RESUMO
Glucose-regulated protein 94 (GRP94) is an endoplasmic reticulum (ER)-resident member of the heat shock protein 90 (HSP90) family. In physiological conditions, it plays a vital role in regulating biological functions, including chaperoning cellular proteins in the ER lumen, maintaining calcium homeostasis, and modulating immune system function. Recently, several reports have shown the functional role and clinical relevance of GRP94 overexpression in the progression and metastasis of several cancers. Therefore, the current review highlights GRP94's physiological and pathophysiological roles in normal and cancer cells. Additionally, the unmet medical needs of small chemical inhibitors and the current development status of monoclonal antibodies specifically targeting GRP94 will be discussed to emphasize the importance of cell surface GRP94 as an emerging therapeutic target in monoclonal antibody therapy for cancer.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Membrana Celular/imunologia , Glicoproteínas de Membrana/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Humanos , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/efeitos dos fármacos , Chaperonas Moleculares/metabolismo , Neoplasias/metabolismoRESUMO
La diabetes Tipo 1 (DT1) es una compleja enfermedad autoinmune con una etiología aún desconocida. La vitamina D ha sido ampliamente estudiada debido a su potencial terapéutico en los potenciales nuevos casos de DT1. Por otra parte, los microARNs (miRs) han sido propuestos como posibles biomarcadores en diversos procesos biológicos como en la apoptosis e inflamación. El objetivo de este estudio fue evaluar el efecto de la suplementación con vitamina D sobre el perfil de expresión del miR-21 y marcadores de apoptosis tales como: BCL2, STAT3, TIPE2 y DAXX, en células mononucleares periféricas provenientes de pacientes con DT1 y sujetos controles. RESULTADOS: El perfil de expresión de miR-21 se encontró disminuido en los pacientes con DT1 en comparación con los controles. La expresión relativa de BCL2 se encontró aumentada en controles al comparar con pacientes DT1 en todas las condiciones experimentales. La expresión relativa de DAXX mostró un perfil de expresión diferencial al comparar pacientes con DT1 versus controles (p=0.006). CONCLUSIÓN: El estímulo con vitamina D parece tener un posible efecto regulador sobre los genes BCL2 y DAXX.
Type 1 diabetes (T1D) is a complex chronic autoimmune disease. Vitamin D has been one of the most studied therapeutic potential outbreaks related to T1D. Specific miRNAs have been proposed as potential biomarkers in several biological processes as apoptosis and inflammation. The aim of this study was to evaluate the effect of vitamin D on the expression profiles of miR-21 and apoptotic markers BCL2, STAT3, TIPE2 and DAXX, in PBMCs from T1D patients and control subjects. RESULTS: miR-21 expression was increased in controls regarding T1D patients. BCL2 was increased in controls compared to T1D patients in all experimental conditions. DAXX showed different expression patterns between T1D patients and controls (p=0.006). CONCLUSION: Vitamin D showed a possible regulation effect on apoptosis markers mainly through the regulation of BCL2 and DAXX
Assuntos
Humanos , Criança , Adolescente , Vitamina D/administração & dosagem , Apoptose , Diabetes Mellitus Tipo 1/metabolismo , Vitamina D/metabolismo , Biomarcadores , Chaperonas Moleculares/efeitos dos fármacos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , MicroRNAs/efeitos dos fármacos , MicroRNAs/genética , MicroRNAs/metabolismo , Fator de Transcrição STAT3/efeitos dos fármacos , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Proteínas Correpressoras/efeitos dos fármacos , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Glucose/administração & dosagemRESUMO
The iron-containing protein, acireductone dioxygenase 1 (ADI1), is a dioxygenase important for polyamine synthesis and proliferation. Using differential proteomics, the studies herein demonstrated that ADI1 was significantly down-regulated by cellular iron depletion. This is important, since ADI1 contains a non-heme, iron-binding site critical for its activity. Examination of multiple human cell-types demonstrated a significant decrease in ADI1 mRNA and protein after incubation with iron chelators. The decrease in ADI1 after iron depletion was reversible upon incubation of cells with the iron salt, ferric ammonium citrate (FAC). A significant decrease in ADI1 mRNA levels was observed after 14 h of iron depletion. In contrast, the chelator-mediated reduction in ADI1 protein occurred earlier after 10 h of iron depletion, suggesting additional post-transcriptional regulation. The proteasome inhibitor, MG-132, prevented the iron chelator-mediated decrease in ADI1 expression, while the lysosomotropic agent, chloroquine, had no effect. These results suggest an iron-dependent, proteasome-mediated, degradation mechanism. Poly r(C)-binding protein (PCBPs) 1 and 2 act as iron delivery chaperones to other iron-containing dioxygenases and were shown herein for the first time to be regulated by iron levels. Silencing of PCBP1, but not PCBP2, led to loss of ADI1 expression. Confocal microscopy co-localization studies and proximity ligation assays both demonstrated decreased interaction of ADI1 with PCBP1 and PCBP2 under conditions of iron depletion using DFO. These data indicate PCBP1 and PCBP2 interact with ADI1, but only PCBP1 plays a role in ADI1 expression. In fact, PCBP2 appeared to play an accessory role, being involved as a potential co-chaperone.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Dioxigenases/metabolismo , Ferro/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sítios de Ligação , Linhagem Celular , Proteínas de Ligação a DNA/genética , Dioxigenases/genética , Regulação para Baixo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Leupeptinas , Potencial da Membrana Mitocondrial , Chaperonas Moleculares/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Proteínas de Ligação a RNA/genética , Espécies Reativas de Oxigênio/metabolismo , Zinco/metabolismoRESUMO
Tumor necrosis factor receptor-associated protein 1 (TRAP1), a molecular chaperone, is a major member of the mitochondrial heat shock protein 90 (Hsp90) family. Studies have shown that TRAP1 can prevent hypoxia-induced damage to cardiomyocytes, maintain cardiomyocytes viability and mitochondrial membrane potential, and protect cardiomyocytes. In addition, it can also protect astrocytes from ischemic damage in vitro. In recent years, there have been many new discoveries in tumors. The abnormal expression of TRAP1 is closely related to the occurrence and development of various tumors. TRAP1 protein seems to be a central regulatory protein, involved in the activation of various oncogenic proteins and signaling pathways, and has a balanced function at tumor transformation and the intersection of different metabolic processes. Targeting its chaperone activity and molecular interactions can destroy the metabolism and survival adaptability of tumor cells, paving the way for the development of highly selective mitochondrial anti-tumor drugs. Moreover, the combination of TRAP1 inhibition and current traditional cancer therapies has shown promising applications. These findings have important implications for the diagnosis and treatment of tumors. Therefore, we reviewed the recently identified functions of the molecular chaperone TRAP1 in cancer development and progression, as well as the discovery and recent advances in selective TRAP1 inhibitors as anticancer drug therapies, opening up new attractive prospects for exploring strategies for targeting TRAP1 as a tumor cell target.
Assuntos
Antineoplásicos/uso terapêutico , Proteínas de Choque Térmico HSP90/efeitos dos fármacos , Chaperonas Moleculares/efeitos dos fármacos , Neoplasias/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico HSP90/fisiologia , Humanos , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/fisiologia , Neoplasias/tratamento farmacológicoRESUMO
One strategy for overcoming infectious diseases caused by drug-resistant fungi involves combining drugs rendered inactive by resistance with agents targeting the drug resistance mechanism. The antifungal activity of n-dodecanol disappears as incubation time passes. In Saccharomyces cerevisiae, anethole, a principal component of anise oil, prolongs the transient antifungal effect of dodecanol by downregulating genes of multidrug efflux pumps, mainly PDR5. However, the detailed mechanisms of dodecanol's antifungal action and the anethole-induced prolonged antifungal action of dodecanol are unknown. Screening of S. cerevisiae strains lacking genes related to Ca2+ homeostasis and signaling identified a pmr1Δ strain lacking Golgi Ca2+-ATPase as more sensitive to dodecanol than the parental strain. Dodecanol and the dodecanol + anethole combination significantly increased intracellular Ca2+ levels in both strains, but the mutant failed to clear intracellular Ca2+ accumulation. Further, dodecanol and the drug combination reduced PMR1 expression and did not lead to specific localization of Pmr1p in the parental strain after 4-h treatment. By contrast with the parental strain, dodecanol did not stimulate PDR5 expression in pmr1Δ. Based on these observations, we propose that the antifungal activity of dodecanol is related to intracellular Ca2+ accumulation, possibly dependent on PMR1 function, with anethole enabling Ca2+ accumulation by restricting dodecanol efflux.
Assuntos
Anisóis/farmacologia , ATPases Transportadoras de Cálcio/genética , Cálcio/metabolismo , Dodecanol/farmacologia , Deleção de Genes , Chaperonas Moleculares/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Derivados de Alilbenzenos , Anisóis/química , Antifúngicos/química , Antifúngicos/farmacologia , ATPases Transportadoras de Cálcio/efeitos dos fármacos , ATPases Transportadoras de Cálcio/metabolismo , Dodecanol/química , Sinergismo Farmacológico , Citometria de Fluxo , Complexo de Golgi/enzimologia , Chaperonas Moleculares/efeitos dos fármacos , Chaperonas Moleculares/metabolismo , RNA Fúngico/química , RNA Fúngico/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/genéticaRESUMO
BACKGROUND: Previous studies demonstrated an important role for connexin 43 (Cx43) in the regulation of apoptosis by influencing mitochondrial functions. This study aimed to investigate the relationship between Cx43 and lipopolysaccharide (LPS)-induced oxidative stress and apoptosis in human umbilical vein endothelial cells (HUVECs). METHODS: Western blot was performed to determine mitochondrial Cx43 (MtCx43) protein level and phosphorylation (p-MtCx43). Gap19, a selective Cx43 inhibitor, was used to examine the effects of Cx43 on LPS-induced oxidative stress and apoptosis in HUVECs. Expression of regulatory genes associated with oxidative stress was examined by quantitative polymerase chain reaction (qPCR) and Western blot. Apoptosis was assessed by flow cytometry. RESULTS: LPS stimulation resulted in increased levels of MtCx43 and p-MtCx43. Interestingly, Gap19 antagonized the upregulation of glutathione S-transferase Zeta 1 (GSTZ1) and cytochrome b alpha beta (CYBB), and the downregulation of antioxidant 1 (ATOX1), glutathione synthetase (GSS) and heme oxygenase 1 (HMOX1) induced by LPS or Cx43 overexpression. Moreover, the increased production of reactive oxygen species (ROS) and apoptosis elicited by LPS or Cx43 overexpression were reduced following treatment with Gap19. CONCLUSIONS: Selective inhibition of Cx43 hemichannels protects HUVECs from LPS-induced apoptosis and this may be via a reduction in oxidative stress production.
Assuntos
Apoptose/efeitos dos fármacos , Conexina 43/antagonistas & inibidores , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Apoptose/genética , Conexina 43/efeitos dos fármacos , Conexina 43/genética , Conexina 43/metabolismo , Proteínas de Transporte de Cobre/efeitos dos fármacos , Proteínas de Transporte de Cobre/genética , Regulação para Baixo , Técnicas de Introdução de Genes , Glutationa Sintase/efeitos dos fármacos , Glutationa Sintase/genética , Glutationa Transferase/efeitos dos fármacos , Glutationa Transferase/genética , Heme Oxigenase-1/efeitos dos fármacos , Heme Oxigenase-1/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Lipopolissacarídeos/farmacologia , Mitocôndrias/metabolismo , Chaperonas Moleculares/efeitos dos fármacos , Chaperonas Moleculares/genética , NADPH Oxidase 2/efeitos dos fármacos , NADPH Oxidase 2/genética , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio/metabolismo , Regulação para CimaRESUMO
Hsp31 protein, belonging to the DJ-1/ThiJ/PfpI superfamily, increases the survival of Escherichia coli under various stresses. While it was reported as a holding chaperone, Hsp31 was also shown to exhibit the glyoxalase III activity in subsequent study. Here, we describe our finding that Hsp31 undergoes a Zn+2-mediated multimerization (HMWZinc), resulting in an enhanced chaperone activity. Furthermore, it was shown that the formation of HMWZinc is reversible such that the oligomer dissociates into the native dimer by EDTA incubation. We attempted to determine the structural change involving the transition between the native dimer and HMWZinc by adding Ni+2, which is Zn+2-mimetic, producing a potential intermediate structure. An analysis of this intermediate revealed a structure with hydrophobic interior exposed, due to an unfolding of the N-terminal loop and the C-terminal ß-to-α region. A treatment with hydrogen peroxide accelerated HMWZinc formation, so that the Hsp31C185E mutant rendered the formation of HMWZinc even at 45 °C. However, the presence of Zn+2 in the catalytic site antagonizes the oxidation of C185, implying a negative role. Our results suggest an unprecedented mechanism of the enhancing chaperone activity by Hsp31, in which the reversible formation of HMWZinc occurs in the presence of heat and Zn+2 ion.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Zinco/farmacologia , Domínio Catalítico , Cromatografia em Gel , Escherichia coli/genética , Proteínas de Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Peróxido de Hidrogênio/farmacologia , Modelos Moleculares , Chaperonas Moleculares/efeitos dos fármacos , Chaperonas Moleculares/genética , Peso Molecular , Mutação , Níquel/farmacologia , Conformação Proteica , Multimerização Proteica/efeitos dos fármacos , Desdobramento de ProteínaRESUMO
Alcohol ingestion decreases postexercise rates of muscle protein synthesis, but the mechanism(s) (e.g., increased protein breakdown) underlying this observation is unknown. Autophagy is an intracellular "recycling" system required for homeostatic substrate and organelle turnover; its dysregulation may provoke apoptosis and lead to muscle atrophy. We investigated the acute effects of alcohol ingestion on autophagic cell signaling responses to a bout of concurrent (combined resistance- and endurance-based) exercise. In a randomized crossover design, eight physically active males completed three experimental trials of concurrent exercise with either postexercise ingestion of alcohol and carbohydrate (12 ± 2 standard drinks; ALC-CHO), energy-matched alcohol and protein (ALC-PRO), or protein (PRO) only. Muscle biopsies were taken at rest and 2 and 8 h postexercise. Select autophagy-related gene (Atg) proteins decreased compared with rest with ALC-CHO (P < 0.05) but not ALC-PRO. There were parallel increases (P < 0.05) in p62 and PINK1 commensurate with a reduction in BNIP3 content, indicating a diminished capacity for mitochondria-specific autophagy (mitophagy) when alcohol and carbohydrate were coingested. DNA fragmentation increased in both alcohol conditions (P < 0.05); however, nuclear AIF accumulation preceded this apoptotic response with ALC-CHO only (P < 0.05). In contrast, increases in the nuclear content of p53, TFEB, and PGC-1α in ALC-PRO were accompanied by markers of mitochondrial biogenesis at the transcriptional (Tfam, SCO2, and NRF-1) and translational (COX-IV, ATPAF1, and VDAC1) level (P < 0.05). We conclude that alcohol ingestion following exercise triggers apoptosis, whereas the anabolic properties of protein coingestion may stimulate mitochondrial biogenesis to protect cellular homeostasis.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Depressores do Sistema Nervoso Central/farmacologia , Carboidratos da Dieta/farmacologia , Proteínas Alimentares/farmacologia , Etanol/farmacologia , Exercício Físico/fisiologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Adolescente , Adulto , Consumo de Bebidas Alcoólicas , Apoptose/fisiologia , Autofagia/fisiologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Estudos Cross-Over , Fragmentação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/efeitos dos fármacos , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Voluntários Saudáveis , Humanos , Masculino , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/efeitos dos fármacos , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Mitofagia/efeitos dos fármacos , Mitofagia/fisiologia , Chaperonas Moleculares/efeitos dos fármacos , Chaperonas Moleculares/metabolismo , Fibras Musculares Esqueléticas/fisiologia , Fator 1 Nuclear Respiratório/efeitos dos fármacos , Fator 1 Nuclear Respiratório/metabolismo , Biogênese de Organelas , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Proteínas Quinases/efeitos dos fármacos , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Ligação a RNA/efeitos dos fármacos , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Canal de Ânion 1 Dependente de Voltagem/efeitos dos fármacos , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Adulto JovemRESUMO
Around 15 years ago, imatinib mesylate (Gleevec(®) or Glivec(®), Novartis, Switzerland) became the very first 'targeted' anticancer drug to be clinically approved. This drug constitutes the quintessential example of a successful precision medicine that has truly changed the fate of patients with Philadelphia-chromosome-positive chronic myeloid leukaemia (CML) and gastrointestinal stromal tumours by targeting the oncogenic drivers of these diseases, BCR-ABL1 and KIT and/or PDGFR, mutations in which lead to gain of function of tyrosine kinase activities. Nonetheless, the aforementioned paradigm might not fully explain the clinical success of this agent in these diseases. Growing evidence indicates that the immune system has a major role both in determining the therapeutic efficacy of imatinib (and other targeted agents) and in restraining the emergence of escape mutations. In this Review, we re-evaluate the therapeutic utility of imatinib in the context of the anticancer immunosurveillance system, and we discuss how this concept might inform on novel combination regimens that include imatinib with immunotherapies.
Assuntos
Antineoplásicos/imunologia , Tumores do Estroma Gastrointestinal/imunologia , Mesilato de Imatinib/imunologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/imunologia , Antígenos de Neoplasias/efeitos dos fármacos , Antígenos de Neoplasias/imunologia , Antineoplásicos/uso terapêutico , Antígenos B7/efeitos dos fármacos , Antígenos B7/imunologia , Aprovação de Drogas , Previsões , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Hematopoese/efeitos dos fármacos , Hematopoese/imunologia , Humanos , Mesilato de Imatinib/uso terapêutico , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/imunologia , Imunidade Celular/efeitos dos fármacos , Vigilância Imunológica/efeitos dos fármacos , Vigilância Imunológica/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Chaperonas Moleculares/efeitos dos fármacos , Chaperonas Moleculares/imunologia , Terapia de Alvo Molecular/métodos , Receptor 3 Desencadeador da Citotoxicidade Natural/efeitos dos fármacos , Receptor 3 Desencadeador da Citotoxicidade Natural/imunologia , Proteínas Tirosina Quinases/efeitos dos fármacos , Proteínas Tirosina Quinases/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Evasão Tumoral/efeitos dos fármacos , Evasão Tumoral/imunologia , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/imunologiaRESUMO
PURPOSE: Cks1, a conformationally heterogenous 9 kDa protein, is markedly overexpressed in cancer cells and contributes to tumor development. Cks1 is an essential component of the SCF-Skp2 ubiquitin ligase complex that targets the Cdk inhibitors p27(Kip1) and p21(Cip1). Cks1 is known to interact with the Hsp90-Cdc37 chaperone machinery, although whether this facilitates its conformational maturation and stability is not known. To test whether abrogating the chaperone function of Hsp90 could destabilize Cks1, we examined the effects of treating different cancer cell lines with the benzoquinone ansamycin 17-allylamino geldanamycin (17-AAG), a compound that selectively binds Hsp90 and potently inhibits its ATP-dependent chaperone activity. METHODS: The effect of Hsp90 inhibition using 17-AAG on Cks1 protein and associated cell cycle proteins including Skp2, p27(Kip1), p21(Cip1), and Cdk1 in cancer cells was determined by Western blotting. Ubiquitination analysis was carried out by transfecting cells with an HA-ubiquitin plasmid and specifically immunoprecipitating Cks1 to examine polyubiquitinated species. Flow cytometry was utilized to examine the effects of Hsp90 inhibition on cell cycle profiles. RESULTS: Here, we demonstrate for the first time that inhibition of Hsp90 utilizing 17-AAG destabilizes Cks1 in cancer cells by promoting its ubiquitination and proteasomal degradation. 17-AAG-induced Cks1 depletion was accompanied by concomitant decreases in Skp2 and Cdk1. 17-AAG treatment also induced G2/M accumulation in MCF-7 breast carcinoma cells, and G1 accumulation in the colon carcinoma lines HCT116 and SW620. CONCLUSIONS: We conclude that perturbing the Hsp90 pathway could provide a useful therapeutic strategy in tumors driven by Cks1 overexpression.
Assuntos
Quinases relacionadas a CDC2 e CDC28/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Trifosfato de Adenosina/antagonistas & inibidores , Antineoplásicos/farmacologia , Benzoquinonas/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Lactamas Macrocíclicas/farmacologia , Chaperonas Moleculares/efeitos dos fármacos , Proteínas Quinases Associadas a Fase S/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
Vitamin E succinate (RRR-alpha-tocopheryl succinate, VES), an efficient inducer of apoptosis, acts as a potent agent for cancer therapy. However, the mechanism by which VES mediates the effects are not yet fully understood. Here we studied the effect of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) on VES-induced apoptosis of SGC-7901 human gastric cancer cells. VES caused cytological changes typical of apoptosis, increased ER dilation and cytosolic Ca(2+) concentration. And endogenous ER stress markers, GRP78 and GRP94 were transcriptionally and translationally altered. In response to VES, induction of CHOP, activation of caspase-4 and JNK were observed. Furthermore, VES also triggered activation of UPR components, including RNA-dependent protein kinase (PKR)-like ER kinase (PERK), activating transcription factor 6 (ATF6), X-box-binding protein 1 (XBP1), and ATF4 in a concentration- and time-dependent manner. Consequently, our results suggest that VES-induced apoptosis is coupled to ER stress and UPR activation in SGC-7901 human gastric cancer cells.
Assuntos
Apoptose/efeitos dos fármacos , Retículo Endoplasmático/patologia , Neoplasias Gástricas/patologia , alfa-Tocoferol/farmacologia , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Divisão Celular/efeitos dos fármacos , Primers do DNA , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/efeitos dos fármacos , Proteínas de Choque Térmico/genética , Humanos , Glicoproteínas de Membrana/efeitos dos fármacos , Glicoproteínas de Membrana/genética , Microscopia Eletrônica , Chaperonas Moleculares/efeitos dos fármacos , Chaperonas Moleculares/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/ultraestrutura , Estresse Mecânico , Células Tumorais CultivadasRESUMO
BACKGROUND: Hsp90 is an essential molecular chaperone that is also a novel anti-cancer drug target. There is growing interest in developing new drugs that modulate Hsp90 activity. METHODOLOGY/PRINCIPAL FINDINGS: Using a virtual screening approach, 4-hydroxytamoxifen, the active metabolite of the anti-estrogen drug tamoxifen, was identified as a putative Hsp90 ligand. Surprisingly, while all drugs targeting Hsp90 inhibit the chaperone ATPase activity, it was found experimentally that 4-hydroxytamoxifen and tamoxifen enhance rather than inhibit Hsp90 ATPase. CONCLUSIONS/SIGNIFICANCE: Hence, tamoxifen and its metabolite are the first members of a new pharmacological class of Hsp90 activators.
Assuntos
Adenosina Trifosfatases/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/agonistas , Tamoxifeno/farmacologia , Adenosina Trifosfatases/metabolismo , Antineoplásicos Hormonais , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos , Humanos , Chaperonas Moleculares/efeitos dos fármacos , Tamoxifeno/análogos & derivadosRESUMO
Corticosteroids are widely used to treat a diversity of pathological conditions including allergic, autoimmune and some infectious diseases. These drugs have complex mechanisms of action involving both genomic and non-genomic mechanisms and interfere with different signal transduction pathways in the cell. The use of corticosteroids to treat critically ill patients with acute respiratory distress syndrome and severe infections, such as sepsis and pneumonia, is still a matter of intense debate in the scientific and medical community with evidence both for and against its use in these patients. Here, we review the basic molecular mechanisms important for corticosteroid action as well as current evidence for their use, or not, in septic patients. We also present an analysis of the reasons why this is still such a controversial point in the literature.
Assuntos
Humanos , Corticosteroides/uso terapêutico , Receptores de Glucocorticoides/efeitos dos fármacos , Síndrome do Desconforto Respiratório/tratamento farmacológico , Choque Séptico/tratamento farmacológico , Ensaios Clínicos como Assunto , Medicina Baseada em Evidências , Genômica , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Chaperonas Moleculares/efeitos dos fármacos , Chaperonas Moleculares/genética , Receptores de Glucocorticoides/genética , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genéticaRESUMO
Subtilase cytotoxin (SubAB) is the prototype of a newly identified family of AB(5) cytotoxins produced by Shiga toxigenic Escherichia coli. SubAB specifically cleaves the essential endoplasmic reticulum (ER) chaperone BiP (GRP78), resulting in the activation of ER stress-induced unfolded protein response (UPR). We have recently shown that the UPR following ER stress can suppress cellular responses to inflammatory stimuli during the later phase, in association with inhibition of NF-kappaB activation. These findings prompted us to hypothesize that SubAB, as a selective UPR inducer, might have beneficial effects on inflammation-associated pathology via a UPR-dependent inhibition of NF-kappaB activation. The pretreatment of a mouse macrophage cell line, RAW264.7, with a subcytotoxic dose of SubAB-triggered UPR and inhibited LPS-induced MCP-1 and TNF-alpha production associated with inhibition of NF-kappaB activation. SubA(A272)B, a SubAB active site mutant that cannot induce UPR, did not show such effects. In addition, pretreatment with a sublethal dose of SubAB, but not SubA(A272)B, protected the mice from LPS-induced endotoxic lethality associated with reduced serum MCP-1 and TNF-alpha levels and also prevented the development of experimental arthritis induced by LPS in mice. Collectively, although SubAB has been identified originally as a toxin associated with the pathogenesis of hemolytic uremic syndrome, the unique ability of SubAB to selectively induce the UPR may have the potential to prevent LPS-associated inflammatory pathology under subcytotoxic conditions.
Assuntos
Retículo Endoplasmático/patologia , Proteínas de Escherichia coli/farmacologia , Inflamação/prevenção & controle , Chaperonas Moleculares/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Subtilisinas/farmacologia , Animais , Artrite Experimental/tratamento farmacológico , Artrite Experimental/prevenção & controle , Linhagem Celular , Citotoxinas , Chaperona BiP do Retículo Endoplasmático , Proteínas de Escherichia coli/administração & dosagem , Inflamação/induzido quimicamente , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Chaperonas Moleculares/metabolismo , NF-kappa B/antagonistas & inibidores , Subtilisinas/administração & dosagemRESUMO
DYT1 is the most common inherited dystonia, a neurological syndrome that causes disabling involuntary muscle contractions. This autosomal dominant disease is caused by a glutamic acid deletion near the carboxy-terminus in the protein torsinA. Cell- and animal-based studies have shown how the DYT1 mutation causes mutant torsinA to redistribute from the endoplasmic reticulum to the nuclear envelope, acting through a dominant negative effect over the wild type protein. As a result, the wild type:mutant torsinA expression ratio would be important for disease pathogenesis, and events that influence it, such as a differential degradation process for each protein, might modulate DYT1 pathobiology. The DYT1 mutation also triggers the formation of abnormal intermolecular disulfide bonds in torsinA, although the significance of this finding is unclear. How the protein quality control machinery handles torsinA, and whether this process is affected by its abnormal oligomerization remain unknown. Here, we first explored how the disease-linked mutation influences the catabolic process of human torsinA, demonstrating that the differences in subcellular localization between both forms of torsinA lead to divergences in their degradation pathways and, whereas torsinA is normally recycled through autophagy, the proteasome is also required for the efficient clearance of the mutated form. Subsequently, we determined that the abnormal disulfide bond-dependent oligomerization of mutant torsinA is not a result of its redistribution to the nuclear envelope, but a direct consequence of the mutation. Finally, we established that the presence of disulfide links in mutant torsinA oligomers interfere with their degradation by the proteasome, thus relying on autophagy as the main pathway for clearance. In conclusion, the abnormal subcellular localization and oligomerization of DYT1-linked torsinA influences its catabolic process, opening the door to the modulation of the wild type:mutant torsinA ratio through pharmacological manipulation of protein degradation pathways.
Assuntos
Regulação da Expressão Gênica/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação/genética , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular , Chlorocebus aethiops , Inibidores de Cisteína Proteinase/farmacologia , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Chaperonas Moleculares/efeitos dos fármacos , Oligopeptídeos/farmacologia , Fatores de Tempo , Transfecção/métodosRESUMO
BACKGROUND: Although hydrogen peroxide may play an important role in the development of cancer, it can be an efficient inducer of apoptosis in cancer cells; the exact mechanism by which this action occurs is not completely understood in oral cancer cells. METHOD: In this study, the mechanisms by which H(2)O(2) inhibited growth and induced apoptosis were differentially investigated using HPV-immortalized human oral keratinocytes (IHOK) and oral cancer cells (HN4). RESULTS: H(2)O(2) treatment sensitively and dose-dependently induced growth inhibition and typical apoptosis in IHOK and HN4 cells, as demonstrated by a decreased level of cell viability, an increased population of cells in the sub-G(0)/G(1) phase, ladder formation of the genomic DNA, chromatin condensation and accumulation of Annexin V(+)/PI(+) cells. Furthermore, the expression of Bax, p53 and p21(WAF1/CIP1) increased, whereas the expression of Bcl-2 decreased in immortalized and malignant keratinocytes that were treated with H(2)O(2). In addition, cytochrome-c from the mitochondria was observed in H(2)O(2)-treated IHOK and oral cancer cells, and this was accompanied by the activation of caspase-3 and -9. Additionally, H(2)O(2) treatment induced upregulation of CHOP, GRP78 and several representative endoplasmic reticulum (ER) stress-responsive proteins, including heme oxygenase-1. CONCLUSION: Overall, these results suggest that H(2)O(2) triggers apoptosis via the mitochondrial and ER stress pathway in IHOK and HN4 cells, and that increasing the cellular levels of H(2)O(2) sufficiently may lead to selective killing of oral cancer cells and therefore be therapeutically useful.
Assuntos
Apoptose/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Queratinócitos/efeitos dos fármacos , Mucosa Bucal/efeitos dos fármacos , Neoplasias Bucais/patologia , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Caspase 3/efeitos dos fármacos , Caspase 9/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/efeitos dos fármacos , Citocromos c/efeitos dos fármacos , DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/efeitos dos fármacos , Heme Oxigenase-1/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/administração & dosagem , Queratinócitos/patologia , Mitocôndrias/efeitos dos fármacos , Chaperonas Moleculares/efeitos dos fármacos , Mucosa Bucal/patologia , Oxidantes/administração & dosagem , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Fator de Transcrição CHOP/efeitos dos fármacos , Proteína Supressora de Tumor p53/efeitos dos fármacos , Proteína X Associada a bcl-2/efeitos dos fármacosRESUMO
Environmental and occupational exposure to heavy metals such as cadmium, mercury and lead results in severe health hazards including prenatal and developmental defects. The deleterious effects of heavy metal ions have hitherto been attributed to their interactions with specific, particularly susceptible native proteins. Here, we report an as yet undescribed mode of heavy metal toxicity. Cd2+, Hg2+ and Pb2+ proved to inhibit very efficiently the spontaneous refolding of chemically denatured proteins by forming high-affinity multidentate complexes with thiol and other functional groups (IC(50) in the nanomolar range). With similar efficacy, the heavy metal ions inhibited the chaperone-assisted refolding of chemically denatured and heat-denatured proteins. Thus, the toxic effects of heavy metal ions may result as well from their interaction with the more readily accessible functional groups of proteins in nascent and other non-native form. The toxic scope of heavy metals seems to be substantially larger than assumed so far.
Assuntos
Cádmio/toxicidade , Chumbo/toxicidade , Mercúrio/toxicidade , Metais Pesados/toxicidade , Dobramento de Proteína , Adenosina Trifosfatases/efeitos dos fármacos , Cátions Bivalentes/farmacologia , Luciferases/química , Luciferases/efeitos dos fármacos , Chaperonas Moleculares/efeitos dos fármacos , Renaturação Proteica/efeitos dos fármacosRESUMO
Galectin (Gal) 1 is a hypoxia-regulated proangiogenic factor that also directly participates in glioblastoma cell migration. To determine how Gal-1 exerts its proangiogenic effects, we investigated Gal-1 signaling in the human Hs683 glioblastoma cell line. Galectin 1 signals through the endoplasmic reticulum transmembrane kinase/ribonuclease inositol-requiring 1alpha, which regulates the expression of oxygen-regulated protein 150. Oxygen-regulated protein 150 controls vascular endothelial growth factor maturation. Galectin 1 also modulates the expression of 7 other hypoxia-related genes (i.e. CTGF, ATF3, PPP1R15A, HSPA5, TRA1, and CYR61) that are implicated in angiogenesis. Decreasing Gal-1 expression in Hs683 orthotopic xenografts in mouse brains by siRNA administration impaired endoplasmic reticulum stress and enhanced the therapeutic benefits of the proautophagic drug temozolomide. These results suggest that decreasing Gal-1 expression (e.g. through brain delivery of nonviral infusions of anti-Gal-1 siRNA in patients) can represent an additional therapeutic strategy for glioblastoma.
Assuntos
Neoplasias Encefálicas/genética , Retículo Endoplasmático/genética , Galectina 1/genética , Glioblastoma/genética , Neovascularização Patológica/genética , Estresse Oxidativo/genética , Interferência de RNA/fisiologia , Animais , Antineoplásicos Alquilantes/farmacologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/terapia , Linhagem Celular Tumoral , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Endorribonucleases/efeitos dos fármacos , Endorribonucleases/genética , Endorribonucleases/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Inativação Gênica/fisiologia , Terapia Genética/métodos , Glioblastoma/metabolismo , Glioblastoma/terapia , Proteínas de Choque Térmico HSP40/efeitos dos fármacos , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70 , Humanos , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Chaperonas Moleculares/efeitos dos fármacos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Neovascularização Patológica/metabolismo , Neovascularização Patológica/terapia , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/efeitos dos fármacos , Proteínas/genética , Proteínas/metabolismo , Interferência de RNA/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/uso terapêutico , Transdução de Sinais/genética , Temozolomida , Transplante HeterólogoRESUMO
In Parkinson's disease, oxidative stress is implicated in protein misfolding and aggregation, which may activate the unfolded protein response by the endoplasmic reticulum (ER). Dopamine (DA) can initiate oxidative stress via H(2)O(2) formation by DA metabolism and by oxidation into DA quinone. We have previously shown that DA quinone induces oxidative protein modification, mitochondrial dysfunction in vitro, and dopaminergic cell toxicity in vivo and in vitro. In this study, we used cysteine- and lysine-reactive fluorescent dyes with 2D difference in-gel electrophoresis, mass spectrometry, and peptide mass fingerprint analysis to identify proteins in PC12 cell mitochondrial-enriched fractions that were altered in abundance following DA exposure (150 muM, 16 h). Quantitative changes in proteins labeled with fluorescent dyes indicated increases in a subset of proteins after DA exposure: calreticulin, ERp29, ERp99, Grp58, Grp78, Grp94 and Orp150 (149-260%), and decreased levels of aldolase A (39-42%). Changes in levels of several proteins detected by 2D difference in-gel electrophoresis were confirmed by western blot. Using this unbiased proteomics approach, our findings demonstrated that in PC12 cells, DA exposure leads to a cellular response indicative of ER stress prior to the onset of cell death, providing a potential link between DA and the unfolded protein response in the pathogenesis of Parkinson's disease.
Assuntos
Dopamina/metabolismo , Retículo Endoplasmático/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Proteínas de Choque Térmico/metabolismo , Neurônios/metabolismo , Estresse Oxidativo/fisiologia , Animais , Calreticulina/efeitos dos fármacos , Calreticulina/metabolismo , Dopamina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Eletroforese em Gel Bidimensional , Retículo Endoplasmático/efeitos dos fármacos , Corantes Fluorescentes , Frutose-Bifosfato Aldolase/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/efeitos dos fármacos , Espectrometria de Massas , Glicoproteínas de Membrana/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/efeitos dos fármacos , Chaperonas Moleculares/metabolismo , Neurônios/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Células PC12 , Doença de Parkinson/metabolismo , Doença de Parkinson/fisiopatologia , Isomerases de Dissulfetos de Proteínas/efeitos dos fármacos , Isomerases de Dissulfetos de Proteínas/metabolismo , Dobramento de Proteína , Proteínas/efeitos dos fármacos , Proteínas/metabolismo , Ratos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
The Hsp70 molecular chaperones are ATPases that play critical roles in the pathogenesis of many human diseases, including breast cancer. Hsp70 ATP hydrolysis is relatively weak but is stimulated by J domain-containing proteins. We identified pyrimidinone-peptoid hybrid molecules that inhibit cell proliferation with greater potency than previously described Hsp70 modulators. In many cases, anti-proliferative activity correlated with inhibition of J domain stimulation of Hsp70.