RESUMEN
OBJECTIVE: To investigate local tumour control after dose-escalation based on [18F]2-fluoro-2-deoxy-d-glucose (FDG) positron emission tomography (PET) obtained before and early during fractionated irradiation. MATERIALS AND METHODS: 85 mice bearing FaDu xenografts underwent FDG-PET twice: first immediately prior to the first 2-Gy fraction of irradiation (PET1_0) and second after 18°Gy (PET2_18). After these 9 fractions, animals were randomly allocated to: (1) continuation of 2-Gy fractions (cumulative dose of 60°Gy; n=31), (2) dose-escalation with 3-Gy fractions (cumulative EQD2-dose 86.25°Gy [α/ß-value: 10]; n=25), or (3) with 4-Gy fractions (cumulative EQD2-dose 116°Gy; n=29). The effects of SUVmax0°Gy, SUVmax18°Gy, and dose on local tumour control were analysed in two ways. First, the Cox proportional hazards model was used with two covariates: continuous SUVmax values and dose. Second, the Kaplan-Meier method was used, with tumours classified according to SUVmax greater than or less than (1) median maximum standardized uptake value (SUVmax) at PET1_0 and PET2_18, or (2) the cut-off value 2.5. RESULTS: The multivariate Cox analysis revealed a significant negative association between higher SUVmax determined before start of treatment and local control (HR=1.59, [95% CI 1.04, 2.42], p=0.031), whereas higher dose had a significant positive effect (HR=0.95, [0.93, 0.98], p<0.001). In contrast, FDG uptake at 18Gy did not correlate with local control (HR=1.14, [0.53, 2.45], p=0.73). Neither FDG uptake prior to irradiation nor at 18Gy correlated with local control irrespective of the delivered dose (log-rank test) when using the median SUVmax values for stratification (SUVmax0Gy: 60Gy: p=0.25, 86.25Gy: p=0.47, 116Gy: p=0.88 and SUVmax18Gy: 60Gy: p=0.42, 86.25Gy: p=0.34, 116Gy: p=0.99). By contrast, stratifying the animals by the cut-off 2.5 at PET1_0 reveals a significant difference in local control for the 60Gy group (p=0.034), but not for the other dose groups. At PET2_18, no significant effect for any dose group was detected. CONCLUSIONS: The multivariate Cox analysis revealed a significantly higher hazard of recurrence for mice with higher SUVmax determined before start of treatment. These results support the hypothesis that patients with high pre-therapeutic FDG uptake should be considered at increased risk of local failure and therefore as possible candidates for dose escalation strategies.
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Carcinoma de Células Escamosas/diagnóstico por imagen , Carcinoma de Células Escamosas/radioterapia , Neoplasias Hipofaríngeas/diagnóstico por imagen , Neoplasias Hipofaríngeas/radioterapia , Tomografía de Emisión de Positrones/métodos , Animales , Carcinoma de Células Escamosas/patología , Fraccionamiento de la Dosis de Radiación , Relación Dosis-Respuesta en la Radiación , Femenino , Fluorodesoxiglucosa F18 , Humanos , Neoplasias Hipofaríngeas/patología , Masculino , Ratones Desnudos , Recurrencia Local de Neoplasia , Modelos de Riesgos Proporcionales , Dosificación Radioterapéutica , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The melanocortin-1 receptor (MC1R) plays an important role in melanoma growth, angiogenesis and metastasis, and is overexpressed in melanoma cells. α-Melanocyte stimulating hormone (α-MSH) and derivatives are known to bind with high affinity at this receptor that provides the potential for selective targeting of melanoma. In this study, one linear α-MSH-derived peptide Nle-Asp-His-D-Phe-Arg-Trp-Gly-NH2 (NAP-NS1) without linker and with εAhx-ß-Ala linker, and a cyclic α-MSH derivative, [Lys-Glu-His-D-Phe-Arg-Trp-Glu]-Arg-Pro-Val-NH2 (NAP-NS2) with εAhx-ß-Ala linker were conjugated with p-SCN-Bn-NOTA and labeled with (64)Cu. Radiochemical and radiopharmacological investigations were performed with regard to transchelation, stability, lipophilicity and in vitro binding assays as well as biodistribution in healthy rats. No transchelation reactions, but high metabolic stability and water solubility were demonstrated. The linear derivatives showed higher affinity than the cyclic one. [(64)Cu]Cu-NOTA-εAhx-ß-Ala-NAP-NS1 ([(64)Cu]Cu-2) displayed rapid cellular association and dissociation in murine B16F10 cell homogenate. All [(64)Cu]Cu-labeled conjugates exhibited affinities in the low nanomolar range in B16F10. [(64)Cu]Cu-2 showed also high affinity in human MeWo and TXM13 cell homogenate. In vivo studies suggested that [(64)Cu]Cu-2 was stable, with about 85 % of intact peptide in rat plasma at 2 h p.i. Biodistribution confirmed the renal pathway as the major elimination route. The uptake of [(64)Cu]Cu-2 in the kidney was 5.9 % ID/g at 5 min p.i. and decreased to 2.0 % ID/g at 60 min p.i. Due to the prospective radiochemical and radiopharmacological properties of the linear α-MSH derivative [(64)Cu]Cu-2, this conjugate is a promising candidate for tracer development in human melanoma imaging.
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Radioisótopos de Cobre/química , Diagnóstico por Imagen/instrumentación , Melanoma/diagnóstico , Radiofármacos/química , alfa-MSH/análogos & derivados , Animales , Radioisótopos de Cobre/administración & dosificación , Radioisótopos de Cobre/farmacocinética , Estabilidad de Medicamentos , Humanos , Masculino , Melanoma/diagnóstico por imagen , Melanoma/metabolismo , Radiofármacos/administración & dosificación , Radiofármacos/farmacocinética , Ratas , Ratas Wistar , Neoplasias Cutáneas , Distribución Tisular , alfa-MSH/administración & dosificación , alfa-MSH/farmacocinética , Melanoma Cutáneo MalignoRESUMEN
This study aimed at in vivo visualization of cyclooxygenase-2 (COX-2) by optical imaging using a representative compound of a class of autofluorescent 2,3-diaryl-substituted indole-based selective COX-2 inhibitors (2,3-diaryl-indole coxibs). COX-2 was successfully visualized in mice models with phorbol myristate ester (TPA)-induced inflammation or bearing xenografted human melanoma cells by 2-[4-(aminosulfonyl)phenyl]-3-(4-methoxyphenyl)-1H-indole (C1). COX-2 protein expression in both TPA-induced inflammatory sites and human melanoma xenografts was confirmed by immunoblotting. Control experiments using surrogate markers, sham injections, and non-COX-2 expressing melanoma cells further confirmed specificity of tissue association of C1. The merging of therapeutic and diagnostic properties of 2,3-diaryl-indole coxibs may widen the range of applications of COX-2-targeted treatment, e.g., for in situ-guided surgery and ex vivo diagnostics.
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Inhibidores de la Ciclooxigenasa 2/química , Inhibidores de la Ciclooxigenasa 2/farmacocinética , Ciclooxigenasa 2/análisis , Indoles/metabolismo , Imagen Óptica/métodos , Sulfonamidas/metabolismo , Animales , Línea Celular Tumoral , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa 2/metabolismo , Femenino , Xenoinjertos , Humanos , Indoles/análisis , Indoles/química , Melanoma/enzimología , Melanoma/patología , Ratones Endogámicos , Sondas Moleculares/análisis , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Sulfonamidas/análisis , Sulfonamidas/química , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Peptides labeled with short-lived positron emitters are of considerable interest as probes for molecular imaging by positron emission tomography. Herein, the regioselective and convenient radiofluorination of a biologically relevant alkyne-modified SWLAY peptide bound on solid support with the radiolabeling building block 1-(3-azidopropyl)-4-(3-fluoropropyl)piperazine ([(18) F]AFP) is described. Peptides including this amino acid sequence are promising candidates for imaging of the erythropoietin-producing hepatoma cell line-A2 receptor (Eph), which is an interesting target for tumor imaging due to its overexpression in various tumor entities. The desired (18) F-peptide could be prepared in a total synthesis time of 140 min including the removal of the catalytic copper species and was obtained with a radiochemical yield of 11 ± 2% (n = 5) and a radiochemical purity >98%. The method's feasibility for a robust and bioorthogonal radiolabeling via the 1,3-dipolar Huisgen cycloaddition was demonstrated. Preliminary radiopharmacological studies regarding the metabolic stability of the peptides in cell culture supernatants and rat plasma were accomplished as well as the cellular association of the (18) F-peptide in erythropoietin-producing hepatoma cell line-A2-overexpressing human melanoma cells in vitro. Furthermore, an initial in vivo positron emission tomography experiment was performed, which showed a fast metabolism of the novel (18) F-peptide.
Asunto(s)
Radioisótopos de Flúor/química , Radiofármacos/síntesis química , Receptor EphA2/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Humanos , Masculino , Oligopéptidos/síntesis química , Oligopéptidos/farmacocinética , Piperazinas/síntesis química , Piperazinas/farmacocinética , Tomografía de Emisión de Positrones , Radiofármacos/farmacocinética , Ratas , Ratas Wistar , Distribución TisularRESUMEN
OBJECTIVE: To investigate the effect of radiation dose-escalation on local control in hypoxic versus non-hypoxic hypoxic tumours defined using [(18)F]fluoromisonidazole ([(18)F]FMISO) PET. MATERIALS AND METHODS: FaDu human squamous cell carcinomas (hSCCs) growing subcutaneously in nude mice were subjected to [(18)F]FMISO PET before irradiation with single doses of 25 or 35Gy under normal blood flow conditions. [(18)F]FMISO hypoxic volume (HV) and maximum standardised uptake value (SUVmax) were used to quantify tracer uptake. The animals were followed up for at least 120days after irradiation. The endpoints were permanent local tumour control and time to local recurrence. RESULTS: HV varied between 38 and 291mm(3) (median 105mm(3)). Non-hypoxic tumours (HV below median) showed significantly better local control after single dose irradiation than hypoxic tumours (HV above median) (p=0.046). The effect of dose was significant and not different in non-hypoxic and in hypoxic tumours (HR=0.82 [95% CI 0.71; 0.93], p=0.002 and HR=0.86 [0.78; 0.95], p=0.001, respectively). Dose escalation resulted in an incremental increase of local tumour control from low-dose hypoxic, over low-dose non-hypoxic and high-dose hypoxic to high-dose non-hypoxic tumours. SUVmax did not reveal significant association with local control at any dose level. CONCLUSIONS: The negative effect of [(18)F]FMISO HV on permanent local tumour control supports the prognostic value of the pre-treatment [(18)F]FMISO HV. Making the assumption that variable [(18)F]FMISO uptake in different FaDu tumours which all have the same genetic background may serve as an experimental model of intratumoural heterogeneity, the data support the concept of dose-escalation with inhomogeneous dose distribution based on pre-treatment [(18)F]FMISO uptake. This result needs to be confirmed in other tumour models and using fractionated radiotherapy schedules.
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Carcinoma de Células Escamosas/radioterapia , Radioisótopos de Flúor/administración & dosificación , Neoplasias de Cabeza y Cuello/radioterapia , Neoplasias Hipofaríngeas/radioterapia , Misonidazol/análogos & derivados , Fármacos Sensibilizantes a Radiaciones/administración & dosificación , Radiofármacos/administración & dosificación , Animales , Carcinoma de Células Escamosas/diagnóstico por imagen , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Hipoxia de la Célula/efectos de la radiación , Línea Celular Tumoral , Relación Dosis-Respuesta en la Radiación , Femenino , Neoplasias de Cabeza y Cuello/diagnóstico por imagen , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Humanos , Neoplasias Hipofaríngeas/diagnóstico por imagen , Neoplasias Hipofaríngeas/metabolismo , Neoplasias Hipofaríngeas/patología , Ratones , Ratones Desnudos , Misonidazol/administración & dosificación , Recurrencia Local de Neoplasia/diagnóstico por imagen , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/patología , Recurrencia Local de Neoplasia/radioterapia , Cintigrafía , Distribución Aleatoria , Carcinoma de Células Escamosas de Cabeza y Cuello , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
S100A4, synthesized and secreted from both tumor and stroma cells, modulates an aggressive tumor phenotype in various cancers by intracellular and extracellular interactions which are not completely understood. Because of the high content of tumor-associated macrophages in melanoma, here, a syngeneic model (coculture of mouse B16-F10 melanoma cells (Mel) and RAW264.7 macrophages (MÏ); administration (i.v.) of Mel and MÏ/Mel in NMRI nu/nu mice) was used to investigate synthesis and secretion of (a) S100A4, (b) S100A4-mediated signaling and activation of NFκB, and (c) S100A4-mediated modulation of Mel invasiveness in vitro (transwell assay, transwell matrigel assay) and in vivo (metastatic lung colonization), respectively. In this model substantial S100A4 synthesis and secretion is demonstrated in MÏ. Macrophage-derived S100A4 promotes Mel invasiveness in a paracrine manner in vitro, which is further substantiated in control experiments using recombinant human S100A4 and Mel stably transfected with mouse S100A4. Moreover, the participation of S100A4-mediated signaling, e.g., via the receptor for advanced glycation endproducts (RAGE), resulting in activation of NFκB was demonstrated in all experimental settings. Finally, we demonstrated that interaction of macrophage-derived S100A4 with Mel results in increased metastatic lung colonization in vivo.
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Macrófagos/metabolismo , Melanoma Experimental/metabolismo , Melanoma Experimental/secundario , Proteínas S100/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Técnicas de Cocultivo , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Melanoma Experimental/patología , Ratones , Ratones Desnudos , FN-kappa B/metabolismo , Invasividad Neoplásica , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Proteína de Unión al Calcio S100A4 , Proteínas S100/genética , Transducción de SeñalRESUMEN
A fluorinated cathepsin inhibitor based on the azadipeptide nitrile chemotype was prepared and selected for positron emission tomography (PET) tracer development owing to its high affinity for the oncologically relevant cathepsins L, S, K and B. Labelling with fluorine-18 was accomplished in an efficient and reliable two-step, one-pot radiosynthesis by using 2-[(18) F]fluoroethylnosylate as a prosthetic agent. The pharmacokinetic properties of the resulting radiotracer compound were studied inâ vitro, exâ vivo and inâ vivo in normal rats by radiometabolite analysis and small-animal positron emission tomography. These investigations revealed rapid conjugate formation of the tracer with glutathione in the blood, which is associated with slow blood clearance. The potential of the developed (18) F-labelled probe to image tumour-associated cathepsin activity was investigated by dynamic small-animal PET imaging in nude mice bearing tumours derived from the human NCI-H292 lung carcinoma cell line. Computational analysis of the obtained image data indicated the time-dependent accumulation of the radiotracer in the tumours. The expression of the target enzymes in the tumours was confirmed by immunohistochemistry with specific antibodies. This indicates that azadipeptide nitriles have the potential to target thiol-dependent cathepsins inâ vivo despite their disadvantageous pharmacokinetics.
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Compuestos Aza/química , Catepsinas/metabolismo , Dipéptidos/química , Radioisótopos de Flúor/química , Nitrilos/química , Radiofármacos/síntesis química , Animales , Catepsinas/química , Línea Celular Tumoral , Dipéptidos/farmacocinética , Semivida , Humanos , Cinética , Ratones , Neoplasias/diagnóstico por imagen , Tomografía de Emisión de Positrones , Radiofármacos/farmacocinética , Ratas , Ratas Wistar , Distribución Tisular , Trasplante HeterólogoRESUMEN
Eph receptor tyrosine kinases and their ephrin ligands are considered to play important roles in melanoma progression and metastasis. Moreover, hypoxia is known to contribute to melanoma metastasis. In this study, the influence of experimental hypoxia on the expression and synthesis of EphA2 and EphB4, and their corresponding ligands ephrinA1, ephrinA5, and ephrinB2 was studied systematically in four human melanoma cell lines in vitro. Melanoma cell monolayer and spheroid cultures were used as both extrinsic and intrinsic hypoxia models. Hypoxic conditions were confirmed by analyzing hypoxia-inducible factors 1α or 2α expression, vascular endothelial growth factor expression, and cellular uptake of [F]fluoromisonidazole. In normoxia, EphA2, EphB4, ephrinA1, ephrinA5, and ephrinB2 expression was detectable in all cell lines to varying extents. Considerable protein synthesis of EphA2 was detected in all cell lines. However, no effect of experimental hypoxia on both Eph/ephrin expression and protein synthesis was observed. This contributes critically to the debate on the hypothesis that hypoxia regulates the Eph/ephrin system in melanoma.
Asunto(s)
Hipoxia de la Célula/fisiología , Efrinas/biosíntesis , Melanoma/metabolismo , Receptor EphA2/biosíntesis , Receptor EphB4/biosíntesis , Neoplasias Cutáneas/metabolismo , Línea Celular Tumoral , Efrina-A1/biosíntesis , Efrina-A1/genética , Efrina-A5/biosíntesis , Efrina-A5/genética , Efrina-B2/biosíntesis , Efrina-B2/genética , Efrinas/genética , Humanos , Ligandos , Melanoma/genética , Unión Proteica , Biosíntesis de Proteínas , Receptor EphA2/genética , Receptor EphB4/genética , Neoplasias Cutáneas/genéticaRESUMEN
This study aimed at visualization of cyclooxygenase-2 (COX-2) protein expression in melanoma cells by confocal laser induced cryofluorescence microscopy using 4-(3-(4-methoxyphenyl)-1H-indol-2-yl)benzene-sulfonamide (C1) representative for a novel class of autofluorescent 2,3-diarylsubstituted indole-based selective COX-2 inhibitors. COX-2 expression was measured in human melanoma cell lines A2058 and MelJuso by immunocytochemistry and immunoblotting. Cellular uptake experiments using varying C1 concentrations down to 0.1 nM (with/without molar excess of celecoxib as control) were performed at 37 °C. Cryofluorescence microscopy was conducted at 20 K. COX-2 protein expression was successfully visualized by C1 in A2058 cells. COX-2-negative MelJuso cells showed no specific accumulation of C1. Control experiments using celecoxib and, additionally, implemented fluorescence spectroscopy confirmed specificity of both cellular uptake and intracellular association of C1. Cryofluorescence microscopy in combination with spectroscopy allowed for visualization of COX-2 protein expression in melanoma cells in vitro using a selective COX-2 inhibitor at very low concentrations.
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Inhibidores de la Ciclooxigenasa 2/química , Ciclooxigenasa 2/análisis , Indoles/química , Melanoma/enzimología , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Sulfonamidas/química , Línea Celular Tumoral , HumanosRESUMEN
Members of the Eph receptor tyrosine kinase family play essential roles in the pathogenesis of cancer and are therefore promising candidates for molecular imaging by positron emission tomography (PET), for example. In this regard, radiochemical access to novel PET radiotracers derived from potent inhibitors that target the EphB4 kinase domain and which bear a benzodioxolylpyrimidine structural motif was developed. A synthetic route was established for a new fluorine-18-containing radiotracer and for the desired precursor based on a high-affinity benzodioxolylpyrimidine receptor tyrosine kinase inhibitor lead structure. The radiotracer [(18)F]15 was obtained in 16 % radiochemical yield with a specific activity of â¼7â GBq µmol(-1) and >95 % radiochemical purity. Due to the implication of EphB4, particularly in the progression, angiogenesis, and metastasis of melanoma, EphB4-overexpressing human melanoma cells were generated and used as a novel inâ vitro model for radiopharmacological evaluation of the radiotracer. We demonstrate that the corresponding non-radioactive reference compound regained its functionality as an inhibitor for both EphB4 receptor tyrosine kinase and Src kinase. EphB4 was significantly inhibited at compound concentrations >1â µM. Cellular uptake studies with [(18)F]15 revealed substantial uptake in both EphB4-overexpressing and control cells. Moreover, NMRI nu/nu mice bearing both EphB4-overexpressing tumors and control tumors were used for radiopharmacological characterization by biodistribution studies exâ vivo and by dynamic small-animal PET experiments inâ vivo. Despite the high metabolic stability of the novel radiotracer observed inâ vivo, no substantial binding or accumulation in EphB4-overexpressing and control tumors was observed. Nevertheless, we point out that the approach presented herein gives convenient access to novel (18)F-labeled benzodioxolylpyrimidines and is a promising strategy for the further development of novel radiotracers for imaging Eph receptor tyrosine kinases in cancer.
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Radioisótopos de Flúor , Melanoma/diagnóstico , Tomografía de Emisión de Positrones/métodos , Pirimidinas , Receptor EphB4/análisis , Receptor EphB4/metabolismo , Animales , Línea Celular Tumoral , Femenino , Radioisótopos de Flúor/química , Radioisótopos de Flúor/metabolismo , Radioisótopos de Flúor/farmacocinética , Humanos , Masculino , Melanoma/genética , Melanoma/patología , Ratones , Ratones Desnudos , Modelos Moleculares , Pirimidinas/química , Pirimidinas/metabolismo , Pirimidinas/farmacocinética , Radiofármacos/química , Radiofármacos/metabolismo , Radiofármacos/farmacocinética , Ratas , Ratas Wistar , Receptor EphB4/genética , Regulación hacia ArribaRESUMEN
Melanoma is the most malignant type of all skin neoplasms. Its worldwide incidence has steadily increased during the past decades, suggesting a probable melanoma 'epidemic'. Although current clinical, morphologic, and histopathologic methods provide insights into disease behavior and outcome, melanoma is still an unpredictable disease. Once in an advanced stage, it remains a disastrous affliction with scarce therapeutic options. Therefore, significant efforts need to be made in finding informative biomarkers or surrogate markers that could aid or improve early diagnosis of melanoma, its correct staging, the discrimination of other pathological conditions as well as indicate patients' prognosis or the most appropriate therapeutic regimes. Ideally these markers are secreted into body fluids and easily amenable to the design of non-invasive clinical tests. A critical view on the current debate on serologic protein markers, e.g., lactate dehydrogenase, tyrosinase, and melanoma inhibiting activity, and some selected non-protein markers, e.g., 5-S-cysteinyl-dopa and circulating nucleic acids, will be offered and novel innovative approaches currently being explored will be discussed. Special emphasis is put on the S100 family of calcium binding proteins that is more and more emerging as a potentially important group of both molecular key players and biomarkers in the etiology, progression, manifestation, and therapy of neoplastic disorders, including malignant melanoma. Notably, S100B and, possibly, other S100 proteins like S100A4 are assumed to fulfill requirements which make them strong biomarker candidates in melanoma. Moreover, S100 proteins receive attention as possible targets of therapeutic intervention moving closer to clinical impact.
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Biomarcadores de Tumor/metabolismo , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Biomarcadores de Tumor/análisis , Humanos , Melanoma/química , Melanoma/diagnóstico , Melanoma/terapia , Proteínas de Neoplasias/análisisRESUMEN
X-ray irradiation influences metastatic properties of tumor cells and, moreover, metastasis and cellular motility can be modified by members of the Eph receptor/ephrin family of receptor tyrosine kinases. We hypothesized that irradiation-induced changes in cellular properties relevant for metastasis in melanoma cells could be mediated by Eph receptor/ephrin signaling. In this pilot study, we analyzed one pre-metastatic (Mel-Juso) and three metastatic human melanoma (Mel-Juso-L3, A375, and A2058) cells lines and predominantly found anti-metastatic effects of X-ray irradiation with impaired cell growth, clonal growth and motility. Additionally, we observed an irradiation-induced increase in adhesion paralleled by a decrease in migration in Mel-Juso and Mel-Juso-L3 cells and, in part, also in A375 cells. We further demonstrate a decrease of EphA2 both in expression and activity at 7 d after irradiation paralleled by an upregulation of EphA3. Analyzing downstream signaling after irradiation, we detected decreased Src kinase phosphorylation, but unchanged focal adhesion kinase (FAK) phosphorylation, indicating, in part, irradiation-induced downregulation of signaling via the EphA2-Src-FAK axis in melanoma cells. However, to which extent this finding contributes to the modification of metastasis-relevant cellular properties remains to be elucidated.
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Melanoma/metabolismo , Receptor EphA1/metabolismo , Western Blotting , Adhesión Celular/genética , Adhesión Celular/efectos de la radiación , Línea Celular Tumoral , Efrinas/genética , Efrinas/metabolismo , Expresión Génica/genética , Expresión Génica/efectos de la radiación , Humanos , Inmunoprecipitación , Fosforilación/genética , Fosforilación/efectos de la radiación , Receptor EphA1/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rayos XRESUMEN
Aneuploidy, an abnormal number of copies of a genomic region, might be a significant source for neuronal complexity, intercellular diversity, and evolution. Genomic instability associated with aneuploidy, however, can also lead to developmental abnormalities and decreased cellular fitness. Here we show that neurons with a more-than-diploid content of DNA are increased in preclinical stages of Alzheimer's disease (AD) and are selectively affected by cell death during progression of the disease. Present findings show that neuronal hyperploidy in AD is associated with a decreased viability. Hyperploidy of neurons thus represents a direct molecular signature of cells prone to death in AD and indicates that a failure of neuronal differentiation is a critical pathogenetic event in AD.
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Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Muerte Celular/genética , Neuronas/patología , Neuronas/fisiología , Poliploidía , Anciano , Anciano de 80 o más Años , Animales , Encéfalo/citología , Encéfalo/patología , Encéfalo/fisiología , Diferenciación Celular/fisiología , Humanos , Neuronas/citologíaRESUMEN
Cyclin-dependent kinases 4 and 6 (Cdk4/6) are important components of cell cycle activation and control in early G(1) phase. Both enzymes and their regulators, e.g., cyclins, play critical roles in embryogenesis, homeostasis, and cancerogenesis. Cdk4/6 are attractive targets for cancer treatment. Recently, numerous selective small molecule inhibitors of Cdk4/6 have been developed. The potential of Cdk4/6 inhibitors, particularly, pyrido[2,3-d]pyrimidine derivatives, as both anti-cancer drugs and 124I- and 18F-radiolabeled tracers for cancer imaging using positron emission tomography is discussed.
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Antineoplásicos/química , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/química , Animales , Antineoplásicos/uso terapéutico , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/metabolismo , Ratones , Neoplasias/diagnóstico , Tomografía de Emisión de Positrones , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/química , Pirimidinas/uso terapéuticoRESUMEN
Eph receptors and their ephrin ligands were identified in the late 1980's. Subsequently, they were linked to different physiological and pathophysiological processes like embryonic development, angiogenesis, and tumorigenesis. In this regard, recent work focused on the distribution and effects of Eph receptors and ephrins on tumor cells and tumor microenvironment. The purpose of this review is to outline the role of these molecules in physiological angiogenesis and pathophysiological tumor angiogenesis. Furthermore, novel therapeutical approaches are discussed as Eph receptors and ephrins represent attractive targets for antiangiogenic therapy.
RESUMEN
PURPOSE: To analyse short term and long term X-ray irradiation effects on proliferation, viability, glucose and amino acid uptake of murine melanoma cells in vitro and metastasis in vivo. MATERIALS AND METHODS: B16-F10 melanoma cells were irradiated with different doses of X-ray irradiation (200 kV) in the range from 1-20 Gy. One, two and three days respectively 7, 14 and 21 days after treatment cells were analysed concerning cell growth, viability, proliferation, cell cycle distribution, glucose and amino acid transport. Moreover the capability of the cells for in vivo metastasis was examined. RESULTS: As short term response on irradiation we detected decreased cell growth, viability and arrest in the G2/M phase of the cell cycle. Long term response involves re-start of proliferation, increased cell growth and glucose uptake but still decreased viability and amino acid transport. In vivo metastasis is lost immediately after irradiation and regained to a low extent beyond two weeks time for recurrence of cells before injection. CONCLUSIONS: In vitro data suggest that surviving melanoma cells compensate the initial irradiation-dependent damage of proliferation within three weeks possibly by increase in glucose uptake. For metastasis in vivo the role of additional mechanisms is strongly suggested.
Asunto(s)
Melanoma Experimental/radioterapia , Aminoácidos/metabolismo , Animales , Secuencia de Bases , Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Ciclina B1/genética , Ciclina B1/metabolismo , Cartilla de ADN/genética , Glucosa/metabolismo , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/secundario , Ratones , Metástasis de la Neoplasia/prevención & control , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Radiofármacos/farmacocinética , Ensayo de Tumor de Célula MadreRESUMEN
PURPOSE: This study investigated the uptake of [(18)F]2-fluoro-2-deoxy-glucose ([(18)F]FDG) in the human tumour xenograft FaDu at early time points after single dose irradiation with Positron-Emission-Tomography (PET), autoradiography and functional histology. MATERIALS AND METHODS: [(18)F]FDG-PET of FaDu hSCC xenografts on nude mice was performed before 25 Gy or 35 Gy single dose irradiation and one, seven or 11 days post irradiation (p.irr.). Before the second PET, mice were injected with pimonidazole (pimo) and bromodeoxyuridine (BrdU). After the PET tumours were excised, sliced and subjected to autoradiography and functional histology staining (pimo, BrdU, Ki67). [(18)F]FDG tumour uptake was quantified in the PET scans by maximal standard uptake value (SUV(max)) and in the autoradiography after co-registration to the histology slices. RESULTS: No differences in the overall [(18)F]FDG uptake between the two dose groups and time points were found with PET or autoradiography. Comparing autoradiography and histology, the [(18)F]FDG uptake was constant in tumour necrosis over time, while it decreased in vital tumour areas and particularly in hypoxic regions. No differences in the [(18)F]FDG uptake between positive and negative areas of Ki67 and BrdU were found. CONCLUSIONS: The decline of [(18)F]FDG uptake in vital tumour and in pimopositive areas as seen in autoradiography, was not reflected by evaluation of SUV(max) determined by PET. These findings suggest that the SUV(max) does not necessarily reflect changes in tumour biology after irradiation.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Fluorodesoxiglucosa F18/metabolismo , Fluorodesoxiglucosa F18/farmacocinética , Dosis de Radiación , Animales , Autorradiografía , Transporte Biológico/efectos de la radiación , Carcinoma de Células Escamosas/diagnóstico por imagen , Hipoxia de la Célula/fisiología , Hipoxia de la Célula/efectos de la radiación , Proliferación Celular/efectos de la radiación , Femenino , Glucosa/metabolismo , Humanos , Ratones , Ratones Desnudos , Tomografía de Emisión de Positrones , Factores de Tiempo , Trasplante HeterólogoRESUMEN
The cyclin-dependent kinase (Cdk)-cyclin D/retinoblastoma (pRb)/E2F cascade, which controls the G1/S transition of cell cycle, has been found to be altered in many neoplasias. Inhibition of this pathway by using, for example, selective Cdk4 inhibitors has been suggested to be a promising approach for cancer therapy. We hypothesized that appropriately radiolabeled Cdk4 inhibitors are suitable probes for tumor imaging and may be helpful studying cell proliferation processes in vivo by positron emission tomography. Herein, we report the synthesis and biological, biochemical, and radiopharmacological characterizations of two (124)I-labeled small molecule Cdk4 inhibitors (8-cyclopentyl-6-iodo-5-methyl-2-(4-piperazin-1-yl-phenylamino)-8H-pyrido[2,3-d]-pyrimidin-7-one (CKIA) and 8-cyclopentyl-6-iodo-5-methyl-2-(5-(piperazin-1-yl)-pyridin-2-yl-amino)-8H-pyrido[2,3-d]pyrimidin-7-one (CKIB)). Our data demonstrate a defined and specific inhibition of tumor cell proliferation through CKIA and CKIB by inhibition of the Cdk4/pRb/E2F pathway emphasizing potential therapeutic benefit of CKIA and CKIB. Furthermore, radiopharmacological properties of [(124)I]CKIA and [(124)I]CKIB observed in human tumor cells are promising prerequisites for in vivo biodistribution and imaging studies.
RESUMEN
Structural variation in the human genome is likely to be an important mechanism for neuronal diversity and brain disease. A combination of multiple different forms of aneuploid cells due to loss or gain of whole chromosomes giving rise to cellular diversity at the genomic level have been described in neurons of the normal and diseased adult human brain. Here, we describe recent advances in molecular neuropathology based on the combination of slide-based cytometry with molecular biological techniques that will contribute to the understanding of genetic neuronal heterogeneity in the CNS and its potential impact on Alzheimer's disease and age-related disorders.
Asunto(s)
Neuronas/patología , Enfermedad de Alzheimer/patología , Aneuploidia , Encefalopatías/patología , Cromosomas/genética , Cromosomas/metabolismo , ADN/metabolismo , Humanos , MosaicismoRESUMEN
Reactivation of the cell cycle, including DNA replication, might play a major role in Alzheimer's disease (AD). A more than diploid DNA content in differentiated neurons might alternatively result from chromosome mis-segregation during mitosis in neuronal progenitor cells. It was our objective to distinguish between these two mechanisms for aneuploidy and to provide evidence for a functional cell cycle in AD. Using slide-based cytometry, chromogenic in situ hybridization, and PCR amplification of alu-repeats, we quantified the DNA amount of identified cortical neurons in normal human brain and AD and analyzed the link between a tetraploid DNA content and expression of the early mitotic marker cyclin B1. In the normal brain, the number of neurons with a more than diploid content amounts to approximately 10%. Less than 1% of neurons contains a tetraploid DNA content. These neurons do not express cyclin B1, most likely representing constitutional tetraploidy. This population of cyclin B1-negative tetraploid neurons, at a reduced number, is also present in AD. In addition, a population of cyclin B1-positive tetraploid neurons of approximately 2% of all neurons was observed in AD. Our results indicate that at least two different mechanisms need to be distinguished giving rise to a tetraploid DNA content in the adult brain. Constitutional aneuploidy in differentiated neurons might be more frequent than previously thought. It is, however, not elevated in AD. In addition, in AD some neurons have re-entered the cell cycle and entirely passed through a functional interphase with a complete DNA replication.