RESUMEN
Structural connectivity (SC), the physical pathways connecting regions in the brain, and functional connectivity (FC), the temporal coactivations, are known to be tightly linked. However, the nature of this relationship is still not understood. In the present study, we examined this relation more closely in six separate human neuroimaging datasets with different acquisition and preprocessing methods. We show that using simple linear associations, the relation between an individual's SC and FC is not subject specific for five of the datasets. Subject specificity of SC-FC fit is achieved only for one of the six datasets, the multimodal Glasser Human Connectome Project (HCP) parcellated dataset. We show that subject specificity of SC-FC correspondence is limited across datasets due to relatively small variability between subjects in SC compared with the larger variability in FC.
RESUMEN
Alzheimer's disease (AD) is marked by cognitive dysfunction emerging from neuropathological processes impacting brain function. AD affects brain dynamics at the local level, such as changes in the balance of inhibitory and excitatory neuronal populations, as well as long-range changes to the global network. Individual differences in these changes as they relate to behaviour are poorly understood. Here, we use a multi-scale neurophysiological model, "The Virtual Brain (TVB)", based on empirical multi-modal neuroimaging data, to study how local and global dynamics correlate with individual differences in cognition. In particular, we modeled individual resting-state functional activity of 124 individuals across the behavioural spectrum from healthy aging, to amnesic Mild Cognitive Impairment (MCI), to AD. The model parameters required to accurately simulate empirical functional brain imaging data correlated significantly with cognition, and exceeded the predictive capacity of empirical connectomes.
Asunto(s)
Enfermedad de Alzheimer/diagnóstico por imagen , Amnesia/diagnóstico por imagen , Encéfalo/diagnóstico por imagen , Disfunción Cognitiva/diagnóstico por imagen , Envejecimiento/fisiología , Enfermedad de Alzheimer/patología , Amnesia/patología , Encéfalo/patología , Cognición/fisiología , Disfunción Cognitiva/patología , Conectoma , Diagnóstico Diferencial , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Modelos NeurológicosRESUMEN
Functional inactivation of the tumour suppressor protein p16(INK4a) constitutes a key event in the multistep process of pancreatic ductal cell transformation. However, the significance of p16 inactivation for complex and tissue-specific aspects of pancreatic cancer progression, such as angiogenesis and metastasis, is less understood. Here, we inducibly re-expressed p16 in vivo in an orthotopic model of pancreatic cancer and examined the impact on these clinically relevant aspects of pancreatic cancer tumour biology. Consistent with previous work in subcutaneous xenograft models, we found p16 capable of reducing primary tumour growth. In addition, p16 restitution resulted in a marked reduction of tumour angiogenesis, largely accounted for by a p16-dependent inhibition of lymphangiogenesis. In excellent agreement with the antilymphangiogenic effect, re-expression of p16 almost completely prevented lymph node metastases of MiaPaca-2 pancreatic tumours. To our knowledge, this is the first report that experimentally links the tumour suppressor p16 to the process of lymphangiogenesis.
Asunto(s)
Carcinoma Ductal Pancreático/genética , Genes p16/fisiología , Linfangiogénesis/genética , Metástasis Linfática/genética , Neoplasias Pancreáticas/genética , Animales , Línea Celular Tumoral , Femenino , Expresión Génica , Humanos , Ratones , Ratones DesnudosRESUMEN
Molecular imaging requires, not only the identification of an appropriate marker, but also its quantitative analysis. We used the Sensitive Particle Acoustic Quantification (SPAQ) technology - a novel ultrasound technique - for detection and quantification of cell adhesion molecules in isolated tissue and in live animals. By conjugating gas-filled microparticles (MPs) with antibodies to intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), we were able to depict and quantify ICAM-1 and VCAM-1 in isolated brain and spinal cord from rats with autoimmune encephalomyelitis (EAE), an established inflammatory disease model of human multiple sclerosis (MS). Depiction and quantification of specific MPs were also feasible in living animals with AT-EAE with similar results. After treatment with methylprednisolone, the measured number of targeted anti-ICAM-1 and VCAM-1-MPs was significantly lower (P<0.01) compared to untreated animals demonstrating the high sensitivity of this imaging technique. Depending on the antibody linked to the surface of the MPs, the technique can be used to quantify the expression of any accessible antigen expressed on the luminal surface of endothelial cells and is therefore a promising tool for the non-invasive and dynamic assessment of disease-related molecules.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Encefalomielitis Autoinmune Experimental/diagnóstico por imagen , Encefalomielitis Autoinmune Experimental/metabolismo , Procesamiento de Imagen Asistido por Computador/métodos , Animales , Antiinflamatorios , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/fisiología , Encéfalo/anatomía & histología , Química Encefálica/efectos de los fármacos , Moléculas de Adhesión Celular/química , Femenino , Inmunohistoquímica , Molécula 1 de Adhesión Intercelular/metabolismo , Metilprednisolona/farmacología , Microesferas , Ratas , Ratas Endogámicas Lew , Médula Espinal/diagnóstico por imagen , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Ultrasonografía Doppler Transcraneal , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Interactions of tumor cells with lymphatic vessels are of paramount importance for tumor progression, however, the underlying molecular mechanisms are poorly understood. Whereas enlarged lymphatic vessels are frequently observed at the periphery of malignant melanomas, it has remained unclear whether intratumoral lymphangiogenesis occurs within these tumors. Here, we demonstrate the presence of intratumoral lymphatics and enlargement of lymphatic vessels at the tumor periphery in vascular endothelial growth factor (VEGF)-C-overexpressing human melanomas transplanted onto nude mice. VEGF-C expression also resulted in enhanced tumor angiogenesis, indicating a coordinated regulation of lymphangiogenesis and angiogenesis in melanoma progression. The specific biological effects of VEGF-C were critically dependent on its proteolytic processing in vivo. Furthermore, VEGF-C induced chemotaxis of macrophages in vitro and in vivo, revealing a potential function of VEGF-C as an immunomodulator. Taken together, our results identify VEGF-C as multifunctional factor involved in regulating tumor lymphangiogenesis, angiogenesis, and immune response.
Asunto(s)
Factores de Crecimiento Endotelial/metabolismo , Sistema Linfático/patología , Melanoma/metabolismo , Melanoma/patología , Animales , División Celular/fisiología , Movimiento Celular/fisiología , Factores de Crecimiento Endotelial/fisiología , Humanos , Sistema Linfático/crecimiento & desarrollo , Macrófagos/fisiología , Melanoma/irrigación sanguínea , Melanoma/fisiopatología , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neovascularización Patológica/fisiopatología , Células Tumorales Cultivadas , Factor C de Crecimiento Endotelial VascularRESUMEN
BACKGROUND & AIMS: The role of vascular endothelial growth factor (VEGF) and its receptors in tumor angiogenesis has been well established. We analyzed the expression pattern and biologic significance of VEGF and its receptors in human pancreatic cancer. METHODS: VEGF, KDR/flk-1, and flt-1 expression were examined by immunohistochemistry, in situ hybridization, reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and receptor phosphorylation. VEGF-stimulated mitogenesis was investigated by mitogen-activated protein kinase (MAPK) phosphorylation, transactivation of a c-fos promoter reporter construct, DNA synthesis assays, and stable transfection of a dominant-negative flk-1 complementary DNA (cDNA) construct. RESULTS: Compared with normal pancreas and chronic pancreatitis, VEGF and its receptors were overexpressed in pancreatic cancer. KDR and flt-1 were detected not only in endothelial cells but also in tumor cells. VEGF expression was observed in all human pancreatic tumor cell lines examined, and the KDR/flk-1 and flt-1 receptor was detected in 2 cell lines. VEGF treatment results in phosphorylation of MAPKs, transactivation of a c-fos promoter construct, and growth stimulation in KDR/flk-1-expressing cell lines, which could be blocked by VEGF antagonists. Furthermore, stable transfection of a dominant-negative flk-1 cDNA significantly inhibited tumor cell growth. CONCLUSIONS: These results not only support the important role of the VEGF/VEGF receptor system in pancreatic tumor biology but also suggest the existence of an autocrine/paracrine mitogenic loop for pancreatic cancer cells.
Asunto(s)
Adenocarcinoma/metabolismo , Factores de Crecimiento Endotelial/metabolismo , Linfocinas/metabolismo , Neoplasias Pancreáticas/metabolismo , Adenocarcinoma/patología , Comunicación Autocrina , División Celular/efectos de los fármacos , Factores de Crecimiento Endotelial/farmacología , Humanos , Linfocinas/farmacología , Mitosis , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Tirosina Quinasas Receptoras/farmacología , Proteínas Tirosina Quinasas Receptoras/uso terapéutico , Receptores de Factores de Crecimiento/metabolismo , Receptores de Factores de Crecimiento/uso terapéutico , Receptores de Factores de Crecimiento Endotelial Vascular , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Receptor 1 de Factores de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
The tumor suppressor gene p16(INK4a) inhibits the kinase activity of the cyclin-dependent kinase 4-6/cyclin D complexes and subsequent phosphorylation of critical substrates necessary for transit through the G1 phase of the cell cycle. Recent studies suggested that control of the G1/S boundary might not be the sole biological function of p16(INK4a). We hypothesized that p16(INK4a) might influence hitherto unknown critical features of a malignant epithelial phenotype, such as anchorage dependence. Here we provide evidence that stable transfection of p16(INK4a) restitutes apoptosis induction upon loss of anchorage (anoikis) in a variety of human cancer cells. Anoikis in p16(INK4a)-transfected cells was evidenced by DNA fragmentation and poly(ADP-ribose) polymerase cleavage upon cultivation on polyhydroxyethylmethacrylate-coated dishes and was associated with suppression of anchorage-independent growth as well as complete loss of tumorigenicity. p16(INK4a)-mediated anoikis was due to selective transcriptional upregulation of the alpha(5) integrin chain of the alpha(5)beta(1) fibronectin receptor as detected by FACS((R)) analysis, immunoprecipitation, Northern blotting, and nuclear run-on assays. Addition of soluble fibronectin and inhibitory alpha(5) antibodies to nonadherent cells completely abolished p16(INK4a)-mediated anoikis, whereas laminin was ineffective. Furthermore, antisense-induced downregulation of the alpha(5) integrin chain in p16(INK4a)-transfected cells restored resistance to anoikis. These data suggest a novel functional interference between a cell cycle-regulating tumor suppressor gene and membrane-bound integrins, thus regulating a hallmark feature of an epithelial transformed phenotype: susceptibility to anoikis.
Asunto(s)
Apoptosis/fisiología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Genes Supresores de Tumor/fisiología , Proteínas Proto-Oncogénicas , Receptores de Fibronectina/metabolismo , Animales , Apoptosis/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , División Celular/fisiología , Quinasa 4 Dependiente de la Ciclina , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Regulación hacia Abajo/fisiología , Fibronectinas/farmacología , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Humanos , Técnicas In Vitro , Riñón/citología , Hígado/citología , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias Pancreáticas , Fenotipo , Piel/citología , Transfección , Células Tumorales Cultivadas , Regulación hacia Arriba/fisiologíaRESUMEN
PTK787/ZK 222584 (1-[4-chloroanilino]-4-[4-pyridylmethyl] phthalazine succinate) is a potent inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases, active in the submicromolar range. It also inhibits other class III kinases, such as the platelet-derived growth factor (PDGF) receptor beta tyrosine kinase, c-Kit, and c-Fms, but at higher concentrations. It is not active against kinases from other receptor families, such as epidermal growth factor receptor, fibroblast growth factor receptor-1, c-Met, and Tie-2, or intracellular kinases such as c-Src, c-Abl, and protein kinase C-alpha. PTK787/ZK 222584 inhibits VEGF-induced autophosphorylation of kinase insert domain-containing receptor (KDR), endothelial cell proliferation, migration, and survival in the nanomolar range in cell-based assays. In concentrations up to 1 microM, PTK787/ZK 222584 does not have any cytotoxic or antiproliferative effect on cells that do not express VEGF receptors. After oral dosing (50 mg/kg) to mice, plasma concentrations of PTK787/ZK 222584 remain above 1 microM for more than 8 h. PTK787/ZK 222584 induces dose-dependent inhibition of VEGF and PDGF-induced angiogenesis in a growth factor implant model, as well as a tumor cell-driven angiogenesis model after once-daily oral dosing (25-100 mg/kg). In the same dose range, it also inhibits the growth of several human carcinomas, grown s.c. in nude mice, as well as a murine renal carcinoma and its metastases in a syngeneic, orthotopic model. Histological examination of tumors revealed inhibition of microvessel formation in the interior of the tumor. PTK787/ZK 222584 is very well tolerated and does not impair wound healing. It also does not have any significant effects on circulating blood cells or bone marrow leukocytes as a single agent or impair hematopoetic recovery after concomitant cytotoxic anti-cancer agent challenge. This novel compound has therapeutic potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Carcinoma/tratamiento farmacológico , Carcinoma/patología , Factores de Crecimiento Endotelial/antagonistas & inhibidores , Linfocinas/antagonistas & inhibidores , Neovascularización Patológica/tratamiento farmacológico , Ftalazinas , Piridinas , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptores de Factores de Crecimiento/antagonistas & inhibidores , Inhibidores de la Angiogénesis/efectos adversos , Inhibidores de la Angiogénesis/sangre , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Carcinoma/irrigación sanguínea , División Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Hematopoyesis/efectos de los fármacos , Humanos , Neoplasias Renales/irrigación sanguínea , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/patología , Leucocitos/citología , Leucocitos/efectos de los fármacos , Linfocinas/farmacología , Ratones , Ratones Desnudos , Metástasis de la Neoplasia/tratamiento farmacológico , Metástasis de la Neoplasia/patología , Trasplante de Neoplasias , Neovascularización Patológica/patología , Fosforilación/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular , Cicatrización de Heridas/efectos de los fármacosRESUMEN
The mechanism of action of anticancer chemotherapeutic agents is mainly thought to be due to a direct inhibition of tumor cell proliferation. The enhanced endothelial cell proliferation rate in tumor specimens raised the question whether therapeutic effects of chemotherapeutic agents might be at least partially attributed to an inhibition of tumor angiogenesis. Meanwhile, numerous anticancer chemotherapeutic agents were tested for their antiangiogenic potential. A few agents seem to exert consistent inhibition of tumor angiogenesis even in drug-resistant tumors. Most recent investigations on the antiangiogenic efficacy of different application schedules suggested the use of a tightly spaced, continuous application of appropriate anticancer chemotherapeutic agents. These application schedules are able to exert a strong antiangiogenic effect as indicated by an increase of apoptosis of tumor endothelial cells. Future clinical trials have to determine the therapeutic benefit of novel combination chemotherapy and alternative application schedules.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Antineoplásicos/uso terapéutico , Neoplasias/irrigación sanguínea , Neoplasias/tratamiento farmacológico , Neovascularización Patológica/prevención & control , Animales , Humanos , Neovascularización Patológica/patología , Neovascularización Patológica/fisiopatologíaRESUMEN
Protein ligands and receptor tyrosine kinases that specifically regulate endothelial cell function are mainly involved in physiological as well as in disease-related angiogenesis. These ligand/receptor systems include the vascular endothelial growth factor (VEGF) and the angiopoietin (Ang) families, and their receptors, the VEGF receptor family and the tyrosine kinase with immunoglobulin-like and epidermal growth factor homology domains (Tie) family. In the present study, the contribution of these endothelium-specific ligand/receptor systems to tumor angiogenesis was evaluated. A375v human melanoma cells, which express at least the angiogenic growth factors VEGF, VEGF-C, and Ang-1, were stably transfected to overexpress the extracellular ligand-binding domains of the endothelium-specific receptor tyrosine kinases fms-like tyrosine kinase-1 (Flt-1), Flt-4, Tie-1, and Tie-2, respectively. In vitro proliferation and colony formation assays confirmed that expression of the extracellular receptor domains inhibited neither tumor cell mitogenesis nor the ability to produce anchorage-independent growth. Nude mouse xenografts revealed that interference with either the VEGF receptor pathway or the Tie-2 pathway resulted in a significant inhibition of tumor growth and tumor angiogenesis. In contrast, interference with the Flt-4 pathway or the Tie-1 pathway was without significant effect. Our results show that both the VEGF receptor pathway and the Tie-2 pathway are essential for A375v melanoma xenograft growth. The inhibition of the VEGF receptor pathway cannot be compensated by the Tie-2 pathway, nor vice versa. These findings suggest that the VEGF receptor pathway and the Tie-2 pathway have to be considered as two independent mediators essential for the process of in vivo angiogenesis.
Asunto(s)
Melanoma/irrigación sanguínea , Melanoma/patología , Neovascularización Patológica/prevención & control , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptores de Factores de Crecimiento/fisiología , Animales , Factores de Crecimiento Endotelial/biosíntesis , Humanos , Linfocinas/biosíntesis , Ratones , Ratones Desnudos , Microcirculación/patología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/fisiología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/genética , Receptor TIE-2 , Receptores de Factores de Crecimiento/antagonistas & inhibidores , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento Endotelial Vascular , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Trasplante Heterólogo , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Receptor 1 de Factores de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Vascular endothelial growth factor (VEGF) directly stimulates endothelial cell proliferation and migration via tyrosine kinase receptors of the split kinase domain family. It mediates vascular growth and angiogenesis in the embryo but also in the adult in a variety of physiological and pathological conditions. The potential binding site of VEGF with its receptor was identified using cellulose-bound overlapping peptides of the extracytosolic part of the human vascular endothelial growth factor receptor II (VEGFR II). Thus, a peptide originating from the third globular domain of the VEGFR II comprising residues 247RTELNVGIDFNWEYP261 was revealed as contiguous sequence stretch, which bound 125I-VEGF165. A systematic replacement with L-amino acids within the peptide representing the putative VEGF-binding site on VEGFR II indicates Asp255 as the hydrophilic key residue for binding. The dimerized peptide (RTELNVGIDFNWEYPAS)2K inhibits VEGF165 binding with an IC50 of 0.5 microM on extracellular VEGFR II fragments and 30 microM on human umbilical vein cells. VEGF165-stimulated autophosphorylation of VEGFR II as well as proliferation and migration of microvascular endothelial cells was inhibited by the monomeric peptide RTELNVGIDFNWEYPASK at a half-maximal concentration of 3-10, 0.1, and 0.1 microM, respectively. We conclude that transduction of the VEGF165 signal can be interrupted with a peptide derived from the third Ig-like domain of VEGFR II by blockade of VEGF165 binding to its receptor.
Asunto(s)
Factores de Crecimiento Endotelial/antagonistas & inhibidores , Linfocinas/antagonistas & inhibidores , Fragmentos de Péptidos/farmacología , Proteínas Tirosina Quinasas Receptoras/química , Receptores de Factores de Crecimiento/química , Adulto , Secuencia de Aminoácidos , Células Cultivadas , Factores de Crecimiento Endotelial/metabolismo , Endotelio Vascular/química , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Humanos , Linfocinas/metabolismo , Datos de Secuencia Molecular , Biblioteca de Péptidos , Mapeo Peptídico , Fosforilación , Unión Proteica , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular , Homología de Secuencia de Aminoácido , Transducción de Señal/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Stable prostacyclin analogues exert a strong inhibitory effect on lymphogenous as well as haematogenous tumor metastasis in a series of tumor lines. The strong inhibition of metastasis was achieved by repeated once-daily i.g. applications. The mechanism of antimetastatic action is related to the expression of functional IP-receptors (PGI-receptors). As cellular assay systems indicated that the IP-receptor mediated signalling is down-regulated upon continuous exposure to prostacyclin or stable derivatives, it has been questioned whether a mode of drug application with constant plasma drug levels may potentially result in a decrease of the antimetastatic effect. We addressed this question using the stable prostacyclin analogue cicaprost in a disease model by comparing i.g. applications given once daily with a continuous administration of equivalent doses via drinking water. Very similar to our previous investigations in the 13762NF MTLn3 rat mammary carcinoma model, cicaprost administered by i.g. application strongly reduced lung and lymph node metastasis. In contrast, administration of equivalent doses via drinking water leading to lower but constant steady-state plasma levels failed to exert inhibitory effects. Plasma and urine levels of cicaprost were measured with a sensitive radioimmunoassay on the last treatment day. Pharmacokinetic evaluation demonstrated a similar bioavailability of cicaprost in both groups. This result first demonstrates a treatment failure of a prostacyclin derivative in a chronic disease model in association with a continuous drug administration leading to constant plasma levels. A desensitization of receptor signalling by constant plasma levels may be a possible mechanism for treatment failure.
Asunto(s)
Antineoplásicos/sangre , Antineoplásicos/uso terapéutico , Epoprostenol/análogos & derivados , Metástasis de la Neoplasia/prevención & control , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ingestión de Líquidos , Esquema de Medicación , Epoprostenol/administración & dosificación , Epoprostenol/sangre , Epoprostenol/uso terapéutico , Femenino , Intubación Gastrointestinal , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Metástasis Linfática/prevención & control , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/patología , Ratas , Ratas Endogámicas F344 , Soluciones/administración & dosificación , Soluciones/uso terapéuticoRESUMEN
The mechanism of action of anticancer chemotherapeutic agents is mainly thought to be due to a direct inhibition of tumor cell proliferation. The enhanced endothelial cell proliferation rate in tumor specimens raised the question of whether therapeutic effects of chemotherapeutic agents might be at least partially attributed to inhibition of tumor angiogenesis. In the present study, we investigated the potential effects of chemotherapeutic agents on human renal carcinoma angiogenesis with the alginate implantation model in mice. For the first time, we also compared results from the angiogenesis model with the inhibitory effects on growth of s.c. xenografts in nude mice. Vincristine and bleomycin exerted strong inhibition of tumor angiogenesis in both carcinoma lines close to the level of the standard antiangiogenic agent O-chloroacetyl-carbamyl-fumagillol (AGM-1470; T/C 22%). Adriamycin reduced angiogenesis of Caki-2 cells (T/C 33%) but had no effect on Caki-1 angiogenesis (T/C 137%). Etoposide and 5-fluorouracil reduced Caki-1 tumor angiogenesis but had no effect on Caki-2. Despite antiangiogenic effects in both carcinoma lines, vincristine, bleomycin, and AGM-1470 significantly reduced only the growth of fast-growing Caki-1 s.c. xenografts but not the slow-growing Caki-2. Antivascular effects by bleomycin and AGM-1470 were also shown by a decrease of microvessel density in nude mouse xenografts. Our findings suggest that chemotherapeutic agents may exert inhibition of tumor angiogenesis, which could be exploitable by combination therapy of fast-growing tumors. The resistance of the slow-growing Caki-2 carcinoma against acute angiogenesis inhibition indicates a need for well-tolerated angiogenesis inhibitors. Our results also suggest the use of fast-growing s.c. xenografts for demonstrating growth inhibition by antiangiogenic compounds. Further characterization of antiangiogenic compounds considered for clinical application should, however, have its focus on slow-growing tumors, which are not accessible for most therapeutic strategies.
Asunto(s)
Antineoplásicos/uso terapéutico , Carcinoma de Células Renales/irrigación sanguínea , Neoplasias Renales/irrigación sanguínea , Neovascularización Patológica/tratamiento farmacológico , Animales , Carcinoma de Células Renales/tratamiento farmacológico , División Celular/efectos de los fármacos , Femenino , Humanos , Neoplasias Renales/tratamiento farmacológico , Ratones , Ratones Desnudos , Ensayo de Capsula SubrrenalRESUMEN
Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.
Asunto(s)
Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/efectos de los fármacos , Linfocinas/farmacología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Sitios de Unión , Unión Competitiva , División Celular/efectos de los fármacos , Células Cultivadas , Clonación Molecular , Dimerización , Factores de Crecimiento Endotelial/biosíntesis , Factores de Crecimiento Endotelial/química , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Variación Genética , Humanos , Cinética , Linfocinas/biosíntesis , Linfocinas/química , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fosforilación , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/efectos de los fármacos , Receptores de Factores de Crecimiento/química , Receptores de Factores de Crecimiento/efectos de los fármacos , Receptores de Factores de Crecimiento Endotelial Vascular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/farmacología , Arterias Umbilicales , Venas Umbilicales , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Changes in integrin expression during malignant transformation have been observed in many tumors. Colon-carcinoma cells show reduced expression or even loss of the alpha5beta1 integrin compared to normal or adenoma cells. To determine the significance of absent alpha5beta1 integrin signaling, we transfected the cDNA coding for the alpha5 integrin sub-unit into the human colon-carcinoma cell line HT29, which constitutively lacks this subunit but does express the beta1 subunit. We show here that the newly expressed fibronectin receptor alpha5beta1 generates multiple signals, causing marked changes in cytoskeletal arrangements within a few minutes of adhesion to fibronectin. Cells expressing the alpha5beta1 integrin exhibit the formation of actin stress fibers and focal adhesions, as well as the induction of tyrosine phosphorylation of several proteins, within 10 min. We identified the focal adhesion kinase pp125FAK and the cytoskeletal protein paxillin as major phosphorylation substrates in these cells. These proteins remained hypophosphorylated when alpha5-negative control cells were plated on fibronectin. The tyrosine kinase pp60c-src, regarded as central in the regulation of cellular proliferation and constitutively over-expressed in HT29 and in colon-carcinoma cells, showed reduced intrinsic kinase activity in unstimulated HT29alpha5 cells. In contrast, fibronectin-induced signaling through alpha5beta1 increased pp60c-src activity. Moreover, immunoprecipitation of pp60c-src from extracts of HT29alpha5 cells cultivated on fibronectin for 20 min revealed complex formation of pp60c-src and tyrosine-phosphorylated pp125FAK. Our data suggest that de novo expression of the alpha5beta1 integrin in HT29 colon-cancer cells restores signaling via pp125FAK and pp60c-src. Thus, loss of this receptor during malignant transformation may contribute to tumor-cell autonomy, while reduced activity of pp60c-src in HT29alpha5-cells may participate directly in growth control.
Asunto(s)
Neoplasias del Colon/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Receptores de Fibronectina/metabolismo , Adhesión Celular , Moléculas de Adhesión Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Células HT29 , Humanos , Paxillin , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , TransfecciónRESUMEN
The integrin alpha5beta1 seems to be the most relevant receptor of tumor cells for binding to fibronectin. Although numerous studies suggest a role of tumor cell fibronectin interaction in tumor metastasis, differential integrin expression on tumor cells has, however, not been correlated with metastatic capabilities. We addressed this question by transfection of the integrin alpha5beta1 cDNA into HT-29 human colon carcinoma cells which led to de novo expression of functional integrin alpha5beta1. Similar to other reports, expression of the integrin alpha5beta1 in HT-29 tumor cells exerted an inhibitory action on cell proliferation as indicated in our study by formation of fewer colonies in soft agar. The tumor growth inhibitory property of the integrin alpha5beta1 was also shown by reduction of subcutaneous xenograft growth in nude mice to approximately 50% of that of control transfectants. For the first time, we found that several clones of integrin alpha5 subunit transfectants displayed dramatically reduced formation of lung colonies and cutaneous metastasis after intravenous injection into nude mice. While most animals inoculated with control transfectant cells formed macroscopically visible lung colonies ranging from 12.6 +/- 2.6 to 22.0 +/- 6.6 (mean colony number +/- SEM), mice inoculated with HT-29 cell clones expressing the integrin alpha5beta1 were almost completely free of lung colonies (ranging from 0.0 +/- 0 to 0.2 +/- 0.1). Our results imply that integrin alpha5beta1 expression inhibits circulating tumor cells in pursuing late steps of the metastatic process as represented by the artificial metastasis (lung colonisation) model.
Asunto(s)
Neoplasias Pulmonares/secundario , Metástasis de la Neoplasia , Receptores de Fibronectina/fisiología , Animales , Antígenos CD/análisis , Antígenos CD/genética , Femenino , Células HT29 , Humanos , Inmunohistoquímica , Integrina alfa5 , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Receptores de Fibronectina/análisis , Receptores de Fibronectina/genética , Transfección , Células Tumorales CultivadasRESUMEN
Vascular endothelial cell growth factor (VEGF) plays a pivotal role in the regulation of angiogenesis by binding to its cognate receptor molecule type II (VEGFr-II, KDR). VEGFr-II is an endothelial cell-specific transmembrane tyrosine kinase important for vascular endothelial cell development and differentiation during embryogenesis, angiogenic processes under physiological conditions, and various diseases. An increasing number of reports indicate that VEGF/VEGFr-II also play a fundamental role for tumor angiogenesis. We present the generation and in vitro characterization of the monoclonal antibodies 2-7-9 and 2-10-1. Both antibodies are highly specific for VEGFr-II as demonstrated by Western blotting and immunoprecipitation. MAbs 2-10-1 and 2-7-9 bind to a disulphide bridge-stabilized epitope within domains 6 and 7 of the human VEGFr-II with an affinity of 8 and 80 nM, respectively. Furthermore, the antibodies are suitable for immunohistochemistry and ELISA techniques. Because both antibodies recognize their epitope on living cells, they also have the potential for drug targeting and diagnostic purposes.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Proteínas Tirosina Quinasas Receptoras/inmunología , Receptores de Factores de Crecimiento/inmunología , Receptores Mitogénicos/inmunología , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Células Cultivadas , Mapeo Epitopo , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Neovascularización Fisiológica , Conformación Proteica , Receptores de Factores de Crecimiento Endotelial Vascular , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunologíaRESUMEN
A remarkable approach to a specific tumor angiogenesis model in vivo is the use of alginate implants encapsulating tumor cells. However, this previously reported approach has often been questioned because of doubts regarding the relevance of hemoglobin at the alginate implant as a parameter of vascularization. In the present investigation, we examined whether or not the use of the blood pool agents FITC-dextran of high molecular weight would significantly improve the determination of vascularization at the alginate implant. In our experiments, we found a rapid distribution of FITC-dextran within the blood circulation of mice after i.v. bolus injection. The amount of FITC-dextran within alginate implants strongly correlated with the number of LL2 carcinoma cells or B16/F10 cells encapsulated. Even a low number of 10(3) cells per alginate implant led to a significantly increased accumulation of FITC-dextran. A more than 10-fold stimulation above that of controls was found with alginate implants containing 10(4) LL2 or B16/F10 tumor cells. Using the investigational compound AGM-1470 in different treatment schedules, we found that quantification of alginate implant anglogenesis with FITC-dextran is a sensitive method for the determination of angiogenesis inhibition. In conclusion, our results demonstrated that the use of FITC-dextran enables highly sensitive, quantitative measurement of blood vessel formation by alginate implants.
Asunto(s)
Alginatos/administración & dosificación , Dextranos , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Hemostáticos/administración & dosificación , Neoplasias/irrigación sanguínea , Neovascularización Patológica/diagnóstico , Animales , Antibióticos Antineoplásicos/farmacología , Carcinoma Pulmonar de Lewis/sangre , Carcinoma Pulmonar de Lewis/irrigación sanguínea , Carcinoma de Células Renales/sangre , Carcinoma de Células Renales/irrigación sanguínea , Ciclohexanos , Dextranos/sangre , Dextranos/química , Portadores de Fármacos , Femenino , Ácido Glucurónico , Ácidos Hexurónicos , Neoplasias Renales/sangre , Neoplasias Renales/irrigación sanguínea , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Microesferas , Peso Molecular , Neoplasias/sangre , Neovascularización Patológica/etiología , Neovascularización Patológica/patología , Neovascularización Patológica/prevención & control , O-(Cloroacetilcarbamoil) Fumagilol , Sesquiterpenos/farmacología , Células Tumorales CultivadasRESUMEN
Cicaprost, a stable prostacyclin analog has been shown to be antimetastatically active in a series of metastasizing rodent tumors. Whereas starting treatment with Cicaprost on the day of tumor implantation was a characteristic feature of our previous investigations, the present study focused on the antimetastatic potency of Cicaprost in animals with established tumor growth. We have previously reported that, in Wistar-Furth rats bearing subcutaneously implanted SMT2A mammary carcinoma, Cicaprost in daily oral doses of 0.1 to 1.0 mg/kg given from the day of tumor implantation to the end of the experiment led to a strong decrease in the number of lung metastases. The 1.0 mg/kg doses reduced the number of lung metastases by about 95% compared with the control. In the present study, we have examined the effect of delaying the start of treatment in animals with established tumor growth, Cicaprost in daily oral doses of 0.1 mg/kg given from Day 10 until Day 32 reduced the number of lung metastases by about 80% compared with the control. In contrast, surgical removal of palpable primary tumors had no effect on lung metastasis. We conclude that Cicaprost exhibits strong antimetastatic activity in the SMT2A rat mammary carcinoma model and interferes not only with mechanisms of tumor cell-blood cell interaction, tumor cell adhesion, and extravasation, but also with steps following extravasation.