RESUMO
The heat shock protein 27 (Hsp27) has emerged as a principal factor of the castration-resistant prostate cancer (CRPC) progression. Also, an antisense oligonucleotide (ASO) against Hsp27 (OGX-427 or apatorsen) has been assessed in different clinical trials. Here, we illustrate that Hsp27 highly regulates the expression of the human DEAD-box protein 5 (DDX5), and we define DDX5 as a novel therapeutic target for CRPC treatment. DDX5 overexpression is strongly correlated with aggressive tumor features, notably with CRPC. DDX5 downregulation using a specific ASO-based inhibitor that acts on DDX5 mRNAs inhibits cell proliferation in preclinical models, and it particularly restores the treatment sensitivity of CRPC. Interestingly, through the identification and analysis of DDX5 protein interaction networks, we have identified some specific functions of DDX5 in CRPC that could contribute actively to tumor progression and therapeutic resistance. We first present the interactions of DDX5 and the Ku70/80 heterodimer and the transcription factor IIH, thereby uncovering DDX5 roles in different DNA repair pathways. Collectively, our study highlights critical functions of DDX5 contributing to CRPC progression and provides preclinical proof of concept that a combination of ASO-directed DDX5 inhibition with a DNA damage-inducing therapy can serve as a highly potential novel strategy to treat CRPC.
Assuntos
Oligonucleotídeos Antissenso , Neoplasias de Próstata Resistentes à Castração , Masculino , Humanos , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/uso terapêutico , Oligonucleotídeos Antissenso/farmacologia , Neoplasias de Próstata Resistentes à Castração/terapia , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , RNA Mensageiro/uso terapêutico , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico HSP27/uso terapêutico , Linhagem Celular Tumoral , RNA Helicases DEAD-box/genéticaRESUMO
OBJECTIVE: To determine whether circulating heat shock proteins HSP27/HSPB1 and HSP90α/HSPC1 may be useful for early prediction of the occurrence of pre-eclampsia in asymptomatic women. METHODS: We have measured by ELISA the levels of HSPB1, HSPC1, and placental protein 13 (PP13) in serum samples from 44 women in the first trimester (10-12 weeks) and second trimester (17-20 weeks) of pregnancy. Western blot and immunohistochemistry for HSPB1 and HSPC1 were performed. RESULTS: HSPB1 serum levels were higher in women with pre-eclampsia than in normotensive pregnant women at the first and second trimester (P = 0.003), whereas PP13 levels decreased in women with pre-eclampsia only in the first trimester of gestation (P = 0.021). We also observed higher HSPB1 levels in patients with early-onset pre-eclampsia in the first and second trimester (P = 0.014). CONCLUSION: This pilot study points out that circulating HSPB1 levels in first and second trimester might be useful for predicting the occurrence of pre-eclampsia in asymptomatic women. Further validation studies are needed to finally establish this protein as a candidate predictive biomarker of pre-eclampsia.
Assuntos
Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico , Chaperonas Moleculares , Pré-Eclâmpsia , Biomarcadores , Feminino , Proteínas de Choque Térmico HSP27/sangue , Proteínas de Choque Térmico/sangue , Humanos , Chaperonas Moleculares/sangue , Projetos Piloto , Placenta/metabolismo , Gravidez , Primeiro Trimestre da Gravidez , Segundo Trimestre da GravidezRESUMO
Human HSP27 is a small heat shock protein that modulates the ability of cells to respond to heat shock and oxidative stress, and also functions as a chaperone independent of ATP, participating in the proteasomal degradation of proteins. The expression of HSP27 is associated with survival in mammalian cells. In cancer cells, it confers resistance to chemotherapy; in neurons, HSP27 has a positive effect on neuronal viability in models of Alzheimer's and Parkinson's diseases. To better understand the mechanism by which HSP27 expression contributes to cell survival, we expressed human HSP27 in the budding yeast Saccharomyces cerevisiae under control of different mutant TEF promoters, that conferred nine levels of graded basal expression, and showed that replicative lifespan and proteasomal activity increase as well as the resistance to oxidative and thermal stresses. The profile of these phenotypes display a dose-response effect characteristic of hormesis, an adaptive phenomenon that is observed when cells are exposed to increasing amounts of stress or toxic substances. The hormetic response correlates with changes in expression levels of HSP27 and also with its oligomeric states when correlated to survival assays. Our results indicate that fine tuning of HSP27 concentration could be used as a strategy for cancer therapy, and also for improving neuronal survival in neurodegenerative diseases.
Assuntos
Proteínas de Choque Térmico HSP27 , Hormese , Saccharomyces cerevisiae , Animais , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico , Resposta ao Choque Térmico , Humanos , Chaperonas Moleculares , Estresse Oxidativo , Saccharomyces cerevisiae/metabolismoRESUMO
Unhealthy lifestyle persistently feeds forward inflammation in metabolic organs thus imposing senescence-associated secretory phenotype (SASP), as observed in obesity and type 2 diabetes. However, SASP blocks physiological resolution of inflammation by suppressing the anti-inflammatory and anti-senescent heat shock (HS) response, i.e., the gene program centered in heat shock factor-1 (HSF1)-dependent expression heat shock proteins (HSPs). As SASP-inducing factors are not removed, leading to the perpetuation of inflammation, we argued that SIRT1-HSF1-HSP axis might also be suppressed in atherosclerosis, which could be reversible by heat treatment (HT), the most powerful HS response trigger. LDLr-/- adult mice were fed on high-fat/high-cholesterol diet from the age of 90 days until the end of study (age of 270 days). After 120 days under atherosclerotic diet, the animals were submitted to either whole-body HT (nâ¯=â¯42; 40⯰C) or sham (nâ¯=â¯59; 37⯰C) treatment (15â¯min/session), under anesthesia, once a week, for 8 weeks, being echographically and metabolically monitored. Aortic expressions of SIRT1, HSF1, HSP27, HSP72 and HSP73 were progressively depressed in atherosclerotic animals, as compared to normal (LDLr+/+; nâ¯=â¯25) healthy counterparts, which was paralleled by increased expression of NF-κB-dependent VCAM1 adhesion molecule. Conversely, HT completely reversed suppression of the above HS response proteins, while markedly inhibiting both VCAM1 expression and NF-κB DNA-binding activity. Also, HT dramatically reduced plasma levels of TG, total cholesterol, LDL-cholesterol, oxidative stress, fasting glucose and insulin resistance while rising HDL-cholesterol levels. HT also decreased body weight gain, visceral fat, cellular infiltration and aortic fatty streaks, and heart ventricular congestive hypertrophy, thereby improving aortic blood flow and myocardial performance (Tei) indices. Remarkably, heat-treated mice stopped dying after the third HT session (= 8 human years), suggesting a curative effect. Therefore, evolution of atherosclerosis is associated with suppression of the anti-inflammatory and anti-senescent SIRT1-HSF1-HSP molecular axis, which is refreshed by chronic heat treatment.
Assuntos
Aorta/metabolismo , Aterosclerose/terapia , Resposta ao Choque Térmico , Hipertermia Induzida , Animais , Aterosclerose/induzido quimicamente , Aterosclerose/genética , Aterosclerose/metabolismo , Colesterol/efeitos adversos , Colesterol/farmacologia , Gorduras na Dieta/efeitos adversos , Gorduras na Dieta/farmacologia , Regulação da Expressão Gênica , Proteínas de Choque Térmico/biossíntese , Temperatura Alta , Masculino , Camundongos , Camundongos Knockout , Receptores de LDL/genética , Receptores de LDL/metabolismo , Sirtuína 1/biossínteseRESUMO
Tibial dyschondroplasia (TD) is a skeletal disorder that occurs in the proximal metaphyses of tibiotarsus and sometimes tarsometatarsus, resulting in the development of avascularized and non-mineralized abnormal cartilage and causing significant economic loss. In this study, we aimed to show the histopathological changes and the relationship between the release of Heat-Shock Protein 27 (HSP-27) and oxidative DNA damage in broiler chickens with tibial dyschondroplasia, using histopathologic and immunohistochemical methods. Our study material consisted of totally 20 animals out of 42 days old 205 Ross 308 broiler chickens, 10 with TD lesions and 10 healthy control subjects. Tissue samples taken from animals performed necropsy was exposed to routine tissue follow-up. Macroscopically, unilateral and bilateral thickening and swelling were observed in the growth plates of tibiotarsal joints of the broiler chickens diagnosed with tibial dyscondroplasia. Histopathologic examination of the tibiotarsal joints of broiler chickens affected by TD revealed an increase in the number of immature chondrocytes, as well as deficiencies in vascularization and calcification. In the immunohistochemical study; HSP-27 and 8-OHDG release was positive in the chondrocytes located on the Proliferative Zone, Maturation Zone and Hypertrophic Zone. However, the positivity was the most profound in the PZ and MZ, while less in the HZ chondrocytes. As a result; we demonstrated by immunohistochemical methods that the increase in the HSP-27 release is parallel to the increase in 8-OHDG release in TD lesioned areas and this may be related to oxidative stress.
Assuntos
Animais , Galinhas/genética , /análise , /genética , Osteocondrodisplasias/enzimologiaRESUMO
Tibial dyschondroplasia (TD) is a skeletal disorder that occurs in the proximal metaphyses of tibiotarsus and sometimes tarsometatarsus, resulting in the development of avascularized and non-mineralized abnormal cartilage and causing significant economic loss. In this study, we aimed to show the histopathological changes and the relationship between the release of Heat-Shock Protein 27 (HSP-27) and oxidative DNA damage in broiler chickens with tibial dyschondroplasia, using histopathologic and immunohistochemical methods. Our study material consisted of totally 20 animals out of 42 days old 205 Ross 308 broiler chickens, 10 with TD lesions and 10 healthy control subjects. Tissue samples taken from animals performed necropsy was exposed to routine tissue follow-up. Macroscopically, unilateral and bilateral thickening and swelling were observed in the growth plates of tibiotarsal joints of the broiler chickens diagnosed with tibial dyscondroplasia. Histopathologic examination of the tibiotarsal joints of broiler chickens affected by TD revealed an increase in the number of immature chondrocytes, as well as deficiencies in vascularization and calcification. In the immunohistochemical study; HSP-27 and 8-OHDG release was positive in the chondrocytes located on the Proliferative Zone, Maturation Zone and Hypertrophic Zone. However, the positivity was the most profound in the PZ and MZ, while less in the HZ chondrocytes. As a result; we demonstrated by immunohistochemical methods that the increase in the HSP-27 release is parallel to the increase in 8-OHDG release in TD lesioned areas and this may be related to oxidative stress.(AU)
Assuntos
Animais , Proteínas de Choque Térmico HSP27/análise , Proteínas de Choque Térmico HSP27/genética , Galinhas/genética , Osteocondrodisplasias/enzimologiaRESUMO
Classical Hodgkin lymphoma (cHL) cells overexpress heat-shock protein 90 (HSP90), an important intracellular signaling hub regulating cell survival, which is emerging as a promising therapeutic target. Here, we report the antitumor effect of celastrol, an anti-inflammatory compound and a recognized HSP90 inhibitor, in Hodgkin and Reed-Sternberg cell lines. Two disparate responses were recorded. In KM-H2 cells, celastrol inhibited cell proliferation, induced G0/G1 arrest, and triggered apoptosis through the activation of caspase-3/7. Conversely, L428 cells exhibited resistance to the compound. A proteomic screening identified a total of 262 differentially expressed proteins in sensitive KM-H2 cells and revealed that celastrol's toxicity involved the suppression of the MAPK/ERK (extracellular signal regulated kinase/mitogen activated protein kinase) pathway. The apoptotic effects were preceded by a decrease in RAS (proto-oncogene protein Ras), p-ERK1/2 (phospho-extracellular signal-regulated Kinase-1/2), and c-Fos (proto-oncogene protein c-Fos) protein levels, as validated by immunoblot analysis. The L428 resistant cells exhibited a marked induction of HSP27 mRNA and protein after celastrol treatment. Our results provide the first evidence that celastrol has antitumor effects in cHL cells through the suppression of the MAPK/ERK pathway. Resistance to celastrol has rarely been described, and our results suggest that in cHL it may be mediated by the upregulation of HSP27. The antitumor properties of celastrol against cHL and whether the disparate responses observed in vitro have clinical correlates deserve further research.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Doença de Hodgkin/metabolismo , Células de Reed-Sternberg/metabolismo , Triterpenos/farmacologia , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Sistema de Sinalização das MAP Quinases , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Triterpenos Pentacíclicos , Proteoma , Proto-Oncogene Mas , Células de Reed-Sternberg/efeitos dos fármacos , Proteínas ras/metabolismoRESUMO
Cadmium (Cd) is a carcinogen with several well-described toxicological effects in humans, but its molecular mechanisms are still not fully understood. Overexpression of heat shock protein 27 (HSP27/HSPB1)-a multifunctional protein chaperone-has been shown to protect cells from oxidative damage and apoptosis triggered by Cd exposure. The aims of this work were to investigate the potential use of extracellular recombinant HSP27 to prevent/counteract Cd-induced cellular toxicity and to evaluate if peroxynitrite was involved in the development of Cd-induced toxicity. Here, we report that the harmful effects of Cd correlated with changes in oxidative stress markers: upregulation of reactive oxygen species, reduction in nitric oxide (NO) bioavailability, increment in lipid peroxidation, peroxynitrite (PN), and protein nitration; intracellular HSP27 was reduced. Treatments with Cd (100 µM) for 24 h or with the peroxynitrite donor, SIN-1, decreased HSP27 levels (~50%), suggesting that PN formation is responsible for the reduction of HSP27. Pre-treatments of the cells either with Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) (a pharmacological inhibitor of NO synthase) or with recombinant HSP27 (rHSP27) attenuated the disruption of the cellular metabolism induced by Cd, increasing in a 55 and 52%, respectively, the cell viability measured by CCK-8. Cd induced necrotic cell death pathways, although apoptosis was also activated; pre-treatment with L-NAME or rHSP27 mitigated cell death. Our findings show for the first time a direct relationship between Cd-induced toxicity and PN production and a role for rHSP27 as a potential therapeutic agent that may counteract Cd toxicity.
Assuntos
Cádmio/toxicidade , Proteínas de Choque Térmico HSP27/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Feminino , Corantes Fluorescentes/química , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/farmacologia , Células HeLa , Humanos , Microscopia de Fluorescência , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Ácido Peroxinitroso/análise , Ácido Peroxinitroso/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Regulação para Cima/efeitos dos fármacos , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologiaRESUMO
Objetivo. Realizar predicciones computacionales de estructura de las proteínas humanas Hsp27, αB cristalina y HspB8. Materiales y métodos. La predicción de la estructura secundaria se obtuvo mediante un consenso de los programas de predicción secundaria GOR 4, nnPred, Sspro, APSSP2, JPredict, Porter, Prof, SOPMA, HNN y Psi-Pred. Los modelos de estructura terciaria se elaboraron a partir de fragmentos homólogos de proteínas con estructura terciaria conocida que fueron obtenidos por múltiples alineamientos. Empleando la secuencia primaria se obtuvieron perfiles de antigenicidad de las proteínas nativas y fueron analizados los perfiles de hidrofobicidad, polaridad, flexibilidad, accesibilidad tanto de las proteínas nativas como de las mutadas. Resultados. Las predicciones de estructura secundaria y terciaria de las proteínas estudiadas muestran que en los tres casos, más del 65% son regiones en coil, 20-25% en hoja plegada y menos del 10% en alfa hélice. Los análisis de estructura primaria muestran que al menos uno de los perfiles estudiados, en cada mutación está alterado. Conclusiones. Los análisis comparativos de estructura sugieren que las mutaciones afectan la solubilidad de las proteínas mutadas y con ello su función como chaperonas moleculares.
Objective: To make computational predictions of the structure of the human proteins Hsp27, αB-crystalline and HspB8. Materials and methods. The prediction of the secondary structure was obtained by a consensus of the programs for secondary prediction GOR 4, nnPred, Sspro, APSSP2, JPredict, Porter, Prof, SOPMA, HNN and Psi-Pred. The models of tertiary structure were built from fragments homologous to proteins with tertiary known structure that were obtained by multiple alignments. Using the primary sequence we obtained the antigenicity profiles of native proteins and we analyzed the profiles of hydrophobicity, polarity, flexibility and accessibility of both native and mutant proteins. Results. Predictions of the secondary and tertiary structures of the studied proteins show that in the three cases, more than 65% are coil regions, 20-25% are folded sheet and less than 10% are alpha helix. Analyses of the primary structure show that at least one of the studied profiles in every mutation is altered. Conclusions. The comparative analyses of structure suggest that mutations affect the solubility of the mutated proteins and hence affect their function as molecular chaperones.
Objetivo. Realizar predições computacionais da estrutura das proteínas humanas Hsp27, αB-cristalina e HspB8. Materiais e métodos. A predição da estrutura secundária foi obtida através de um consenso dos programas de predição secundária GOR 4, nnPred, Sspro, APSSP2, JPredict, Porter, Prof, SOPMA, HNN e Psi-Pred. Os modelos de estrutura terciária foram desenvolvidos a partir de fragmentos homólogos de proteínas com estrutura terciária conhecida que foram obtidos por alinhamentos múltiplos. Utilizando a seqüência primária foram obtidos perfis de antigenicidade das proteínas nativas e foram analisados os perfis de hidrofobicidade, polaridade, flexibilidade e acessibilidade, tanto da proteína nativa, como das mutantes. Resultados. As predições de estrutura secundária e terciária das proteínas estudadas mostram que nos três casos, mais de 65% são regiões em coil, 20-25% de folha pregueada e inferior a 10% em alfa-hélice. A análise da estrutura primária mostra que pelo menos um dos perfis estudados, em cada mutação está alterado. Conclusões. A análise comparativa da estrutura sugere que as mutações afetam a solubilidade das proteínas mutantes e, assim, sua função como chaperones moleculares.
RESUMO
Cimicifuga foetida, an Asian Cimicifuga species, has been employed as a cooling and detoxification agent in traditional Chinese medicine since ancient times. For this herb, two cycloartane triterpene glycosides isolated from the rhizomes have demonstrated cytotoxicity on rat tumor and human cancer cell lines. Since human Hsp27 is increased in various human cancers and exhibits cytoprotective activity that affects tumorigenesis and the susceptibility of tumours to cancer treatment, the purpose of this research was to study the expression of this protein in MCF-7 breast cancer cells. To accomplish this aim, MCF-7 cells were exposed to different concentrations of Cimicifuga foetida extract showing a reduction in cell number measured by the sulforhodamine assay. In addition, the expression of Hsp-27 mRNA detected by RT-PCR and Hsp-27 protein detected by immnofluorescence was present in all conditions, except when using the highest concentration of Cimicifuga foetida extract (2,000 jig /L). We conclude that Hsp 27 expression at 2,000 jig /L Cimicifuga foetida extract is diminished. This is the first report showing the Hsp-27 expression after exposure to Cimicifuga foetida extract in MCF-7 cells.