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PURPOSE: This study was performed to investigate the chemical stability of different dyes used in chromovitrectomy and the influence of various product parameters on it. METHODS: Buffered dye solutions were prepared containing 1.5 g/L acid violet 17, 0.25 g/L brilliant blue G, 1.3 g/L bromophenol blue, and 1.5 g/L trypan blue, combined with deuterium oxide, polyethylene glycol 3350, and D-mannitol as additives. For accelerated storage testing, samples were incubated for 400 h at 80°C corresponding to 2 years according to the Van 't Hoff equation. After different incubation times samples were taken for UV/Vis spectroscopy, pH measurement, and osmometry. RESULTS: Depending on dye, additive, and packaging, different solutions exhibit differences in chemical stability and hence shelf life. Packaging in syringes instead of vials increases dye stability. Additives may negatively influence important parameters, e.g. polyethylene glycol 3350 increases osmolality beyond the physiological range. Notably, acid violet 17 is chemically unstable except in D-mannitol-containing buffer, packed in syringes. However, simultaneously, D-mannitol leads to a pH shift below 7.0. CONCLUSION: In summary, dye solutions filled in syringes should be preferred to vials to slow down oxidative degradation. Especially acid violet 17 solutions should be used with caution because the addition of D-mannitol may contribute to pH values beyond the physiological range.
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PURPOSE: Developing new blends of heavier-than-water silicone oil tamponade agents containing high molecular weight polydimethylsiloxane polymer for use in vitreoretinal surgery. MATERIALS AND METHODS: The viscoelastic properties of heavier-than-water silicone oil blends (30.5% F6H8 + 69.5% polydimethylsiloxane) containing high molecular weight polymer additive at increasing concentrations were measured using a controlled-stress rheometer (TA Instruments Rheolyst AR 1000 N). Emulsification of the blends was induced using a sonication device and a pluronic surfactant as a strong emulsifier. The percentage emulsion area was photographed and measured using ImageJ software. In a second in vitro emulsification assessment, silicone oil blends were dispersed using a high shear homogenizer and the oil-in-water droplets were counted using a coulter counter particle analyser. RESULTS: The addition of the high molecular weight polymer increased shear viscosity and viscoelasticity of the oil blends, which were measureable and to some extent predictable. The in vitro emulsification models produced contradictory results. This demonstrates the difficulty of designing and using in vitro models to evaluate the emulsification tendency of tamponade agents in vivo. CONCLUSION: Addition of a high molecular weight polymer to heavy silicone oil can increase the viscoelasticity. These findings might contribute to the development of emulsification resistant heavy silicone oils.
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Emulsiones , Polímeros/química , Aceites de Silicona/química , Peso Molecular , Reología , Agua/químicaRESUMEN
Many types of cancer, for example glioblastoma, show resistance against current anti-cancer treatments. One reason is that they are not capable to effectively activate their intracellular cell death pathways. Novel treatments designed to overcome these deficiencies in cancer cells present promising concepts to eradicate chemotherapy-resistant cancer cells. One of these approaches includes the membrane seeking compound erucylphosphohomocholine (ErPC3) which is part of the latest generation of alkylphospholipid analogs developed over the last two-and-a-half decades. ErPC3 exerts potent antineoplastic effects in animal models and against established cancer cell lines including, for example, glioblastoma and different types of leukemia, while sparing their normal counterparts. Starting with a historical survey, we report here on the anticancer activity of ErPC3 and on ErPC3's established mechanisms of action. We cover the current knowledge on the induction of mitochondrial apoptosis by ErPC3, including its interaction with the 18 kDa translocator protein (TSPO). In addition we discuss other signaling pathways modulated by ErPC3. Interaction with the TSPO leads to activation of the mitochondrial apoptosis cascade. This includes cardiolipin oxidation at mitochondrial levels, collapse of the mitochondrial membrane potential, and release of cytochrome c, the initiating steps of the mitochondrial apoptosis cascade. Other pathways modulated by ErPC3 include different kinases for the PI3K/Akt/mTOR and the MAP kinase pathways. Furthermore, ErPC3's cytotoxic actions may include its effects on phosphatidylcholine synthesis to inhibit the endoplasmic reticulum enzyme CTP:phosphocholine cytidyltransferase. These basic research data hopefully will lead to effective approaches toward exploitation of ErPC3 for the treatment of cancer.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Ácidos Erucicos/farmacología , Proteínas Mitocondriales/metabolismo , Neoplasias/tratamiento farmacológico , Fosforilcolina/análogos & derivados , Receptores de GABA/metabolismo , Animales , Antineoplásicos/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Membrana Celular/metabolismo , Ácidos Erucicos/uso terapéutico , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Mitocondrias/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Fosforilcolina/farmacología , Fosforilcolina/uso terapéutico , Transducción de SeñalRESUMEN
Class-3 semaphorins are secreted axon guidance factors. Some of these semaphorins have recently been characterized as suppressors of tumor progression. To determine if class-3 semaphorins can be used to inhibit the development of glioblastoma-multiforme tumors, we expressed recombinant sema-3A, 3B, 3D, 3E, 3F or 3G in U87MG glioblastoma cells. Sema3A and sema3B expressing cells contracted and changed shape persistently while cells expressing other semaphorins did not. Sema3A and sema3F differed from other semaphorins including sema3B as they also inhibited the proliferation of the cells and the formation of soft agar colonies. With the exception of sema3G and sema3B, expression of these semaphorins in U87MG cells inhibited significantly tumor development from subcutaneously implanted cells. Strong inhibition of tumor development was also observed following implantation of U87MG cells expressing each of the class-3 semaphorins in the cortex of mouse brains. Sema3D and sema3E displayed the strongest inhibitory effects and their expression in U373MG or in U87MG glioblastoma cells implanted in the brains of mice prolonged the survival of the mice by more then two folds. Furthermore, most of the mice that died prior to the end of the experiment did not develop detectable tumors and many of the mice survived to the end of the experiment. Most of the semaphorins that we have used here with the exception of sema3D were characterized previously as inhibitors of angiogenesis. Our results indicate that sema3D also functions as an inhibitor of angiogenesis and suggest that the anti-tumorigenic effects are due primarily to inhibition of tumor angiogenesis. These results indicate that class-3 semaphorins such as sema3D and sema3E could perhaps be used to treat glioblastoma patients.
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Encéfalo/metabolismo , Encéfalo/patología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Glioblastoma/metabolismo , Glioblastoma/patología , Semaforinas/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Semaforinas/genéticaRESUMEN
OBJECTIVE: Previously, several important roles for glutamate have been described for the biology of primary brain tumors. For example, glutamate has been suggested to promote glioma cell proliferation by the activation of the 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid (AMPA) subtype of glutamate receptors. In the present study, we determined the potential regulatory roles of the 18-kDa translocator protein (TSPO) in the glutamatergic system in relation to cell death of brain tumor cells through knockdown of the TSPO by genetic manipulation. MATERIALS AND METHODS: With microarray analysis and validation of gene expression of particular genes using real-time PCR, we found effects because of small inhibitory RNA knockdown of the TSPO in human U118MG glioblastoma cells on gene expression of glutamate receptors, glutamate transporters, and enzymes for glutamate metabolism. We also applied antisense RNA to silence TSPO in rat C6 glioblastoma cells and assayed the effects on DNA fragmentation, indicative of apoptosis, because of glutamate exposure. RESULTS: In particular, the effects of TSPO silencing in human U118MG cells related to glutamate metabolism indicate a net effect of a reduction in glutamate levels, which may potentially protect the cells in question from cell death. The TSPO knockdown in C6 cells showed that TSPO is required for the induction of apoptosis because of glutamate exposure. CONCLUSION: These findings show that interactions between the TSPO and the glutamatergic system may play a role in tumor development of glioblastoma cells. This may also have implications for our understanding of the involvement of the TSPO in secondary brain damage and neurodegenerative diseases.
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Supervivencia Celular , Ácido Glutámico , Receptores de GABA , Receptores de Glutamato , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Glioblastoma , Ácido Glutámico/genética , Ácido Glutámico/metabolismo , Ácido Glutámico/farmacología , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN Interferente Pequeño , Ratas , Receptores de GABA/genética , Receptores de GABA/metabolismo , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismoRESUMEN
BACKGROUND: It is known that the mitochondrial 18 kDa translocator protein (TSPO) is present in almost all peripheral tissues and also in glial cells in the brain. TSPO levels are typically enhanced in correlation with tumorigenesis of cancer cells including glioblastoma. Relevant for angiogenesis, TSPO is also present in almost all cells of the cardiovascular system. METHODS: We studied the effect of TSPO knockdown by siRNA on various aspects of tumor growth of U118MG glioblastoma cells in two in-vivo models: a nude mouse model with intracerebral implants of U118MG glioblastoma cells and implantation of U118MG glioblastoma cells on the chorionallantoic membrane (CAM) of chicken embryos. In vitro, we further assayed the influence of TSPO on the invasive potential of U118MG cells. RESULTS: TSPO knockdown increased tumor growth in both in-vivo models compared with the scrambled siRNA control. Angiogenesis was also increased by TSPO knockdown as determined by a CAM assay. TSPO knockdown led to a decrease in adhesion to the proteins of the extracellular matrix, including fibronectin, collagen I, collagen IV, laminin I, and fibrinogen. TSPO knockdown also led to an enhancement in the migratory capability of U118MG cells, as determined in a modified Boyden chamber. Application of the TSPO ligand 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK 11195) at a concentration of 25 µmol/l in the in-vitro models yielded results similar to those obtained on TSPO knockdown. We found no effects of PK 11195 on TSPO protein expression. Interestingly, at low nmol/l concentrations (around 1 nmol/l), PK 11195 enhanced adhesion to collagen I, suggesting a bimodal concentration effect of PK 11195. CONCLUSION: Intact TSPO appears to be able to counteract the invasive and angiogenic characteristics related to the aggressiveness of U118MG glioblastoma cells in vivo and in vitro.
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Proliferación Celular , Glioblastoma/metabolismo , Glioblastoma/patología , Receptores de GABA/metabolismo , Animales , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Embrión de Pollo , Glioblastoma/irrigación sanguínea , Humanos , Isoquinolinas/farmacología , Ligandos , Masculino , Ratones , Ratones Desnudos , Neovascularización Patológica , ARN Interferente Pequeño/metabolismoRESUMEN
PURPOSE: To develop new blends of emulsification-resistant silicone oil based on high molecular weight (HMW) silicone oil for use as an endotamponade in vitreoretinal surgery. METHODS: Viscosity and elasticity of various silicone oil blends (Siluron 1000, Siluron 2000, Siluron 5000, 7% HMW + Siluron 1000, 10% HMW + Siluron 1000, and 15% HMW + Siluron 1000; Fluoron GmbH, Ulm, Germany) were measured using a piezoelectric axial vibrator. Emulsification was induced using a sonication device. Pluronic 10%, plasma, and serum were used as emulsifiers. The emulsion area was photographed and measured using ImageJ software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html). RESULTS: Viscosity increased proportionally to HMW concentrations. Fluid elasticity was optimum using 10% HMW. Emulsification was at a minimum when using 10% or 15% HMW blends. CONCLUSIONS: A new silicone oil-based tamponade was developed with a viscosity similar to Siluron 5000 (at 37°C) but with significantly less emulsification tendency than Siluron 5000 or Siluron 2000. HMW concentration increases the fluid elasticity, thereby reducing the emulsification tendency.
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Emulsiones/química , Endotaponamiento , Aceites de Silicona/química , Sustancias Viscoelásticas/química , Cirugía Vitreorretiniana , Elasticidad , Peso Molecular , ViscosidadRESUMEN
Even with current standard-of-care therapies, the prognosis for patients with malignant gliomas is very poor and several new treatment modalities for glioblastomas are currently under investigation. Given the role of TGF-ß in gliomas, anti-TGF-ß strategies against gliomas are currently being investigated. Biodistribution of intravenously injected AF680-labeled 1D11, a pan-neutralizing TGF-ß antibody, was monitored in mice bearing either subcutaneous or orthotopic gliomas using in vivo imaging and fluorescence microscopy. AF680-labeled 1D11 entered both the subcutaneous and intracranial tumors and the antibody was detected within the tumor tissue for several days whereas only low fluorescence was found in organs. The effects of 1D11 on subcutaneous versus orthotopic U87MG and GL261 gliomas in immunocompetent C57BL/6J versus immunodeficient CD1-Foxn1nu mice were observed by direct tumor size measurement, H&E staining and immunohistochemistry. Treatment of immunocompetent mice bearing subcutaneous GL261 tumors with 1D11 resulted in complete remission. In immune deficient mice, the growth of subcutaneous GL261 tumors was increased following treatment with 1D11. Intracranially implanted gliomas in C57Bl/6J mice showed no size reduction after 1D11 treatment but there was reduced invasion of the glioma cells into the adjacent normal brain. Together these data demonstrate that TGF-ß plays different roles in combating the tumor depending on subcutaneous versus orthotopic implantation site.
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Anticuerpos Monoclonales/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Glioma/tratamiento farmacológico , Factor de Crecimiento Transformador beta/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacocinética , Especificidad de Anticuerpos , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Glioma/inmunología , Glioma/patología , Humanos , Inmunocompetencia , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Microscopía Fluorescente , Invasividad Neoplásica , Espectroscopía Infrarroja Corta , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: The aim of the present study was to gain insight into the penetration, biodistribution, and fate of globulins in the brain after 2-O-hexyldiglycerol-induced blood-brain barrier opening. PROCEDURES: The spatial distribution of fluorescence probes was investigated after blood-brain barrier opening with intracarotid 2-O-hexyldiglycerol injection. Fluorescence intensity was visualized by microscopy (mice and rats) and by in vivo time-domain optical imaging. RESULTS: There was an increased 2-O-hexyldiglycerol-mediated transfer of fluorescence-labeled globulins into the ipsilateral hemisphere. Sequential in vivo measurements revealed that the increase in protein concentration lasted at least 96 h after administration. Ex vivo detection of tissue fluorescence confirmed the results obtained in vivo. CONCLUSION: Globulins enter the healthy brain in conjunction with 2-O-hexyldiglycerol. Sequential in vivo near-infrared fluorescence measurements enable the visualization of the spatial distribution of antibodies in the brain of living small animals.
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Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Glicerol/análogos & derivados , Glicerol/farmacología , Imagenología Tridimensional/métodos , Espectroscopía Infrarroja Corta/métodos , gammaglobulinas/metabolismo , Animales , Fluoresceína/metabolismo , Colorantes Fluorescentes/metabolismo , Ratones , Peso Molecular , Ratas , Ratas Wistar , Factores de Tiempo , Distribución Tisular/efectos de los fármacosRESUMEN
Erucylphosphohomocholine (ErPC3, Erufosine) was reported previously to induce apoptosis in otherwise highly apoptosis-resistant malignant glioma cell lines while sparing their non-tumorigenic counterparts. We also previously found that the mitochondrial 18 kDa Translocator Protein (TSPO) is required for apoptosis induction by ErPC3. These previous studies also suggested involvement of reactive oxygen species (ROS). In the present study we further investigated the potential involvement of ROS generation, the participation of the mitochondrial respiration chain, and the role of the mitochondrial F(O)F(1)-ATP(synth)ase in the pro-apoptotic effects of ErPC3 on U87MG and U118MG human glioblastoma cell lines. For this purpose, cells were treated with the ROS chelator butylated hydroxyanisole (BHA), the mitochondrial respiration chain inhibitors rotenone, antimycin A, myxothiazol, and the uncoupler CCCP. Also oligomycin and piceatannol were studied as inhibitors of the F(O) and F(1) subunits of the mitochondrial F(O)F(1)-ATP(synth)ase, respectively. BHA was able to attenuate apoptosis induction by ErPC3, including mitochondrial ROS generation as determined with cardiolipin oxidation, as well as collapse of the mitochondrial membrane potential (Deltapsi(m)). Similarly, we found that oligomycin attenuated apoptosis and collapse of the Deltapsi(m), normally induced by ErPC3, including the accompanying reductions in cellular ATP levels. Other inhibitors of the mitochondrial respiration chain, as well as piceatannol, did not show such effects. Consequently, our findings strongly point to a role for the F(O) subunit of the mitochondrial F(O)F(1)-ATP(synth)ase in ErPC3-induced apoptosis and dissipation of Deltapsi(m) as well as ROS generation by ErPC3 and TSPO.
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Antineoplásicos/toxicidad , Apoptosis , Neoplasias Encefálicas/metabolismo , Ácidos Erucicos/toxicidad , Glioblastoma/metabolismo , Fosforilcolina/análogos & derivados , ATPasas de Translocación de Protón/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Adenosina Trifosfato/metabolismo , Neoplasias Encefálicas/enzimología , Hidroxianisol Butilado/farmacología , Caspasas/metabolismo , Línea Celular Tumoral , Ciclosporina/farmacología , Transporte de Electrón/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Glioblastoma/enzimología , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/antagonistas & inhibidores , Poro de Transición de la Permeabilidad Mitocondrial , Oligomicinas/farmacología , Fosforilcolina/toxicidad , ATPasas de Translocación de Protón/antagonistas & inhibidores , Receptores de GABA/metabolismo , Estilbenos/farmacologíaRESUMEN
Stanniocalcin 2 (STC2) is a secreted glycoprotein of as yet unknown functions. We investigated STC2 in human neuroblastoma, the most common solid extra-cranial tumor of infancy. In primary tumor samples, we found that expression of STC2 is associated with the metastatic Stages 4 and 4s and MYCN expression. In vitro, however, we demonstrate that cell proliferation is reduced by STC2 due to an increase in the basal apoptosis rate of the transfected cells. On the other hand, in vitro assays showed that STC2-transfected neuroblastoma cells have an increased invasive potential and display higher activity of collagen-degrading matrix metalloproteinase 2 (MMP2). Using experimental tumors on the chick chorioallantoic membrane (CAM), we observed that STC2 expressing cells show signs of emigration from the solid tumor and destroy blood vessels of the CAM, giving rise to massively bleeding tumors. Erosion of blood vessels was also seen when purified STC2 protein was applied on the CAM. Taken together, we demonstrate a dual role for STC2 in neuroblastoma. It reduces proliferation of tumor cells in vitro, but increases the invasive potential and induces bleeding, and thereby may facilitate early metastasis. The potential of STC2 as a surrogate marker for metastatic neuroblastoma calls for further investigation.
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Glicoproteínas/fisiología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Neuroblastoma/patología , Animales , Línea Celular Tumoral , Proliferación Celular , Embrión de Pollo , Glicoproteínas/genética , Tejido de Granulación/patología , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Invasividad Neoplásica , Neuroblastoma/secundario , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: We have previously shown that the anti-neoplastic agent erucylphosphohomocholine (ErPC3) requires the mitochondrial 18 kDa Translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor (PBR), to induce cell death via the mitochondrial apoptosis pathway. METHODS: With the aid of the dye JC-1 and cyclosporin A, applied to glioblastoma cells, we now investigated the significance of opening of the mitochondrial permeability transition pore (MPTP) for ErPC3-induced apoptosis in interaction with the TSPO ligands, PK 11195 and Ro5 4864. Furthermore, we measured cytochrome c release, and caspase-9 and -3 activation in this paradigm. RESULTS: The human glioblastoma cell lines, U87MG, A172 and U118MG express the MPTP-associated TSPO, voltage-dependent anion channel and adenine nucleotide transporter. Indeed, ErPC3-induced apoptosis was inhibited by the MPTP blocker cyclosporin A and by PK 11195 and Ro5 4864 in a concentration-dependent manner. Furthermore, PK 11195 and Ro5 4864 inhibited collapse of the mitochondrial membrane potential, cytochrome c release, and caspase-9 and -3 activation caused by ErPC3 treatment. CONCLUSIONS: This study shows that PK 11195 and Ro5 4864 inhibit the pro-apoptotic function of ErPC3 by blocking its capacity to cause a collapse of the mitochondrial membrane potential. Thus, the TSPO may serve to open the MPTP in response to anti-cancer drugs such as ErPC3.
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Antineoplásicos/farmacología , Apoptosis/fisiología , Ácidos Erucicos/farmacología , Glioblastoma/metabolismo , Fosforilcolina/análogos & derivados , Receptores de GABA/metabolismo , Apoptosis/efectos de los fármacos , Benzodiazepinonas/farmacología , Western Blotting , Caspasas/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citocromos c/efectos de los fármacos , Humanos , Isoquinolinas/farmacología , Ligandos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Fosforilcolina/farmacologíaRESUMEN
Alkylphosphocholines are a new class of anticancer agents. The mechanisms by which these drugs display their antitumor activities are not known. In this work, we show that erucylphosphohomocholine, a new antineoplastic compound, significantly decreased ATP synthesis in isolated rat liver mitochondria at a concentration of 50 microm or higher via permeabilization of the inner membrane. At a concentration of 25 microm, it induced a moderate swelling of mitochondria, a slight decrease of the inner membrane potential, and an increase in state 4 respiration without an essential influence on state 3 respiration or the outer membrane permeability to cytochrome c. We found that cyclosporin A did not prevent mitochondrial swelling induced by 25-100 microm erucylphosphohomocholine. Moreover, cyclosporin A induced a fast drop of the inner membrane potential in the presence of 25-50 microm erucylphosphohomocholine that seems to be due to a strong synergistic inhibition of the respiratory activity. The ratio of uncoupled to state 3 respiration rates increased from 1.3 +/- 0.1 with 25 microm erucylphosphohomocholine and from 1.5 +/- 0.1 with 1 microm cyclosporin A to 4.5 +/- 0.3 in the presence of both drugs. On the other hand, oligomycin or cyclosporin A protected certain cancer cell lines against erucylphosphohomocholine-induced apoptosis. This protection might be related to a prevention of cellular ATP hydrolysis by permeabilized mitochondria and to the inhibition of the classical permeability transition pore, respectively. Our findings provide new insight into the mechanisms by which these unusual alterations of mitochondria might be involved in anticancer activity of alkylphosphocholines.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Ciclosporina/administración & dosificación , Sinergismo Farmacológico , Ácidos Erucicos/administración & dosificación , Mitocondrias/metabolismo , Consumo de Oxígeno , Fosforilcolina/análogos & derivados , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Ciclosporina/farmacología , Ácidos Erucicos/farmacología , Hidrólisis , Hígado/metabolismo , Masculino , Potenciales de la Membrana , Mitocondrias/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Oligomicinas/farmacología , Fosforilcolina/administración & dosificación , Fosforilcolina/química , Fosforilcolina/farmacología , RatasRESUMEN
Galectins are evolutionarily conserved beta-galactoside-binding lectins which recognize specific glycoconjugates on the cell surface and the extracellular matrix. Accumulating evidence indicates that these proteins are involved in a variety of physiological and pathological processes including tumor growth and metastasis. Up-regulated expression of galectin-1 is a hallmark of a variety of malignant tumors. Here, we examined the expression of galectin-1 in glioma cell lines, the influence of ionizing irradiation and the intracellular and extracellular effects of this protein on tumor cell proliferation and migration. Galectin-1 was detected in both A172 and U118 glioma cells by immunoblot analysis. Ionizing irradiation induced a statistically significant up-regulation in glioma cell lines. RNA-interference-mediated silencing resulted in a significant suppression of the proliferation of the A172 cells, while the addition of recombinant galectin-1 had no effect. On the other hand, the migratory capacity of both cell lines was reduced after galectin-1 down-regulation, and up-regulated by the addition of exogenous galectin-1. Our results provide evidence of a role for galectin-1 in the regulation of glioma cell proliferation and migration. While an intracellular mechanism seemed to prevail in galectin-1-mediated regulation of tumor cell proliferation, the control of cell migration was exerted by both intracellular and extracellular mechanisms. In addition, this protein was up-regulated by ionizing radiation, indicating that the blockade of this protein should be performed before radiotherapy to avoid any undesired stimulating effects. Given the multifactorial role of galectin-1 in the regulation of tumor escape and metastasis, we conclude that targeting galectin-1 may have therapeutic benefits in the treatment of malignant glioma.
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Galectina 1/metabolismo , Galectina 1/farmacología , Proteínas Recombinantes/farmacología , Western Blotting , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Galectina 1/genética , Expresión Génica , Glioma/genética , Glioma/metabolismo , Glioma/patología , Humanos , Interferencia de ARN , ARN Interferente Pequeño/genética , Radiación Ionizante , Proteínas Recombinantes/metabolismo , Transfección , Regulación hacia Arriba/efectos de la radiaciónRESUMEN
Short-chain alkylglycerols have been described to increase the penetration of drugs and macromolecules across the blood-brain barrier (BBB) into the central nervous system (CNS) and were considered to be of potential value in the pharmaceutical treatment of CNS disorders. Due to the lack of information on the pharmacological behavior of these compounds in vivo, pharmacokinetics and biodistribution of [14C]- and [3H]-labeled 1-O-pentylglycerol (49 mg/kg, 100 mM) was investigated in normal male Wistar rats after intravenous as well as intracarotid administration. There was a rapid and predominant renal elimination of 1-O-pentylglycerol and more than 70% of administered dose was found in the urine within 270 min. Analysis of the pharmacokinetic parameters after a single i.v. bolus injection of 1-O-pentylglycerol resulted in a peak blood concentration of 0.58+/-0.06 micromol/ml, an initial half life of 23+/-7 min and a terminal half life of 18.8+/-4.1 h. No accumulation of 1-O-pentylglycerol was observed in the brain or other organs while highest concentrations were found in liver and thymus. This was confirmed by autoradiographic studies. Five minutes after intracarotid administration, high radioactivity was found in the ipsilateral brain, whereas after 30 min radioactivity in the brain has dramatically decreased. Autoradiographic images gave evidence of biliary excretion in addition to the renal elimination. There were no signs of cleavage of the O-alkyl bond in vivo as demonstrated by HPLC analysis. In conclusion, 1-O-pentylglycerol is characterized by pharmacological properties appearing very favorable for in vivo use as a permeabilizing drug for increased drug delivery to the brain.
Asunto(s)
Barrera Hematoencefálica , Glicerol/análogos & derivados , Glicerol/farmacocinética , Animales , Autorradiografía , Arterias Carótidas , Glicerol/administración & dosificación , Inyecciones Intraarteriales , Inyecciones Intravenosas , Masculino , Ratas , Ratas Wistar , Distribución TisularRESUMEN
Peripheral-type benzodiazepine receptors (PBR) are constituted by three protein components, the isoquinoline binding protein (IBP), the voltage-dependent anion channel (VDAC), and the adenine nucleotide transporter (ANT). Recently, we found that high levels of PBR ligand binding in glioma cell lines correlate with in vitro tumorigenicity. To study whether enhanced PBR expression is causative or in response to cancer, we genetically modified C6 glioma cells. Antisense knockdown of the IBP resulted in more than 50% reductions in PBR ligand binding both in the mitochondrial and whole cell fractions, accompanied by similar reductions in IBP levels in these respective fractions. The IBP knockdown was accompanied by a 25% increase in cell number in confluent cultures. This correlated with an 8-fold increase in in vitro tumorigenicity, as assessed by anchorage independent growth. Cell cycle analysis indicated that knockdown of the IBP resulted in a 60% reduction in the number of cells in the pre-G1 apoptosis phase. This paralleled the reduction seen in apoptosis and cell death shown by DNA fragmentation and Trypan blue assays, respectively. Furthermore, knockdown of the IBP appeared to prevent induction of apoptosis by the antineoplastic agent, erucylphosphocholine. In addition, IBP knockdown prevented processing of the caspase 3 component of the apoptosis cascade by the erucylphosphocholine congener, erucylphospho-N,N,N-trimethylammonium. In conclusion, our results suggest that enhanced IBP expression, including enhanced PBR ligand binding, such as occurring in untreated C6 glioma cells, may provide a mechanism to increase apoptotic rates of cancer cells.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/genética , Receptores de GABA-A/química , Receptores de GABA-A/genética , Animales , Apoptosis/genética , Western Blotting , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/biosíntesis , Caspasa 3 , Caspasas/metabolismo , Recuento de Células , Línea Celular Tumoral , Células Clonales , ADN sin Sentido/química , Antagonistas de Receptores de GABA-A , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Procesamiento Proteico-Postraduccional , Ratas , Receptores de GABA-A/biosíntesis , Transfección , Azul de TripanoRESUMEN
Erucylphosphocholine (ErPC) is a promising antineoplastic drug for the treatment of malignant brain tumors. It exerts strong anticancer activity and induces apoptosis even in chemoresistant glioma cells. In the present study, A172 and U373MG glioma cells were treated with ErPC to explore the contribution of MAP kinase family members ERK, JNK and p38 kinase to ErPC-induced cell death. The exposure to ErPC led to activation of JNK and concurrent inhibition of ERK in both cell lines. Using specific MAP kinase inhibitors we confirmed that in U373MG cells ERK was blocked and JNK was activated upon ErPC treatment. Both effects were dependent on caspase activation. In A172 cells, ErPC treatment resulted in an activation of the JNK pathway, whereas the situation with respect to ERK signalling was more complex. We conclude that inhibition of the ERK pathway by ErPC may be related to antiproliferative effects, while activation of the JNK pathway may be responsible for its pro-apoptotic action.
Asunto(s)
Neoplasias Encefálicas/enzimología , Neoplasias Encefálicas/patología , Quinasas MAP Reguladas por Señal Extracelular/farmacología , Glioblastoma/enzimología , Glioblastoma/patología , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacología , Proliferación Celular/efectos de los fármacos , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/farmacología , Transducción de Señal , Células Tumorales CultivadasRESUMEN
1. The blood-brain barrier (BBB) represents the major impediment to successful delivery of therapeutic agents to target tissue within the central nervous system. Intracarotid alkylglycerols have been shown to increase the transfer of chemotherapeutics across the BBB. 2. We investigated the spatial distribution of intracarotid fluorescein sodium and intravenous lissamine-rhodamine B200 (RB 200)-albumin in the brain of normal and C6 glioma-bearing rats after intracarotid co-administration of 1-O-pentylglycerol (200 mm). To elucidate the mechanisms involved in the alkylglycerol-mediated BBB opening, intraluminal accumulation of fluorescein isothiocyanate (FITC)-dextran 40,000 was studied in freshly isolated rat brain capillaries using confocal microscopy during incubation with different alkylglycerols. Furthermore, 1-O-pentylglycerol-induced increase in delivery of methotrexate (MTX) to the brain was evaluated in nude mice. 3. Microscopic evaluation showed a marked 1-O-pentylglycerol-induced extravasation of fluorescein and RB 200-albumin in the ipsilateral normal brain. In glioma-bearing rats, increased tissue fluorescence was found in both tumor tissue and brain surrounding tumor. Confocal microscopy revealed a time- and concentration-dependent accumulation of FITC-dextran 40,000 within the lumina of isolated rat brain capillaries during incubation with 1-O-pentylglycerol and 2-O-hexyldiglycerol, indicating enhanced paracellular transfer via tight junctions. Intracarotid co-administration of MTX and 1-O-pentylglycerol (200 mm) in nude mice resulted in a significant increase in MTX concentrations in the ipsilateral brain as compared to controls without 1-O-pentylglycerol (P<0.005). 4. In conclusion, 1-O-pentylglycerol increases delivery of small and large compounds to normal brain and brain tumors and this effect is mediated at least in part by enhanced permeability of tight junctions.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Neoplasias Encefálicas/metabolismo , Encéfalo/efectos de los fármacos , Glioma/metabolismo , Glicerol/análogos & derivados , Glicerol/farmacología , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Neoplasias Encefálicas/patología , Capilares , Fluoresceína , Colorantes Fluorescentes , Glioma/patología , Glicerol/administración & dosificación , Inyecciones Intraarteriales , Inyecciones Intraventriculares , Masculino , Metotrexato/administración & dosificación , Metotrexato/farmacocinética , Ratones , Ratones Desnudos , Microscopía Confocal , Trasplante de Neoplasias , Ratas , Ratas Wistar , Distribución TisularRESUMEN
1 The intracarotid administration of alkylglycerols has been reported previously by us to be a novel strategy for increased delivery of various chemotherapeutic drugs to the normal brain and brain tumors in rats. 2 Effectiveness and structure-activity relations of the most promising pentyl- and hexylglycerol derivatives have been elucidated in vivo by analyzing the transfer of methotrexate (MTX) across the blood-brain barrier (BBB) in normal rats. The effects were compared with BBB disruption using hypertonic mannitol or intracarotid infusion of bradykinin. Furthermore, toxicity of the alkylglycerols has been studied in long-term experiments. 3 Apart from 1-O-pentyldiglycerol, all alkylglycerols induced a concentration-dependent increase in MTX delivery to the brain varying from 1.1 to more than 300-fold compared to intra-arterial MTX alone. Enhanced barrier permeability rapidly approached baseline values within 5 and 120 min at the latest. Chemical structure, concentration, time schedule of injections and combination of different alkylglycerols were identified as instruments suited to regulate the MTX accumulation within a wide range. Mannitol 1.4 M resulted in very high MTX levels in the brain as observed using the highest concentrations of alkylglycerols. Intracarotid infusion of bradykinin had only a minor effect on the BBB. Using 1-O-pentylglycerol or 2-O-hexyldiglycerol, both cell culture experiments and long-term in vivo analyses including clinical, laboratory and histopathological evaluations revealed no signs of toxicity. 4 In summary, intracarotid short-chain alkylglycerols constitute a very effective and low toxic strategy for transient opening of the BBB to overcome the limited access of cytotoxic drugs to the brain.
Asunto(s)
Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Glicerol/análogos & derivados , Glicerol/administración & dosificación , Glicerol/farmacocinética , Éteres de Glicerilo/administración & dosificación , Éteres de Glicerilo/farmacocinética , Metotrexato/farmacocinética , Animales , Barrera Hematoencefálica/fisiopatología , Bradiquinina/administración & dosificación , Bradiquinina/farmacocinética , Encéfalo/ultraestructura , Arterias Carótidas , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Portadores de Fármacos , Sinergismo Farmacológico , Quimioterapia Combinada , Femenino , Glicerol/química , Humanos , Inyecciones Intraarteriales , Masculino , Manitol/administración & dosificación , Manitol/farmacocinética , Metotrexato/administración & dosificación , Metotrexato/química , Monocitos/efectos de los fármacos , Permeabilidad , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Relación Estructura-Actividad , Factores de Tiempo , Vena Cava SuperiorRESUMEN
Erucylphosphocholine (ErPC) is a promising anti-neoplastic drug for the treatment of malignant brain tumours. It exerts strong anti-cancer activity in vivo and in vitro and induces apoptosis even in chemoresistant glioma cell lines. The purpose of this study was to expand on our previous observations on the potential mechanisms of ErPC-mediated apoptosis with a focus on death receptor activation and the caspase network. A172 and T98G glioma cells were treated with ErPC for up to 48 h. ErPC effects on the expression of the tumour necrosis factor (TNF) and TNF-related apoptosis-inducing ligand (TRAIL) receptor system, and on caspase activation were determined. ErPC had no effect on the expression of TNFalpha or TRAIL. Inhibition of the TNF or TRAIL signalling pathway with antagonistic antibodies or fusion proteins did not affect apoptosis induced by ErPC, and a dominant-negative FADD construct did not abolish ErPC-induced effects. Western blot analysis indicated that ErPC-triggered apoptosis resulted in a time-dependent processing of caspases-3, -7, -8 and -9 into their respective active subunits. Co-treatment of A172 cells with different caspase inhibitors prevented apoptosis but did not abrogate cell death. These data suggest that A172 cells might have an additional caspase-independent pathway that insures cell death and guarantees killing of those tumour cells whose caspase pathway is incomplete.