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Specific inhibition of ALK5 provides a novel method for controlling the development of cancers and fibrotic diseases. In this work, a novel series of N-(3-fluorobenzyl)-4-(1-(methyl-d3)-1H-indazol-5-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-amine (11), a potential clinical candidate, was synthesized by strategic incorporation of deuterium at potential metabolic soft spots and identified as ALK5 inhibitors. This compound has a low potential for CYP-mediated drug-drug interactions as a CYP450 inhibitor (IC50 = >10 µM) and showed potent inhibitory effects in cellular assay (IC50 = 3.5 ± 0.4 nM). The pharmacokinetic evaluation of 11 in mice demonstrated moderate clearance (29.0 mL/min/kg) and also revealed high oral bioavailability in mice (F = 67.6%).
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Proteínas Serina-Treonina Quinases , Receptores de Fatores de Crescimento Transformadores beta , Camundongos , Animais , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Aminas , Indazóis/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Relação Estrutura-Atividade , Inibidores de Proteínas Quinases/farmacologiaRESUMO
EWS (Ewing sarcoma) encodes an RNA/ssDNA binding protein that is frequently rearranged in a number of different cancers by chromosomal translocations. Physiologically, EWS has diverse and essential roles in various organ development and cellular processes. In this study, we uncovered a new role of EWS in mitochondrial homeostasis and energy metabolism. Loss of EWS leads to a significant decrease in mitochondria abundance and activity, which is caused by a rapid degradation of Peroxisome proliferator-activated receptor γ Coactivator (PGC-1α), a central regulator of mitochondria biogenesis, function, and cellular energy metabolism. EWS inactivation leads to increased ubiquitination and proteolysis of PGC-1α via proteasome pathway. Complementation of EWS in Ews-deficient cells restores PGC-1α and mitochondrial abundance. We found that expression of E3 ubiquitin ligase, FBXW7 (F-box/WD40 domain protein 7), is increased in the absence of Ews and depletion of Fbxw7 in Ews-null cells restores PGC-1α expression and mitochondrial density. Consistent with these findings, mitochondrial abundance and activity are significantly reduced in brown fat and skeletal muscles of Ews-deficient mice. Furthermore, expression of mitochondrial biogenesis, respiration and fatty acid ß-oxidation genes is significantly reduced in the liver of Ews-null mice. These results demonstrate a novel role of EWS in mitochondrial and cellular energy homeostasis by controlling PGC-1α protein stability, and further implicate altered mitochondrial and energy metabolism in cancers harboring the EWS translocation.
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Mitocôndrias/metabolismo , Proteína EWS de Ligação a RNA/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Tecido Adiposo Marrom/metabolismo , Animais , DNA Mitocondrial/metabolismo , Metabolismo Energético , Proteínas F-Box/metabolismo , Proteína 7 com Repetições F-Box-WD , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Perfilação da Expressão Gênica , Células HEK293 , Homeostase , Humanos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Músculo Esquelético/metabolismo , Oxigênio/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Conformação Proteica , Proteína EWS de Ligação a RNA/metabolismo , Ubiquitina/química , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Although p21(WAF1/CIP1) is known to be elevated during replicative senescence of human embryonic fibroblasts (HEFs), the mechanism for p21 up-regulation has not been elucidated clearly. In order to explore the mechanism, we analyzed expression of p21 mRNA and protein and luciferase activity of full-length p21 promoter. The result demonstrated that p21 up-regulation was accomplished largely at transcription level. The promoter assay using serially-deleted p21 promoter constructs revealed that p53 binding site was the most important site and Sp1 binding sites were necessary but not sufficient for transcriptional activation of p21. In addition, p53 protein was shown to interact with Sp1 protein. The interaction was increased in aged fibroblasts and was regulated by phosphorylation of p53 and Sp1. DNA binding activity of p53 was significantly elevated in aged fibroblasts but that of Sp1 was not. DNA binding activities of p53 and Sp1 were also regulated by phosphorylation. Phosphorylation of p53 at serine-15 and of Sp1 at serines appears to be involved. Taken together, the result demonstrated that p21 transcription during replicative senescence of HEFs is up-regulated by increase in DNA binding activity and interaction between p53 and Sp1 via phosphorylation.
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Senescência Celular/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Fibroblastos/metabolismo , Fator de Transcrição Sp1/metabolismo , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Sítios de Ligação , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação da Expressão Gênica , Humanos , Fosforilação , Regiões Promotoras Genéticas , Ligação Proteica , Regulação para CimaRESUMO
There is growing evidence that long-term central nervous system (CNS) inflammation exacerbates secondary deterioration of brain structures and functions and is one of the major determinants of disease outcome and progression. In acute CNS injury, brain microglia are among the first cells to respond and play a critical role in neural repair and regeneration. However, microglial activation can also impede CNS repair and amplify tissue damage, and phenotypic transformation may be responsible for this dual role. Mesenchymal stem cell (MSC)-derived exosomes (Exos) are promising therapeutic agents for the treatment of acute CNS injuries due to their immunomodulatory and regenerative properties. MSC-Exos are nanoscale membrane vesicles that are actively released by cells and are used clinically as circulating biomarkers for disease diagnosis and prognosis. MSC-Exos can be neuroprotective in several acute CNS models, including for stroke and traumatic brain injury, showing great clinical potential. This review summarized the classification of acute CNS injury disorders and discussed the prominent role of microglial activation in acute CNS inflammation and the specific role of MSC-Exos in regulating pro-inflammatory microglia in neuroinflammatory repair following acute CNS injury. Finally, this review explored the potential mechanisms and factors associated with MSC-Exos in modulating the phenotypic balance of microglia, focusing on the interplay between CNS inflammation, the brain, and injury aspects, with an emphasis on potential strategies and therapeutic interventions for improving functional recovery from early CNS inflammation caused by acute CNS injury.
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BACKGROUND: Heatstroke (HS) is a serious disease caused by central nervous system (CNS) injuries, such as delirium, convulsion, and coma. Currently, mesenchymal stem cells (MSCs) have demonstrated novel neuroprotective effects; therefore, this research explores the neuroprotective effects and mechanisms of MSCs against HS injury. METHODS: HS rat models were induced in a 40°C and 65% humidity environment until the rectal temperature reached 42°C. The verified HS injury model rats were divided into the HS and MSCs-treated groups. Each rat in the treated group was infused with 1x106 MSCs suspended in 0.3 ml physiological saline via the tail vein. The HS- or MSCs-treated rats were further divided into early-stage (3d) and late-stage (28d). HS rat models were induced by a high-temperature and high-humidity environment at a specific time, the mortality was analyzed, and an automatic biochemical analyzer measured levels of liver and kidney function indicators in the blood. The neurons' morphologic changes were observed through Nissl staining, and neurological deficit scores were performed. Moreover, the levels of inflammatory factors in brain tissue were measured using a multi-cytokine detection platform, and the expression of BDNF, phosphorylated TrkB and P38 were detected by the Western Bolt. RESULTS: MSCs injection significantly reduced mortality and alleviated liver and kidney function. Moreover, the neurological deficit and neuronic edema of the hippocampus caused by HS at 3d and 28d were significantly ameliorated by MSCs administration. Specifically, the injection of MSCs inhibited high levels of interleukin (IL)-1ß, IL-6, tumor necrosis factor-α (TNF-α), and IL-17A caused by HS but elevated the levels of IL-10 and IL-13 in the early period (3d); while in the later period (28d), MSCs significantly increased the levels of IL-10 and IL-13 continuously and inhibited the high level of IL-17A. Furthermore, MSCs injection increased the expressions of BDNF and phosphorylated TrkB (BDNF receptor), meanwhile inhibiting the expression of phosphorylated P38 (inflammatory factor) in the brains of HS rats in the early period (3d) but had no significant influence on the later period (28d). CONCLUSION: These results suggested that MSCs injection may provide therapeutic effects for HS in rats by improving liver and kidney function and reducing CNS damage. Moreover, MSCs injection inhibited the brain inflammatory response of HS rats, and the BDNF-TrkB and P38/MAPK signal pathways may be involved, providing a potential mechanism for HS therapy by MSCs administration.
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Golpe de Calor , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Fármacos Neuroprotetores , Ratos , Animais , Interleucina-10/metabolismo , Interleucina-17/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Interleucina-13/metabolismo , Encéfalo , Golpe de Calor/terapia , Golpe de Calor/metabolismo , Golpe de Calor/patologia , Células-Tronco Mesenquimais/metabolismo , Transplante de Células-Tronco Mesenquimais/métodosRESUMO
BACKGROUND: Sustained yet intractable immunosuppression is commonly observed in septic patients, resulting in aggravated clinical outcomes. However, due to the substantial heterogeneity within septic patients, precise indicators in deciphering clinical trajectories and immunological alterations for septic patients remain largely lacking. METHODS: We adopted cross-species, single-cell RNA sequencing (scRNA-seq) analysis based on two published datasets containing circulating immune cell profile of septic patients as well as immune cell atlas of murine model of sepsis. Flow cytometry, laser scanning confocal microscopy (LSCM) imaging and Western blotting were applied to identify the presence of S100A9+ monocytes at protein level. To interrogate the immunosuppressive function of this subset, splenic monocytes isolated from septic wild-type or S100a9-/- mice were co-cultured with naïve CD4+ T cells, followed by proliferative assay. Pharmacological inhibition of S100A9 was implemented using Paquinimod via oral gavage. RESULTS: ScRNA-seq analysis of human sepsis revealed substantial heterogeneity in monocyte compartments following the onset of sepsis, for which distinct monocyte subsets were enriched in disparate subclusters of septic patients. We identified a unique monocyte subset characterized by high expression of S100A family genes and low expression of human leukocyte antigen DR (HLA-DR), which were prominently enriched in septic patients and might exert immunosuppressive function. By combining single-cell transcriptomics of murine model of sepsis with in vivo experiments, we uncovered a similar subtype of monocyte significantly associated with late sepsis and immunocompromised status of septic mice, corresponding to HLA-DRlowS100Ahigh monocytes in human sepsis. Moreover, we found that S100A9+ monocytes exhibited profound immunosuppressive function on CD4+ T cell immune response and blockade of S100A9 using Paquinimod could partially reverse sepsis-induced immunosuppression. CONCLUSIONS: This study identifies HLA-DRlowS100Ahigh monocytes correlated with immunosuppressive state upon septic challenge, inhibition of which can markedly mitigate sepsis-induced immune depression, thereby providing a novel therapeutic strategy for the management of sepsis.
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Monócitos , Sepse , Humanos , Animais , Camundongos , Monócitos/química , Monócitos/metabolismo , Modelos Animais de Doenças , Antígenos HLA-DR/análise , Antígenos HLA-DR/metabolismo , Sepse/genéticaRESUMO
BACKGROUND: The aim of the study was to investigate the procalcitonin-to-cortisol ratio (P/C ratio) as a prognostic predictor among septic patients with abdominal source. METHODS: We retrospectively enrolled 132 post-surgery patients between 18 and 90 years old with sepsis of the abdominal source. On the second day of sepsis onset, cortisol, procalcitonin (PCT), Acute Physiology and Chronic Health Evaluation (APACHE) II score, Sequential Organ Failure Assessment (SOFA) score, C-response protein (CRP), and other baseline characteristics were collected. In addition, the length of ICU stay, length of mechanical ventilation (MV) days, length of shock days, and 28-day mortality were also recorded. Univariate analysis was performed to screen potential risk factors. Stratified analysis was used to identify the interaction among the risk factors. Multivariate analysis was also utilized to demonstrate the relationship between the risk factors and mortality. The receiver operator characteristic (ROC) curve analysis was conducted to evaluate the risk factors. A restricted cubic spline (RCS) demonstrated the association between survival outcome and the P/C ratio variation. RESULTS: A total of twenty-nine patients died, and 103 patients survived within 28 d. There were significant differences in cortisol, PCT, P/C ratio, interleukin (IL)-6, SOFA, and APACHE II scores between the survival and non-survival groups. No significant interaction was observed in the stratified analysis. Logistic regression analysis revealed that P/C ratio (P=0.033) was significantly related to 28-day mortality. Based on ROC curves, P/C ratio (AUC=0.919) had a higher AUC value than cortisol or PCT. RCS analysis depicted a positive relationship between survival possibility and P/C ratio decrement. CONCLUSION: P/C ratio might be a potential prognostic predictor in septic patients with abdominal sources.
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Desmoplastic Small Round Cell Tumor (DSRCT) is a rare and aggressive pediatric cancer driven by the EWSR1-WT1 fusion oncogene. Combinations of chemotherapy, radiation and surgery are not curative, and the 5-years survival rate is less than 25%. One potential explanation for refractoriness is the existence of a cancer stem cell (CSC) subpopulation able escape current treatment modalities. However, no study to-date has examined the role of CSCs in DSRCT or established in vitro culture conditions to model this subpopulation. In this study, we investigated the role of stemness markers in DSRCT survival and metastasis, finding that elevated levels of SOX2 and NANOG are associated with worse survival in sarcoma patients and are elevated in metastatic DSRCT tumors. We further develop the first in vitro DSRCT CSC model which forms tumorspheres, expresses increased levels of stemness markers (SOX2, NANOG, KLF4, and OCT4), and resists doxorubicin chemotherapy treatment. This model is an important addition to the DSRCT tool kit and will enable investigation of this critical DSRCT subpopulation. Despite lower sensitivity to chemotherapy, the DSRCT CSC model remained sensitive to knockdown of the EWSR1-WT1 fusion protein, suggesting that future therapies directed against this oncogenic driver have the potential to treat both DSRCT bulk tumor and CSCs.
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Desmoplastic Small Round Cell Tumor (DSRCT) is a rare and aggressive malignant cancer caused by a chromosomal translocation t(11;22)(p13;q12) that produces an oncogenic transcription factor, EWSR1-WT1. EWSR1-WT1 is essential for the initiation and progression of DSRCT. However, the precise mechanism by which EWSR1-WT1 drives DSRCT oncogenesis remains unresolved. Through our integrative gene expression analysis, we identified Salt Inducible Kinase 1 (SIK1) as a direct target of EWSR1-WT1. SIK1 as a member of the AMPK related kinase is involved in many biological processes. We showed that depletion of SIK1 causes inhibition of tumor cell growth, similar to the growth inhibition observed when EWSR1-WT1 is depleted. We further showed that silencing SIK1 leads to cessation of DNA replication in DSRCT cells and inhibition of tumor growth in vivo. Lastly, combined inhibition of SIK1 and CHEK1with small molecule inhibitors, YKL-05-099 and prexasertib, respectively, showed enhanced cytotoxicity in DSRCT cells compared to inhibition of either kinases alone. This work identified SIK1 as a new potential therapeutic target in DSRCT and the efficacy of SIK1 inhibition may be improved when combined with other intervention strategies.
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Since the detailed comparison of DNA repair activities among mammalian embryonic fibroblast cells with different replicative life spans has not been investigated, we tested DNA repair activities in embryonic fibroblast cells derived from mammals including human, dog, rat, and mouse. The cell viability after treatment of four DNA damage agents appeared to be decreased in the order of human embryonic fibroblasts (HEFs) > dog embryonic fibroblasts (DEFs) > rat embryonic fibroblasts (REFs) > mouse embryonic fibroblasts (MEFs) although statistical significance was lacking. The amounts of strand breaks and AP (apurinic/apyrimidinic) sites also appear to be decreased in the order of HEFs > DEFs > REFs ≥ MEFs after treatment of DNA damage agents. The DNA repair activities and rates including base excision repair (BER), nucleotide excision repair (NER) and double-strand break repair (DSBR) including non-homologous end-joining (NHEJ) decreased again in the order of HEFs > DEFs > REFs ≥ MEFs. BER and NHEJ activities in 3% O(2) also decreased in the order of HEFs > DEFs > REFs > MEFs. This order in DNA repair activity appears to be coincident with that of replicative life span of fibroblasts and that of life span of mammals. These results indicate that higher DNA repair activity is related with longer replicative life span in embryonic fibroblast cells.
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Proliferação de Células , Senescência Celular , Dano ao DNA , Reparo do DNA , Fibroblastos/metabolismo , Animais , Camptotecina/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Reparo do DNA/efeitos dos fármacos , Cães , Relação Dose-Resposta a Droga , Embrião de Mamíferos/citologia , Etoposídeo/farmacologia , Fibroblastos/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Camundongos , Camundongos Endogâmicos ICR , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Heat stroke (HS) is a serious, life-threatening disease. However, there is no scoring system for HS so far. This research is to establish a scoring system that can quantitatively assess the severity of exertional heat stroke (EHS). METHODS: Data were collected from a total of 170 exertional heat stroke (EHS) patients between 2005 and 2016 from 52 hospitals in China. Univariate statistical methods and comparison of the area under the receiver operating characteristic (ROC) curve (AUC) were used to screen exertional heat stroke score (EHSS) parameters, including but not limited body temperature (T), Glasgow Coma Scale (GCS) and others. By comparing the sizes of the AUCs of the APACHE II, SOFA and EHSS assessments, the effectiveness of EHSS in evaluating the prognosis of EHS patients was verified. RESULTS: Through screening with a series of methods, as described above, the present study determined 12 parameters - body temperature (T), GCS, pH, lactate (Lac), platelet count (PLT), prothrombin time (PT), fibrinogen (Fib), troponin I (TnI), aspartate aminotransferase (AST), total bilirubin (TBIL), creatinine (Cr) and acute gastrointestinal injury (AGI) classification - as EHSS parameters. It is a 0-47 point system designed to reflect increasing severity of heat stroke. Low (EHSS< 20) and high scores (EHSS> 35) showed 100% survival and 100% mortality, respectively. We found that AUCEHSS > AUCSOFA > AUCAPACHE II. CONCLUSION: A total of 12 parameters - T, GCS, pH, Lac, PLT, PT, Fib, TnI, AST, TBIL, Cr and gastrointestinal AGI classification - are the EHSS parameters with the best effectiveness in evaluating the prognosis of EHS patients. As EHSS score increases, the mortality rate of EHS patients gradually increases.
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Golpe de Calor/classificação , Esforço Físico/fisiologia , Índice de Gravidade de Doença , APACHE , Adulto , China , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
A new toxic protein named malanin was isolated from seeds of Malania olei fera by hydrophobic chromatography, whose cytotoxic activities against carcinoma cells were very strong. The conformational changes of malanin at various temperatures, pH, organic solvents, surfactant, denaturant and fluorescence quenching solvents were studied by fluorescence spectra. The fluorescence spectra of malanin excited at 280 nm and 295 nm showed a maximum at 340 nm. The emission spectra of malanin showed that Trp residues were located by a great degree in the hydrophobic area. Addition of SDS, CH5N3 x HSCN, acrylamide and KI led to changes in the molecular conformation of malanin, and caused the fluorescence quenching of Trp residues. The red-shifted emission band of malanin after adding CH5 N3 x HSCN showed that Trp residues were exposed in polar solvents.
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Glicoproteínas/química , Olacaceae , Proteínas de Plantas/química , Toxinas Biológicas/química , Amidas/química , Amidas/farmacologia , Glicoproteínas/metabolismo , Guanidina/farmacologia , Concentração de Íons de Hidrogênio , Proteínas de Plantas/metabolismo , Conformação Proteica/efeitos dos fármacos , Desnaturação Proteica/efeitos dos fármacos , Dodecilsulfato de Sódio/farmacologia , Soluções , Solventes/farmacologia , Espectrometria de Fluorescência , Temperatura , Tiocianatos/farmacologia , Toxinas Biológicas/metabolismoRESUMO
OBJECTIVE: To investigate the dose-effect relationship and time-effect relationship of astragalus injection on the protein expression of nitric-oxide synthase (NOS) and extracellular signal-regulated kinase (ERK) in cultured cardiac myocytes. METHODS: (1) Ventricular myocytes of Sprague-Dawley rats were isolated, cultured, and incubated with astragalus injection of the concentrations of 0, 20, 100, 200, or 400 ml/L for 8 days. Cell growth curve was drawn. Automatic biochemical analyzer was used to detect the level of creatine kinase (CK). (2) Western blotting was used 2 h after incubation to observe the protein expression of ERK and nitric oxide synthase (NOS). (3) Myocytes were incubated with astragalus injection (200 ml/L) or with astragalus and PD98059, an ERK inhibitor, for 5, 15, 30, 60, and 120 min respectively, and Western blotting was used to detect the protein expression of ERK and NOS. RESULTS: Astragalus of different concentrations showed no effect on the cell proliferation. Only the CK level of the astragalus 200 ml/L group was significantly higher than that of the control group (P < 0.05). Astragalus showed no dose-dependent effect on the ERK expression, and dose-dependently reduced the NOS expression level. 15 min after the co-incubation with astragalus 200 ml/L the ERK level of the myocytes decreased transiently, and then began to increase gradually since 15 min after the co-incubation; the NOS level began to increase since 60 min after the co-incubation (P < 0.05). After the addition of PD98059 the ERK expression significantly decreased (P < 0.05). CONCLUSION: Astragalus injection has modulation effect on ERK and NOS expression. This effect is enhanced time-dependently at appropriate Astragalus concentration, but attenuated by high dose Astragalus injection.
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Astrágalo/química , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Preparações de Plantas/farmacologia , Animais , Western Blotting , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Flavonoides/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Óxido Nítrico Sintase/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
Systemic inflammatory response syndrome (SIRS) is an important process associated with the pathogenesis of multiple organ failure resulting from heat stroke (HS). Alterations in the levels of circulating cytokines during the progression of SIRS have been well established. However, only a small number of studies have demonstrated the responses of lymphocytes during HS, and no studies have investigated immune-regulatory cells, such as regulatory T cells (Tregs). Tregs have been revealed to be important in numerous inflammation-associated diseases, and have exhibited promising therapeutic effects in both experimental and clinical trials. In the present study, the splenic Treg response in a classic HS mouse model was investigated, and the results demonstrated that total numbers of splenic Tregs were significantly decreased at 0, 24 and 72 h time intervals post-heat stress. Furthermore, the immunosuppressive capacity of splenic Tregs on cluster of differentiation (CD)4+T cell expansion was revealed to be suppressed following heat stress. In addition, HS was demonstrated to downregulate the expression levels of surface inhibitory molecules (CD39, CD73 and cytotoxic T-lymphocyte associated protein 4), as well as anti-inflammatory cytokines [interleukin (IL)-10, transforming growth factor-ß and IL-35], in Tregs. It was hypothesized that the aforementioned Treg responses may contribute to SIRS during HS. To the best of our knowledge, the present study is the first study to investigate the response of Tregs to HS, and the results demonstrated that there were significant alterations regarding to the total number, and function, of splenic Tregs, as well as the expression levels of inhibitory surface molecules and secretory cytokines. These results may highlight a novel mechanism underlying the pathogenesis of HS, as well as identify a potential therapeutic target for SIRS in patients suffering from HS.
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Previous studies have shown that SIRT2 plays a role in mitosis through deacetylating specific downstream targets. However, the upstream regulation of SIRT2 activity has been relatively unexplored. In this study, we provide evidence that NAD(P)H:quinone oxidoreductase 1 (NQO1) interacts with and activates SIRT2 in an NAD-dependent manner. Strong protein-protein interaction and co-localization of the two proteins during mitosis is required to maintain an active NQO1-SIRT2 axis which is critical for successful completion of mitosis. This is evident by the observed delay in mitotic exit in cells upon NQO1 inhibition. Mechanistically, this phenotype can be explained by the decrease in APC/C complex activity resulting from decreased SIRT2 deacetylation activity. Furthermore, we show that this newly established role of NQO1 has an impact on how cancer cells may respond to mitotic stress. In this regard, both pharmacologic and genetic NQO1 inhibition increases sensitivity to anti-mitotic drugs functioning as microtubule poisons by inducing mitotic arrest and allowing cells to accumulate cell death signals. Therefore, the significant prognostic value of NQO1 in predicting outcome of cancer patients might be explained in part due to the functional contribution of NQO1-SIRT2 axis to mitotic stress. Altogether, this novel mechanism of action further supports the pleiotropic biological effects exerted by NQO1 in addition to its antioxidant function and it might provide the basis for expanding the therapeutic potential of NQO1 inhibition towards increasing sensitivity to standard treatments.
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Antioxidantes/metabolismo , Mitose/genética , NAD(P)H Desidrogenase (Quinona)/genética , Neoplasias/genética , Sirtuína 2/genética , Morte Celular/genética , Proliferação de Células/genética , Humanos , Células MCF-7 , Microtúbulos/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Mapas de Interação de Proteínas/genética , Transdução de Sinais/genéticaRESUMO
Primary neuronal culture is a powerful tool to study neuronal development, aging, and degeneration. However, cultured neurons show signs of cell death after 2 or 3 weeks. Although the mechanism underlying this phenomenon has not been elucidated, several preventive methods have been identified. Here we show that the neuronal loss in primary cortical culture involves calpain activation and subsequent neuronal cell death. Neuronal loss during cultivation showed destruction of neurites and synapses, and a decrease in neuron numbers. mu-Calpain and m-calpain were initially activated and accumulated by increased RNA expression. This neuronal death exhibited neurodegenerative features, such as conversion of p35 to p25, which is important in the developmental process and in the pathogenesis of Alzheimer's disease. But, postnatal and aged rat cortex did not show calpain activation and prolonged processing of p35 to p25, in contrast to the long-term culture of cortical neurons. In addition, the inhibition of calpains by ALLM or ALLN blocked the conversion of p35 to p25, indicating that the calpain activity is essential for the neurodegenerative features of cell death. Taken together, this study shows that the neuronal loss in primary cortical cultures involves neurodegeneration-like cell death through the activation of calpains and the subsequent processing of p35 to p25, but not developmental apoptosis or aging. Our results suggest that the long term primary culture of cortical neurons represent a valuable model of neurodegeneration, such as Alzheimer's disease.
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Apoptose , Calpaína/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fosfotransferases/metabolismo , Animais , Calpaína/antagonistas & inibidores , Calpaína/genética , Inibidores de Caspase , Caspases/metabolismo , Forma Celular , Células Cultivadas , Ratos , Fatores de Tempo , Transcrição Gênica/genéticaRESUMO
Ewing sarcoma (ES) involves a tumor-specific chromosomal translocation that produces the EWS-FLI1 protein, which is required for the growth of ES cells both in vitro and in vivo. However, an EWS-FLI1-driven transgenic mouse model is not currently available. Here, we present data from six independent laboratories seeking an alternative approach to express EWS-FLI1 in different murine tissues. We used the Runx2, Col1a2.3, Col1a3.6, Prx1, CAG, Nse, NEFL, Dermo1, P0, Sox9 and Osterix promoters to target EWS-FLI1 or Cre expression. Additional approaches included the induction of an endogenous chromosomal translocation, in utero knock-in, and the injection of Cre-expressing adenovirus to induce EWS-FLI1 expression locally in multiple lineages. Most models resulted in embryonic lethality or developmental defects. EWS-FLI1-induced apoptosis, promoter leakiness, the lack of potential cofactors, and the difficulty of expressing EWS-FLI1 in specific sites were considered the primary reasons for the failed attempts to create a transgenic mouse model of ES.
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Modelos Animais de Doenças , Proteínas de Fusão Oncogênica/genética , Regiões Promotoras Genéticas , Proteína Proto-Oncogênica c-fli-1/genética , Proteína EWS de Ligação a RNA/genética , Sarcoma de Ewing/patologia , Adenoviridae/genética , Animais , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Técnicas de Introdução de Genes , Humanos , Camundongos , Camundongos Transgênicos , Transplante de Neoplasias , Sarcoma de Ewing/genéticaRESUMO
Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further supported by recent findings in high-resolution mass spectrometry based proteomics showing that many new proteins and sites within these proteins can be acetylated. However the identity of the enzymes regulating these proteins and sites is often unknown. Among these enzymes, sirtuins, which belong to the class III histone lysine deacetylases, have attracted great interest as enzymes regulating the acetylome under different physiological or pathophysiological conditions. Here we describe methods to link SIRT2, the cytoplasmic sirtuin, with its substrates including both in vitro and in vivo deacetylation assays. These assays can be applied in studies focused on other members of the sirtuin family to unravel the specific role of sirtuins and are necessary in order to establish the regulatory interplay of specific deacetylases with their substrates as a first step to better understand the role of protein acetylation. Furthermore, such assays can be used to distinguish functional acetylation sites on a protein from what may be non-regulatory acetylated lysines, as well as to examine the interplay between a deacetylase and its substrate in a physiological context.
Assuntos
Acetilação , Sirtuínas/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Lisina/química , Espectrometria de Massas , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas/metabolismo , ProteômicaRESUMO
BACKGROUND: Exertional heat stroke is a fatal condition and remains a health problem. This paper evaluates the publication trend regarding exertional heat stroke research between 1996 and 2015 using a bibliometric method. METHOD: Articles regarding exertional heat stroke research published between 1996 and December 2015 were searched for in the SCI-EXPANDED database of Web of Science. The search results were analyzed with regard to publication year; publication quantity regarding countries/regions, and authors; citation frequency; and journal distribution. CiteSpace (v3.6) was used for a document co-citation visualization analysis. RESULTS: In total, 289 publications on heat stroke were located. After selection, 209 original articles conducted across 28 countries/regions and published in 83 journals were included in the analysis. The USA, Isreal, and France were the most common locations for exertional heat stroke studies. The CiteSpace visualization cluster analysis showed that exertional heat stroke-related mortality and protective measures were constant concerns of research. CONCLUSIONS: Research related to exertional heat stroke has been continuous concerned. USA is still the leading country in this field.