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Background Astrocytes maintain central nerve system homeostasis and are relatively resistant to cell death. Dysfunction of cell death mechanisms may underlie glioblastoma genesis and resistance to cancer therapy; therefore more detailed understanding of astrocytic death modalities is needed in order to design effective therapy. The purpose of this study was to determine the effect of VAS2870, a pan-NADPH oxidase inhibitor, on staurosporine-induced cell death in astrocytes. Materials and methods Cultured rat astrocytes were treated with staurosporine as activator of cell death. Cell viability, production of reactive oxygen species (ROS), and mitochondrial potential were examined using flow cytometric analysis, while chemiluminescence analysis was performed to assess caspase 3/7 activity and cellular ATP. Results We show here for the first time, that VAS2870 is able to prevent staurosporine-induced cell death. Staurosporine exerts its toxic effect through increased generation of ROS, while VAS2870 reduces the level of ROS. Further, VAS2870 partially restores mitochondrial inner membrane potential and level of ATP in staurosporine treated cells. Conclusions Staurosporine induces cell death in cultured rat astrocytes through oxidative stress. Generation of ROS, mitochondrial membrane potential and energy level are sensitive to VAS2870, which suggests NADPH oxidases as an important effector of cell death. Consequently, NADPH oxidases activation pathway could be an important target to modulate astrocytic death.
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Astrocitos/efectos de los fármacos , Benzoxazoles/farmacología , Muerte Celular/efectos de los fármacos , NADPH Oxidasas/antagonistas & inhibidores , Estaurosporina/antagonistas & inhibidores , Triazoles/farmacología , Adenosina Trifosfato/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/fisiología , Caspasa 3/biosíntesis , Caspasa 7/biosíntesis , Supervivencia Celular/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Citometría de Flujo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Estaurosporina/farmacologíaRESUMEN
A specific activation of metabotropic glutamate receptor 7 (mGluR7) has been shown to be neuroprotective in various models of neuronal cell damage, however, its role in glia cell survival has not been studied, yet. Thus, we performed comparative experiments estimating protective effects of the mGluR7 allosteric agonist AMN082 in glia, neuronal and neuronal-glia cell cultures against various harmful stimuli. First, the transcript levels of mGluR7 and other subtypes of group II and III mGluRs in cortical neuronal, neuronal-glia and glia cell cultures have been measured by qPCR method. Next, we demonstrated that AMN082 with similar efficiency attenuated the glia cell damage evoked by staurosporine (St) and doxorubicin (Dox). The AMN082-mediated glioprotection was mGluR7-dependent and associated with decreased DNA fragmentation without involvement of caspase-3 inhibition. Moreover, the inhibitors of PI3K/Akt and MAPK/ERK1/2 pathways blocked the protective effect of AMN082. In neuronal and neuronal-glia cell cultures in the model of glutamate (Glu)- but not St-evoked cell damage, we showed a significant glia contribution to mGluR7-mediated neuroprotection. Finally, by using glia and neuronal cells derived from mGluR7+/+ and mGluR7-/- mice we demonstrated a higher cell-damaging effect of St and Dox in mGluR7-deficient glia but not in neurons (cerebellar granule cells). Our present data showed for the first time a glioprotective potential of AMN082 underlain by mechanisms involving the activation of PI3K/Akt and MAPK/ERK1/2 pathways and pro-survival role of mGluR7 in glia cells. These findings together with the confirmed neuroprotective properties of AMN082 justify further research on mGluR7-targeted therapies for various CNS disorders.
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Astrocitos/citología , Astrocitos/efectos de los fármacos , Compuestos de Bencidrilo/farmacología , Supervivencia Celular/efectos de los fármacos , Neuroprotección/efectos de los fármacos , Receptores de Glutamato Metabotrópico/agonistas , Animales , Compuestos de Bencidrilo/antagonistas & inhibidores , Caspasa 3/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/metabolismo , Técnicas de Cocultivo , Fragmentación del ADN/efectos de los fármacos , Doxorrubicina/efectos adversos , Doxorrubicina/antagonistas & inhibidores , Inhibidores Enzimáticos , Ratones Noqueados , Neuronas/efectos de los fármacos , Receptores de Glutamato Metabotrópico/biosíntesis , Receptores de Glutamato Metabotrópico/genética , Transducción de Señal , Estaurosporina/antagonistas & inhibidoresRESUMEN
The current medical and surgical therapies for neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease offer symptomatic relief but do not provide a cure. Thus, small synthetic compounds that protect neuronal cells from degeneration are critically needed to prevent and treat these. Oxidative stress has been implicated in various pathophysiological conditions, including neurodegenerative diseases. In a search for neuroprotective agents against oxidative stress using the murine hippocampal HT22â¯cell line, we found a novel oxindole compound, GIF-0726-r, which prevented oxidative stress-induced cell death, including glutamate-induced oxytosis and erastin-induced ferroptosis. This compound also exerted a protective effect on tunicamycin-induced ER stress to a lesser extent but had no effect on campthothecin-, etoposide- or staurosporine-induced apoptosis. In addition, GIF-0726-r was also found to be effective after the occurrence of oxidative stress. GIF-0726-r was capable of inhibiting reactive oxygen species accumulation and Ca2+ influx, a presumed executor in cell death, and was capable of activating the antioxidant response element, which is a cis-acting regulatory element in promoter regions of several genes encoding phase II detoxification enzymes and antioxidant proteins. These results suggest that GIF-0726-r is a low-molecular-weight compound that prevents neuronal cell death through attenuation of oxidative stress. Among the more than 200 derivatives of the GIF-0726-r synthesized, we identified the 11 most potent activators of the antioxidant response element and characterized their neuroprotective activity in HT22â¯cells.
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Muerte Celular/efectos de los fármacos , Hipocampo/citología , Neuronas/citología , Neuronas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Oxindoles/farmacología , Animales , Elementos de Respuesta Antioxidante/efectos de los fármacos , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Camptotecina/antagonistas & inhibidores , Camptotecina/farmacología , Línea Celular , Etopósido/antagonistas & inhibidores , Etopósido/farmacología , Ratones , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Especies Reactivas de Oxígeno/metabolismo , Estaurosporina/antagonistas & inhibidores , Estaurosporina/farmacología , Tunicamicina/antagonistas & inhibidores , Tunicamicina/farmacologíaRESUMEN
BACKGROUND AND PURPOSE: Small conductance calcium-activated potassium (KCa 2.x) channels have a widely accepted canonical function in regulating cellular excitability. In this study, we address a potential non-canonical function of KCa 2.x channels in breast cancer cell survival, using in vitro models. EXPERIMENTAL APPROACH: The expression of all KCa 2.x channel isoforms was initially probed using RT-PCR, Western blotting and microarray analysis in five widely studied breast cancer cell lines. In order to assess the effect of pharmacological blockade and siRNA-mediated knockdown of KCa 2.x channels on these cell lines, we utilized MTS proliferation assays and also followed the corresponding expression of apoptotic markers. KEY RESULTS: All of the breast cancer cell lines, regardless of their lineage or endocrine responsiveness, were highly sensitive to KCa 2.x channel blockade. UCL1684 caused cytotoxicity, with LD50 values in the low nanomolar range, in all cell lines. The role of KCa 2.x channels was confirmed using pharmacological inhibition and siRNA-mediated knockdown. This reduced cell viability and also reduced expression of Bcl-2 but increased expression of active caspase-7 and caspase-9. Complementary to these results, a variety of cell lines can be protected from apoptosis induced by staurosporine using the KCa 2.x channel activator CyPPA. CONCLUSIONS AND IMPLICATIONS: In addition to a well-established role for KCa 2.x channels in migration, blockade of these channels was potently cytotoxic in breast cancer cell lines, pointing to modulation of KCa 2.x channels as a potential therapeutic approach to breast cancer.
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Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Técnicas de Silenciamiento del Gen , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/deficiencia , Alcanos/toxicidad , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/biosíntesis , Neoplasias de la Mama/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Dosificación Letal Mediana , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/deficiencia , Pirazoles/farmacología , Pirimidinas/farmacología , Compuestos de Quinolinio/toxicidad , ARN Interferente Pequeño/farmacología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/biosíntesis , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/genética , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Estaurosporina/antagonistas & inhibidores , Estaurosporina/farmacologíaRESUMEN
Many herbs, and recently their biomass from in vitro cultures, are essential for the treatment of diseases. The aim of this study was to determine the optimal growth of Bacopa monnieri (water hyssop) in an in vitro culture and to examine if extracts of the B. monnieri biomass from the in vitro culture would affect hydrogen peroxide- and staurosporine-induced injury of the human neuroblastoma SH-SY5Y cell line. It has been found that B. monnieri at concentrations of 25, 50, and 100 µg/mL inhibited both hydrogen peroxide-induced efflux of lactate dehydrogenase from damaged cells to culture medium and increased cell viability determined by an MTT assay. Moreover, B. monnieri at concentrations of 10, 25, and 50 µg/mL decreased staurosporine-induced activity of an executive apoptotic enzyme-caspase-3 and protected mitochondrial membrane potential. The obtained data indicate that the biomass from the in vitro culture of B. monnieri prevented SH-SY5Y cell damage related to oxidative stress and had the ability to inhibit the apoptotic process. Thus, this study supports the traditional use of B. monnieri as a neuroprotective therapy, and further in vivo studies on the effects of this preparation on morphology and function of nerve cells could lead to its wider application.
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Bacopa/química , Peróxido de Hidrógeno/antagonistas & inhibidores , Fármacos Neuroprotectores/farmacología , Extractos Vegetales/farmacología , Estaurosporina/antagonistas & inhibidores , Línea Celular Tumoral , Humanos , NeuroblastomaRESUMEN
The main objectives of the present study were to investigate the biocompatibility of polyelectrolyte-coated nanocapsules and to evaluate the neuroprotective action of the nanoencapsulated water-insoluble neuroprotective drug-undecylenic acid (UDA), in vitro. Core-shell nanocapsules were synthesized using nanoemulsification and the layer-by-layer (LbL) technique (by saturation method). The average size of synthesized nanocapsules was around 80 nm and the concentration was 2.5 × 10(10) particles/ml. Their zeta potential values ranged from less than -30 mV for the ones with external polyanion layers through -4 mV for the PEG-ylated layers to more than 30 mV for the polycation layers. Biocompatibility of synthesized nanocarriers was evaluated in the SH-SY5Y human neuroblastoma cell line using cell viability/toxicity assays (MTT reduction, LDH release). The results obtained showed that synthesized nanocapsules coated with PLL and PGA (also PEG-ylated) were non-toxic to SH-SY5Y cells, therefore, they were used as nanocarriers for UDA. Moreover, studies with ROD/FITC-labeled polyelectrolytes demonstrated approximately 20% cellular uptake of synthetized nanocapsules. Further studies showed that nanoencapsulated form of UDA was biocompatible and protected SH-SY5Y cells against the staurosporine-induced damage in lower concentrations than those of the same drug added directly to the culture medium. These data suggest that designed nanocapsules might serve as novel, promising delivery systems for neuroprotective agents.
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Electrólitos/química , Nanocápsulas/química , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/farmacología , Ácidos Undecilénicos/química , Ácidos Undecilénicos/farmacología , Materiales Biocompatibles/síntesis química , Materiales Biocompatibles/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Portadores de Fármacos , Estabilidad de Medicamentos , Humanos , Ensayo de Materiales , Fármacos Neuroprotectores/síntesis química , Tamaño de la Partícula , Estaurosporina/antagonistas & inhibidores , Estaurosporina/toxicidad , Ácidos Undecilénicos/síntesis químicaRESUMEN
Endochondral ossification is the result of chondrocyte differentiation, hypertrophy, death and replacement by bone. The careful timing and progression of this process is important for normal skeletal bone growth and development, as well as fracture repair. Apoptosis Signal-Regulating Kinase 1 (ASK1) is a mitogen-activated protein kinase (MAPK), which is activated by reactive oxygen species and other cellular stress events. Activation of ASK1 initiates a signaling cascade known to regulate diverse cellular events including cytokine and growth factor signaling, cell cycle regulation, cellular differentiation, hypertrophy, survival and apoptosis. ASK1 is highly expressed in hypertrophic chondrocytes, but the role of ASK1 in skeletal tissues has not been investigated. Herein, we report that ASK1 knockout (KO) mice display alterations in normal growth plate morphology, which include a shorter proliferative zone and a lengthened hypertrophic zone. These changes in growth plate dynamics result in accelerated long bone mineralization and an increased formation of trabecular bone, which can be attributed to an increased resistance of terminally differentiated chondrocytes to undergo cell death. Interestingly, under normal cell culture conditions, mouse embryonic fibroblasts (MEFs) derived from ASK1 KO mice show no differences in either MAPK signaling or osteogenic or chondrogenic differentiation when compared with wild-type (WT) MEFs. However, when cultured with stress activators, H2O2 or staurosporine, the KO cells show enhanced survival, an associated decrease in the activation of proteins involved in death signaling pathways and a reduction in markers of terminal differentiation. Furthermore, in both WT mice treated with the ASK1 inhibitor, NQDI-1, and ASK1 KO mice endochondral bone formation was increased in an ectopic ossification model. These findings highlight a previously unrealized role for ASK1 in regulating endochondral bone formation. Inhibition of ASK1 has clinical potential to treat fractures or to slow osteoarthritic progression by enhancing chondrocyte survival and slowing hypertrophy.
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Aporfinas/farmacología , Huesos/metabolismo , Condrocitos/metabolismo , MAP Quinasa Quinasa Quinasa 5/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Quinolinas/farmacología , Animales , Apoptosis/efectos de los fármacos , Huesos/citología , Huesos/efectos de los fármacos , Calcificación Fisiológica/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Condrocitos/citología , Condrocitos/efectos de los fármacos , Condrogénesis/efectos de los fármacos , Condrogénesis/genética , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Peróxido de Hidrógeno/antagonistas & inhibidores , Peróxido de Hidrógeno/farmacología , MAP Quinasa Quinasa Quinasa 5/genética , MAP Quinasa Quinasa Quinasa 5/metabolismo , Ratones , Ratones Noqueados , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Transducción de Señal , Estaurosporina/antagonistas & inhibidores , Estaurosporina/farmacologíaRESUMEN
Caspases are currently known as the central executioners of the apoptotic pathways. Inhibition of apoptosis and promotion of normal cell survival by caspase inhibitors would be a tremendous benefit for reducing the side effects of cancer therapy and for control of neurodegenerative disorders such as Parkinson's, Alzheimer's, and Huntington's diseases. The objective of this study was to discover small-molecule caspase inhibitors with which to achieve cytoprotective effect. We completed the high-throughput screening of Bionet's 37,500-compound library (Key Organics Limited, Camelford, Cornwall, UK) against caspase-1, -3, and -9 and successfully identified 43 initial hit compounds. The 43 hit compounds were further tested for cytoprotective activity against staurosporine-induced cell death in NIH3T3 cells. Nineteen compounds were found to have significant cytoprotective effects in cell viability assays. One of the compounds, RBC1023, was demonstrated to protect NIH3T3 cells from staurosporine-induced caspase-3 cleavage and activation. RBC1023 was also shown to protect against staurosporine-induced impairment of mitochondrial membrane potential. DNA microarray analysis demonstrated that staurosporine treatment induced broad global gene expression alterations, and RBC1023 co-treatment significantly restored these changes, especially of the genes that are related to cell growth and survival signaling such as Egr1, Cdc25c, cdkn3, Rhob, Nek2, and Taok1. Collectively, RBC1023 protects NIH3T3 cells against staurosporine-induced apoptosis via inhibiting caspase activity, restoring mitochondrial membrane potential, and possibly upregulating some cell survival-related gene expressions and pathways.
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Apoptosis/efectos de los fármacos , Inhibidores de Caspasas/farmacología , Caspasas/metabolismo , Citoprotección/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Estaurosporina/antagonistas & inhibidores , Animales , Inhibidores de Caspasas/síntesis química , Inhibidores de Caspasas/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Células 3T3 NIH , Análisis de Secuencia por Matrices de Oligonucleótidos , Células PC12 , Ratas , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Estaurosporina/farmacología , Relación Estructura-Actividad , TranscriptomaRESUMEN
Chloride (Clâ») channels participate in the regulation of cardiac function in response to stress although the underlying regulatory mechanism remains poorly understood. This study was designed to examine the impact of the pro-apoptotic stimulus staurosporine (STS) on the volume-sensitive outwardly rectifying Clâ» current (I(Cl,Vol)) in cardiomyocytes and possible regulatory mechanism involved with a focus on phosphatidylinositol-3 kinase (PI3K)/Akt. Primary cultured rat neonatal cardiomyocytes were subjected to hypotonic and isotonic environment in the presence or absence of STS prior to whole-cell voltage-clamp evaluation of Clâ» current. Whole-cell recordings revealed that STS activated an outwardly rectifying Clâ» current with phenotypic properties reminiscent of I(Cl,Vol). These currents were outwardly rectifying with a time-dependent inactivation at positive potentials and were sensitive to 4,4'-diisothiocya-natostilbene- 2,2'- disulfonicacid (DIDS), a non-selective Clâ» channel blocker, and 4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-on-5-yl)oxybutyric acid (DCPIB), a selective VSOR Clâ» channel blocker. DIDS and DCPIB inhibited I(Cl,Vol) by 92.6% ± 7.3% and 78.4% ± 5.5%, respectively. Our data further revealed that the PI3K inhibitor LY294002 facilitated the current with the peak amplitude of 19.54 ± 2.70 pA/pF. To the contrary, insulin partially inhibited the current amplitude with the peak current amplitude of 15.4 ± 2.13 pA/pF. Taken together, our data depicted staurosporine is capable of activating I(Cl,Vol) channel in cardiomyocytes via possibly a PI3K/Akt-dependent mechanism.
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Agonistas de los Canales de Cloruro , Moduladores del Transporte de Membrana/farmacología , Miocitos Cardíacos/efectos de los fármacos , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Estaurosporina/farmacología , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Células Cultivadas , Canales de Cloruro/antagonistas & inhibidores , Canales de Cloruro/metabolismo , Inhibidores Enzimáticos/farmacología , Hipoglucemiantes/farmacología , Soluciones Hipotónicas , Insulina/farmacología , Potenciales de la Membrana/efectos de los fármacos , Moduladores del Transporte de Membrana/agonistas , Moduladores del Transporte de Membrana/antagonistas & inhibidores , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Fosfatidilinositol 3-Quinasa/química , Inhibidores de las Quinasa Fosfoinosítidos-3 , Ratas , Ratas Sprague-Dawley , Estaurosporina/agonistas , Estaurosporina/antagonistas & inhibidoresRESUMEN
A major consequence of Parkinson's disease (PD) involves the loss of dopaminergic neurons in the substantia nigra (SN) and a subsequent loss of dopamine (DA) in the striatum. We have shown that glial cell line-derived neurotrophic factor (GDNF) shows robust restorative and protective effects for DA neurons in rats, non-human primates and possibly in humans. Despite GDNF's therapeutic potential, its clinical value has been questioned due to its limited diffusion to target areas from its large size and chemical structure. Several comparatively smaller peptides are thought to be generated from the prosequence. A five amino-acid peptide, dopamine neuron stimulating peptide-5 (DNSP-5), has been proposed to demonstrate biological activity relevant to neurodegenerative disease. We tested the in vitro effects of DNSP-5 in primary dopaminergic neurons dissected from the ventral mesencephalon of E14 Sprague Dawley rat fetuses. Cells were treated with several doses (0.03, 0.1, 1.0, 10.0 ng/mL) of GDNF, DNSP-5, or an equivalent volume of citrate buffer (vehicle). Morphological features of tyrosine hydroxylase positive neurons were quantified for each dose. DNSP-5 significantly increased (p < 0.001) all differentiation parameters compared to citrate vehicle (at one or more dose). For in vivo studies, a unilateral DNSP-5 treatment (30 µg) was administered directly to the SN. Microdialysis in the ipsilateral striatum was performed 28 days after treatment to determine extracellular levels of DA and its primary metabolites (3,4-dihydroxyphenylacetic acid and homovanillic acid). A single treatment significantly increased (~66%) extracellular DA levels compared to vehicle, while DA metabolites were unchanged. Finally, the protective effects of DNSP-5 against staurosporine-induced cytotoxicity were investigated in a neuronal cell line showing substantial protection by DNSP-5. Altogether, these studies strongly indicate biological activity of DNSP-5 and suggest that DNSP-5 has neurotrophic-like properties that may be relevant to the treatment of neurodegenerative diseases like PD.
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Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Neuropéptidos/farmacología , Oligopéptidos/farmacología , Animales , Bencimidazoles , Química Encefálica/efectos de los fármacos , Carbocianinas , Diferenciación Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Dopamina/metabolismo , Relación Dosis-Respuesta a Droga , Electroquímica , Colorantes Fluorescentes , Factor Neurotrófico Derivado de la Línea Celular Glial/química , Factor Neurotrófico Derivado de la Línea Celular Glial/farmacología , Indicadores y Reactivos , Infusiones Intravenosas , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mesencéfalo/citología , Mesencéfalo/efectos de los fármacos , Microdiálisis , Células PC12 , Ratas , Ratas Endogámicas F344 , Ratas Sprague-Dawley , Estaurosporina/antagonistas & inhibidores , Estaurosporina/toxicidadRESUMEN
OBJECTIVE: Curcumin is extracted from the turmeric plant (Curcuma longa Linn.) and is widely used as a food additive and traditional medicine. The present study investigated the activity of curcumin against staurosporine (STS) toxicity in cell culture. METHODS: Rat hippocampal neurons in primary culture were exposed to STS (20 µmol/L) and treated with curcumin (20 µmol/L). Cell viability was tested by MTT assay and reactive oxygen species (ROS) were measured using the MitoSOX™ red mitochondrial superoxide indicator. Western blot was used to assess changes in the levels of caspase-3 (Csp3), heat shock protein 70 (Hsp70) and Akt. RESULTS: The results showed that curcumin protects against STS-induced cytotoxicity in rat hippocampal neurons. Csp3, Hsp70, Akt and ROS activation may be involved in this protection. CONCLUSION: Curcumin could be a potential drug for combination with STS in cancer treatment to reduce the unwanted cytotoxicity of STS.
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Curcumina/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Estaurosporina/antagonistas & inhibidores , Estaurosporina/toxicidad , Animales , Animales Recién Nacidos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Neuronas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Pituitary adenylate cyclase-activating peptide (PACAP) is a neuroprotective peptide which exerts its effects mainly through the cAMP-protein kinase A (PKA) pathway. Here, we show that in cortical neurons, PACAP-induced PKA signaling exerts a major part of its neuroprotective effects indirectly, by triggering action potential (AP) firing. Treatment of cortical neurons with PACAP induces a rapid and sustained PKA-dependent increase in AP firing and associated intracellular Ca(2+) transients, which are essential for the anti-apoptotic actions of PACAP. Transient exposure to PACAP induces long-lasting neuroprotection in the face of apoptotic insults which is reliant on AP firing and the activation of cAMP response element (CRE) binding protein (CREB)-mediated gene expression. Although direct, activity-independent PKA signaling is sufficient to trigger phosphorylation on CREB's activating serine-133 site, this is insufficient for activation of CREB-mediated gene expression. Full activation is dependent on CREB-regulated transcription co-activator 1 (CRTC1), whose PACAP-induced nuclear import is dependent on firing activity-dependent calcineurin signaling. Over-expression of CRTC1 is sufficient to rescue PACAP-induced CRE-mediated gene expression in the face of activity-blockade, while dominant negative CRTC1 interferes with PACAP-induced, CREB-mediated neuroprotection. Thus, the enhancement of AP firing may play a significant role in the neuroprotective actions of PACAP and other adenylate cyclase-coupled ligands.
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Fármacos Neuroprotectores , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/fisiología , Potenciales de Acción/fisiología , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Calcineurina/fisiología , Calcio/metabolismo , Muerte Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/fisiología , Medios de Cultivo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Fenómenos Electrofisiológicos , Técnicas de Placa-Clamp , Fosforilación , Ratas , Ratas Sprague-Dawley , Estaurosporina/antagonistas & inhibidores , Estaurosporina/toxicidad , TransfecciónRESUMEN
We previously demonstrated that αB-crystallin and protease-activated receptor (PAR) are involved in protection of astrocytes against C2-ceramide- and staurosporine-induced cell death [Li et al. (2009) J. Neurochem.110, 1433-1444]. Here, we further investigated the mechanism of cytoprotection by αB-crystallin. Our current data revealed that after down-regulation of αB-crystallin by siRNA, cell death caused by C2-ceramide and staurosporine is increased. Furthermore, we investigated the mechanism of cytoprotection of astrocytes by intracellular αB-crystallin. Application of specific inhibitors of p38 and extracellular regulated kinase (ERK) abrogates the protection of astrocytes by over-expression of αB-crystallin. Thus, p38 and ERK contribute to protective processes by αB-crystallin. To reveal the molecular mechanism of αB-crystallin-mediated cytoprotection, we mimicked phosphorylation or unphosphorylation of αB-crystallin. In these experiments, we found that the phosphorylation of αB-crystallin at Ser45 and Ser59 is required for protection. Ser19 phosphorylation of αB-crystallin does not contribute to protection. Moreover, we detected that PAR-2 activation increases the phosphorylation level of αB-crystallin at Ser59, but does not affect the expression level of αB-crystallin. Thus, endogenous αB-crystallin has protective capacity employing a mechanism, which involves regulation of the phosphorylation status of αB-crystallin and p38 and ERK activity. Moreover, we report that PAR-2 activation evokes the phosphorylation of αB-crystallin to increase astrocytes survival.
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Astrocitos/efectos de los fármacos , Fármacos Neuroprotectores , Serina/química , Esfingosina/análogos & derivados , Estaurosporina/antagonistas & inhibidores , Estaurosporina/toxicidad , Cadena B de alfa-Cristalina/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología , Animales , Western Blotting , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Proteínas Quinasas JNK Activadas por Mitógenos/fisiología , Fosforilación , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Receptor PAR-2/genética , Receptor PAR-2/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esfingosina/antagonistas & inhibidores , Esfingosina/toxicidad , Cadena B de alfa-Cristalina/química , Proteínas Quinasas p38 Activadas por Mitógenos/químicaRESUMEN
Shigella flexneri is a gram-negative, facultative intracellular pathogen that invades the colonic epithelium and causes bacillary dysentery. We previously demonstrated that S. flexneri inhibits staurosporine-induced apoptosis in infected epithelial cells and that a DeltamxiE mutant is unable to inhibit apoptosis. Therefore, we hypothesized that an MxiE-regulated gene was responsible for protection of epithelial cells from apoptosis. Analysis of all MxiE-regulated genes yielded no mutants that lacked the ability to prevent apoptosis. Spa15, which is defined as a type III secretion system chaperone, was analyzed since it associates with MxiE. A Deltaspa15 mutant was unable to prevent staurosporine-induced apoptosis. C-terminal hemagglutinin-tagged spa15 was secreted by S. flexneri within 2 h in the Congo red secretion assay, and secretion was dependent on the type III secretion system. Spa15 was also secreted by Shigella in infected epithelial cells, as verified by immunofluorescence analysis. Spa15 secretion was decreased in the DeltamxiE mutant, which demonstrates why this mutant is unable to prevent staurosporine-induced apoptosis. Our data are the first to show that Spa15 is secreted in a type III secretion system-dependent fashion, and the absence of Spa15 in the Deltaspa15 mutant results in the loss of protection from staurosporine-induced apoptosis in epithelial cells. Thus, Spa15 contributes to the intracellular survival of Shigella by blocking apoptosis in the infected host cell.
Asunto(s)
Apoptosis , Proteínas Bacterianas/metabolismo , Chaperonas Moleculares/metabolismo , Shigella flexneri/patogenicidad , Estaurosporina/antagonistas & inhibidores , Estaurosporina/toxicidad , Proteínas Bacterianas/genética , Células Epiteliales/microbiología , Eliminación de Gen , Chaperonas Moleculares/genética , Shigella flexneri/genéticaRESUMEN
NG108-15 cells differentiate into neurons by 1 mM sodium butyrate (NaB) treatment. Differentiated cells resulted more resistant to staurosporine (STS) than proliferating cells. In particular, STS treatment decreased Bcl-2 and Bcl-x(L) content in mitochondria of proliferating cells, but not in mitochondria of differentiated cells. Bad was phosphorylated and downregulated only in differentiated cells. Bax accumulated in the mitochondria of proliferating but not differentiated cells. Mitochondrial release of cytochrome c was observed in proliferating cells, whereas mitochondria of differentiated cells retained cytochrome c. Proliferating cells treated with STS accumulated Endo G and AIF in the nucleus. By contrast, differentiated cells did not show such nuclear accumulation. Treatment of differentiated cells with Insulin-like Growth Factor-1 (IGF-1) and STS resulted in a 17.1% increase of cell viability. The survival role of IGF-1 was demonstrated by treating differentiated cells with an anti-IGF-1 neutralizing antibody. Such treatment significantly increased STS-induced cell death. Electrophysiology studies showed that in STS-treated cells membrane potential oscillations were reduced in amplitude and did not give rise to spontaneous action potentials (APs). However, the percentage of cells yielding overshooting APs returned to control values after STS removal. It is concluded that neuronal differentiation of NG108-15 cells induces resistance to apoptotic cell death and that IGF-1 plays a central role in sustaining this mechanism.
Asunto(s)
Apoptosis , Factor I del Crecimiento Similar a la Insulina/metabolismo , Neuronas/citología , Estaurosporina/farmacología , Animales , Factor Inductor de la Apoptosis/metabolismo , Diferenciación Celular , Línea Celular , Electrofisiología , Endodesoxirribonucleasas/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Ratones , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Estaurosporina/antagonistas & inhibidores , Proteína X Asociada a bcl-2/metabolismo , Proteína Letal Asociada a bcl/metabolismo , Proteína bcl-X/metabolismoRESUMEN
The fibroplasia noted during wound repair is resolved by fibroblast cell death. How fibroblasts undergo death and how this is prevented by trophic growth factors present during the regenerative phase are unknown at the molecular level. We examined a model of staurosporine-induced apoptosis in fibroblasts. We demonstrated that epidermal growth factor (EGF) stimulation of fibroblast NR6WT expressing human EGF receptors blocks staurosporine-induced apoptosis by inhibiting the activation of caspase-3. The survival effect of EGF on rescuing apoptotic NR6WT involves signaling pathways that derive from PI3K and Rac; the blockade of apoptosis is abolished when PI3K and Rac signals are inhibited simultaneously. Furthermore, by using KP372-1, a specific Akt inhibitor, we found that downstream of Akt signaling pathways is absolutely required for the EGF rescue from staurosporine-induced apoptosis in NR6WT. Interestingly, EGF prevention of apoptosis induced by tumor necrosis factor-alpha in the face of cycloheximide blockade of protein translation occurs via a different set of pathways as the simultaneous inhibition of extracellular signal-regulated kinase, Rac, and PI3K signaling did not eliminate EGF from rescuing fibroblasts in the face of this cytokine. These findings indicate that EGF receptor activation provides survival response against staurosporine-induced apoptosis through signal pathways of PI3K and Rac, which then may prevent the activation of caspase-3.
Asunto(s)
Apoptosis/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Fibroblastos/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Animales , Apoptosis/fisiología , Caspasas/metabolismo , Células Cultivadas , Humanos , Immunoblotting , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Plásmidos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Estaurosporina/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
Memantine, a clinically used N-methyl-D-aspartate (NMDA)-receptor antagonist, has been shown to prevent apoptotic neuronal damage connected with the over-activity of NMDA receptors. In the present study, we examined the effect of memantine on staurosporine-, salsolinol- and doxorubicin-induced apoptosis in the SH-SY5Y cell line which does not possess functional NMDA receptors. Electrophysiological recordings and toxicity studies showed no response to NMDA-evoked currents in this cell line, irrespective of the stage of its neuronal differentiation. Memantine (0.1-2 microM) attenuated staurosporine-induced apoptosis as evidenced by reversal of the changes in mitochondrial membrane potential (DeltaPsi(m)) and decreased caspase-3 activity, lactate dehydrogenase (LDH) release and DNA fragmentation. Wortmannin (10 nM) and LY 294002 (10 microM) (inhibitors of phosphatidylinositol-3-kinase, PI3-K) reversed the inhibitory effect of memantine on the staurosporine-induced LDH release, suggesting that the PI3-K/Akt prosurvival pathway is a possible target for antiapoptotic action of memantine. Memantine at low micromolar concentrations also attenuated salsolinol- and doxorubicin-induced LDH release and DNA fragmentation, but only in the case of salsolinol was this effect accompanied by a decrease in caspase-3 activity. The present data indicate that memantine attenuates the toxic effects of various proapoptotic agents and the cytoprotective effect of memantine does not seem to be connected with its action on NMDA receptor but rather with its influence on intracellular pathways engaged in cellular survival/apoptotic processes.
Asunto(s)
Antibióticos Antineoplásicos/antagonistas & inhibidores , Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Doxorrubicina/antagonistas & inhibidores , Doxorrubicina/toxicidad , Inhibidores Enzimáticos/toxicidad , Agonistas de Aminoácidos Excitadores/farmacología , Isoquinolinas/antagonistas & inhibidores , Isoquinolinas/toxicidad , Memantina/farmacología , Estaurosporina/antagonistas & inhibidores , Estaurosporina/toxicidad , Androstadienos/farmacología , Caspasa 3/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cromonas/farmacología , Electrofisiología , Humanos , L-Lactato Deshidrogenasa/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Morfolinas/farmacología , N-Metilaspartato/toxicidad , Técnicas de Placa-Clamp , Tretinoina/farmacología , WortmaninaRESUMEN
The AGAPEPAEPAQPGVY proline-rich peptide (PRP-1) was isolated from neurosecretory granules of the bovine neurohypophysis; it is produced by N. supraopticus and N. paraventricularis. It has been shown that PRP-1 has many potentially beneficial biological effects including immunoregulatory, hematopoietic, antimicrobial and anti-neurodegenerative properties. Here we investigated the influence of PRP-1 on staurosporine-induced apoptosis of postnatal hippocampal cells and on doxorubicin-induced bone marrow granulocyte- and monocyte apoptosis. The intention was to further characterize the effect of PRP-1 on the survival rate of neurons and in context with myelopoiesis. We demonstrate that PRP-1 significantly reduced apoptosis of postnatal hippocampal cells induced by staurosporine. The protective effect of PRP-1 against apoptotic cell death was shown to be both time- and dose-dependent. Neuroprotection was more pronounced after prolonged pretreatment of the cells with PRP-1 before the induction of apoptosis with staurosporine. The related peptide [arg(8)]vasopressin did not reveal neuroprotection. PRP-1 also significantly reduced apoptosis of bone marrow monocytes and granulocytes induced by doxorubicin. This protective effect lasted for 2-4 h and was not detectable anymore after 24 h when PRP-1 and doxorubicin were added simultaneously. Previously obtained data and results of the current studies suggested that the hypothalamic PRP-1 possibly represents an endogenous peptide whose primary functions are to regulate myelopoiesis and neuron survival as we provide evidence that PRP can differentially reduce both staurosporine- and doxorubicin-induced hippocampal and bone marrow cell apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Células de la Médula Ósea/efectos de los fármacos , Neuronas/citología , Péptidos/farmacología , Animales , Doxorrubicina/farmacología , Hipocampo/citología , Hipocampo/efectos de los fármacos , Neuronas/efectos de los fármacos , Dominios Proteicos Ricos en Prolina , Ratas , Estaurosporina/antagonistas & inhibidores , Estaurosporina/farmacologíaRESUMEN
Recently, we reported that GM-CSF showed therapeutic effects on the spinal cord injury (SCI) in rat model possibly via its anti-apoptotic activity in the nervous system. This study investigated the molecular mechanism of its anti-apoptotic and neuroprotective effects in N2a neuroblastoma cells and in rat SCI model. GM-CSF inhibited staurosporine-induced cytotoxicity and apoptosis of N2a cells. Single administration of GM-CSF either intraperitoneally or locally using a gelfoam, clearly reduced the apoptotic events in the surrounding region of the injury site in rat SCI model. Immunohistochemical analysis showed that apoptosis of cells occurred mainly in the neurons, but not significantly in the astrocytes in the surrounding regions. In both N2a cells and in rat SCI model, GM-CSF actually reduced the expression of pro-apoptotic proteins (p53, p21(WAF1/CIP1) and Bax), while further induced that of an anti-apoptotic protein (Bcl-2). In the Basso-Beattie-Bresnahan (BBB) locomotor test, the single GM-CSF administration showed better behavioral recovery than the untreated control only at early times within 1 week after injury. Overall, GM-CSF was shown to exert its neuroprotective effect on the neural injury by regulating the expression of apoptosis related genes, providing the molecular basis on its anti-apoptotic activity. Longer administration of GM-CSF appeared to be necessary for the sustained functional recovery from SCI.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/efectos de los fármacos , Apoptosis/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Degeneración Nerviosa/tratamiento farmacológico , Neuronas/efectos de los fármacos , Traumatismos de la Médula Espinal/tratamiento farmacológico , Animales , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis/metabolismo , Mapeo Encefálico , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Inhibidores Enzimáticos/toxicidad , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Masculino , Ratones , Degeneración Nerviosa/etiología , Degeneración Nerviosa/fisiopatología , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Proteínas Proto-Oncogénicas c-bcl-2/agonistas , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Ratas Sprague-Dawley , Recuperación de la Función/efectos de los fármacos , Recuperación de la Función/fisiología , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/fisiopatología , Estaurosporina/antagonistas & inhibidores , Estaurosporina/toxicidad , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología , Proteína X Asociada a bcl-2/antagonistas & inhibidores , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Neurotoxicity in primary neurons was induced using hypoxia/hypoglycemia (H/H), veratridine (10microM), staurosporine (1microM) or glutamate (100microM), which resulted in 72%, 67%, 75% and 66% neuronal injury, respectively. 3-Aminopyridine-2-carboxaldehyde thiosemicarbazone (PAN-811; 10microM; Panacea Pharmaceuticals, Gaithersburg, MD) pretreatment for 24 h provided maximal neuroprotection of 89%, 42%, 47% and 89% against these toxicities, respectively. Glutamate or H/H treatment of cells increased cytosolic cytochrome c levels, which was blocked by pretreatment of cells with PAN-811. Pretreatment of neurons with PAN-811 produced a time-dependent increase in the protein level of Bcl-2, which was evident even after glutamate or H/H treatments. An up-regulation in the expression of the p53 and Bax genes was also observed following exposure to these neurotoxic insults; however, this increase was not suppressed by PAN-811 pretreatment. Functional inhibition of Bcl-2 by HA14-1 reduced the neuroprotective efficacy of PAN-811. PAN-811 treatment also abolished glutamate or H/H-mediated internucleosomal DNA fragmentation.