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1.
Biochimie ; 190: 91-110, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34298080

RESUMO

Deregulation of apoptosis is associated with various pathologies, such as neurodegenerative disorders at one end of the spectrum and cancer at the other end. Generally speaking, differentiated cells like cardiomyocytes, skeletal myocytes and neurons exhibit low levels of Apaf-1 (Apoptotic protease activating factor 1) protein suggesting that down-regulation of Apaf-1 is an important event contributing to the resistance of these cells to apoptosis. Nonetheless, upregulation of Apaf-1 has not emerged as a common phenomenon in pathologies associated with enhanced neuronal cell death, i.e., neurodegenerative diseases. In cancer, on the other hand, Apaf-1 downregulation is a common phenomenon, which occurs through various mechanisms including mRNA hyper-methylation, gene methylation, Apaf-1 localization in lipid rafts, inhibition by microRNAs, phosphorylation, and interaction with specific inhibitors. Due to the diversity of these mechanisms and involvement of other factors, defining the exact contribution of Apaf-1 to the development of cancer in general and neurodegenerative disorders, in particular, is complicated. The current review is an attempt to provide a comprehensive image of Apaf-1's contribution to the pathologies observed in cancer and neurodegenerative diseases with the emphasis on the therapeutic aspects of Apaf-1 as an important target in these pathologies.


Assuntos
Fator Apoptótico 1 Ativador de Proteases/metabolismo , Neoplasias/etiologia , Doenças Neurodegenerativas/etiologia , Animais , Apoptossomas/efeitos dos fármacos , Fator Apoptótico 1 Ativador de Proteases/agonistas , Fator Apoptótico 1 Ativador de Proteases/antagonistas & inibidores , Fator Apoptótico 1 Ativador de Proteases/genética , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo
3.
Oxid Med Cell Longev ; 2017: 9747296, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29279737

RESUMO

(2S,3S,4S,5R,6R)-6-(4-((4-guanidinobutoxy)carbonyl)-2,6-dihydroxyphenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (ZYZ-488) was discovered as a novel inhibitor of apoptotic protease activating factor-1 (Apaf-1). In present work, a surface plasmon resonance (SPR) assay confirms the direct binding between ZYZ-488 and Apaf-1 and this interaction was found to be able to block the recruitment of procaspase-9 by Apaf-1. This study also shows that the treatment of MI (myocardial infarction) mice with this novel Apaf-1 inhibitor remarkably reduces the infarct size, improves cardiac functions, and attenuates the histopathology changes caused by MI. Meanwhile, here it is shown that ZYZ-488 decreases myocardial enzyme release, inhibits cardiomyocyte apoptosis, and suppresses the activation of the downstream cascade of caspases. Moreover, in silico prediction validated the drug-like properties of ZYZ-488. In conclusion, our findings present the first piece of evidence indicating the interaction between Apaf-1 and procaspase-9 as a novel therapeutic target in myocardial infarction and suggesting ZYZ-488 as a promising therapeutic option for myocardial infarction disease.


Assuntos
Fator Apoptótico 1 Ativador de Proteases/antagonistas & inibidores , Cardiotônicos/uso terapêutico , Caspase 9/metabolismo , Guanidinas/uso terapêutico , Infarto do Miocárdio/patologia , Piranos/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Aspartato Aminotransferases/metabolismo , Cardiotônicos/química , Cardiotônicos/farmacologia , Linhagem Celular , Creatina Quinase/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Ecocardiografia , Guanidinas/química , Guanidinas/farmacologia , L-Lactato Desidrogenase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/prevenção & controle , Infarto do Miocárdio/veterinária , Miocárdio/enzimologia , Ligação Proteica , Precursores de Proteínas/metabolismo , Piranos/química , Piranos/farmacologia , Ratos , Ressonância de Plasmônio de Superfície
4.
Biomed Pharmacother ; 96: 153-164, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28982084

RESUMO

Increasing studies have indicated that the dysregulated microRNAs (miRNAs) are associated with tumorigenesis, development and even the poor prognosis of a variety of tumors, including the non-small-cell lung cancer (NSCLC). Here in our study, we found that miRNA-484 was expressed highly in NSCLC clinical tumor samples in comparison to the matched adjacent tissues. In addition, high and low expression of miRNA-484 was observed in NSCLC cell lines and lung normal cells, respectively. Furthermore, the capability of migration and proliferation changed accompanied with the altered expression of miR-484 in NSCLC. Apoptotic protease activating factor-1 (APAF-1), frequently down-regulated in a number of types of cancer, was found to be reduced in NSCLC tissue samples or NSCLC cell lines along with high expression of miR-484, which were inversely expressed in Apaf-1 over-expressed tissues or cells. Moreover, miR-484 triggered the migration and proliferation, and simultaneously reduced the cleavage of poly (ADP-ribose) polymerase-2 (PARP-2) and Caspase-3 of A549 cells, which could be suppressed by the improvement of Apaf-1. And the inhibition of Apaf-1 could reverse the function caused by miR-484 in A549 cells, suggesting that Apaf-1 was targeted by miR-484 directly and it could be acted as a potential therapeutic target against NSCLC. In conclusion, the reductive Apaf-1 regulated by miR-484 accelerated the NSCLC cell progression associated with the inhibition of apoptosis via down-regulating Caspase-3 and PARP cleavage.


Assuntos
Apoptose/fisiologia , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Progressão da Doença , Neoplasias Pulmonares/metabolismo , MicroRNAs/biossíntese , Células A549 , Animais , Apoptose/efeitos dos fármacos , Fator Apoptótico 1 Ativador de Proteases/antagonistas & inibidores , Carcinoma Pulmonar de Células não Pequenas/genética , Humanos , Neoplasias Pulmonares/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/administração & dosagem , MicroRNAs/genética , Ratos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
5.
Sci Rep ; 6: 29820, 2016 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-27443636

RESUMO

Apaf-1 is a central component in the apoptosis regulatory network for the treatment of apoptosis related diseases. Excessive Apaf-1 activity induced by myocardial ischemia causes cell injury. No drug targeted to Apaf-1 for treating myocardial ischemia has been reported to the best of our knowledge. In the present work, we synthesized a novel compound, ZYZ-488, which exhibited significant cardioprotective property in significantly increasing the viability of hypoxia-induced H9c2 cardiomyocytes and reducing CK and LDH leakage. Further study suggested the protective activity of ZYZ-488 dependent on its anti-apoptosis effect. This anti-apoptotic effect is most probably related to its disturbing the interaction between Apaf-1 and procaspase-9 as the target fishing and molecular docking indicated. The suppression on the activation of procaspase-9 and procaspase-3 with ZYZ-488 strongly suggested that compound ZYZ-488 could be a novel inhibitor of Apaf-1. In conclusion, ZYZ-488 as a novel small molecule competitive inhibitor of Apaf-1, with the great potential for treating cardiac ischemia.


Assuntos
Apoptose/efeitos dos fármacos , Fator Apoptótico 1 Ativador de Proteases/antagonistas & inibidores , Cardiotônicos , Isquemia Miocárdica/tratamento farmacológico , Miócitos Cardíacos/metabolismo , Animais , Fator Apoptótico 1 Ativador de Proteases/química , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Cardiotônicos/síntese química , Cardiotônicos/química , Cardiotônicos/farmacologia , Caspase 3/química , Caspase 3/metabolismo , Caspase 9/química , Caspase 9/metabolismo , Linhagem Celular , Simulação de Acoplamento Molecular , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Miócitos Cardíacos/patologia , Ratos
6.
PLoS One ; 9(10): e110979, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25330150

RESUMO

BACKGROUND: Excessive apoptosis induces unwanted cell death and promotes pathological conditions. Drug discovery efforts aimed at decreasing apoptotic damage initially targeted the inhibition of effector caspases. Although such inhibitors were effective, safety problems led to slow pharmacological development. Therefore, apoptosis inhibition is still considered an unmet medical need. METHODOLOGY AND PRINCIPAL FINDINGS: The interaction between Apaf-1 and the inhibitors was confirmed by NMR. Target specificity was evaluated in cellular models by siRNa based approaches. Cell recovery was confirmed by MTT, clonogenicity and flow cytometry assays. The efficiency of the compounds as antiapoptotic agents was tested in cellular and in vivo models of protection upon cisplatin induced ototoxicity in a zebrafish model and from hypoxia and reperfusion kidney damage in a rat model of hot ischemia. CONCLUSIONS: Apaf-1 inhibitors decreased Cytc release and apoptosome-mediated activation of procaspase-9 preventing cell and tissue damage in ex vivo experiments and in vivo animal models of apoptotic damage. Our results provide evidence that Apaf-1 pharmacological inhibition has therapeutic potential for the treatment of apoptosis-related diseases.


Assuntos
Antineoplásicos/efeitos adversos , Fator Apoptótico 1 Ativador de Proteases/antagonistas & inibidores , Cisplatino/efeitos adversos , Perda Auditiva , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Nefropatias/metabolismo , Traumatismo por Reperfusão/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Peixe-Zebra/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Morte Celular/efeitos dos fármacos , Cisplatino/farmacologia , Modelos Animais de Doenças , Células HeLa , Perda Auditiva/induzido quimicamente , Perda Auditiva/metabolismo , Perda Auditiva/patologia , Compostos Heterocíclicos de 4 ou mais Anéis/química , Humanos , Rim/metabolismo , Rim/patologia , Nefropatias/patologia , Masculino , Camundongos , Ratos , Traumatismo por Reperfusão/patologia , Proteínas de Peixe-Zebra/metabolismo
7.
Neuroscience ; 262: 83-91, 2014 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-24412373

RESUMO

Polyglutamine expansions in some proteins associated with neurodegenerative diseases, such as Huntington's disease or several ataxias, lead to insoluble aggregates in the cell. These aggregates accumulate through a mechanism that is not yet fully understood, but it activates cell death pathways and contributes to kill the cell. Here, we show that apoptotic protease activating factor 1 (Apaf1) down-regulation, or treatment with pharmacological Apaf1 inhibitor SVT016426, decreases both polyglutamine-induced aggregation and polyglutamine-induced apoptotic cell death in different cellular models. We demonstrate that Apaf1 binds to both Htt and to heat shock protein chaperone Hsp70, and that this interaction is altered in the presence of the pharmacological inhibitor of Apaf1. Based on our findings, we hypothesize that Apaf1 enhances polyglutamine aggregation by reducing the cellular protein levels of available functional Hsp70.


Assuntos
Apoptose/fisiologia , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Fator Apoptótico 1 Ativador de Proteases/antagonistas & inibidores , Fator Apoptótico 1 Ativador de Proteases/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/fisiologia , Fibroblastos , Células HeLa , Compostos Heterocíclicos/farmacologia , Humanos , Proteína Huntingtina , Doença de Huntington/tratamento farmacológico , Doença de Huntington/genética , Doença de Huntington/fisiopatologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo
8.
PLoS One ; 8(2): e56881, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23437261

RESUMO

BACKGROUND: Owing to their important function in regulating cell death, pharmacological inhibition of Bcl-2 proteins by dubbed BH3-mimetics is a promising strategy for apoptosis induction or sensitization to chemotherapy. However, the role of Apaf-1, the main protein constituent of the apoptosome, in the process has yet not been analyzed. Furthermore as new chemotherapeutics develop, the possible chemotherapy-induced toxicity to rapidly dividing normal cells, especially sensitive differentiated cells, has to be considered. Such undesirable effects would probably be ameliorated by selectively and locally inhibiting apoptosis in defined sensitive cells. METHODOLOGY AND PRINCIPAL FINDINGS: Mouse embryonic fibroblasts (MEFS) from Apaf-1 knock out mouse (MEFS KO Apaf-1) and Bax/Bak double KO (MEFS KO Bax/Bak), MEFS from wild-type mouse (MEFS wt) and human cervix adenocarcinoma (HeLa) cells were used to comparatively investigate the signaling cell death-induced pathways of BH3-mimetics, like ABT737 and GX15-070, with DNA damage-inducing agent cisplatin (cis-diammineplatinum(II) dichloride, CDDP). The study was performed in the absence or presence of apoptosis inhibitors namely, caspase inhibitors or apoptosome inhibitors. BH3-mimetic ABT737 required of Apaf-1 to exert its apoptosis-inducing effect. In contrast, BH3-mimetic GX15-070 and DNA damage-inducing CDDP induced cell death in the absence of both Bax/Bak and Apaf-1. GX15-070 induced autophagy-based cell death in all the cell lines analyzed. MEFS wt cells were protected from the cytotoxic effects of ABT737 and CDDP by chemical inhibition of the apoptosome through QM31, but not by using general caspase inhibitors. CONCLUSIONS: BH3-mimetic ABT737 not only requires Bax/Bak to exert its apoptosis-inducing effect, but also Apaf-1, while GX15-070 and CDDP induce different modalities of cell death in the absence of Bax/Bak or Apaf-1. Inclusion of specific Apaf-1 inhibitors in topical and well-localized administrations, but not in systemic ones, to avoid interferences with chemotherapeutics would be of interest to prevent chemotherapeutic-induced unwanted cell death which could improve cancer patient care.


Assuntos
Apoptose/efeitos dos fármacos , Cisplatino/farmacologia , Mimetismo Molecular , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Pirróis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Fator Apoptótico 1 Ativador de Proteases/antagonistas & inibidores , Fator Apoptótico 1 Ativador de Proteases/genética , Caspase 3/metabolismo , Fibroblastos , Células HeLa , Humanos , Indóis , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
9.
Bioorg Med Chem Lett ; 22(23): 7097-9, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-23079529

RESUMO

Apoptosis is the process of programmed cell death and plays a fundamental role in several human diseases. We have previously reported the synthesis of the perhydro-1,4-diazepine-2,5-dione and 1,4-piperazine-2,5-dione derivatives as racemic mixtures. Compounds 1 and 2 showed a potent in vitro and in cellular extracts antiapoptotic activity. In view that the chiral discrimination has been an issue in the development and use of pharmaceutical drugs, the present contribution reports the synthesis of enantiopure peptidomimetics 1 and 2. The biological evaluation of these enantiomers as apoptosis inhibitors is also reported.


Assuntos
Azepinas/química , Piperazinas/química , Apoptose/efeitos dos fármacos , Apoptossomas/antagonistas & inibidores , Apoptossomas/metabolismo , Fator Apoptótico 1 Ativador de Proteases/antagonistas & inibidores , Fator Apoptótico 1 Ativador de Proteases/genética , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Azepinas/síntese química , Azepinas/farmacologia , Células HEK293 , Humanos , Peptidomiméticos , Piperazina , Piperazinas/síntese química , Piperazinas/farmacologia , Ligação Proteica , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estereoisomerismo
10.
Cell Cycle ; 11(11): 2149-58, 2012 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-22592527

RESUMO

Reversine is a small synthetic molecule that inhibits multiple mitotic kinases, including MPS1 as well as Aurora kinase A and B (AURKA and AURKB). Here, we investigated the effects of reversine on p53-deficient vs p53-proficient cancer cells. We found that low doses (~0.5 µM) of reversine, which selectively inhibit MPS1 and hence impair the spindle assembly checkpoint, kill human TP53 (-/-) colon carcinoma cells less efficiently than their wild-type counterparts. In sharp contrast, high doses (~5 µM) of reversine induced hyperploidization and apoptosis to a much larger extent in TP53 (-/-) than in TP53 (+/+) cells. Such a selective cytotoxicity could not be reproduced by the knockdown of MPS1, AURKA and AURKB, neither alone nor in combination, suggesting that it involves multiple (rather than a few) molecular targets of reversine. Videomicroscopy-based cell fate profiling revealed that, in response to high-dose reversine, TP53 (-/-) (but not TP53 (+/+) ) cells undergo several consecutive rounds of abortive mitosis, resulting in the generation of hyperpolyploid cells that are prone to succumb to apoptosis upon the activation of mitotic catastrophe. In line with this notion, the depletion of anti-apoptotic proteins of the BCL-2 family sensitized TP53 (-/-) cells to the toxic effects of high-dose reversine. Moreover, the knockdown of BAX or APAF-1, as well as the chemical inhibition of caspases, limited the death of TP53 (-/-) cells in response to high-dose reversine. Altogether, these results suggest that p53-deficient cells are particularly sensitive to the simultaneous inhibition of multiple kinases, including MPS1, as it occurs in response to high-dose reversine.


Assuntos
Apoptose/efeitos dos fármacos , Morfolinas/toxicidade , Purinas/toxicidade , Proteína Supressora de Tumor p53/metabolismo , Fator Apoptótico 1 Ativador de Proteases/antagonistas & inibidores , Fator Apoptótico 1 Ativador de Proteases/genética , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Aurora Quinase A , Aurora Quinase B , Aurora Quinases , Inibidores de Caspase , Caspases/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células HCT116 , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína X Associada a bcl-2/antagonistas & inibidores , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
11.
Biochemistry ; 49(9): 1923-30, 2010 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-20121050

RESUMO

Structurally diverse small molecules, including 5-(2-benzofuryl)-4-phenyl-1,2,4-triazole-3-thiol (BETT), have been identified via high-throughput screening as activators of caspase-3 in HeLa cell extracts. However, little is known about their mechanism of action. In this study, we investigate how BETT regulates prothymosin alpha (ProT), a nuclear protein previously shown to play essential roles in apoptosis. We first showed that Apaf-1 is the direct target protein of BETT. We further demonstrated that BETT relieved ProT-mediated inhibition of apoptosome formation by blocking the interaction between Apaf-1 and ProT. Using two-dimensional (1)H-(15)N heteronuclear single-quantum correlation (HSQC) experiments, we were also able to examine the interaction between Apaf-1 and (15)N-labeled ProT alpha. Furthermore, we were able to reconstitute the entire caspase-3 activation pathway using purified ProT, Apaf-1, procaspase-9, procaspase-3, Hsp70, cytochrome c, PHAPI, CAS, and regulatory compounds to mimic stress-induced apoptosis in vitro. Together, these studies would lead to novel and specific methods for the prevention, diagnosis, and treatment of human cancer.


Assuntos
Apoptossomas/metabolismo , Fator Apoptótico 1 Ativador de Proteases/antagonistas & inibidores , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Precursores de Proteínas/antagonistas & inibidores , Precursores de Proteínas/fisiologia , Timosina/análogos & derivados , Triazóis/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptossomas/química , Apoptossomas/efeitos dos fármacos , Fator Apoptótico 1 Ativador de Proteases/química , Caspase 3/metabolismo , Inibidores de Caspase , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Células HeLa , Humanos , Mitocôndrias/química , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Ligação Proteica , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Spodoptera , Timosina/antagonistas & inibidores , Timosina/genética , Timosina/metabolismo , Timosina/fisiologia
12.
Stroke ; 41(1): 166-72, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19910549

RESUMO

BACKGROUND AND PURPOSE: Emerging evidence suggests that mitochondrial damage-mediated neuronal apoptosis is a major contributor to neonatal hypoxic-ischemic (H-I) brain injury. This study was performed to determine whether targeted inhibition of the apoptotic protease activating factor-1 (Apaf-1) signaling pathway downstream of mitochondrial damage confers neuroprotection in rodent models of neonatal H-I. METHODS: H-I was induced in 7-day-old (P7) transgenic mice overexpressing the specific Apaf-1-inhibitory protein AIP. Apaf-1 inhibition was also achieved in P7 rats by protein transduction-enhanced delivery of recombinant AIP. Pups were euthanized 6 to 24 hours after H-I for assessing caspase activation and mitochondrial release of cytochrome c and AIF, and 7 days after H-I for analyzing brain tissue damage. Sensorimotor functions were assessed in rats up to 4 weeks after H-I. RESULTS: Transgenic overexpression of AIP protected against H-I brain injury, resulting in attenuated activation of caspase-9 and caspase-3, and attenuated brain tissue loss. In neonatal H-I rats, intraperitoneal injection of TAT-AIP, but not the control proteins TAT-GFP or AIP, decreased caspase activation and brain damage and improved neurological functions. Neuroprotection conferred by AIP was also associated with significantly reduced release of cytochrome c and AIF from mitochondria. CONCLUSIONS: The Apaf-1 signaling pathway, which transmits cell death signals after mitochondrial damage to effector caspases, may be a legitimate therapeutic target for the treatment of neonatal H-I brain injury.


Assuntos
Fator Apoptótico 1 Ativador de Proteases/antagonistas & inibidores , Hipóxia-Isquemia Encefálica/prevenção & controle , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Transdução de Sinais/fisiologia , Animais , Animais Recém-Nascidos , Fator Apoptótico 1 Ativador de Proteases/fisiologia , Modelos Animais de Doenças , Hipóxia-Isquemia Encefálica/enzimologia , Hipóxia-Isquemia Encefálica/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ratos , Transdução de Sinais/genética
13.
Apoptosis ; 14(2): 182-90, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19152031

RESUMO

QM31 represents a new class of cytoprotective agents that inhibit the formation of the apoptosome, the caspase activation complex composed by Apaf-1, cytochrome c, dATP and caspase-9. Here, we analyzed the cellular effects of QM31, as compared to the prototypic caspase inhibitor Z-VAD-fmk. QM31 was as efficient as Z-VAD-fmk in suppressing caspase-3 activation, and conferred a similar cytoprotective effect. In contrast to Z-VAD-fmk, QM31 inhibited the release of cytochrome c from mitochondria, an unforeseen property that may contribute to its pronounced cytoprotective activity. Moreover, QM31 suppressed the Apaf-1-dependent intra-S-phase DNA damage checkpoint. These results suggest that QM31 can interfere with the two known functions of Apaf-1, namely apoptosome assembly/activation and intra-S-phase cell cycle arrest. Moreover, QM31 can inhibit mitochondrial outer membrane permeabilization, an effect that is independent from its action on Apaf-1.


Assuntos
Fator Apoptótico 1 Ativador de Proteases/antagonistas & inibidores , Azepinas/farmacologia , Dano ao DNA , Mitocôndrias/efeitos dos fármacos , Peptoides/farmacologia , Fase S/efeitos dos fármacos , Clorometilcetonas de Aminoácidos/farmacologia , Fator Apoptótico 1 Ativador de Proteases/deficiência , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Imunofluorescência , Humanos , Neoplasias Pulmonares/patologia , RNA Interferente Pequeno/metabolismo , Transfecção
14.
J Occup Health ; 50(2): 169-80, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18403868

RESUMO

Di (2-ethylhexyl) phthalate (DEHP) exposure is thought to lead to hepatocellular hypertrophy and hyperplasia in rodents mediated via peroxisome proliferator-activated receptor alpha (PPAR alpha). A recent study revealed that long-term exposure to relatively low-dose DEHP (0.05%) caused liver tumors including hepatocellular carcinomas, hepatocellular adenomas, and chologiocellular carcinomas at a higher incidence in Ppar alpha-null mice (25.8%) than in wild-type mice (10.0%). Using tissues with hepatocellular adenoma, microarray (Affymetrix MOE430A) as well as, in part, real-time quantitative PCR analysis was conducted to elucidate the mechanisms of the adenoma formation resulting from DEHP exposure in both genotyped mice. The microarray profiles showed that the up- or down-regulated genes were quite different between hepatocellular adenoma tissues of wild-type and Ppar alpha-null mice exposed to DEHP. The gene expressions of apoptotic peptidase activating factor 1 (Apaf1) and DNA-damage-inducible 45 alpha (Gadd45a) were increased in the hepatocellular adenoma tissues of wild-type mice exposed to DEHP, whereas they were unchanged in corresponding tissues of Ppar alpha-null mice. On the other hand, the expressions of cyclin B2 and myeloid cell leukemia sequence 1 were increased only in the hepatocellular adenoma tissues of Ppar alpha-null mice. Taken together, DEHP may induce hepatocellular adenomas, in part, via suppression of G2/M arrest regulated by Gadd45a and caspase 3-dependent apoptosis in Ppar alpha-null mice, but these genes may not be involved in tumorigenesis in the wild-type mice. In contrast, the expression level of Met was notably increased in the liver adenoma tissue of wild-type mice, which may suggest the involvement of Met in DEHP-induced tumorigenesis in wild-type mice.


Assuntos
Dietilexilftalato/toxicidade , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/genética , Fígado/efeitos dos fármacos , PPAR alfa/deficiência , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Fator Apoptótico 1 Ativador de Proteases/antagonistas & inibidores , Fator Apoptótico 1 Ativador de Proteases/biossíntese , Caspase 3/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/biossíntese , Divisão Celular/genética , Dietilexilftalato/administração & dosagem , Fase G2/genética , Perfilação da Expressão Gênica , Hepatócitos/efeitos dos fármacos , Hiperplasia , Fígado/patologia , Neoplasias Hepáticas Experimentais/patologia , Camundongos , Camundongos Knockout , Análise em Microsséries , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/biossíntese , PPAR alfa/genética , PPAR alfa/metabolismo , Plastificantes/toxicidade , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , RNA Mensageiro/genética
15.
Mol Cell ; 30(2): 239-47, 2008 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-18439902

RESUMO

During apoptosis, cytochrome c is released from mitochondria to the cytosol, where it binds Apaf-1. The Apaf-1/cytochrome c complex then oligomerizes either into heptameric caspase-9-activating apoptosome, which subsequently activates caspase-3 and caspase-7, or bigger inactive aggregates, depending on the availability of nucleotide dATP/ATP. A tumor suppressor protein, PHAPI, enhances caspase-9 activation by promoting apoptosome formation through an unknown mechanism. We report here the identification of cellular apoptosis susceptibility protein (CAS) and heat shock protein 70 (Hsp70) as mediators of PHAPI activity. PHAPI, CAS, and Hsp70 function together to accelerate nucleotide exchange on Apaf-1 and prevent inactive Apaf-1/cytochrome c aggregation. CAS expression is induced by multiple apoptotic stimuli including UV irradiation. Knockdown of CAS by RNA interference (RNAi) in cells attenuates apoptosis induced by UV light and causes endogenous Apaf-1 to form aggregates. These studies indicated that PHAPI, CAS, and Hsp70 play an important regulatory role during apoptosis.


Assuntos
Apoptose , Apoptossomas/metabolismo , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Proteína de Suscetibilidade a Apoptose Celular/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fator Apoptótico 1 Ativador de Proteases/antagonistas & inibidores , Caspase 9/metabolismo , Inibidores de Caspase , Proteína de Suscetibilidade a Apoptose Celular/antagonistas & inibidores , Proteína de Suscetibilidade a Apoptose Celular/genética , Ativação Enzimática , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Nucleares , Nucleotídeos/metabolismo , Interferência de RNA , Proteínas de Ligação a RNA
16.
J Med Chem ; 51(3): 521-9, 2008 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-18197610

RESUMO

The programmed cell death or apoptosis plays both physiological and pathological roles in biology. Anomalous activation of apoptosis has been associated with malignancies. The intrinsic mitochondrial pathway of apoptosis activation occurs through a multiprotein complex named the apoptosome. We have discovered molecules that bind to a central protein component of the apoptosome, Apaf-1, and inhibits its activity. These new first-in-class apoptosome inhibitors have been further improved by modifications directed to enhance their cellular penetration to yield compounds that decrease cell death, both in cellular models of apoptosis and in neonatal rat cardiomyocytes under hypoxic conditions.


Assuntos
Apoptose/efeitos dos fármacos , Apoptossomas/antagonistas & inibidores , Fator Apoptótico 1 Ativador de Proteases/antagonistas & inibidores , Peptoides/síntese química , Animais , Animais Recém-Nascidos , Apoptossomas/metabolismo , Proteínas de Transporte/química , Hipóxia Celular , Peptídeos Penetradores de Células , Células Cultivadas , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Conformação Molecular , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Fragmentos de Peptídeos/química , Peptoides/química , Peptoides/farmacologia , Ácido Poliglutâmico/química , Ligação Proteica , Ratos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química
17.
BMC Dev Biol ; 7: 134, 2007 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-18053235

RESUMO

BACKGROUND: The middle ear of mammals is composed of three endochondrial ossicles, the stapes, incus and malleus. Joints link the malleus to the incus and the incus to the stapes. In the mouse the first arch derived malleus and incus are formed from a single Sox9 and Type II collagen expressing condensation that later subdivides to give rise to two separate ossicles. In contrast the stapes forms from a separate condensation derived from the second branchial arch. Fusion of the malleus and incus is observed in a number of human syndromes and results in conductive hearing loss. Understanding how this joint forms during normal development is thus an important step in furthering our understanding of such defects. RESULTS: We show that the developing incudomalleal joint is characterised by a lack of proliferation and discrete areas of apoptosis. Apoptosis has been suggested to aid in the removal of pre-cartilaginous cells from the joint region, allowing for the physical separation of the cartilaginous elements, however, we show that joint initiation is unaffected by blocking apoptosis. There is also no evidence of cell migration out of the presumptive joint region, as observed by labelling of joint and ossicle cells in culture. Using Type II collagen lacZ reporter mice, however, it is evident that cells in the presumptive joint region remain in place and downregulate cartilage markers. CONCLUSION: The malleus and incus first appear as a single united condensation expressing early cartilage markers. The incudomalleal joint region forms by cells in the presumptive joint region switching off cartilage markers and turning on joint markers. Failure in this process may result in fusion of this joint, as observed in human syndromes such as Branchio-Oto-Renal Syndrome or Treacher Collins Syndrome.


Assuntos
Apoptose , Cartilagem Articular/embriologia , Orelha Média/embriologia , Animais , Fator Apoptótico 1 Ativador de Proteases/antagonistas & inibidores , Biomarcadores , Cartilagem Articular/citologia , Caspase 3 , Caspase 9 , Inibidores de Caspase , Movimento Celular , Regulação para Baixo , Orelha Média/citologia , Embrião de Mamíferos , Marcação In Situ das Extremidades Cortadas , Articulações/citologia , Articulações/embriologia , Camundongos , Camundongos Mutantes , Técnicas de Cultura de Órgãos , Antígeno Nuclear de Célula em Proliferação/análise
18.
Acta Pharmacol Sin ; 28(8): 1097-104, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17640469

RESUMO

AIM: To investigate the effects of lithium (Li) and prostaglandin A1 (PGA1) on the expression of heat shock factor 1 (HSF-1), heat shock proteins (HSP), and apoptosis protease activating factor-1 (Apaf-1) induced by permanent focal ischemia in rats. METHODS: The rats were pretreated with a subcutaneous (sc) injection of Li for 2 d or a single intracerebral ventricle (icv) administration of PGA1 for 15 min before ischemic insult, or a combination of Li (sc, 1 mEq/kg, 2 d) and PGA1 (icv, 15 min prior to ischemic insult). Brain ischemia was induced by the permanent middle cerebral artery occlusion (pMCAO). Twenty-four hours after the occlusion, the expression of HSF-1, HSP, and Apaf-1 in the ischemic striatum were examined with Western blot analysis. RESULTS: The expression of HSF-1, heme oxygenase-1 (HO-1), HSP90alpha, and Apaf-1 were significantly increased, but the expression of HSP90beta was significantly decreased 24 h after the pMCAO. PGA1 and Li and their combination significantly enhanced the ischemia-induced elevation in the levels of HSF-1, HO-1, and HSP90alpha, and recovered HSP90beta expression, but decreased Apaf-1 levels in the ischemic striatum. CONCLUSION: The present study demonstrates that PGA1 and Li have synergistic effects on the enhancement of the expression of HSP, suggesting that the synergistic effects of PGA1 and Li in the rat model of permanent focal cerebral ischemia may be mediated by the enhancement expression of HSP expression and the downregulation of Apaf-1. Our studies suggest that combined PGA1 and Li may have potential clinical value for the treatment of stroke.


Assuntos
Isquemia Encefálica/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Choque Térmico HSP90/biossíntese , Heme Oxigenase (Desciclizante)/biossíntese , Lítio/farmacologia , Prostaglandinas A/farmacologia , Fatores de Transcrição/biossíntese , Animais , Fator Apoptótico 1 Ativador de Proteases/antagonistas & inibidores , Isquemia Encefálica/tratamento farmacológico , Modelos Animais de Doenças , Fatores de Transcrição de Choque Térmico , Masculino , Ratos , Ratos Sprague-Dawley
19.
Peptides ; 28(5): 958-68, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17408805

RESUMO

We have identified a family of peptoids that inhibits in vitro the activity of the apoptosome, a macromolecular complex that activates mitochondrial-dependent apoptosis pathways. The analysis of peptide-based cell compatible delivery systems of the most active peptoid is presented. The active peptoid was then fused to cell penetrating peptides (CPP) as penetratin (PEN-peptoid) and HIV-1 TAT (TAT-peptoid). PEN-peptoid showed greater cell viability and as a consequence better efficiency as an apoptosis inhibitor than the TAT-peptoid. The intracellular trafficking of both inhibitors was studied by flow cytometry and confocal fluorescence microscopy. Finally, the influence of the cargo (peptoid) molecules on the conformational behavior of the CPP in buffers and in membrane mimetic environments was analyzed using circular dichroism (CD) spectroscopy.


Assuntos
Apoptose/efeitos dos fármacos , Fator Apoptótico 1 Ativador de Proteases/antagonistas & inibidores , Mitocôndrias/metabolismo , Peptídeos/farmacologia , Peptoides/farmacologia , Sequência de Aminoácidos , Apoptossomas/antagonistas & inibidores , Proteínas de Transporte/química , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Peptídeos Penetradores de Células , Dicroísmo Circular , Citometria de Fluxo , Produtos do Gene tat/química , Humanos , Microscopia Confocal , Mitocôndrias/efeitos dos fármacos , Dados de Sequência Molecular , Estrutura Molecular , Peptídeos/química , Peptídeos/farmacocinética , Peptoides/química , Peptoides/farmacocinética , Conformação Proteica
20.
Cell Cycle ; 6(5): 589-94, 2007 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-17361096

RESUMO

Chronic myelogenous leukemia (CML) is a stem cell disorder that eventually progresses to a blast crisis phase (BC) characterized by distorted apoptotic pathways. The exact mechanism leading to failure in apoptotic pathways during CML progression is unclear. In view of the central role of p53 and apaf1 in the apoptotic machinery we examined six human paired chronic and BC phases samples for their expression. Real-time PCR (RQ-PCR) experiments showed an elevation of p53 mRNA in all patients during transition to BC. However, elevation of apaf1 during BC was observed in five patients only. In contrast, one patient displayed a significant 11.5-fold reduction of apaf1 expression during the transition to BC. No apaf1 promoter methylation was observed. The reduced apaf1 expression was accompanied by a trans-dominant point mutation (H179R) in one p53 allele and the loss of the other. This mutant p53, when tested using functionality assays, was unable to activate apaf1, consequently explaining the reduced expression observed in this patient. Furthermore, the same mutant failed to activate either genes involved in apoptotic or cell cycle arrest pathways, and can be considered as a complete loss of function mutation. This specific mutation was reported in several types of cancer, but was not implicated in CML. To conclude, in this study we have demonstrated mRNA elevation of p53 and apaf1 during CML blast crisis, indicating that genes and proteins involved in cellular apoptosis might be involved in disease progression/response to therapy. Moreover, the mutated p53 discovered in the patient exhibiting lowered apaf1 expression provides, in a clinical case, the first correlation between p53 and apaf1 transcription regulation in humans.


Assuntos
Fator Apoptótico 1 Ativador de Proteases/antagonistas & inibidores , Fator Apoptótico 1 Ativador de Proteases/genética , Crise Blástica/genética , Regulação Leucêmica da Expressão Gênica/fisiologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Mutação Puntual , Proteína Supressora de Tumor p53/genética , Fator Apoptótico 1 Ativador de Proteases/biossíntese , Arginina/genética , Crise Blástica/metabolismo , Histidina/genética , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo
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