RESUMO
Apoptosis-inducing factor (AIF) is a flavoprotein and essential partner of the CHCHD4 redox protein during the mitochondrial intermembrane space import machinery. Mammalian AIF has three cysteine residues, which have received little attention. Previous reports have evidenced a redox interaction between AIF and thioredoxin 1 (Trx1), particularly after oxidant conditions. Therefore, we asked whether the cysteine residues of the human AIF could be oxidized. Our data showed that endogenous AIF could be oxidized to disulfide-linked conjugates (DLC). Overexpressed WT AIF in HEK293T cells, as well as recombinant WT AIF, formed DLC. Expression of C256S, C317S or C441S AIF mutants severely inhibited DLC formation in cells exposed to oxidants. In vitro, DLC formation was completely precluded with C256S and C441S AIF mutants and partially inhibited with the C317S mutant. DLC was shown to enhance cellular susceptibility to apoptosis induced by staurosporine, likely by preventing AIF to maintain mitochondrial oxidative phosphorylation. Cells with decreased expression of Trx1 produced more AIF DLC than those with normal Trx1 levels, and in vitro, Trx1 was able to decrease the amount of AIF DLC. Finally, confocal analysis, as well as immunoblotting of mitochondrial fraction, indicated that a fraction of Trx1 is present in mitochondria. Overall, these data provide evidence that all three cysteine residues of AIF can be oxidized to DLC, which can be disrupted by mitochondrial Trx1.
Assuntos
Fator de Indução de Apoptose , Apoptose , Dissulfetos , Substituição de Aminoácidos , Fator de Indução de Apoptose/química , Fator de Indução de Apoptose/genética , Fator de Indução de Apoptose/metabolismo , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Células HEK293 , Células HeLa , Humanos , Mutação de Sentido Incorreto , Oxirredução , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estaurosporina/farmacologiaRESUMO
The chronic consumption of alcohol causes a worsening of the events that follow the cerebral ischemia. These events are regulated through the expression of several genes and microRNAs. The aimof this work was To analyze and describe the expression profile of PARP and AIF and miRNA-9 proteins in rats submitted to focal cerebral ischemia, associated or not with chronic alcoholism model. Methods: Twenty adult Wistar rats, subdivided into: control; ischemic; alcoholic and ischemic / alcoholized for immunohistochemical analysis and miRNA-9 gene expression. Results: There was a reduction in the protein expression of PARP-1 and a positive marking for AIF in the ischemic / alcoholized group. The miRNA-9 did not obtain significant expression. The association of ischemia with chronic alcohol use promoted a tendency to low expression of miRNA-9, low expression of PARP-1 and high expression of AIF, indicating an interference in the protective effect of miRNA-9 be observed in the other groups.
El consumo crónico de alcohol provoca un empeoramiento de los eventos que siguen a la isquemia cerebral. Estos eventos están regulados a través de la expresión de varios genes y microRNA. El objetivo de este trabajo fue analizar y describir el perfil de expresión de las proteínas PARP y AIF y microRNA-9 en ratas sometidas a isquemia cerebral focal, asociadas o no, con el modelo de alcoholismo crónico. Veinte ratas Wistar adultas se dividieron en: grupo control, isquémico alcohólico, e isquémico / alcoholizado para análisis inmunohistoquímico y expresión de genes microRNA-9. Resultados: Hubo una reducción en la expresión de proteínas de PARP-1 y un marcado positivo para AIF en el grupo isquémico / alcoholizado. No se observó una expresión significativa en el microRNA-9. La asociación de la isquemia con el consumo crónico de alcohol promovió una tendencia a la baja expresión de microRNA-9, baja expresión de PARP1 y alta expresión de AIF, lo que indica una interferencia en el efecto protector de microRNA-9 en los otros grupos.
Assuntos
Animais , Ratos , Isquemia Encefálica/metabolismo , Alcoolismo/metabolismo , Imuno-Histoquímica , Isquemia Encefálica/genética , Ratos Wistar , MicroRNAs/metabolismo , Modelos Animais de Doenças , Alcoolismo/genética , Fator de Indução de Apoptose/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismoRESUMO
Ceramide (Cer) has a key role inducing cell death and has been proposed as a messenger in photoreceptor cell death in the retina. Here, we explored the pathways induced by C2-acetylsphingosine (C2-Cer), a cell-permeable Cer, to elicit photoreceptor death. Treating pure retina neuronal cultures with 10 µM C2-Cer for 6 h selectively induced photoreceptor death, decreasing mitochondrial membrane potential and increasing the formation of reactive oxygen species (ROS). In contrast, amacrine neurons preserved their viability. Noteworthy, the amount of TUNEL-labeled cells and photoreceptors expressing cleaved caspase-3 remained constant and pretreatment with a pan-caspase inhibitor did not prevent C2-Cer-induced death. C2-Cer provoked polyADP ribosyl polymerase-1 (PARP-1) overactivation. Inhibiting PARP-1 decreased C2-Cer-induced photoreceptor death; C2-Cer increased polyADP ribose polymer (PAR) levels and induced the translocation of apoptosis inducing factor (AIF) from mitochondria to photoreceptor nuclei, which was prevented by PARP-1 inhibition. Pretreatment with a calpain and cathepsin inhibitor and with a calpain inhibitor reduced photoreceptor death, whereas selective cathepsin inhibitors granted no protection. Combined pretreatment with a PARP-1 and a calpain inhibitor evidenced the same protection as each inhibitor by itself. Neither autophagy nor necroptosis was involved in C2-Cer-elicited death; no increase in LDH release was observed upon C2-Cer treatment and pretreatment with inhibitors of necroptosis and autophagy did not rescue photoreceptors. These results suggest that C2-Cer induced photoreceptor death by a novel, caspase-independent mechanism, involving activation of PARP-1, decline of mitochondrial membrane potential, calpain activation, and AIF translocation, all of which are biochemical features of parthanatos.
Assuntos
Ceramidas/farmacologia , Parthanatos/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/patologia , Animais , Fator de Indução de Apoptose/metabolismo , Calpaína/metabolismo , Caspases/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Modelos Biológicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Transporte Proteico/efeitos dos fármacos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismoRESUMO
Whether fructose (FRU), as the sole energy source, confers a metabolic advantage on cancer cells against noxious stimuli is unknown. The aim of this study was to evaluate the effects of low (11 mM), moderate (25 mM), and high (55 mM) FRU concentrations alone or in combination with rotenone (ROT) or doxorubicin (DOX) in Jurkat cells, an acute lymphoblastic leukemia cell model. Glucose (GLU) was used as a control. Using different cell analysis techniques, we demonstrated that FRU was predominantly metabolized via oxidative phosphorylation (â¼95%) (i.e., lactate production was reduced >120-fold), resulting in endogenous oxidative stress-induced conditions. The cells were characterized by generation of O2â¢- (43%)/ H2 O2 (40%) and activation of NF-κB (â¼95-fold increase, fi), c-Jun-N terminal kinase (JNK), p53 (40-fi), and c-Jun (9-fi). In addition, we observed a loss of ΔΨm (10%), activation of caspase-3 (50-fi) and apoptosis-inducing factor (AIF, 2-fi), and condensation and fragmentation of the nuclei [20% by acridine orange/ethidium bromide/Hoechst (AO/EB/H) staining, 15% by flow cytometry] compared to those of GLU 11 at 24 h. Although DOX killed Jurkat cells independent of sugar content in the culture medium, leukemic cells in low, but not high, FRU were extremely sensitive to ROT. Taken together, our findings suggest that Jurkat cells are more susceptible to cell death if forced to shift from GLU metabolism (i.e., aerobic glycolysis) to FRU metabolism (i.e., oxidative phosphorylation) after treatment with mitochondria-targeting molecules. These observations may help elucidate the cell death mechanism of leukemic cells cultured in FRU.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Frutose/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Apoptose/fisiologia , Morte Celular/efeitos dos fármacos , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacologia , Frutose/administração & dosagem , Glucose/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Células Jurkat , NF-kappa B/metabolismo , Estresse Oxidativo/fisiologia , Fosforilação , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Rotenona/administração & dosagem , Rotenona/farmacologia , Transdução de Sinais/efeitos dos fármacos , Superóxidos/metabolismoRESUMO
The aim of the present study was to investigate the effects of different periods of ovariectomy and 17ß-estradiol (E2) replacement on the expression of Cytochrome C, apoptosis inducing factor (AIF) and Endonuclease-G (Endo-G) in mitochondrial and cytosolic fractions obtained from hippocampus of the adult female rats. In addition, the expression of phosphorylated CREB (phospho-CREB) was also analyzed in hippocampus. Ovariectomy or E2 treatment did not change the expression of Cytochrome C and AIF. Ovariectomy (15, 21 and 36 days) decreased the expression of Endo-G in the mitochondrial fractions and increased it in the cytosolic fractions obtained from hippocampus. The treatment with E2 after 15 days of ovariectomy for 7 days or 21 days, and throughout the post-ovariectomy period prevented the effects of ovariectomy on Endo-G expression. Our results suggest that ovariectomy-induced apoptotic cell death in hippocampal tissue could be mediated by Endo-G, but not by AIF, via a caspase-independent apoptotic pathway. Furthermore, ovariectomy decreased the expression of phospho-CREB and the treatment with E2 prevented these effects. In conclusion, E2 may help maintain long-term neuronal viability by regulating the expression of members of the Bcl-2 family. Regulation of Endo-G released from mitochondria, but not of Cytochrome C and AIF, is also involved in the neuroprotective actions of E2. Furthermore, CREB may be involved in the expression of Bcl-2. These data provide new understanding into the mechanisms involved in the neuroprotective role of estrogen.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Endodesoxirribonucleases/metabolismo , Estradiol/farmacologia , Terapia de Reposição de Estrogênios , Hipocampo/metabolismo , Ovariectomia , Animais , Fator de Indução de Apoptose/metabolismo , Citocromos c/metabolismo , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/enzimologia , Fosforilação/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Fucan is a term used to denominate a family of sulfated polysaccharides rich in sulfated l-fucose. Heterofucan SF-1.5v was extracted from the brown seaweed Sargassum filipendula by proteolytic digestion followed by sequential acetone precipitation. This fucan showed antiproliferative activity on Hela cells and induced apoptosis. However, SF-1.5v was not able to activate caspases. Moreover, SF-1.5v induced glycogen synthase kinase (GSK) activation, but this protein is not involved in the heterofucan SF-1.5v induced apoptosis mechanism. In addition, ERK, p38, p53, pAKT and NFκB were not affected by the presence of SF-1.5v. We determined that SF-1.5v induces apoptosis in HeLa mainly by mitochondrial release of apoptosis-inducing factor (AIF) into cytosol. In addition, SF-1.5v decreases the expression of anti-apoptotic protein Bcl-2 and increased expression of apoptogenic protein Bax. These results are significant in that they provide a mechanistic framework for further exploring the use of SF-1.5v as a novel chemotherapeutics against human cervical cancer.
Assuntos
Apoptose/efeitos dos fármacos , Fucose/química , Polissacarídeos/farmacologia , Sargassum/química , Fator de Indução de Apoptose/efeitos dos fármacos , Fator de Indução de Apoptose/metabolismo , Proliferação de Células/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Polissacarídeos/isolamento & purificação , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/patologiaRESUMO
Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that mediates both NADH-oxidizing and caspase-independent apoptosis. Further, the proapoptotic activity of AIF is located in the C-terminus of AIF, although the precise minimum sequence responsible for apoptosis induction remains to be investigated. In the present study, we generated two truncated AIFs, AIFΔ1-480-FLAG, which is a FLAG-tagged C-terminal peptide comprising amino acids from 481 to 613, and AIF360-480 containing amino acids from 360 to 480 of AIF. We used confocal microscopy to demonstrate that both the truncated proteins are expressed and located in the cytoplasm of transfected cells. AIFΔ1-480 but not AIF360-480 induces apoptosis in transfected cells. We also found that the expression of AIFΔ1-480 could initiate the release of cytochrome c from the mitochondria. The suppression of caspase-9 via siRNA blocked the proapoptotic activity of AIFΔ1-480. Therefore, AIFΔ 1-480 is sufficient for inducing caspase-9-dependent apoptotic signaling, probably by promoting the release of cytochrome c. At last, we generated a chimeric immuno-AIFΔ 1-480 protein, which comprised an HER2 antibody, a Pseudomonas exotoxin A translocation domain and AIFΔ 1-480. Human Jurkat cells transfected with the immuno-AIFΔl-480 gene could express and secrete the chimeric protein, which selectively recognize and kill HER2-overexpressing tumor cells. Our study demonstrates the feasibility of the immuno-AIFΔl-480 gene as a novel approach to treating HER2-overexpressing cancers.