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1.
Nat Commun ; 10(1): 2091, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31064994

RESUMO

Caspase-1 activated in inflammasomes triggers a programmed necrosis called pyroptosis, which is mediated by gasdermin D (GSDMD). However, GSDMD-deficient cells are still susceptible to caspase-1-mediated cell death. Therefore, here, we investigate the mechanism of caspase-1-initiated cell death in GSDMD-deficient cells. Inflammasome stimuli induce apoptosis accompanied by caspase-3 activation in GSDMD-deficient macrophages, which largely relies on caspase-1. Chemical dimerization of caspase-1 induces pyroptosis in GSDMD-sufficient cells, but apoptosis in GSDMD-deficient cells. Caspase-1-induced apoptosis involves the Bid-caspase-9-caspase-3 axis, which can be followed by GSDME-dependent secondary necrosis/pyroptosis. However, Bid ablation does not completely abolish the cell death, suggesting the existence of an additional mechanism. Furthermore, cortical neurons and mast cells exhibit little or low GSDMD expression and undergo apoptosis after oxygen glucose deprivation and nigericin stimulation, respectively, in a caspase-1- and Bid-dependent manner. This study clarifies the molecular mechanism and biological roles of caspase-1-induced apoptosis in GSDMD-low/null cell types.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Caspase 1/fisiologia , Inflamassomos/imunologia , Piroptose/imunologia , Receptores Estrogênicos/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Córtex Cerebral/citologia , Embrião de Mamíferos , Técnicas de Inativação de Genes , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Nigericina/farmacologia , Cultura Primária de Células , Piroptose/efeitos dos fármacos , Células RAW 264.7 , Salmonella typhimurium/imunologia
2.
J Agric Food Chem ; 67(16): 4578-4587, 2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-30933511

RESUMO

The objective of this study was to investigate the mechanism underlying lysosome-mediated apoptosis, the cross-talk between the lysosomes and mitochondria, and the effect of the pathway on bovine longissimus muscle tenderness during 7 d post-mortem aging through the observation and analysis of longissimus dorsi (LD) muscles of six crossbred cattle. Results showed that an elevated reactive oxygen species level ( P < 0.05) can damage lysosomal membrane stability ( P < 0.05) through accumulating redox-active iron of bovine muscle during post-mortem aging. In addition, the activities of cathepsins B and D increased with post-mortem aging ( P < 0.05). Moreover, cathepsin B and D activated Bid and Bax in the mitochondria ( P < 0.05). Activated Bid and Bax triggered mitochondrial membrane permeability ( P < 0.05) and further activated caspase-9 and caspase-3 ( P < 0.05), leading to apoptosis. Ultimately, the tenderness of bovine muscle was improved during post-mortem aging ( P < 0.05). Importantly, cathepsin D plays a crucial role in the lysosomal-mitochondrial apoptotic pathway and tenderness in post-mortem muscle. These findings provide new insights into the apoptotic pathway of bovine muscle during post-mortem aging.


Assuntos
Bovinos/metabolismo , Lisossomos/metabolismo , Carne/análise , Mitocôndrias/metabolismo , Músculo Esquelético/química , Animais , Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspases/genética , Caspases/metabolismo , Catepsina B/genética , Catepsina B/metabolismo , Catepsina D/genética , Catepsina D/metabolismo , Bovinos/genética , Ferro/metabolismo , Lisossomos/genética , Masculino , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo
3.
Artif Cells Nanomed Biotechnol ; 47(1): 501-511, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30810413

RESUMO

The present research seeks to investigate the process of mixing targeted gene delivery and transcriptional targeting. We have conjugated Polyethylenimine polymers (PEI) and molecules of poly (ethylene glycol). The next step was covalent attachment of anti-HER2 variables domains of camelid heavy chains antibodies (VHHs) or nanobodies (Nbs) to the distal terminals of NHS-PEG3500 in PEI-PEG nanoparticles. The whole procedure yielded PEI-PEG-Nb immunoconjugates. Having determined the properties of polyplexes, steps were taken to investigate the most efficient ratio of PEI polymers to pDNA molecules (N/P) so that the greatest rate of transfection may be obtained. This immune targeted nano biopolymer could condense the gene constructs that coded a transcriptionally targeted truncated -Bid (tBid) killer gene which was controlled by the breast cancer-specific MUC1 promoter. The favourable physicochemical properties matching both the size and zeta potential were observed in engineered polyplexes. Elevated transfection efficiency in HER2 positive cell lines using Nb-modified polyplexes were shown by the results of flow cytometry as compared against non-modified particles. 1.6 and 4.8 fold higher transfection efficiencies were observed in in vitro gene expression researches which used PEI-PEG-Nb/pGL4.50 compared to the situation when native PEI polymers were utilized in both BT-474 and SK-BR-3, respectively. A 2.22 and 3.62 fold rise in the level of tBid gene expression in BT-474 and SK-BR-3 cell lines relative to unmodified PEI treated cells was the result of transfection with PEI-PEG-Nb/pMUC1-tBid, respectively. In those HER2-positive cells which were transfected by targeted polyplexes, higher levels of cell death were observed. This fact points not only to the effective targeted delivery, but it is also indicative of transcriptional targeting efficiency of tBid killer gene when its expression is controlled by MUC1 promoter.


Assuntos
Antineoplásicos Imunológicos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Técnicas de Transferência de Genes , Terapia Genética , Polietilenoglicóis , Polietilenoimina/análogos & derivados , Receptor ErbB-2/antagonistas & inibidores , Anticorpos de Domínio Único , Transcrição Genética/efeitos dos fármacos , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/farmacologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/biossíntese , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/terapia , Feminino , Humanos , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Polietilenoimina/química , Polietilenoimina/farmacologia , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/farmacologia
5.
Biochim Biophys Acta Biomembr ; 1861(1): 268-280, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29958826

RESUMO

BCL-2-associated X (BAX) protein acts as a gatekeeper in regulating mitochondria-dependent apoptosis. Under cellular stress, BAX becomes activated and transforms into a lethal oligomer that causes mitochondrial outer membrane permeabilization (MOMP). Previous studies have identified several structural features of the membrane-associated BAX oligomer; they include the formation of the BH3-in-groove dimer, the collapse of the helical hairpin α5-α6, and the membrane insertion of α9 helix. However, it remains unclear as to the role of lipid environment in determining the conformation and the pore-forming activity of the BAX oligomers. Here we study molecular details of the membrane-associated BAX in various lipid environments using fluorescence and ESR techniques. We identify the inactive versus active forms of membrane-associated BAX, only the latter of which can induce stable and large membrane pores that are sufficient in size to pass apoptogenic factors. We reveal that the presence of CL is crucial to promoting the association between BAX dimers, hence the active oligomers. Without the presence of CL, BAX dimers assemble into an inactive oligomer that lacks the ability to form stable pores in the membrane. This study suggests an important role of CL in determining the formation of active BAX oligomers.


Assuntos
Cardiolipinas/metabolismo , Proteína X Associada a bcl-2/metabolismo , Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Difusão , Recuperação de Fluorescência Após Fotodegradação , Células HCT116 , Humanos , Lipídeos/química , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Mutagênese , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/metabolismo , Solventes/química
6.
Oncogene ; 38(1): 47-59, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30076411

RESUMO

Estrogen dependence is major driver of ER + breast cancer, which is associated with PI3K mutation. PI3K inhibition (PI3Ki) can restore dependence on ER signaling for some hormone therapy-resistant ER + breast cancers, but is ineffective in others. Here we show that short-term supplementation with estrogen strongly enhanced Pik3caH1047R-induced mammary tumorigenesis in mice that resulted exclusively in ER + tumors, demonstrating the cooperation of the hormone and the oncogene in tumor development. Similar to human ER + breast cancers that are endocrine-dependent or endocrine-independent at diagnosis, tumor lines from this model retained ER expression but were sensitive or resistant to hormonal therapies. PI3Ki did not induce cell death but did cause upregulation of the pro-apoptotic gene BIM. BH3 mimetics or PI3Ki were unable to restore hormone sensitivity in several resistant mouse and human tumor lines. Importantly however, combination of PI3Ki and BH3 mimetics had a profound, BIM-dependent cytotoxic effect in PIK3CA-mutant cancer cells while sparing normal cells. We propose that addition of BH3 mimetics offers a therapeutic strategy to markedly improve the cytotoxic activity of PI3Ki in hormonal therapy-resistant and ER-independent PIK3CA-mutant breast cancer.


Assuntos
Compostos de Anilina/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/antagonistas & inibidores , Proteína 11 Semelhante a Bcl-2/agonistas , Estradiol , Receptor alfa de Estrogênio/fisiologia , Neoplasias Mamárias Experimentais/tratamento farmacológico , Proteínas de Neoplasias/fisiologia , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Neuropeptídeos/antagonistas & inibidores , Sulfonamidas/farmacologia , Tiazóis/farmacologia , Compostos de Anilina/administração & dosagem , Animais , Antineoplásicos Hormonais/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteína 11 Semelhante a Bcl-2/biossíntese , Proteína 11 Semelhante a Bcl-2/genética , Proteína 11 Semelhante a Bcl-2/fisiologia , Linhagem Celular Tumoral , Cocarcinogênese , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Estradiol/toxicidade , Receptor alfa de Estrogênio/efeitos dos fármacos , Feminino , Fulvestranto/administração & dosagem , Fulvestranto/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Introdução de Genes , Neoplasias Mamárias Experimentais/induzido quimicamente , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Nus , Mutação de Sentido Incorreto , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Neoplasias Hormônio-Dependentes/induzido quimicamente , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/patologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/fisiologia , Sulfonamidas/administração & dosagem , Tiazóis/administração & dosagem
7.
Colloids Surf B Biointerfaces ; 173: 400-406, 2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30321797

RESUMO

Cochlear implants, the only way to recover from severe/profound hearing loss, may cause adverse effects, among which reactions to silicone materials coating implant electrodes, leading to apoptosis and necrosis of spiral ganglion cells. Our aim was to evaluate whether three polydimethylsiloxane (PDMS) compounds (hexadimethylsiloxane, octamethyltrisiloxane, decamethylcyclopentasiloxane) used in silicone rods could exert toxic effects on an in vitro neuronal cell model (PC12). Cell viability, morphology and mRNA expression levels of apoptotic markers were evaluated on PC12 cells at different PDMS dilutions up to 6 days of exposure. The results showed that at the highest concentrations tested cell viability was reduced by hexadimethylsiloxane and octamethyltrisiloxane at all times of exposure, but only from 72 h onwards by decamethylcyclopentasiloxane. The number of neurites per cell was not affected by hexadimethylsiloxane, but was significantly reduced from 24 h onwards by octamethyltrisiloxane and decamethylcyclopentasiloxane. Neurite length was reduced by hexadimethylsiloxane only at 24 h, and by octamethyltrisiloxane and decamethylcyclopentasiloxane at all exposure intervals. In controls exposed to silicone or glass rods cell viability was reduced only after 24 h, but neurite number and length was never reduced at any exposure interval. Biomolecular investigations showed that apoptotic markers did not change in any experimental condition, suggesting that PDMS are biocompatible. The reduction of cell viability and neurite number and length caused by exposure to these compounds was probably caused by a PDMS surface film formed over the cell medium, preventing air exchange, and not by the release of cytotoxic molecules.


Assuntos
Apoptose/efeitos dos fármacos , Materiais Biocompatíveis/farmacologia , Dimetilpolisiloxanos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Crescimento Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Proteína 11 Semelhante a Bcl-2/genética , Proteína 11 Semelhante a Bcl-2/metabolismo , Caspases/genética , Caspases/metabolismo , Diferenciação Celular , Sobrevivência Celular/efeitos dos fármacos , Neurônios/citologia , Neurônios/metabolismo , Células PC12 , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismo
8.
Cell Tissue Res ; 375(3): 733-742, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30338379

RESUMO

Development of the human placenta is critical for a successful pregnancy. The placenta allows the exchange of oxygen and carbon dioxide and is crucial to manage acid-base balance within a narrow pH. It is known that low pH levels are a risk of apoptosis in several tissues. However, there has been little discussion about the effect of acidic stress in the placenta. Leptin is produced by the placenta with a trophic autocrine effect. Previous results of our group have demonstrated that leptin prevents apoptosis of trophoblast cells under different stress conditions such as serum deprivation and hyperthermia. The purpose of the present work is to evaluate acidic stress consequences in trophoblast explant survival and to determine leptin action in these conditions. For this objective, term human trophoblast explants were cultured at physiological pH (pH 7.4) and at acidic pH (pH 6.8) in the presence or absence of leptin. Western blot assays were performed to study the abundance of active caspase-3 and the p89 fragment of PARP-1. Pro-apoptotic and pro-survival members of Bcl-2 family, as Bax, t-Bid, and Bcl-2, were studied. Moreover, p53 pathway was also evaluated including Mdm-2, the main p53 regulator. Active caspase-3 and cleaved PARP-1 abundances were increased at low extracellular pH. Moreover, t-Bid levels were also augmented as well as p53 expression and phosphorylation on S46. Leptin treatment prevents the consequences of acidosis, decreasing p53 expression and increasing Mdm-2 expression. In summary, this work demonstrated for first time that low pH induces apoptosis of human trophoblast explants involving apoptotic intrinsic pathway, and leptin impairs this effect.


Assuntos
Ácidos/toxicidade , Apoptose/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Leptina/farmacologia , Placenta/citologia , Estresse Fisiológico/efeitos dos fármacos , Adulto , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Feminino , Humanos , Concentração de Íons de Hidrogênio , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Gravidez , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Trofoblastos/citologia , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismo
9.
Methods Mol Biol ; 1877: 61-76, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30535998

RESUMO

Apoptosis (programmed cell death) is activated by a wide variety of cellular stresses or insults and is vital for proper mammalian development as well as the maintenance of organismal homeostasis. The apoptosis pathway is also engaged by many common types of anticancer therapies and ionizing radiation, which contributes to the regressions of tumors or the toxic side effects of treatment. Due to the importance of maintaining healthy cell survival or the efficient clearance of cancer cells, the BH3 profiling assay was developed to functionally measure the state of the apoptosis pathway in any given cells. This assay involves the exposure of cellular mitochondria, where the BCL-2 family of proteins resides and controls the commitment to apoptosis, to proapoptotic BH3 peptides that mimic the activity of endogenous proapoptotic proteins. By using either activator or sensitizer peptides, the level of mitochondrial apoptotic priming (proximity to the threshold at which a cell commits to cell death) or dependence on prosurvival BCL-2 family proteins can be determined. Described here are two methods of BH3 profiling that can enable a user to make these functional measurements, which can be useful for predicting cellular responses to proapoptotic stressors or therapeutics (BH3 mimetics) that inhibit the activity of prosurvival proteins.


Assuntos
Apoptose/fisiologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Células HeLa , Humanos , Mitocôndrias/metabolismo , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
10.
Methods Mol Biol ; 1877: 111-119, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30536001

RESUMO

Apoptosis, a form of programmed cell death that is important for development and homeostasis, is regulated by the BCL-2 family of proteins. Over twenty BCL-2 family members have been classified in three groups based on structural homology and function. The multidomain antiapoptotic proteins promote survival, whereas the multidomain and the BH3-only proapoptotic members induce cell death. Because the interaction among the BCL-2 family members occurs primarily at the mitochondrial outer membrane, biochemical assays using artificial liposomes have been developed to study the functional relationship between these proteins. The liposomal permeabilization assay is a cell-free system that relies on the ability of multidomain pro-apoptotic members to promote membrane permeabilization upon activation. By encapsulating a fluorophore and a quencher into liposomes, the degree of permeabilization can be quantified by the increase in fluorescence intensity as the fluorophore and quencher dissociate. The liposomal permeabilization assay has been used to delineate interactions among BCL-2 family members as well as to characterize peptides, small molecules, and lipids that modulate the function of BCL-2 family of proteins. Here, we describe in detail the permeabilization of liposomes induced by the interaction between BAX and BH3-only activator tBID.


Assuntos
Lipossomos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Apoptose/fisiologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Humanos , Lipídeos/farmacologia , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Peptídeos/metabolismo , Permeabilidade , Bibliotecas de Moléculas Pequenas/metabolismo , Proteína X Associada a bcl-2/metabolismo
11.
Methods Mol Biol ; 1877: 185-200, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30536007

RESUMO

Mitochondrial outer membrane permeabilization (MOMP) is a crucial initiating event in apoptosis that activates the caspase cascade to execute cell demise. The effector B-cell lymphoma 2 (BCL-2) antagonist killer (BAK) forms mitochondrial apoptotic pores to mediate MOMP. In healthy cells, BAK resides at the outer mitochondrial membrane as a dormant monomer. Upon direct interactions with the BCL-2 homology 3 (BH3)-only proapoptotic proteins during apoptosis, BAK undergoes conformational changes to form the active species associated with apoptotic pores. We describe methods to purify mitochondria for MOMP assays and to detect conformational changes in native BAK associated with MOMP by using limited proteolysis and cross-linking analyses.


Assuntos
Mitocôndrias/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Animais , Apoptose/fisiologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Linhagem Celular , Humanos , Camundongos , Membranas Mitocondriais/metabolismo , Conformação Molecular , Permeabilidade , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
12.
Methods Mol Biol ; 1877: 247-256, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30536011

RESUMO

During apoptosis, mitochondria permeabilize the outer membranes to release apoptogenic proteins from the intermembrane space. This process termed mitochondrial outer membrane permeabilization (MOMP) is regulated by Bcl-2 family proteins. Bax is an effector proapoptotic molecule that permeabilizes the lipid membranes when it is activated by activator BH3-only proteins. We investigated this critical event by developing simple but faithful vesicle systems-outer membrane vesicles (OMVs) and liposomes-to visualize the pores in the membrane by using cryo-electron microscopy (cryo-EM). We have revealed the morphology of the pore, determined the localization of Bax labeled with nanogold and have performed image analysis to help understand the mechanisms of pore formation induced by Bax.


Assuntos
Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteína X Associada a bcl-2/metabolismo , Apoptose/fisiologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Microscopia Crioeletrônica/métodos , Humanos , Lipídeos de Membrana/metabolismo , Permeabilidade , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
13.
Blood ; 133(2): 107-120, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30413413

RESUMO

Hematopoiesis is a dynamic system that requires balanced cell division, differentiation, and death. The 2 major modes of programmed cell death, apoptosis and necroptosis, share molecular machinery but diverge in outcome with important implications for the microenvironment; apoptotic cells are removed in an immune silent process, whereas necroptotic cells leak cellular contents that incite inflammation. Given the importance of cytokine-directed cues for hematopoietic cell survival and differentiation, the impact on hematopoietic homeostasis of biasing cell death fate to necroptosis is substantial and poorly understood. Here, we present a mouse model with increased bone marrow necroptosis. Deletion of the proapoptotic Bcl-2 family members Bax and Bak inhibits bone marrow apoptosis. Further deletion of the BH3-only member Bid (to generate Vav CreBaxBakBid triple-knockout [TKO] mice) leads to unrestrained bone marrow necroptosis driven by increased Rip1 kinase (Ripk1). TKO mice display loss of progenitor cells, leading to increased cytokine production and increased stem cell proliferation and exhaustion and culminating in bone marrow failure. Genetically restoring Ripk1 to wild-type levels restores peripheral red cell counts as well as normal cytokine production. TKO bone marrow is hypercellular with abnormal differentiation, resembling the human disorder myelodysplastic syndrome (MDS), and we demonstrate increased necroptosis in MDS bone marrow. Finally, we show that Bid impacts necroptotic signaling through modulation of caspase-8-mediated Ripk1 degradation. Thus, we demonstrate that dysregulated necroptosis in hematopoiesis promotes bone marrow progenitor cell death that incites inflammation, impairs hematopoietic stem cells, and recapitulates the salient features of the bone marrow failure disorder MDS.


Assuntos
Doenças da Medula Óssea/etiologia , Medula Óssea/patologia , Células-Tronco Hematopoéticas/patologia , Inflamação/etiologia , Síndromes Mielodisplásicas/etiologia , Necrose , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/fisiologia , Medula Óssea/metabolismo , Doenças da Medula Óssea/metabolismo , Doenças da Medula Óssea/patologia , Células Cultivadas , Citocinas/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia , Proteína Killer-Antagonista Homóloga a bcl-2/fisiologia
14.
Cell Death Dis ; 9(11): 1112, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30385739

RESUMO

Due to the lack of effective treatments for glioblastoma (GBM), we here studied the responsiveness of GBM cell lines to the combination of death ligand, TRAIL and the IAP antagonist, TL32711 (Birinapant). Responses were highly heterogeneous, with synergistic apoptosis as well as treatment resistance observed. Caspase-8 and Bid, together with caspase-3, form a nonlinear signalling hub that efficiently induced apoptosis in responder cell lines. Cells resistant to TRAIL/TL32711 expressed low amounts of procaspase-8 and Bid and poorly activated caspase-3. We therefore hypothesised that improving caspase-8 activation or sensitising mitochondria to truncated Bid (tBid) could convert non-responder GBM cell lines to responders. Mathematical simulations of both strategies predicted mitochondrial sensitization to tBid would outperform enhancing caspase-8 activation. Indeed, antagonising Bcl-2 by ABT-199 allowed TRAIL/TL32711 response synergies to manifest in otherwise TRAIL resistant cell lines. These findings were further corroborated in experiments with a translationally relevant hexavalent TRAIL variant. Our study therefore demonstrates that a high caspase-8/Bid signature is associated with synergistic TRAIL/TL32711-induced apoptosis in GBM cells and outlines Bcl-2 antagonism as a highly potent intervention to sensitize highly TRAIL-resistant GBM cells to TRAIL/TL32711 combination treatment.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Dipeptídeos/farmacologia , Regulação Neoplásica da Expressão Gênica , Indóis/farmacologia , Proteínas Inibidoras de Apoptose/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Sulfonamidas/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/antagonistas & inibidores , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neuroglia/patologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais
15.
Elife ; 72018 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-30281024

RESUMO

Bcl-2 family proteins reorganize mitochondrial membranes during apoptosis, to form pores and rearrange cristae. In vitro and in vivo analysis integrated with human genetics reveals a novel homeostatic mitochondrial function for Bcl-2 family protein Bid. Loss of full-length Bid results in apoptosis-independent, irregular cristae with decreased respiration. Bid-/- mice display stress-induced myocardial dysfunction and damage. A gene-based approach applied to a biobank, validated in two independent GWAS studies, reveals that decreased genetically determined BID expression associates with myocardial infarction (MI) susceptibility. Patients in the bottom 5% of the expression distribution exhibit >4 fold increased MI risk. Carrier status with nonsynonymous variation in Bid's membrane binding domain, BidM148T, associates with MI predisposition. Furthermore, Bid but not BidM148T associates with Mcl-1Matrix, previously implicated in cristae stability; decreased MCL-1 expression associates with MI. Our results identify a role for Bid in homeostatic mitochondrial cristae reorganization, that we link to human cardiac disease.


Assuntos
Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Genômica , Cardiopatias/genética , Cardiopatias/prevenção & controle , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Animais , Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/química , Proteína Beclina-1/metabolismo , Respiração Celular , Fibrose , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Cardiopatias/patologia , Ventrículos do Coração/patologia , Humanos , Camundongos Endogâmicos C57BL , ATPases Mitocondriais Próton-Translocadoras , Mutação/genética , Células Progenitoras Mieloides/metabolismo , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Miócitos Cardíacos/ultraestrutura , Polimorfismo de Nucleotídeo Único/genética , Multimerização Proteica , Estrutura Secundária de Proteína , Subunidades Proteicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reprodutibilidade dos Testes , Regulação para Cima
16.
PLoS One ; 13(9): e0203268, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30192809

RESUMO

Emerging evidence has shown that oocytes from diabetic ovaries exhibit delayed maturation, mitochondrial dysfunction and meiotic defects, which are related increased apoptosis. The main objective of the present study was to analyze the apoptosis pathways activated during follicular loss at multiple time points in a diabetic mouse model. Twenty BALB/c mice were used in this study, and diabetes mellitus was induced by streptozotocin injection. Three diabetic and two control animals were sacrificed on days 15, 20, 70 and 80 posttreatment. The ovaries were then removed; one was used for follicular counting, TUNEL, immunohistochemistry and immunofluorescence, while the other was used for Western blot analysis. The proteins studied were BAX, BCL2, t-BID, FAS, FASL, active caspase 8, active caspase 9 and active caspase 3. Follicular apoptosis decreased over time, with the highest values observed at 15 days posttreatment. Granulosa cells were positive for active caspase 3, which showed constant expression levels at all time points. FAS, FASL, t-BID and active caspase 8 showed strong cytoplasmic immunostaining in the oocytes and granulosa cells of the diabetic mice, with significant increases observed at 15, 20 and 70 days posttreatment. BAX expression was slightly higher in the diabetic mouse ovaries than in the control ovaries at 15, 20 and 70 days posttreatment, whereas the highest active caspase 9 expression was at observed 20 days posttreatment. Low BCL2 protein levels were detected in the diabetic mouse ovaries at all time points. This study describes for the first time the behavior of apoptosis-related proteins in the diabetic mouse ovary and shows not only that the FAS/FASL pathway contributes to follicular loss but also that antral follicles are the most affected.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Diabetes Mellitus Experimental/metabolismo , Ovário/metabolismo , Animais , Apoptose/fisiologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspase 3/metabolismo , Caspase 8/metabolismo , Caspase 9/metabolismo , Diabetes Mellitus Experimental/patologia , Proteína Ligante Fas/metabolismo , Feminino , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Folículo Ovariano/metabolismo , Folículo Ovariano/patologia , Ovário/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fatores de Tempo , Proteína X Associada a bcl-2/metabolismo , Receptor fas/metabolismo
17.
Chem Biol Interact ; 294: 9-17, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30125548

RESUMO

Among five major anthocyanin compounds, delphinidin exhibited the most potent and selective cytocidal effect against HL-60, a trivalent arsenic (As(III))-resistant cell line. Co-treatment with delphinidin and As(III) resulted in the reduction of IC50 value for As(III) from 11.2 to 1.5 µM, which was considered as clinically achieved concentrations of As(III). The combination treatment strongly preferred to selectively enhance the cytotoxicity of As(III) against HL-60 cells rather than human peripheral blood mononuclear cells. The induction of apoptosis as evidenced by the increase of sub-G1 cells, DNA fragmentation, annexin V-positive cells and the activation of caspase-8, -9 and -3 was observed in HL-60 cells co-treated with As(III) and delphinidin. Similar to the activation pattern of caspases, a substantial decrease in the expression level of Bid along with the loss of mitochondrial membrane potential was also observed. These results suggested that the combination treatment triggered a convergence of the intrinsic and extrinsic pathways of apoptosis via the activation of caspase-8 and cleaved Bid. Delphinidin itself significantly decreased the intracellular GSH ([i]GSH) and nuclear factor-κB (NF-κB) binding activity, and further returned As(III)-triggered increment of [i]GSH and enhancement of NF-κB binding activity to control level. Additionally, buthionine sulfoximine, a GSH depletor; JSH-23, a NF-κB inhibitor, also mimicked the capacity of delphinidin to significantly induce the reduction of [i]GSH along with the potentiation of As(III) cytotoxicity in HL-60 cells. These observations suggested that delphinidin-induced sensitization of HL-60 cells to As(III) was caused by the reduction of [i]GSH, which was probably associated with the inhibitory effect of delphinidin on NF-κB binding activity. These findings further suggest that delphinidin-induced sensitization of HL-60 cells to As(III) may lead to dose reduction of As(III) in clinical application, and ultimately contribute to minimizing its side effects.


Assuntos
Antocianinas/química , Apoptose/efeitos dos fármacos , Arsenitos/farmacologia , Antocianinas/farmacologia , Arsenitos/química , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspases/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Sinergismo Farmacológico , Glutationa/metabolismo , Células HL-60 , Humanos , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , NF-kappa B/química , NF-kappa B/metabolismo
18.
Molecules ; 23(7)2018 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-29996473

RESUMO

In this study, 2,3-dihydro-1H-indolizinium alkaloid-prosopilosidine (PPD), that was isolated from Prosopis glandulosa, was evaluated against C. neoformans in a murine model of cryptococcosis. In vitro and in vivo toxicity of indolizidines were also evaluated. Mice were infected via the tail vein with live C. neoformans. Twenty-four hours post-infection, the mice were treated with PPD once a day (i.p.) or twice a day (bid) orally, or with amphotericin B (Amp B) intraperitoneally (IP), or with fluconazole (Flu) orally for 5 days. The brains of all of the animals were aseptically removed and the numbers of live C. neoformans were recovered. In vitro toxicity of indolizidine alkaloids was determined in HepG2 cells. PPD showed to be potent in vivo activity against C. neoformans at a dose of 0.0625 mg/kg by eliminating ~76% of the organisms compared to ~83% with Amp B (1.5 mg/kg). In addition, PPD was found to be equally efficacious, but less toxic, at either 0.125 or 0.0625 mg/kg compared to Amp B (1.5 mg/kg) when it was administered bid (twice a day) by an i.p. route. When tested by an oral route, PPD (10 mg/kg) showed potent activity in this murine model of cryptococcosis with ~82% of organisms eliminated from the brain tissue, whereas Flu (15 mg/kg) reduced ~90% of the infection. In vitro results suggest that quaternary indolizidines were less toxic as compared to those of tertiary bases. PPD (20 mg/kg) did not cause any alteration in the plasma chemistry profiles. These results indicated that PPD was active in eliminating cryptococcal infection by oral and i.p. routes at lower doses compared to Amp B. or Flu.


Assuntos
Criptococose/tratamento farmacológico , Criptococose/microbiologia , Cryptococcus neoformans/fisiologia , Indolizidinas/uso terapêutico , Prosopis/química , Administração Oral , Alcaloides/administração & dosagem , Alcaloides/química , Alcaloides/farmacologia , Alcaloides/uso terapêutico , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Peso Corporal/efeitos dos fármacos , Criptococose/sangue , Cryptococcus neoformans/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Células Hep G2 , Humanos , Indolizidinas/administração & dosagem , Indolizidinas/sangue , Indolizidinas/química , Camundongos , Resultado do Tratamento
19.
Biophys J ; 115(3): 533-542, 2018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-30017071

RESUMO

The interactions of Bcl-2 family proteins with intracellular lipids are essential for the regulation of apoptosis, a mechanism of programmed cell death that is central to the health and development of multicellular organisms. Bid and its caspase-8 cleavage product, tBid, promote the permeabilization of the mitochondrial outer membrane and sequester antiapoptotic Bcl-2 proteins to counter their cytoprotective activity. Bid and tBid also promote lipid exchange, a characteristic trait of apoptosis. Here, we show that tBid is capable of associating with phospholipids to form soluble, nanometer-sized lipoprotein particles that retain binding affinity for the antiapoptotic protein Bcl-xL. The tBid lipoprotein particles form with a lipid/protein stoichiometry in the range of 20/1 and have a diameter of ∼11.5 nm. Lipoparticle-bound tBid retains an α-helical structure and binds Bcl-xL through its third Bcl-2 homology motif, forming a soluble, lipid-associated heteroprotein complex. The results shed light on the role of lipids in mediating Bcl-2 protein mobility and interactions.


Assuntos
Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Lipoproteínas/metabolismo , Deleção de Sequência , Sequência de Aminoácidos , Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/química , Lipoproteínas/química , Permeabilidade , Ligação Proteica , Conformação Proteica em alfa-Hélice , Solubilidade
20.
Cell Physiol Biochem ; 47(5): 2046-2055, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29969781

RESUMO

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is one of the most common human malignant diseases in the world, and the mechanisms underlying HCC carcinogenesis and progression need further investigation. MicroRNAs play important roles in the development of cancer, and miR-500a is suggested to be deregulated in some types of cancer. However, the underlying molecular mechanisms of miR-500a in HCC remain unknown. METHODS: The expression of miR-500a in HCC was analyzed in The Cancer Genome Atlas (TCGA) database and examined in 33 pairs of HCC tissues and matched nontumor tissues. The correlation between miR-500a expression and prognosis of HCC patients was analyzed from the survival data in TCGA. The mechanism of miR-500a upregulation in HCC was detected using chromatin immunoprecipitation-quantitative real-time PCR. The roles of miR-500a in HCC development were examined using a cell counting kit-8 assay in vitro and growth of transplanted tumors in nude mice in vivo. Apoptosis of HCC was detected using Annexin V/propidium iodide staining. The expression of BH3-interacting death agonist (BID) protein was examined using western blot analysis. RESULTS: miR-500a expression was upregulated in HCC tissues, and high miR-500a expression was significantly correlated with the poor prognosis of HCC patients. Histone modifications in the promoter region of miR-500a may be responsible for its increased expression. Inhibition of miR-500a in HCC cell lines significantly promoted apoptosis, as well as inhibiting the proliferation of HCC cells and growth of transplanted tumors in nude mice. miR-500a directly targeted the 3' untranslated region of BID mRNA, and inhibition of miR-500a-promoted apoptosis was almost completely abolished by the administration of ABT-199 via the BID-mitochondria pathway. CONCLUSION: Our results suggest that histone modifications in the promoter region of miR-500a may be responsible for the increased expression of miR-500a in HCC, which promotes cancer progression by targeting BID, indicating that miR-500a may be a potential prognostic predictor and therapeutic target for HCC patients.


Assuntos
Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Carcinoma Hepatocelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/metabolismo , MicroRNAs/metabolismo , Proteínas de Neoplasias/metabolismo , RNA Neoplásico/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Células HEK293 , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , MicroRNAs/genética , Proteínas de Neoplasias/genética , RNA Neoplásico/genética
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