RESUMO
The angiogenic process is central in the pathogenesis of various diseases. The in vitro and in vivo monitoring of the neovascular process is essential for the development and evaluation of angiogenesis inhibitors or stimulators. Since no single method exists that can assess angiogenesis in a robust, reliable, and reproducible fashion, researchers often use a combination of assays to circumvent this problem. The experimental details of the most commonly in vitro, ex vivo, and in vivo assays are presented here.
Assuntos
Bioensaio/métodos , Neovascularização Patológica , Neovascularização Fisiológica , Animais , Aorta/fisiologia , Membrana Corioalantoide/irrigação sanguínea , Técnicas de Cocultura , Colágeno/metabolismo , Córnea/irrigação sanguínea , Combinação de Medicamentos , Células Endoteliais/citologia , Feminino , Fibroblastos/citologia , Humanos , Recém-Nascido , Laminina/metabolismo , Masculino , Camundongos , Proteoglicanas/metabolismo , Ratos , Retinopatia da Prematuridade/fisiopatologiaRESUMO
Leukocyte adhesion to the vascular wall is a critical early step in the pathogenesis of inflammatory diseases and is mediated in part by the leukocyte integrin, VLA-4, which binds to endothelial vascular cell adhesion molecule (VCAM) -1. Here, we investigate VLA-4's role in endotoxin-induced uveitis (EIU). At various time points (6-48 h) after EIU induction, the severity of the inflammation was evaluated by quantifying cell and protein content in the aqueous fluid, firm leukocyte adhesion in the retinal vessels, and the number of extravasated leukocytes into the vitreous. Functional activation of VLA-4 in vivo was investigated in our previously introduced autoperfused micro flow chamber assay. Firm adhesion of EIU leukocytes to immobilized VCAM-1 under physiological blood flow conditions was significantly increased compared with normal controls (P<0.05), suggesting an important role for VLA-4 in EIU. VLA-4 blockade in vivo significantly suppressed all uveitis-related inflammatory parameters studied, decreasing the clinical score by 45% (P<0.01), protein content in the aqueous fluid by 21% (P<0.01), retinal leukostasis by 68% (P<0.01), and leukocyte accumulation in the vitreous by 75% (P<0.01). Our data provide novel evidence for functional up-regulation of VLA-4 during EIU and suggest VLA-4 blockade as a promising therapeutic strategy for treatment of acute inflammatory eye diseases.
Assuntos
Endotoxinas/toxicidade , Integrina alfa4beta1/metabolismo , Integrinas/metabolismo , Uveíte/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Western Blotting , Adesão Celular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Proteínas do Olho/metabolismo , Integrina alfa4beta1/imunologia , Integrina alfa4beta1/fisiologia , Integrinas/fisiologia , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Ratos , Fatores de Tempo , Regulação para Cima/efeitos dos fármacos , Uveíte/induzido quimicamente , Uveíte/fisiopatologia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
AIM: To report long term efficacy and complications of retinectomy as an intraocular pressure lowering procedure for intractable glaucoma. METHODS: This was a consecutive interventional case series. In 44 consecutive eyes (39 patients, 22 men and 17 women) retinectomy was performed to lower the intraocular pressure (IOP) in patients with uncontrolled IOP (>35 mm Hg for more than 4 months) despite conventional filtering surgery and drug treatment. Pars plana vitrectomy was performed and the peripheral retina was surgically excised to various degrees. The procedure was concluded by an intraocular gas tamponade of 20% C(3)F(8). Included were patients with neovascular glaucoma (12 eyes), infantile and juvenile glaucoma (three eyes), secondary glaucoma due to aphakia (13 eyes), severe ocular trauma (seven eyes), uveitis (seven eyes), and glaucoma in Ehlers-Danlos syndrome (two). RESULTS: All patients underwent successful surgical retinectomy. All patients were followed for 5 years. Mean postoperative IOP after 4 years was 15.7 (SD 9.4) mm Hg, representing a decrease of IOP by 61% compared to the preoperative level (41.2 (9.4) mm Hg). In 52.3% of eyes long term regulation of IOP could be achieved without complications. Retinectomy was least effective in neovascular glaucoma because of central retinal vein occlusion (CRVO). Eyes with glaucoma secondary to uveitis showed a tendency towards low IOP levels with subsequent phthisis bulbi. The initial visual acuity of all patients was lower than 20/50 (mean 1.8 (0.8) logMAR) in the treated eye. Final visual acuity was 2.3 (0.6) logMAR. 21 out of 44 cases developed retinal complications (retinal detachment or proliferative vitreoretinopathy (PVR)) after surgery, requiring silicone tamponade in 11 eyes (52%) either for persistent low IOP or for PVR. Nine eyes developed phthisis, seven of which were enucleated during the follow up. CONCLUSIONS: Long term results after retinectomy demonstrate its efficacy in otherwise intractable glaucoma. Efficacy and safety of retinectomy are dependent on the underlying disease.
Assuntos
Glaucoma/cirurgia , Retina/cirurgia , Adulto , Idoso , Doença Crônica , Feminino , Glaucoma/fisiopatologia , Glaucoma Neovascular/fisiopatologia , Glaucoma Neovascular/cirurgia , Humanos , Pressão Intraocular/fisiologia , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Cuidados Pós-Operatórios , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/cirurgia , Estudos Prospectivos , Reoperação , Doenças Retinianas/etiologia , Doenças Retinianas/cirurgia , Óleos de Silicone/uso terapêutico , Resultado do Tratamento , Transtornos da Visão/etiologia , Acuidade Visual/fisiologiaRESUMO
The tumor necrosis factor (TNF) family comprises several ligands, such as the prototype TNF-alpha, the Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL/Apo2L), which trigger apoptosis in susceptible cells by activating respective cell-surface receptors. The study of these cell death pathways has attracted significant attention in several fields, including that of thyroid cancer, because they participate in immune system function, as an arm of cell-mediated cytotoxicity, and because recombinant ligands are available for pharmacological use. TNF-alpha is a pluripotent cytokine that induces both pro-apoptotic and anti-apoptotic effects on thyroid carcinoma cells. FasL triggers apoptosis in other tumor types, but thyroid carcinoma cells are resistant to this effect. On the other hand, TRAIL potently and selectively kills thyroid carcinoma cells. Consequently, TRAIL is the only member of the family with significant anticancer activity and an acceptable toxicity profile to be used as a novel therapy for thyroid cancer. The caspase inhibitor FLIP plays a significant role in negatively regulating receptor-induced apoptosis. Thelper 1-type cytokines, such as interferon-gamma, TNF-alpha and interleukin-1beta increase the sensitivity of both normal and neoplastic thyrocytes to FasL and TRAIL. On the other hand, IGF-I and other growth/survival factors produced in the local tumor microenvironment activate the phosphatidylinositol 3-kinase/Akt kinase pathway and exert an anti-apoptotic effect by upregulating several apoptosis inhibitors, including FLIP. Pharmacological modulation of apoptosis induced by FasL and TRAIL/Apo2L holds promise of therapeutic applications in human malignancies.
Assuntos
Glicoproteínas de Membrana/fisiologia , Neoplasias da Glândula Tireoide/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Proteínas de Bactérias/metabolismo , Comunicação Celular , Proteína Ligante Fas , Humanos , Interferon gama/metabolismo , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/uso terapêutico , Receptores do Fator de Necrose Tumoral/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF , Células Th1/imunologia , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/imunologia , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/uso terapêutico , Receptor fas/metabolismoRESUMO
PURPOSE: Studies have demonstrated a causal role for specific molecules in the pathogenesis of diabetic retinopathy. Among the implicated mediators are growth factors such as vascular endothelial growth factor (VEGF) as well as adhesion molecules and proliferation- and apoptosis-related genes. However, a coordinated large-scale investigation of gene expression in the diabetic retina has not yet been reported. Here the retinal gene expression profile of diabetic and nondiabetic animals using cDNA microarrays were analyzed and compared. METHODS: Long-Evans rats were made diabetic with streptozotocin. Retinal gene expression was analyzed over 3 weeks using high-density nylon filter-based cDNA arrays. Genes were sorted into clusters according to their temporal expression profiles. They were also grouped according to their potential pathophysiological significance. The in vivo gene expression profiles of selected genes were verified via RNase protection assay. RESULTS: The rat GeneFilter contains a total of 5147 genes, of which 1691 are known genes and 3456 are expressed sequence tags (ESTs). On day 3, the expression of 27 known genes was increased by more than twofold. On days 7 and 21, the corresponding numbers were 60 and 12, respectively. A transient upregulation (>2-fold) in expression was seen in 627 of 5147 total genes. A subset of 926 genes exhibited a modest (<2-fold) decrease in expression. No genes showed a greater than twofold decrease in expression. Overall, the identity of the genes that were upregulated suggests that the response of the retina to the diabetic challenge contains an inflammatory component. Moreover, most regulatory activity occurs during the first week of diabetes. CONCLUSIONS: The development of a rational therapy for diabetic retinopathy will be assisted by detailed knowledge regarding the molecular pathophysiology of the disease. Here, an expression profile of an underlying retinal inflammatory process in early diabetes was extracted. Beyond providing insight into the general nature of the response to a pathogenic challenge, gene expression profiling may also allow the efficient identification of potential drug targets and markers for monitoring the course of disease.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética/metabolismo , Proteínas do Olho/genética , Expressão Gênica , Retina/metabolismo , Animais , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Ratos , Ratos Long-Evans , Regulação para CimaRESUMO
PURPOSE: To determine the efficacy of the angiogenic inhibitor TNP-470 on inflammatory corneal neovascularization. Topical and systemic delivery of the drug were investigated in a murine model as well as inhibition of endothelial cell proliferation in vitro and in vivo. METHODS: The effect of TNP-470 on VEGF- and bFGF-stimulated bovine capillary endothelial (BCE) cell proliferation was evaluated in vitro. Corneal neovascularization was induced in vivo by mechanical debridement of the corneal and limbal epithelium with 0.15 M NaOH on C57BL6 mice. TNP-470 was administered systemically at 30 mg/kg body weight (BW) every other day or topically three times daily in a concentration of 5 ng/ml dissolved in methylcellulose. Vessel length was investigated on day 7. VEGF protein content in murine corneas was analyzed by ELISA on days 2, 4, and 7 of treatment. A modified bromouridine (BrdU) ELISA was used to quantify endothelial cell proliferation. RESULTS: TNP-470 exerted a dose-dependent inhibition of bFGF- and VEGF-induced endothelial cell proliferation in vitro. Both systemic and topical application of TNP-470 led to a significant reduction of inflammatory corneal neovascularization (P < 1 x 10(-5)). BrdU labeling showed that TNP-470 inhibited endothelial cell proliferation. VEGF protein levels were reduced by systemic TNP-470 treatment. CONCLUSIONS: These results suggest that TNP-470 reduces inflammatory corneal angiogenesis by directly inhibiting endothelial cell proliferation. Topical and systemic treatment with TNP-470 reduces VEGF levels that are responsible for vessel growth during the neovascularization process.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Neovascularização da Córnea/prevenção & controle , Sesquiterpenos/uso terapêutico , Administração Tópica , Inibidores da Angiogênese/farmacologia , Animais , Bromodesoxiuridina , Divisão Celular/efeitos dos fármacos , Neovascularização da Córnea/patologia , Cicloexanos , Replicação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fatores de Crescimento Endotelial/farmacologia , Endotélio Corneano/efeitos dos fármacos , Endotélio Corneano/patologia , Endotélio Vascular/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Fator 2 de Crescimento de Fibroblastos/farmacologia , Injeções Subcutâneas , Linfocinas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , O-(Cloroacetilcarbamoil)fumagilol , Sesquiterpenos/farmacologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
FasL and TRAIL/Apo2L participate in cell-mediated cytotoxicity by inducing apoptosis in susceptible cells via respective cell surface receptors. Normal and neoplastic thyroid tissues are resistant to FasL-induced apoptosis but are sensitized by Th-1-type cytokines. In Hashimoto's thyroiditis, both FasL and its receptor, Fas, are strongly upregulated and their interaction leads to the suicidal/fratricidal death of thyrocytes. In Graves' disease, FasL expression in thyroid follicular cells is induced by thionamides and kills infiltrating lymphocytes. In this condition, Th-2-type cytokines upregulate the anti-apoptotic molecules FLIP and Bcl-x(L) and protect thyrocytes from apoptosis. FasL is expressed by neoplastic thyrocytes and induces apoptosis of infiltrating lymphocytes. TRAIL/Apo2L kills thyroid carcinoma cells but spares normal thyrocytes, thus providing a potential therapy for thyroid cancer.
Assuntos
Apoptose/fisiologia , Glicoproteínas de Membrana/fisiologia , Doenças da Glândula Tireoide/etiologia , Fator de Necrose Tumoral alfa/fisiologia , Proteínas Reguladoras de Apoptose , Proteína Ligante Fas , Doença de Graves/metabolismo , Humanos , Ligante Indutor de Apoptose Relacionado a TNF , Neoplasias da Glândula Tireoide/fisiopatologia , Receptor fas/metabolismoRESUMO
PURPOSE: To determine the efficacy and safety of naked plasmid gene therapy to the corneal stroma and epithelium. METHODS: Naked plasmid DNA was injected under pressure into the cornea of mice. The expression of genes coding for beta galactosidase (beta-gal), enhanced green fluorescent protein (EGFP), vascular endothelial growth factor (VEGF), and soluble Flt-1 (s-Flt) was recorded and measured with regard to dose, time course, and bioactivity. RESULTS: LacZ gene expression of the protein beta-gal was demonstrated as early as 1 hour, with expression persisting for 10 days. Plasmid-injected corneas remained clear and free of inflammation. EGFP was bicistronically expressed with VEGF to demonstrate the practicality of simultaneous in vivo analysis of gene expression and growth factor bioactivity. Corneal injection of a plasmid containing VEGF cDNA induced corneal and anterior chamber neovascularization. Moreover, corneal injection of plasmid containing the cDNA for the soluble form of the VEGF receptor Flt-1 effectively prevented corneal neovascularization. CONCLUSIONS: The cornea is readily accessible for gene therapy in the laboratory and in the clinic. The method described is safe, effective, titratable, and easily monitored. Naked DNA delivery to the cornea has the potential to alter the treatment of a wide variety of corneal and anterior segment diseases.
Assuntos
Córnea/metabolismo , Neovascularização da Córnea/prevenção & controle , DNA/administração & dosagem , Plasmídeos/genética , Transfecção/métodos , Animais , Córnea/patologia , Neovascularização da Córnea/induzido quimicamente , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Relação Dose-Resposta a Droga , Fatores de Crescimento Endotelial/biossíntese , Fatores de Crescimento Endotelial/genética , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Terapia Genética/métodos , Proteínas de Fluorescência Verde , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Linfocinas/biossíntese , Linfocinas/genética , Camundongos , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/biossíntese , Receptores Proteína Tirosina Quinases/genética , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
Fas-mediated apoptosis proceeds though mitochondria-dependent or -independent pathways and is deficient in drug-resistant cells. Neuroblastoma, a common pediatric malignancy, often develops drug-resistance and has a silenced caspase 8 (FLICE) gene, which has been associated with Fas- and drug-resistance. We report that besides caspase 8, which was absent in approximately one-third of 26 neuroblastoma cases in this study, other proteins such as bcl-2 and FLICE-inhibitory protein (FLIP), are equally important in conferring Fas-resistance to neuroblastoma cells. Both bcl-2 and FLIP were frequently expressed in neuroblastoma tissues. Our in vitro studies showed that FLIP was recruited to the death-inducing signaling complex and interfered with the recruitment of caspase 8 in neuroblastoma cells. bcl-2 inhibited the activation of the mitochondria; but it also lowered the free cytoplasmic levels of caspase 8 by binding and sequestering it, thus acting through a novel antiapoptotic mechanism upstream of the mitochondria. In vitro down-regulation of bcl-2 with antisense oligonucleotides allowed the release of cytochrome c from mitochondria and the activation of caspases 8 and 3 upon Fas activation as well as sensitized neuroblastoma cells to Fas-mediated apoptosis. Down-regulation of FLIP had only a modest apoptotic effect because of the coexistent mitochondrial block. However, combined treatment with bcl-2 and FLIP antisense oligonucleotides had a statistically significant synergistic effect reversing Fas-resistance in neuroblastoma cells in vitro. These data indicate that Fas-mediated apoptosis in neuroblastoma cells is mitochondria-dependent and inhibited both at the mitochondrial level and at the level of caspase 8 activation. Thus, gene-targeting therapies for bcl-2 and FLIP may reverse Fas-resistance and prove useful in the treatment of drug-resistant neuroblastomas.
Assuntos
Apoptose/fisiologia , Proteínas de Transporte/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Mitocôndrias/fisiologia , Neuroblastoma/patologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Receptor fas/fisiologia , Adolescente , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Proteínas de Transporte/genética , Caspase 8 , Caspase 9 , Inibidores de Caspase , Caspases/biossíntese , Caspases/metabolismo , Criança , Pré-Escolar , Grupo dos Citocromos c/metabolismo , Ativação Enzimática , Humanos , Lactente , Recém-Nascido , Neuroblastoma/metabolismo , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
We have previously shown that corticotropin-releasing hormone plays an important proinflammatory role in the induction of experimental autoimmune uveoretinitis. In this study, we examined the role of apoptosis in the destruction of the retina during experimental autoimmune uveoretinitis, and the role of corticotropin-releasing hormone as a local regulator of Fas and Fas Ligand expression in this condition. We evaluated apoptosis by the terminal deoxynucleotidyl transferase dUTP nick end labeling method and Fas and Fas Ligand presence by immunohistochemistry. We examined formalin-fixed, paraffin-embedded eye sections from female Lewis rats or B10.A mice immunized with the major pathogenetic epitope (R16 peptide) of the interphotoreceptor retinoid-binding protein. Female B10.A mice similarly immunized were treated with intraperitoneal injections of the rabbit anti-corticotropin-releasing hormone antibody TS-2 or nonimmune rabbit serum. The percentage of retinal cells undergoing apoptosis and the expression of Fas and Fas Ligand were increased in inflamed retinas in immunized Lewis rats and B10.A mice, compared to controls. Retinas from immunized B10.A mice treated with anti-corticotropin-releasing hormone antibody showed significantly lower apoptosis and Fas and Fas Ligand expression than placebo-treated animals. In conclusion, retinal cells in experimental autoimmune uveoretinitis undergo apoptosis associated with concurrent upregulation of Fas and Fas Ligand. The local presence of corticotropin-releasing hormone appears to be of pivotal importance in this process.
Assuntos
Apoptose/fisiologia , Doenças Autoimunes do Sistema Nervoso/patologia , Hormônio Liberador da Corticotropina/fisiologia , Retinite/patologia , Uveíte/patologia , Receptor fas/fisiologia , Animais , Doenças Autoimunes do Sistema Nervoso/metabolismo , DNA Nucleotidiltransferases/fisiologia , Feminino , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos , Ratos , Ratos Endogâmicos Lew , Retinite/metabolismo , Regulação para Cima , Uveíte/metabolismoRESUMO
Gem is a small GTP-binding protein within the Ras superfamily whose function has not been determined. We report here that ectopic Gem expression is sufficient to stimulate cell flattening and neurite extension in N1E-115 and SH-SY5Y neuroblastoma cells, suggesting a role for Gem in cytoskeletal rearrangement and/or morphological differentiation of neurons. Consistent with this potential function, in clinical samples of neuroblastoma, Gem protein was most highly expressed within cells which had differentiated to express ganglionic morphology. Gem was also observed in developing trigeminal nerve ganglia in 12.5 day mouse embryos, demonstrating that Gem expression is a property of normal ganglionic development. Although Gem expression is rare in epithelial and hematopoietic cancer cell lines, constitutive Gem levels were detected in several neuroblastoma cell lines and could be further induced as much as 10-fold following treatment with PMA or the acetylcholine muscarinic agonist, carbachol.
Assuntos
Ganglioneuroblastoma/patologia , Proteínas Imediatamente Precoces/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas de Neoplasias/metabolismo , Neuroblastoma/patologia , Animais , Carbacol/farmacologia , Diferenciação Celular , Agonistas Colinérgicos/farmacologia , Humanos , Proteínas Imediatamente Precoces/genética , Melanoma/patologia , Camundongos , Proteínas Monoméricas de Ligação ao GTP/genética , Receptores Muscarínicos/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Gânglio Trigeminal/química , Células Tumorais CultivadasRESUMO
In this study, we investigated the sensitivity of Ewing's sarcoma family tumors (ESFTs) of children and adolescents to the tumor necrosis factor-related apoptosis-inducing Ligand (TRAIL). TRAIL binds to death receptors (DRs) DR4, DR5, DcR1, and DcR2. Either DR4 or DR5 can induce apoptosis, whereas DcR1 and DcR2 are considered inhibitory receptors. Nine of 10 ESFT cell lines, including several that were Fas resistant, underwent apoptosis with TRAIL through activation of caspase-10, capase-8 (FLICE), caspase-3, and caspase-9. In contrast to the Fas signaling pathway, caspase-10, but not caspase-8 or the Fas-associated death domain-containing molecule, was recruited to the TRAIL receptor-associated signaling complex. We found that 9 of 10 ESFT cell lines expressed both DR4 and DR5 by Western blotting, whereas the TRAIL-resistant line expressed only DR4. However, DR4 was absent from the cell surface in the resistant and two additional lines (three of five tested lines), suggesting that it may have been nonfunctional. On the contrary, DR5 was located on the cell surface in all four sensitive lines tested, being absent only from the cell surface of the resistant line that was also DR5-negative by Western blotting. In agreement with these findings, the resistance of the line was overcome by restoration of DR5 levels by transfection. Levels of DcR1 and DcR2 or levels of the FLICE-inhibitory protein (FLIP) did not correlate with TRAIL resistance, and protein synthesis inhibition did not sensitize the TRAIL-resistant line to TRAIL. Because these data suggested that sensitivity of ESFTs to TRAIL was mainly based on the presence of DR4/DR5, we investigated the presence of these receptors in 32 ESFT tissue sections by immunohistochemistry. We found that 23 of 32 tumor tissues (72%) expressed both receptors, 8 of 32 (25%) expressed one receptor only, and 1 was negative for both. Our finding of wide expression of DR4/DR5 in ESFT in vivo, in combination with their high sensitivity to TRAIL in vitro and the reported lack of toxicity of TRAIL in mice and monkeys, suggests that TRAIL may be a novel effective agent in the treatment of ESFTs.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/farmacologia , Receptores do Fator de Necrose Tumoral/biossíntese , Sarcoma de Ewing/patologia , Fator de Necrose Tumoral alfa/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Proteínas de Transporte/biossíntese , Caspases/metabolismo , Membrana Celular/metabolismo , Cicloeximida/farmacologia , Ativação Enzimática , Humanos , Isoenzimas/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/genética , Proteínas Recombinantes/farmacologia , Sarcoma de Ewing/tratamento farmacológico , Sarcoma de Ewing/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF , Transfecção , Células Tumorais CultivadasRESUMO
Choroidal neovascularization in age-related macular degeneration is a frequent and poorly treatable cause of vision loss in elderly Caucasians. This choroidal neovascularization has been associated with the expression of vascular endothelial growth factor (VEGF). In current animal models choroidal neovascularization is induced by subretinal injection of growth factors or vectors encoding growth factors such as VEGF, or by disruption of the Bruch's membrane/retinal pigment epithelium complex with laser treatment. We wished to establish a transgenic murine model of age-related macular degeneration, in which the overexpression of VEGF by the retinal pigment epithelium induces choroidal neovascularization. A construct consisting of a tissue-specific murine retinal pigment epithelium promoter (RPE(65) promoter) coupled to murine VEGF(164) cDNA with a rabbit beta-globin-3' UTR was introduced into the genome of albino mice. Transgene mRNA was expressed in the retinal pigment epithelium at all ages peaking at 4 months. The expression of VEGF protein was increased in both the retinal pigment epithelium and choroid. An increase of intravascular adherent leukocytes and vessel leakage was observed. Histopathology revealed intrachoroidal neovascularization that did not penetrate through an intact Bruch's membrane. These results support the hypothesis that additional insults to the integrity of Bruch's membrane are required to induce growth of choroidal vessels into the subretinal space as seen in age-related macular degeneration. This model may be useful to screen for inhibitors of choroidal vessel growth.
Assuntos
Corioide/irrigação sanguínea , Modelos Animais de Doenças , Fatores de Crescimento Endotelial/genética , Linfocinas/genética , Degeneração Macular/etiologia , Neovascularização Patológica , Epitélio Pigmentado Ocular/irrigação sanguínea , Fatores Etários , Animais , Bromodesoxiuridina/química , Permeabilidade Capilar , Adesão Celular , Divisão Celular , Corioide/metabolismo , Corioide/patologia , Corantes/metabolismo , Fatores de Crescimento Endotelial/biossíntese , Azul Evans/metabolismo , Leucócitos/imunologia , Linfocinas/biossíntese , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Epitélio Pigmentado Ocular/metabolismo , Biossíntese de Proteínas , Transcrição Gênica , Transgenes , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Recent evidence suggests that one mechanism whereby cytotoxic drugs, such as doxorubicin, kill tumors is the induction or up-regulation of Fas ligand (FasL) expression on the tumor cell surface. The ensuing engagement of Fas by FasL on adjacent cells leads to apoptosis. However, despite cytotoxic drug-induced FasL expression, Fas-sensitive tumors frequently resist chemotherapy, suggesting that they may possess a mechanism that prevents or inactivates Fas-FasL interactions. In the present work, we addressed the involvement of the FasL/Fas signaling pathway in doxorubicin-induced apoptosis and the ability of matrix metalloproteinases (MMPs) to proteolytically cleave FasL in tumor cells. Doxorubicin-induced apoptosis was inhibited by expression of soluble Fas or incubation of the tumor cells with MMP-7 but not with MMP-2 or MMP-9. Resistance to doxorubicin was also induced by expression in the tumor cells of constitutively active MMP-7 but not of a catalytically inactive mutant. Conversely, inhibition of MMP-7 expression in tumor cells by transfection of MMP-7 cDNA in antisense orientation resulted in sensitization to doxorubicin. MMP-7 efficiently cleaved recombinant FasL in vitro and reduced cell surface FasL expression. Our observations provide evidence that one mechanism whereby MMP-7 may promote tumor survival and resistance to doxorubicin is by cleaving FasL and reducing its effectiveness in triggering Fas-mediated apoptosis.
Assuntos
Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Metaloproteinase 7 da Matriz/metabolismo , Glicoproteínas de Membrana/metabolismo , Apoptose/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Neoplasias do Colo/prevenção & controle , Resistencia a Medicamentos Antineoplásicos , Proteína Ligante Fas , Humanos , Hidrólise , Immunoblotting , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Sarcoma de Ewing/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Receptor fas/metabolismoRESUMO
Matrix metalloproteinases (MMPs) regulate the turnover of extracellular matrix (ECM) components and play an important role in embryo development, morphogenesis and tissue remodelling, as well as in tumour invasion and metastasis. Synthetic MMP inhibitors (MMPIs) were designed to prevent tumour cell-induced changes in ECM and thereby achieve antitumour activity. Several MMPIs have entered clinical trials but the preliminary results did not meet the expectations. Recent evidence suggests that MMPs may have more diverse roles than originally believed, influencing angiogenesis, cytokine secretion, as well as tumour cell growth and survival. In particular, synthetic MMPIs may directly induce apoptosis of cancer cells via their inhibitory effect on the shedding of Fas Ligand (FasL), a transmembrane member of the TNF superfamily that kills susceptible cells through its receptor, Fas. Several types of cancers have been shown to express FasL and to shed it from their surface as a soluble form, which is significantly less potent in promoting apoptosis. MMP-7 was recently reported to catalyse this process. Conversely, inhibition of FasL-shedding by a synthetic MMPI results in apoptosis of Fas-sensitive cancer cells. More importantly, DNA-damaging anticancer agents, such as adriamycin, kill cancer cells, at least in part, by upregulating FasL. By inhibiting the proteolytic cleavage of FasL, MMPIs can potentiate the killing effect of traditional chemotherapeutic drugs. These studies therefore demonstrate a direct link between DNA-damaging chemotherapeutic drugs, the apoptosis-inducing Fas/FasL system and the proteolytic activity of MMPs and have important therapeutic implications. For example, the proteolytic activity of MMP-7, which is broadly expressed in primary and especially metastatic human malignancies, may contribute to tumour resistance to cytotoxic agents; targeting and inactivating MMP-7 may, therefore, enhance the efficacy of conventional cancer chemotherapy.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inibidores de Metaloproteinases de Matriz , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Animais , Antineoplásicos/farmacologia , Apoptose/fisiologia , Ensaios Clínicos como Assunto/estatística & dados numéricos , Proteína Ligante Fas , Humanos , Metaloproteinase 7 da Matriz/metabolismo , Metaloproteinases da Matriz/metabolismo , Glicoproteínas de Membrana/metabolismo , Neoplasias/enzimologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/enzimologia , Receptor fas/metabolismoRESUMO
TNF-related apoptosis inducing ligand/Apo2 ligand (TRAIL/Apo2L) is a member of the TNF superfamily of death ligands that selectively induces apoptosis in tumour cells of diverse origins. In this report, we have reviewed recent studies examining TRAIL/Apo2L-induced apoptosis in multiple myeloma (MM), a B-cell malignancy which, in spite of its initial sensitivity to steroids, cytotoxic and high-dose chemotherapy, remains incurable. Recently, we demonstrated that TRAIL/Apo2L induces apoptosis of steroid- and chemotherapy-sensitive and resistant MM cell lines. Moreover, TRAIL/Apo2L selectively induced apoptosis of patient MM tumour cells while sparing non-malignant bone marrow and peripheral blood mononuclear cells. In addition, TRAIL/Apo2L inhibited the growth of human plasmacytomas xenografted into mice. Importantly, TRAIL/Apo2L-induced apoptosis was unaffected by IL-6, a potent growth and survival factor for MM cells which, as we and others have previously shown, blocks various pro-apoptotic signals including Fas ligand, which like TRAIL/Apo2L is also a member of the TNF family of ligands. In view of the potential clinical application of TRAIL/Apo2L to the treatment of MM, we have attempted to discern intracellular mechanisms of action and resistance for TRAIL/Apo2L in MM, along with strategies to increase sensitivity and overcome resistance of MM cells to TRAIL/Apo2L. These studies demonstrated that doxorubicin, an agent which is commonly used to treat MM patients, upregulated the expression of the DR5 death-signalling TRAIL receptor and synergistically enhanced the pro-apoptotic effect of TRAIL on MM cells. Moreover, NF-kappaB inhibitors such as SN50 (a cell permeable inhibitor of NF-kappaB nuclear translocation) as well as the proteasome inhibitor PS-341, which is currently in Phase II clinical trials, also enhanced the pro-apoptotic activity of TRAIL/Apo2L in MM cells. Lastly, TRAIL/Apo2L-induced apoptosis in MM cells was dependent on caspase-8 activation and inhibited by the caspase regulatory proteins FLIP and cIAP2. These studies provide a framework for the use of TRAIL/Apo2L as a single agent or as part of combination therapy for the treatment of MM.
Assuntos
Antineoplásicos/uso terapêutico , Glicoproteínas de Membrana/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Neoplasias/tratamento farmacológico , Fator de Necrose Tumoral alfa/uso terapêutico , Animais , Antibióticos Antineoplásicos/uso terapêutico , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Inibidores de Caspase , Doxorrubicina/uso terapêutico , Inibidores Enzimáticos/farmacologia , Humanos , Glicoproteínas de Membrana/farmacologia , Mieloma Múltiplo/patologia , Neoplasias/patologia , Transdução de Sinais/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Fas Ligand (FasL) is a member of the TNF superfamily that induces apoptosis in susceptible cells upon cross-linking of its own receptor, Fas (Apo-1/CD95). FasL-induced apoptosis contributes to immune homeostasis and cell-mediated cytotoxicity. Several groups have suggested that it also participates in the mechanism of action of DNA-damaging anticancer drugs. However, others have disputed this hypothesis, based largely on the inability of exogenously added anti-Fas/FasL reagents to attenuate drug-induced apoptosis in their studies. In this minireview, we discuss the most recent evidence for and against the involvement of FasL/Fas in the sensitivity and resistance to chemotherapy in a variety of models. In our own model of Ewing's sarcoma (ES), we have extensively investigated the involvement of the FasL/Fas pathway in doxorubicin (Dox)-induced apoptosis. We have generated clones of the Fas-sensitive, Dox-sensitive ES cell line SK-N-MC that were either Fas-resistant or FasL-deficient, and found that they were significantly resistant to Dox. Cleavage of FasL by MMP-7 (matrilysin) protected the parental SK-N-MC cells from Dox, whereas inhibition of MMP-7 activity increased their sensitivity. Transfection of a construct encoding soluble (decoy) Fas protected SK-N-MC cells from Dox. However, incubation with anti-Fas or anti-FasL neutralizing antibodies or exogenous addition of pre-synthesized recombinant soluble Fas decoy protein had no protective effect. This raises the possibility that the proposed Fas/FasL suicidal interaction may take place in an intracellular compartment and thus is not accessible to exogenously added reagents. Therefore, commercially available Fas/FasL neutralizing reagents may not be a reliable indicator of the involvement of the Fas pathway in anticancer-drug-induced apoptosis and experiments using these agents should be carefully re-evaluated. The combined use of MMP inhibitors with conventional, cytotoxic chemotherapy may hold therapeutic benefit.
Assuntos
Antineoplásicos/uso terapêutico , Glicoproteínas de Membrana/fisiologia , Sarcoma de Ewing/tratamento farmacológico , Receptor fas/fisiologia , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteína Ligante Fas , Humanos , Sarcoma de Ewing/patologiaRESUMO
Thionamides are used in the treatment of Graves' disease (GD) and act mainly by inhibiting the organification of iodide, but also lower the levels of thyroid autoantibodies, sometimes leading to long-term remission. Fas ligand (FasL) induces apoptosis of susceptible cells by cross-linking its own receptor, Fas. While Fas is present in a wide variety of normal tissues, FasL expression is limited mainly to cells of the immune system, where it acts as an effector molecule of cell-mediated cytotoxicity, and to the placenta, brain, eye, and testis where it presumably contributes to their immune-privileged status by eliminating infiltrating lymphocytes. We examined immunohistochemically the presence of FasL in thyroid tissue from 15 glands of thionamide-treated GD patients and in 8 normal thyroid control specimens. We also investigated the presence of FasL in thionamide-treated thyrocytes in vitro and their ability to induce Fas-mediated apoptosis in lymphocytes. We found that FasL expression was very weak to undetectable in normal thyroid tissue and cultured thyrocytes, whereas it was strong in thionamide-treated GD glands and cultured thyrocytes. Methimazole-treated thyrocytes induced FasL-dependent apoptosis in cocultured lymphocytes, whereas methimazole treatment of lymphocytes grown in the absence of thyrocytes had no such effect. We conclude that FasL is highly expressed in follicular cells of thyroid glands obtained from thionamide-treated Graves' patients and may contribute to the immunomodulatory effect of thionamides in this disease.
Assuntos
Antitireóideos/uso terapêutico , Doença de Graves/metabolismo , Glicoproteínas de Membrana/análise , Glândula Tireoide/química , Adulto , Apoptose/efeitos dos fármacos , Carbimazol/uso terapêutico , Células Cultivadas , Técnicas de Cocultura , Proteína Ligante Fas , Feminino , Doença de Graves/patologia , Humanos , Immunoblotting , Imuno-Histoquímica , Células Jurkat , Masculino , Glicoproteínas de Membrana/genética , Metimazol/uso terapêutico , Pessoa de Meia-Idade , RNA Mensageiro/análise , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Glândula Tireoide/patologiaRESUMO
Fas (APO-1/CD95) is a transmembrane protein of the tumor necrosis factor (TNF)/nerve growth factor receptor superfamily that induces apoptosis in susceptible normal and neoplastic cells upon cross-linking by its ligand (FasL). TNF-related apoptosis-inducing ligand (TRAIL) is a more recently identified member of the TNF superfamily that has been shown to selectively kill neoplastic cells by engaging two cell-surface receptors, DR4 and DR5. Two additional TRAIL receptors (DcR1 and DcR2) do not transmit an apoptotic signal and have been proposed to confer protection from TRAIL-induced apoptosis. We addressed the expression of Fas, DR4, and DR5 in thyroid carcinoma cell lines and in 31 thyroid carcinoma specimens by Western blot analysis and immunohistochemistry, respectively, and tested the sensitivity of thyroid carcinoma cell lines to Fas- and TRAIL-induced apoptosis. Fas was found to be expressed in most thyroid carcinoma cell lines and tissue specimens. Although cross-linking of Fas did not induce apoptosis in thyroid carcinoma cell lines, Fas-mediated apoptosis did occur in the presence of the protein synthesis inhibitor cycloheximide, suggesting the presence of a short-lived inhibitor of the Fas pathway in these cells. Cross-linking of Fas failed to induce recruitment and activation of caspase 8, whereas transfection of a constitutively active caspase 8 construct effectively killed the SW579 papillary carcinoma cell line, arguing that the action of the putative inhibitor occurs upstream of caspase 8. By contrast, recombinant TRAIL induced apoptosis in 10 of 12 thyroid carcinoma cell lines tested, by activating caspase-10 at the receptor level and triggering a caspase-mediated apoptotic cascade. Resistance to TRAIL did not correlate with DcR1 or DcR2 protein expression and was overcome by protein synthesis inhibition in 50% of the resistant cell lines. One medullary carcinoma cell line was resistant to Fas-and TRAIL-induced apoptosis, even in the presence of cycloheximide, and to transfection of constitutively active caspase-8, suggesting a different regulation of the apoptotic pathway. Our observations indicate that TRAIL effectively kills carcinomas that originate from the follicular epithelium of the thyroid gland, by inducing caspase-mediated apoptosis, and may provide a potentially potent therapeutic reagent against thyroid cancer.
Assuntos
Apoptose/fisiologia , Glicoproteínas de Membrana/fisiologia , Neoplasias da Glândula Tireoide/patologia , Fator de Necrose Tumoral alfa/fisiologia , Receptor fas/fisiologia , Adulto , Idoso , Proteínas Reguladoras de Apoptose , Western Blotting , Antígenos CD8/biossíntese , Antígenos CD8/genética , Carcinoma Papilar/imunologia , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patologia , Caspases/biossíntese , Caspases/genética , Caspases/metabolismo , Caspases/farmacologia , Ativação Enzimática , Feminino , Humanos , Interferon gama/farmacologia , Masculino , Pessoa de Meia-Idade , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/biossíntese , Receptores do Fator de Necrose Tumoral/fisiologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Transdução de Sinais/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF , Neoplasias da Glândula Tireoide/imunologia , Neoplasias da Glândula Tireoide/metabolismo , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologia , Receptor fas/biossínteseRESUMO
BACKGROUND: Fas ligand (FasL) is a transmembrane protein that induces apoptosis (programmed cell death) in susceptible cells by interacting with its receptor, Fas. Transmembrane FasL is cleaved by a metalloproteinase enzyme into a soluble form that is released into the extracellular medium. Tumors of the Ewing's sarcoma family express functional transmembrane FasL and release soluble FasL. This cleavage is inhibited by a matrix metalloproteinase inhibitor (MMPI). We therefore hypothesized that MMPIs can lead to apoptosis of tumor cells by inducing accumulation of transmembrane FasL. METHODS: Ewing's sarcoma and neuroblastoma cell lines were treated with two synthetic MMPIs (BB-3103 and A-151011) and examined for apoptosis and expression of FasL and Fas. RESULTS: Although MMPIs increase levels of FasL and Fas proteins on the surface of all tumor cells studied, they induced apoptosis in Fas-sensitive but not in Fas-resistant cell lines; the induction of apoptosis was inhibited by a Fas-neutralizing antibody. The increase in protein expression was not associated with enhanced transcription. Treatment with an MMPI sensitized the Ewing's sarcoma cells to Fas-activating antibody and to doxorubicin-induced apoptosis. CONCLUSIONS: MMPIs cause accumulation of transmembrane FasL by inhibiting its cleavage, accumulation of Fas (probably secondarily to FasL cleavage inhibition), and decreased levels of soluble FasL. These effects lead to apoptosis in Fas-sensitive cell lines. The observed cooperative action of MMPIs and doxorubicin suggests a possible role of MMPIs in combination treatments with standard apoptosis-inducing chemotherapeutic agents.