RESUMEN
The increasing incidence of multidrug resistant uropathogenic E. coli (MDR-UPEC), the most common opportunistic pathogen in urinary tract infections (UTI) pose a global health problem and demands searching for alternative therapeutics. Antibiotics generate oxidative stress in bacteria which results in overexpression of the universal stress protein, UspA that helps in bacterial survival. An in silico study showed that two compounds ZINC000104153710, and ZINC000000217308 effectively bound bacterial UspA. This study aimed to determine the activity of ZINC000104153710, and ZINC000000217308 against bacterial UspA function in MDR-UPEC in vitro. Twenty-five highly MDR-UPEC were screened against ZINC000104153710, and, ZINC000000217308 either alone or in combination with the bactericidal antibiotics; ciprofloxacin (CIP), ceftazidime(CAZ), gentamicin(GEN) respectively by determining minimum inhibitory concentrations (MICs) using a broth microdilution assay. Additionally, the effect of ZINC000104153710, and ZINC000000217308 in the absence and presence of antibiotics on the bacteria was monitored by bacterial growth curve assays, ROS production, structure of the organism by scanning electron microscopy (FESEM) and quantitating UspA using a western blot technique. A 2-8 fold reduction in MIC values against ZINC000104153710, and ZINC000000217308 was observed against all 25 MDR-UPEC isolates in the presence of antibiotics with no alteration in intracellular ROS production. Discrete changes in cell morphology was evident in bacteria treated with ZINC000104153710 or ZINC000000217308 and antibiotics individually by FESEM compared with untreated control. Reduction in the level of UspA protein in bacteria treated with combination of ZINC000104153710 or ZINC000000217308 with individual antibiotics established their ability to inhibit UspA whose expression was elevated in presence of antibiotics alone. Therefore this study validated ZINC000104153710, and ZINC000000217308 as potent inhibitors of bacterial UspA function and indicated their potential as alternative therapeutics to combat the MDR-UPEC.
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Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Infecciones por Escherichia coli/tratamiento farmacológico , Proteínas de Choque Térmico/antagonistas & inhibidores , Infecciones Urinarias/tratamiento farmacológico , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/orina , Humanos , Pruebas de Sensibilidad Microbiana , Especies Reactivas de Oxígeno , Infecciones Urinarias/microbiología , Infecciones Urinarias/orinaRESUMEN
BACKGROUND: Diabetes Mellitus (DM) is characterized by hyperglycemia (high blood glucose levels) which is due to the destruction of insulin-producing ß-cells in the islets of Langerhans in the pancreas. It is associated with oxidative and endoplasmic reticulum stress. The plant alkaloid Palmatine has been previously reported to possess antidiabetic and antioxidant properties as well as other protective properties against kidney and liver tissue damage. OBJECTIVE: Here, we investigated the ability of Palmatine to reduce the up-regulation of chaperone proteins Glucose Regulatory Protein 78 (GRP78), and Calreticulin (CALR) protein in a Streptozotocin (STZ)-induced diabetic rat model. METHODS: Streptozotocin (STZ) induced diabetes in Sprague Dawley rats treated with 2mg/kg of Palmatine for 12 weeks after the elevation of plasma glucose levels above 11mmol/L post-STZ administration. Proteins were extracted from the pancreas after treatment and Two-Dimensional gel electrophoresis (2-DE), PDQuest 2-D analysis software genomic solutions and mass spectrometer were used to analyze differentially expressed protein. Mass Spectrometry (MS/MS), Multidimensional Protein Identification Technology (MudPIT) was used for protein identification. RESULTS: There was an up-regulation of the expression of chaperone proteins CALR and GRP78 and down-regulation of the expression of antioxidant and protection proteins peroxidoxin 4 (Prdx4), protein disulfide isomerase (PDIA2/3), Glutathione-S-Transferase (GSTs), and Serum Albumin (ALB) in non-diabetic rats. Palmatine treatment down-regulated the expression of chaperone proteins CALR and GRP78 and up-regulated the expression of Prdx4, PDIA2/3, GST, and ALB. CONCLUSION: Palmatine may have activated antioxidant proteins, which protected the cells against reactive oxygen species and endoplasmic stress. The result is in consonance with our previous report on Palmatine.
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Alcaloides de Berberina/uso terapéutico , Calreticulina/antagonistas & inhibidores , Diabetes Mellitus Experimental/tratamiento farmacológico , Proteínas de Choque Térmico/antagonistas & inhibidores , Hipoglucemiantes/uso terapéutico , Regulación hacia Arriba/efectos de los fármacos , Animales , Alcaloides de Berberina/farmacología , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Calreticulina/biosíntesis , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/inducido químicamente , Chaperón BiP del Retículo Endoplásmico , Proteínas de Choque Térmico/biosíntesis , Hipoglucemiantes/farmacología , Masculino , Ratas , Ratas Sprague-Dawley , Estreptozocina/toxicidad , Espectrometría de Masas en Tándem/métodos , Regulación hacia Arriba/fisiologíaRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Endoplasmic reticulum (ER) stress plays a role in the pathogenesis of diabetes mellitus, contributing to pancreatic dysfunction and insulin resistance. Ameliorating ER stress may be a viable therapeutic approach in the proper management of diabetes mellitus. Cymbopogon citratus (C.citratus) has been used in traditional medicine in the management of diabetes mellitus. Although well known for its anti-diabetic effect, the mechanism underlying this effect remains unclear. AIM OF THE STUDY: This study was designed to investigate the effect of C. citratus methanolic leaves extract on ER stress induced by streptozotocin (STZ) in wistar rats. MATERIALS AND METHODS: STZ (60 mg/kg) was used to induce ER stress in the pancreas of rats. The rats were administered C. citratus methanolic leaves extract via gastric gavage at doses 100, 200 and 400 mg/kg for two weeks while metformin (100 mg/kg) was used as positive control. Fasting blood glucose (FBG), expression of ER-stress related genes (GRP78, CHOP, ATF4, TRB3, PERK, IRE1), antioxidant (Nrf2 and AhR) and pro-inflammatory (TNF-α) genes were determined. Possible compounds responsible for this effect were also predicted through molecular docking. RESULTS: Induction of ER stress using STZ significantly increased FBG while administration of C. citratus methanolic extract restored it to normal control level (p < 0.05). Significant down-regulation of ER stress genes was observed upon treatment of ER stress induced rats with C. citratus methanolic extract when compared to ER-stress untreated rats. Significant up-regulation (p < 0.05) of genes coding for Nrf2 and AhR was also noticed upon treatment of ER stress induced rats with C. citratus methanolic extract. Molecular docking suggests that apigenin targets GRP78 with binding affinity of -9.3 kcal/mol while kaempferol and quercetin target Keap1 with binding affinity of -9.5 kcal/mol and may be responsible for this ameliorative effect on ER stress. CONCLUSION: These observations suggest that C. citratus mitigate ER stress induced by STZ via its down-regulative effect on GRP78 and up-regulative effect on NRF2 signaling.
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Cymbopogon , Estrés del Retículo Endoplásmico/efectos de los fármacos , Proteínas de Choque Térmico/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Extractos Vegetales/farmacología , Estreptozocina/toxicidad , Animales , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Estrés del Retículo Endoplásmico/fisiología , Proteínas de Choque Térmico/antagonistas & inhibidores , Factor 2 Relacionado con NF-E2/agonistas , Extractos Vegetales/aislamiento & purificación , Hojas de la Planta , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Listeria monocytogenes (Lm) is a widespread foodborne intracellular pathogen that invades a variety of cells, causing abortions and severe human diseases. After internalization into host cells, pore-forming cytolysin listeriolysin O (LLO) disrupts the phagosome, which allows the bacterium to survive and colonize the cytoplasm, providing the bacterium the chance to infect neighboring cells. Betulin is an extracted natural compound from birch bark with diverse pharmacological activities. Here, we showed that LLO-induced rabbit red blood cell lysis in vitro was inhibited by preincubation with betulin, which suppressed the oligomerization process. Infectious assays performed with human monocyte macrophages indicated that betulin significantly protected cells against Lm-induced cell injury. In addition, Balb/c mice were used to perform a general infection, and betulin administration obviously inhibited organ damage and bacterial burden in livers and spleens of infected mice. In conclusion, betulin obviously inhibited Lm-induced cell injury in vitro and protected against infection in vivo through an antivirulence effect. Our results showed betulin as a new candidate against listeriosis by targeting LLO and highlight the potential of natural product-based medicine to be applied in the treatment of pathogenic infections.
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Toxinas Bacterianas/antagonistas & inhibidores , Proteínas de Choque Térmico/antagonistas & inhibidores , Proteínas Hemolisinas/antagonistas & inhibidores , Listeriosis/tratamiento farmacológico , Triterpenos/farmacología , Animales , Eritrocitos/efectos de los fármacos , Femenino , Hemólisis/efectos de los fármacos , Humanos , Listeria monocytogenes , Hígado/microbiología , Hígado/patología , Macrófagos/efectos de los fármacos , Ratones Endogámicos BALB C , Conejos , Bazo/microbiología , Bazo/patología , Células THP-1RESUMEN
Cancer metastasis represents a multistep process, including alteration of cell adhesion/motility in the microenvironment and sustained angiogenesis, which is essential for supporting cancer growth in tissues that are distant from the primary tumor. There is growing evidence suggesting that heat shock proteins (HSPs) (also known as heat stress proteins), which constitute a family of stress-inducible proteins, may be involved in the pathogenesis of cancer. Curcumin (diferuloylmethane) is a potent anti-inflammatory, antioxidant, antimicrobial, and antitumor agent. Curcumin has been shown to regulate different members of HSPs including HSP27, HSP40, HSP60, HSP70, and HSP90 in cancer. Here, we present extent findings suggesting that curcumin may act as a potential therapeutic agent for the treatment of cancer through its regulation of HSPs.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Curcumina/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas de Neoplasias/genética , Neoplasias/tratamiento farmacológico , Neovascularización Patológica/prevención & control , Chaperonina 60/antagonistas & inhibidores , Chaperonina 60/genética , Chaperonina 60/metabolismo , Proteínas del Choque Térmico HSP40/agonistas , Proteínas del Choque Térmico HSP40/genética , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/agonistas , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/antagonistas & inhibidores , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Metástasis Linfática , Proteínas Mitocondriales/antagonistas & inhibidores , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Chaperonas Moleculares/antagonistas & inhibidores , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Transducción de Señal , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/genéticaRESUMEN
Harsh climate induces physiological stress thus compromising organismal survival. Our previous studies demonstrated that curcumin (CUR) supplementation increased survival of turtle under heat stress (HS). Here, we span this work to investigate the survival and lifespan of HS Drosophila fed a diet supplemented with CUR. For this purpose, female and male flies were fed basal diet (N) and CUR diet (0.2 mg/g), and exposed to three conditions: 25°C and 29°C continuously, and 34 °C for 2 h at days 1, 4, and 7, then kept at 25 °C. Lifespan analysis showed that, compared to N-25 °C flies, the mean lifespans of N-29 °C and N-34 °C flies were decreased significantly by 8.5-15.7% in males, and 3.7-7.9% in females. Conversely, in the CUR-supplemented diet, mean lifespans of C-29 °C and C-34 °C flies were significantly extended by 8.7-16.4% in males, and by 8.9-12.8% in females, compared to that of temperature-matched flies fed basal diets. The MDA levels of C-34 °C flies were significantly lower than those of N-34 °C flies, indicating CUR reduced oxidative stress caused by HS. Furthermore, CUR palliated the increased oxidative stress caused by HS, by increasing the expression of SOD1, CAT, and PHGPx and decreasing the expression of Hsp70 and Hsp83. Our results indicated that CUR supplementation increases the survival rate of Drosophila by enhancing thermal tolerance. © 2018 BioFactors, 44(6):577-587, 2018.
Asunto(s)
Antioxidantes/farmacología , Curcumina/farmacología , Suplementos Dietéticos , Drosophila melanogaster/efectos de los fármacos , Longevidad/efectos de los fármacos , Termotolerancia/efectos de los fármacos , Animales , Catalasa/genética , Catalasa/metabolismo , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/antagonistas & inhibidores , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Respuesta al Choque Térmico/efectos de los fármacos , Longevidad/fisiología , Masculino , Malondialdehído/antagonistas & inhibidores , Malondialdehído/metabolismo , Estrés Oxidativo/efectos de los fármacos , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Termotolerancia/genéticaRESUMEN
INTRODUCTION: Cisplatin is used as a standard chemotherapeutic agent for head and neck cancer treatment. However, some head and neck cancers have cisplatin resistance, leading to difficulty in treatment and poor prognosis. Overcoming cisplatin resistance remains an important strategy to improve prognoses for head and neck cancer patients. OBJECTIVE: Elucidation of the mechanisms underlying cisplatin resistance can suggest novel targets to enhance the anticancer effects of cisplatin for treating head and neck cancers. MATERIAL AND METHODS: We used a cisplatin-resistant human maxillary cancer cell line, IMC-3CR to analyse the cisplatin resistance mechanisms. Cisplatin-induced genes were analysed in IMC-3CR cells using PCR array. Among the genes with expression increased by cisplatin, we specifically examined SESN1. SESN family reportedly regenerates peroxiredoxin and suppresses oxidative DNA injury by reactive oxygen species (ROS), which can be induced by chemotherapeutic agents such as cisplatin, radiation, and hyperthermia. The function of SESN1 in cisplatin resistance and ROS generation were analysed using specific RNAi. RESULTS: Results show that SESN1 was induced by cisplatin treatment in IMC-3CR cells. Suppression of SESN1 by RNAi induced apoptosis and reduced cell viability through enhancement of ROS after cisplatin treatment. Moreover, suppression of SESN1 enhanced the cell-killing effects of hyperthermia with increased ROS, but did not affect the cell-killing effects of radiation. CONCLUSIONS: This study demonstrated the participation of SESN1 in cisplatin and hyperthermia resistance of human head and neck cancers. SESN1 is a novel molecular target to overcome cisplatin resistance and hyperthermia resistance and improve head and neck cancer treatment.
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Cisplatino/farmacología , Proteínas de Choque Térmico/antagonistas & inhibidores , Hipertermia Inducida/métodos , Neoplasias Maxilares/terapia , Especies Reactivas de Oxígeno/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Resistencia a Antineoplásicos , Expresión Génica/efectos de los fármacos , Proteínas de Choque Térmico/biosíntesis , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Neoplasias Maxilares/genética , Neoplasias Maxilares/metabolismo , Neoplasias Maxilares/patología , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , TransfecciónRESUMEN
BACKGROUND/AIMS: Protodioscin (PD) is a steroidal saponin with anti-cancer effects on a number of cancer cells, but the anti-tumor effects and mechanism of action of PD on human cervical cancer cells is unclear. METHODS: We determined cell viability using the MTT assay. Cell death, mitochondrial membrane potential (MMP), intracellular reactive oxygen species (ROS) generation, and endoplasmic reticulum (ER) stress were measured on a flow cytometry. Caspase activation, ER stress, and MMP-dependent apoptosis proteins in cervical cancer cells in response to PD were determined by Western blot analysis. The ability of ATF4 binding to ChIP promoter was measured using the ChIP assay. RESULTS: We demonstrated that PD inhibits cell viability, causes a loss of mitochondrial function, and induces apoptosis, as evidenced by up-regulation of caspase-8, -3, -9, -PARP, and Bax activation, and down-regulation of Bcl-2 expression. PD was shown to induce ROS and the ER stress pathway, including GRP78, p-eIF-2α, ATF4, and CHOP. Pre-treatment with NAC, a ROS production inhibitor, significantly reduced ER stress and apoptosis-related proteins induced by PD. Transfection of GRP78/CHOP-siRNA effectively inhibited PD-induced ER stress-dependent apoptosis. Moreover, treatment with PD significantly increased p38 and JNK activation. Co-administration of a JNK inhibitor (SP600125) or p38 inhibitor (SB203580) abolished cell death and ER stress effects during PD treatment. In addition, PD induced the expression of nuclear ATF4 and CHOP, as well as the binding ability of ATF4 to the CHOP promoter. CONCLUSION: Our results suggest that PD is a promising therapeutic agent for the treatment of human cervical cancer.
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Diosgenina/análogos & derivados , Estrés del Retículo Endoplásmico/efectos de los fármacos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Saponinas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Acetilcisteína/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Diosgenina/química , Diosgenina/farmacología , Regulación hacia Abajo/efectos de los fármacos , Medicamentos Herbarios Chinos/farmacología , Chaperón BiP del Retículo Endoplásmico , Femenino , Células HeLa , Proteínas de Choque Térmico/antagonistas & inhibidores , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Interferencia de ARN , Saponinas/química , Factor de Transcripción CHOP/antagonistas & inhibidores , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
SCOPE: We investigated the role of endoplasmic reticulum (ER) stress in the protective effects of EGCG against the neuronal apoptosis in Aß1-42 -induced SH-SY5Y cells and APP/PS1 transgenic mice. METHODS AND RESULTS: Cell viability (CCK8 assay), flow cytometry, Hoechst 33258 staining, immunohistochemistry, transmission electron microscopy (TEM), and western blotting were used. EGCG prevented Aß1-42-induced toxicity in SH-SY5Y cells, increased cell viability, and decreased apoptosis in a dose-dependent manner. In a subsequent mechanism study, it was found that this effect contributed to the down-regulation of GRP78, CHOP, cleaved-caspase-12 and -3. Moreover, EGCG also reduced the cytotoxicity induced by tunicamycin (TM) and thapsigargin (TG), two ER stress activators. Consistent with the in vitro study, EGCG inhibited neuronal apoptosis in the cortex of APP/PS1 transgenic mice, with the mitigation of ER abnormal ultrastructural swelling and the downregulation of ER-stress-associated proteins. CONCLUSION: These results indicate that EGCG attenuates the neurotoxicity in Alzheimer's disease (AD) via a novel mechanism that involves inhibition of ER-stress-associated neuronal apoptosis in vitro and in vivo, suggesting the tremendous potential of EGCG for use in a nutritional preventive strategy against AD.
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Péptidos beta-Amiloides/antagonistas & inhibidores , Apoptosis , Catequina/análogos & derivados , Suplementos Dietéticos , Estrés del Retículo Endoplásmico , Neuronas/metabolismo , Fármacos Neuroprotectores/metabolismo , Fragmentos de Péptidos/antagonistas & inhibidores , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/prevención & control , Péptidos beta-Amiloides/metabolismo , Animales , Caspasa 12/química , Caspasa 12/genética , Caspasa 12/metabolismo , Caspasa 3/química , Caspasa 3/genética , Caspasa 3/metabolismo , Catequina/metabolismo , Catequina/uso terapéutico , Línea Celular Tumoral , Supervivencia Celular , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Corteza Cerebral/ultraestructura , Chaperón BiP del Retículo Endoplásmico , Proteínas de Choque Térmico/agonistas , Proteínas de Choque Térmico/antagonistas & inhibidores , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Proteínas del Tejido Nervioso/agonistas , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/patología , Neuronas/ultraestructura , Fármacos Neuroprotectores/uso terapéutico , Nootrópicos/metabolismo , Nootrópicos/uso terapéutico , Fragmentos de Péptidos/metabolismo , Distribución Aleatoria , Factor de Transcripción CHOP/agonistas , Factor de Transcripción CHOP/antagonistas & inhibidores , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismoRESUMEN
SCOPE: Glutamate excitotoxicity has been observed in association with neurodegenerative disorders. This study aimed to investigate whether a phycoerythrin-derived tryptic peptide of Pyropia yezoensis (PYP) reduces glutamate-induced excitotoxicity and neuronal senescence in primary rat hippocampal neurons. METHODS AND RESULTS: Glutamate exposure (100 µm) decreased cell viability and increased expression of endoplasmic reticulum (ER) stress response protein glucose-regulated protein 78 (GRP78) starting at 60 min following glutamate exposure, which was prevented by pretreating the neurons with PYP (1 µg mL-1 ). The glutamate-induced increase in GRP78 expression was downregulated by blocking N-methyl-d-aspartate (NMDA) receptor with MK801 (10 µm) and inhibiting c-Jun N-terminal kinase (JNK) phosphorylation with SP600125 (10 µm). Moreover, phosphorylation of JNK was decreased by blockade of NMDA receptor. The PYP pretreatment downregulated glutamate-induced increase in GRP78 expression and JNK phosphorylation, and this effect was abolished by inhibiting tropomyosin-related kinase B (TrkB) receptor, phosphatidylinositiol 3-kinase, and extracellular signal-regulated kinase (ERK)1/2 using cyclotraxin B (200 nm), LY294002 (20 µm), and SL327 (10 µm), respectively. In addition, PYP downregulated increase in GRP78 expression, senescence-associated ß-galactosidase activity, and neurite degeneration in aging hippocampal neurons. CONCLUSION: These findings indicate that activation of TrkB receptor-mediated ERK1/2 by PYP attenuates glutamate-induced ER stress, which may improve the survival of hippocampal neurons with age.
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Estrés del Retículo Endoplásmico , Hipocampo/metabolismo , Neuronas/metabolismo , Fármacos Neuroprotectores/metabolismo , Fragmentos de Péptidos/metabolismo , Ficoeritrina/metabolismo , Receptor trkB/agonistas , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Senescencia Celular/efectos de los fármacos , Suplementos Dietéticos , Embrión de Mamíferos/citología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Antagonistas de Aminoácidos Excitadores/metabolismo , Antagonistas de Aminoácidos Excitadores/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Ácido Glutámico/envenenamiento , Proteínas de Choque Térmico/agonistas , Proteínas de Choque Térmico/antagonistas & inhibidores , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Hipocampo/citología , Hipocampo/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas del Tejido Nervioso/agonistas , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Nootrópicos/metabolismo , Fragmentos de Péptidos/química , Ficoeritrina/química , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Receptor trkB/antagonistas & inhibidores , Receptor trkB/metabolismo , Rhodophyta/enzimologíaRESUMEN
Membrane type-1 matrix metalloproteinase (MT1-MMP) is a transmembrane MMP which triggers intracellular signaling and regulates extracellular matrix proteolysis, two functions that are critical for tumor-associated angiogenesis and inflammation. While green tea catechins, particularly epigallocatechin gallate (EGCG), are considered very effective in preventing MT1-MMP-mediated functions, lack of structure-function studies and evidence regarding their direct interaction with MT1-MMP-mediated biological activities remain. Here, we assessed the impact in both cellular and biophysical assays of four ungallated catechins along with their gallated counterparts on MT1-MMP-mediated functions and molecular binding partners. Concanavalin-A (ConA) was used to trigger MT1-MMP-mediated proMMP-2 activation, expression of MT1-MMP and of endoplasmic reticulum stress biomarker GRP78 in U87 glioblastoma cells. We found that ConA-mediated MT1-MMP induction was inhibited by EGCG and catechin gallate (CG), that GRP78 induction was inhibited by EGCG, CG, and gallocatechin gallate (GCG), whereas proMMP-2 activation was inhibited by EGCG and GCG. Surface plasmon resonance was used to assess direct interaction between catechins and MT1-MMP interactors. We found that gallated catechins interacted better than their ungallated analogs with MT1-MMP as well as with MT1-MMP binding partners MMP-2, TIMP-2, MTCBP-1 and LRP1-clusterIV. Overall, current structure-function evidence supports a role for the galloyl moiety in both direct and indirect interactions of green tea catechins with MT1-MMP-mediated oncogenic processes.
Asunto(s)
Catequina/análogos & derivados , Metaloproteinasa 14 de la Matriz/metabolismo , Té/química , Carcinogénesis/efectos de los fármacos , Catequina/metabolismo , Catequina/farmacología , Línea Celular Tumoral , Concanavalina A/farmacología , Chaperón BiP del Retículo Endoplásmico , Precursores Enzimáticos/antagonistas & inhibidores , Gelatinasas/antagonistas & inhibidores , Glioblastoma/patología , Proteínas de Choque Térmico/antagonistas & inhibidores , Humanos , Metaloproteinasa 14 de la Matriz/farmacología , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Unión Proteica , Relación Estructura-ActividadRESUMEN
Translation of drug candidates into clinical settings requires demonstration of preclinical efficacy and formal toxicology analysis for filling an Investigational New Drug (IND) application with the US Food and Drug Administration (FDA). Here, we investigate the membrane-associated glucose response protein 78 (GRP78) as a therapeutic target in leukemia and lymphoma. We evaluated the efficacy of the GRP78-targeted proapoptotic drug bone metastasis targeting peptidomimetic 78 (BMTP-78), a member of the D(KLAKLAK)2-containing class of agents. BMTP-78 was validated in cells from patients with acute myeloid leukemia and in a panel of human leukemia and lymphoma cell lines, where it induced dose-dependent cytotoxicity in all samples tested. Based on the in vitro efficacy of BMTP-78, we performed formal good laboratory practice toxicology studies in both rodents (mice and rats) and nonhuman primates (cynomolgus and rhesus monkeys). These analyses represent required steps towards an IND application of BMTP-78 for theranostic first-in-human clinical trials.
Asunto(s)
Evaluación Preclínica de Medicamentos , Proteínas de Choque Térmico/genética , Leucemia/tratamiento farmacológico , Linfoma/tratamiento farmacológico , Peptidomiméticos/administración & dosificación , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Chaperón BiP del Retículo Endoplásmico , Proteínas de Choque Térmico/antagonistas & inhibidores , Humanos , Leucemia/patología , Linfoma/patología , Macaca fascicularis , Macaca mulatta , Ratones , Terapia Molecular Dirigida , Peptidomiméticos/efectos adversos , Primates , Ratas , Estados Unidos , United States Food and Drug AdministrationRESUMEN
Heat shock proteins (HSPs) are a large family of chaperones that are involved in protein folding and maturation of a variety of "client" proteins protecting them from degradation, oxidative stress, hypoxia, and thermal stress. Hence, they are significant regulators of cellular proliferation, differentiation and strongly implicated in the molecular orchestration of cancer development and progression as many of their clients are well established oncoproteins in multiple tumor types. Interestingly, tumor cells are more HSP chaperonage-dependent than normal cells for proliferation and survival because the oncoproteins in cancer cells are often misfolded and require augmented chaperonage activity for correction. This led to the development of several inhibitors of HSP90 and other HSPs that have shown promise both preclinically and clinically in the treatment of cancer. In this article, we comprehensively review the roles of some of the important HSPs in cancer, and how targeting them could be efficacious, especially when traditional cancer therapies fail.
Asunto(s)
Proteínas de Choque Térmico/metabolismo , Neoplasias/metabolismo , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Ensayos Clínicos como Asunto , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Regulación Neoplásica de la Expresión Génica , Proteínas de Choque Térmico/antagonistas & inhibidores , Proteínas de Choque Térmico/genética , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Terapia Molecular Dirigida , Familia de Multigenes , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Transducción de Señal , Estrés FisiológicoRESUMEN
The foodborne pathogenic bacterial species Listeria monocytogenes (L. monocytogenes) has caused incalculable damages to public health, and its successful infection requires various virulence factors, including Listeriolysin O (LLO). By forming pores in phagosomal membranes and even in some organelles, LLO plays an indispensable role in the ability of L. monocytogenes to escape from host immune attacks. Because of its critical role, LLO offers an appropriate therapeutic target against L. monocytogenes infection. Here, lutein, a natural small molecule existing widely in fruits and vegetables, is demonstrated as an effective inhibitor of LLO that works by blocking its oligomerization during invasion without showing significant bacteriostatic activity. Further assays applying lutein in cell culture models of invasion and in animal models showed that lutein could effectively inhibit L. monocytogenes infection. Overall, our results indicate that lutein may represent a promising and novel therapeutic agent against L. monocytogenes infection.
Asunto(s)
Antibacterianos/farmacología , Toxinas Bacterianas/antagonistas & inhibidores , Proteínas de Choque Térmico/antagonistas & inhibidores , Proteínas Hemolisinas/antagonistas & inhibidores , Listeria monocytogenes/efectos de los fármacos , Listeriosis/tratamiento farmacológico , Luteína/farmacología , Animales , Eritrocitos/efectos de los fármacos , Femenino , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Células RAW 264.7 , ConejosRESUMEN
Glucose-regulated protein 78 (GRP78) is the key regulator of endoplasmic reticular (ER) function. Expression of GRP78 was correlated with malignancy in different cancers. However, the role of GRP78 in the cytotoxic effect of curcumin on colon cancer cells is still unclear. A silencing RNA (siRNA) technique was used to knock down GRP78 expression. The anticancer effects of curcumin were assessed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, a flow cytometric cell cycle analysis, and a terminal dexynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. HT-29 cells expressed lower GRP78 compared with DLD-1 cells. The MTT assay revealed that HT-29 cells were more resistant to curcumin treatment than DLD-1 cells. GRP78KD cells showed more resistance to curcumin treatment compared with scrambled control cells. Overexpressed GRP78 in HT-29 cells increased the sensitivity to curcumin treatment. According to the cell cycle analysis and TUNEL assay, we found that apoptosis dramatically increased in scrambled control cells compared to GRP78KD DLD-1 cells after curcumin treatment. Finally, we evaluated levels of Bcl-2, BAX, and Bad and found that an increase of Bcl-2 level was observed in GRP78KD cells treated with curcumin. Those results were consistent with the increasing of resistance to curcumin after silencing of GRP78. The levels of GRP78 expression might determine the therapeutic efficacy of curcumin against colon cancer cells.
Asunto(s)
Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Curcumina/administración & dosificación , Proteínas de Choque Térmico/genética , Apoptosis/efectos de los fármacos , Ciclo Celular , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Neoplasias del Colon/patología , Retículo Endoplásmico/efectos de los fármacos , Chaperón BiP del Retículo Endoplásmico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Células HT29 , Proteínas de Choque Térmico/antagonistas & inhibidores , HumanosRESUMEN
The co-chaperonin GroES (Hsp10) works with chaperonin GroEL (Hsp60) to facilitate the folding reactions of various substrate proteins. Upon forming a specific disordered state in guanidine hydrochloride, GroES is able to self-assemble into amyloid fibrils similar to those observed in various neurodegenerative diseases. GroES therefore is a suitable model system to understand the mechanism of amyloid fibril formation. Here, we determined the cytotoxicity of intermediate GroES species formed during fibrillation. We found that neuronal cell death was provoked by soluble intermediate aggregates of GroES, rather than mature fibrils. The data suggest that amyloid fibril formation and its associated toxicity toward cell might be an inherent property of proteins irrespective of their correlation with specific diseases. Furthermore, with the presence of anthocyanins that are abundant in bilberry, we could inhibit both fibril formation and the toxicity of intermediates. Addition of bilberry anthocyanins dissolved the toxic intermediates and fibrils, and the toxicity of the intermediates was thus neutralized. Our results suggest that anthocyanins may display a general and potent inhibitory effect on the amyloid fibril formation of various conformational disease-causing proteins.
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Amiloide/antagonistas & inhibidores , Antocianinas/farmacología , Proteínas de Escherichia coli/antagonistas & inhibidores , Frutas/química , Proteínas de Choque Térmico/antagonistas & inhibidores , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Vaccinium myrtillus/química , Amiloide/efectos adversos , Amiloide/metabolismo , Amiloide/ultraestructura , Animales , Antiparkinsonianos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Suplementos Dietéticos/análisis , Proteínas de Escherichia coli/efectos adversos , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestructura , Proteínas de Choque Térmico/efectos adversos , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/ultraestructura , Potenciales de la Membrana/efectos de los fármacos , Ratones , Microscopía Electrónica de Transmisión , Peso Molecular , Neuronas/metabolismo , Neuronas/ultraestructura , Nootrópicos/farmacología , Extractos Vegetales/química , Pliegue de Proteína/efectos de los fármacos , SolubilidadRESUMEN
Inhibition of ClpB, the bacterial representative of the heat-shock protein 100 family that is associated with virulence of several pathogens, could be an effective strategy to develop new antimicrobial agents. Using a high-throughput screening method, we have identified several compounds that bind to different conformations of ClpB and analyzed their effect on the ATPase and chaperone activities of the protein. Two of them inhibit these functional properties as well as the growth of Gram negative bacteria (E. coli), displaying antimicrobial activity under thermal or oxidative stress conditions. This activity is abolished upon deletion of ClpB, indicating that the action of these compounds is related to the stress cellular response in which ClpB is involved. Moreover, their moderate toxicity in human cell lines suggests that they might provide promising leads against bacterial growth.
Asunto(s)
Antiinfecciosos/química , Antiinfecciosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos , Proteínas de Choque Térmico/antagonistas & inhibidores , Compuestos Orgánicos/química , Compuestos Orgánicos/farmacología , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/deficiencia , Adenosina Trifosfatasas/genética , Animales , Antiinfecciosos/toxicidad , Proteínas Bacterianas/genética , Células CHO , Cricetinae , Cricetulus , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Eliminación de Gen , Células HeLa , Proteínas de Choque Térmico/deficiencia , Ensayos Analíticos de Alto Rendimiento , Humanos , Ligandos , Compuestos Orgánicos/toxicidad , Estrés Oxidativo/efectos de los fármacos , TemperaturaRESUMEN
Protein-protein interactions (PPIs) are important in all aspects of cellular function, and there is interest in finding inhibitors of these contacts. However, PPIs with weak affinities and/or large interfaces have traditionally been more resistant to the discovery of inhibitors, partly because it is more challenging to develop high-throughput screening (HTS) methods that permit direct measurements of these physical interactions. Here, we explored whether the functional consequences of a weak PPI might be used as a surrogate for binding. As a model, we used the bacterial ATPase DnaK and its partners DnaJ and GrpE. Both DnaJ and GrpE bind DnaK and catalytically accelerate its ATP cycling, so we used stimulated nucleotide turnover to indirectly report on these PPIs. In pilot screens, we identified compounds that block activation of DnaK by either DnaJ or GrpE. Interestingly, at least one of these molecules blocked binding of DnaK to DnaJ, while another compound disrupted allostery between DnaK and GrpE without altering the physical interaction. These findings suggest that the activity of a reconstituted multiprotein complex might be used in some cases to identify allosteric inhibitors of challenging PPIs.
Asunto(s)
Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efectos de los fármacos , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Mapas de Interacción de Proteínas/efectos de los fármacos , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos/métodos , Escherichia coli/metabolismo , Infecciones por Escherichia coli/tratamiento farmacológico , Proteínas del Choque Térmico HSP40/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas de Choque Térmico/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Modelos MolecularesRESUMEN
BACKGROUND: Glucose regulated protein 78 (GRP78) functions as a sensor of endoplasmic reticulum (ER) stress. The aim of this study was to test the hypothesis that molecules that bind to GRP78 induce the unfolded protein response (UPR) and enhance cell death in combination with ER stress inducers. METHODS: Differential scanning calorimetry (DSC), measurement of cell death by flow cytometry and the induction of ER stress markers using western blotting. RESULTS: Epigallocatechin gallate (EGCG), a flavonoid component of Green Tea Camellia sinensis, and honokiol (HNK), a Magnolia grandiflora derivative, bind to unfolded conformations of the GRP78 ATPase domain. Epigallocatechin gallate and HNK induced death in six neuroectodermal tumour cell lines tested. Levels of death to HNK were twice that for EGCG; half-maximal effective doses were similar but EGCG sensitivity varied more widely between cell types. Honokiol induced ER stress and UPR as predicted from its ability to interact with GRP78, but EGCG was less effective. With respect to cell death, HNK had synergistic effects on melanoma and glioblastoma cells with the ER stress inducers fenretinide or bortezomib, but only additive (fenretinide) or inhibitory (bortezomib) effects on neuroblastoma cells. CONCLUSION: Honokiol induces apoptosis due to ER stress from an interaction with GRP78. The data are consistent with DSC results that suggest that HNK binds to GRP78 more effectively than EGCG. Therefore, HNK may warrant development as an antitumour drug.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Apoptosis/efectos de los fármacos , Compuestos de Bifenilo/uso terapéutico , Catequina/análogos & derivados , Proteínas de Choque Térmico/metabolismo , Lignanos/uso terapéutico , Neoplasias/tratamiento farmacológico , Antineoplásicos Fitogénicos/metabolismo , Compuestos de Bifenilo/metabolismo , Catequina/metabolismo , Catequina/uso terapéutico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Proteínas de Choque Térmico/antagonistas & inhibidores , Humanos , Lignanos/metabolismo , Terapia Molecular Dirigida , Peso Molecular , Neoplasias/patología , Unión Proteica/efectos de los fármacosRESUMEN
Effects of inhibitors of the heat shock protein 90 (HSP90) chaperone activity and inhibitors of the heat shock protein (HSP) expression on sensitivity of HeLa tumor cells to hyperthermia were studied. It was found that nanomolar concentrations of inhibitors of the HSP90 activity (17AAG or radicicol) slowed down chaperone-dependent reactivation of a thermo-labile reporter (luciferase) in heat-stressed HeLa cells and slightly enhanced their death following incubation for 60 min at 43 degrees C. Herein, the inhibitors of HSP90 activity stimulated de novo induction of additional chaperones (HSP70 and HSP27) that significantly increased the intracellular HSP levels. If the cells were treated with 17AAG or radicicol along with an inhibitor of the HSP induction (e.g. quercetin or triptolid, or NZ28), this fully prevented the increase in intracellular chaperone levels resulting from the inhibition of HSP90 activity and subsequent heating. Importantly, in the case of conjunction of all the three treatments (an inhibitor of the HSP90 activity + an inhibitor of the HSP induction + 43 degrees C for 60 min), the reporter reactivation was retarded yet stronger while the cell death was sharply (2-3-fold) enhanced. Such an enhancement of the cytotoxicity appears to occur owing to the "chaperone deficiency" when prior to heat stress both the functional activity of constitutive HSP90 and the expression of additional (inducible) chaperones are blocked in the cells.