaminoglycans. Chondroitin sulfate proteoglycans were the main proteoglycans observed in hyaline cartilage, fibrocartilage, and dense connective tissue. Ultrastructurally, they were seen as electron-dense granules and filaments. The granules, rich in chondroitin sulfate chains, were exclusively found in hyaline cartilage, whereas filaments were present in cartilage, fibrocartilage, and dense connective tissue. The latter were classified by size and susceptibility to enzyme digestion into F1, F2 and F3 filaments: F1 filaments were small, thin, and collagen fibril-associated; F2 filaments were thick, heavily stained, and localized around individual collagen fibrils and between bundles of collagen fibrils; and F3 filaments were scattered throughout elastic fiber surfaces. Considering their localization, susceptibility to chondroitinase AC and immunohistochemical detection, the symphysial F1 filaments were found to be preferentially decorin substituted with chondroitin sulfate side chains. The F2 filaments were also susceptible to chondroitinase AC treatment, whereas F3 filaments could be digested by heparitinase. The data thus obtained on the localization and identification of pubic symphyseal proteoglycans in virgin mice may be useful in the study of structural modifications that occur throughout pregnancy.