RESUMO
Antecedentes/Objetivo: El objetivo de este estudio fue determinar si se producía un incremento de la expresión de Bax (proapoptótico) y una disminución de la expresión de Blc-2A1 (antiapoptótico) en el intestino de los recién nacidos con enterocolitis necrosante. Materiales y métodos: Comparamos a ocho pacientes recién nacidos de manera consecutiva sometidos a resección intestinal debido a enterocolitis necrosante con ocho recién nacidos sometidos a resección intestinal debido a atresia ileal. La evaluación histopatológica de la lesión tisular y la apoptosis se realizó mediante microscopía óptica y el método TUNEL. El nivel de ARNm en los genes apoptóticos (CASP3, CASP6, CASP7, Bax, BIRC2) y antiapoptóticos se evaluó con el método de matriz de RCP (PCR array). La expresión de proteínas se evaluó mediante inmunohistoquímica. Resultados: Los puntajes de las lesiones tisulares y los puntajes medios de apoptosis fueron significativamente más altos en el grupo con enterocolitis necrosante en comparación con el grupo de referencia (p < 0,01). La expresión de los genes proapoptóticos aumentó significativamente en el grupo con enterocolitis necrosante frente al grupo de referencia (p < 0,01). La expresión del gen Bcl-2A1 (antiapoptótico) disminuyó significativamente en el grupo con enterocolitis necrosante (p < 0,01). La expresión de las proteínas Bax y CASP3 aumentó significativamente en el grupo con enterocolitis necrosante (p < 0,01). Conclusión: Según nuestros datos, la alteración del equilibrio entre la expresión de Bax (proapoptótico) y la expresión de Bcl-2A1 (antiapoptótico) en el lugar de la lesión es un posible mecanismo de la patogenia en recién nacidos que presentan enterocolitis necrosante.
Background/Aim. The aim of the present study was to find out if there is an increase in the expression of pro-apoptotic Bax and reduction in expression of anti-apoptotic Blc-2A1 in newborn intestines with necrotizing enterocolitis (NEC). Material and Methods. We compared 8 consecutive newborn patients undergoing bowel resection for NEC with 8 neonates undergoing intestinal resection for ileal atresia. Histopathological evaluation of tissue injury and apoptosis was performed by using light microscopic examination and TUNEL method. The mRNA level of apoptotic (CASP3, CASP6, CASP7, Bax, BIRC2) and anti-apoptotic genes were evaluated by PCR array method. Protein expression was assessed by immunohistochemistry. Results. Tissue injury scores and mean apoptosis scores were significantly higher in NEC group when compared with control group (p <0.01). Expression of pro-apoptotic genes were significantly increased in NEC group when compared with control group (p <0.01). Expression of anti-apoptotic Bcl-2A1 gene was significantly decreased in NEC group, (p <0.01). Protein expression of Bax and CASP3 was significantly increased in NEC group, (p <0.01). Conclusion. Our data in humannewborns suggest that alteration of the balance between pro-apoptotic Bax expression and anti-apoptotic Bcl-2A1 expression in the site of injury is a possible mechanism in the pathogenesis of NEC.
Assuntos
Humanos , Recém-Nascido , Antígenos de Histocompatibilidade Menor/biossíntese , Antígenos de Histocompatibilidade Menor/fisiologia , Apoptose/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Enterocolite Necrosante/metabolismo , Proteína X Associada a bcl-2/biossíntese , Proteína X Associada a bcl-2/fisiologiaRESUMO
BACKGROUND/AIM: The aim of the present study was to find out if there is an increase in the expression of pro-apoptotic Bax and reduction in expression of anti-apoptotic Blc-2A1 in newborn intestines with necrotizing enterocolitis (NEC). MATERIALS AND METHODS: We compared 8 consecutive newborn patients undergoing bowel resection for NEC with 8 neonates undergoing intestinal resection for ileal atresia. Histopathological evaluation of tissue injury and apoptosis was performed by using light microscopic examination and TUNEL method. The mRNA level of apoptotic (CASP3, CASP6, CASP7, Bax, BIRC2) and anti-apoptotic genes were evaluated by PCR array method. Protein expression was assessed by immunohistochemistry. RESULTS: We compared 8 consecutive newborn patients undergoing bowel resection for NEC with 8 neonates undergoing intestinal resection for ileal atresia. Histopathological evaluation of tissue injury and apoptosis was performed by using light microscopic examination and TUNEL method. The mRNA level of apoptotic (CASP3, CASP6, CASP7, Bax, BIRC2) and anti-apoptotic genes were evaluated by PCR array method. Protein expression was assessed by immunohistochemistry. CONCLUSIONS: Our data in humannewborns suggest that alteration of the balance between pro-apoptotic Bax expression and anti-apoptotic Bcl-2A1 expression in the site of injury is a possible mechanism in the pathogenesis of NEC.
ANTECEDENTES/OBJETIVO: El objetivo de este estudio fue determinar si se producía un incremento de la expresión de Bax (proapoptótico) y una disminución de la expresión de Blc-2A1 (antiapoptótico) en el intestino de los recién nacidos con enterocolitis necrosante. MATERIALES Y MÉTODOS: Comparamos a ocho pacientes recién nacidos de manera consecutiva sometidos a resección intestinal debido a enterocolitis necrosante con ocho recién nacidos sometidos a resección intestinal debido a atresia ileal. La evaluación histopatológica de la lesión tisular y la apoptosis se realizó mediante microscopía óptica y el método TUNEL. El nivel de ARNm en los genes apoptóticos (CASP3, CASP6, CASP7, Bax, BIRC2) y antiapoptóticos se evaluó con el método de matriz de RCP (PCR array). La expresión de proteínas se evaluó mediante inmunohistoquímica. RESULTADOS: Los puntajes de las lesiones tisulares y los puntajes medios de apoptosis fueron significativamente más altos en el grupo con enterocolitis necrosante en comparación con el grupo de referencia (p < 0,01). La expresión de los genes proapoptóticos aumentó significativamente en el grupo con enterocolitis necrosante frente al grupo de referencia (p < 0,01). La expresión del gen Bcl-2A1 (antiapoptótico) disminuyó significativamente en el grupo con enterocolitis necrosante (p < 0,01). La expresión de las proteínas Bax y CASP3 aumentó significativamente en el grupo con enterocolitis necrosante (p < 0,01). CONCLUSIONES: Según nuestros datos, la alteración del equilibrio entre la expresión de Bax (proapoptótico) y la expresión de Bcl-2A1 (antiapoptótico) en el lugar de la lesión es un posible mecanismo de la patogenia en recién nacidos que presentan enterocolitis necrosante.
Assuntos
Apoptose/fisiologia , Enterocolite Necrosante/metabolismo , Antígenos de Histocompatibilidade Menor/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteína X Associada a bcl-2/biossíntese , Feminino , Humanos , Recém-Nascido , Masculino , Antígenos de Histocompatibilidade Menor/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Proteína X Associada a bcl-2/fisiologiaRESUMO
BCR-ABL kinase has been observed to be potentially related to leukemic cell development. Adult patients with acute lymphoblastic leukemia (ALL) were evaluated to determine whether presence/absence of BCR-ABL induced differences in activation of Src, PI3K/Akt and NF-κB or in the expression of anti-apoptotic proteins such as BCL-2 and c-IAP1. BCR-ABL positive patients showed a significantly higher activation of Src and Akt compared with BCR-ABL negative patients and healthy donors. BCR-ABL negative patients also showed a significant activation of Src and low levels of Akt activation compared with healthy donors. Both patient groups had increased NF-κB activation and overexpression of BCL-2 and c-IAP1. This is the first study to evaluate concurrently in ALL patients presence/absence of BCR-ABL in relation to activation of Src, Akt and NF-κB and the expression of anti-apoptotic proteins. Results suggest that these proteins may be involved in an anti-apoptotic signaling pathway.
Assuntos
Apoptose/genética , Proteínas de Fusão bcr-abl/metabolismo , Proteínas Inibidoras de Apoptose/fisiologia , NF-kappa B/fisiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogênicas c-akt/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Proteínas Proto-Oncogênicas pp60(c-src)/fisiologia , Adulto , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/fisiologia , Exame de Medula Óssea , Estudos de Casos e Controles , Feminino , Proteínas de Fusão bcr-abl/genética , Regulação Leucêmica da Expressão Gênica , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Masculino , NF-kappa B/genética , NF-kappa B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Transdução de Sinais/genéticaRESUMO
Endoplasmic reticulum (ER) and mitochondria are intracellular organelles and their interactions are directly involved in different processes such as Ca(2+) signaling in cell survival and death mechanisms. Bcl-2 is an anti-apoptotic protein intrinsically related to ER and mitochondria, modulating Ca(2+) content in these organelles. We investigated the effects of Bcl-2 overexpression on ER and mitochondrial Ca(2+) dynamics in PC12 cells. Bcl-2 overexpressing and control cells were loaded with Fura 2/AM and stimulated with different drugs. Results showed that in Bcl-2 cells, ACh induced a lower Ca(2+) response compared to control. Ca(2+) release induced by TG was decreased in Bcl-2 cells, however, it was greater in Caff induced Ca(2+) rise. In addition, FCCP induced a higher Ca(2+) release in Bcl-2 cells. These results suggest that Bcl-2 overexpression modulate the ER Ca(2+) pools differently and the release of ER Ca(2+) may increase mitochondrial Ca(2+) accumulation. These alterations of intracellular Ca(2+) stores are important mechanisms for the control of Ca(2+) signaling.
Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Acetilcolina/farmacologia , Animais , Cafeína/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Células PC12 , Ratos , Tapsigargina/farmacologiaRESUMO
Apoptotic pathways are providing important saveguard mechanisms in protection from cancer by eliminating altered and often harmful cells. The disturbances of cell proliferation, differentiation and apoptosis are also found on specific signal-transduction pathways within the tumour cells and between these and the immune system. The article focuses attention on the evolution of the melanocytic naevi in the direction of a dysplastic or tumour cell. The determination of single molecules as prognostic parameters within cancer genesis seems to be problematic. New hopes are being placed on the treatment with TW-37, ABT-737 and TAT-Bim, which, to an extent, are able to support the programmed cell death. The clinical importance of these innovative therapies remains to be seen and should therefore, be viewed with considerable criticism.
Assuntos
Evasão Tumoral/fisiologia , Ciclo Celular/fisiologia , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/patologia , Evasão Tumoral/imunologiaRESUMO
Coxiella burnetii is an obligate intracellular bacterium that generates large vacuoles in which this pathogen replicates and survives. We have previously demonstrated that C. burnetii interacts with the autophagic pathway as a strategy for its survival and replication. Coxiella displays an anti-apoptotic activity to maintain host cell viability, leading to a persistent infection. Our recent study reveals that Beclin 1 is recruited to the Coxiella-membrane vacuole favoring its development and bacterial replication. In contrast, the anti-apoptotic protein Bcl-2 alters the normal development of the Coxiella-replicative compartment. In addition, our results indicate that C. burnetii infection modulates autophagy and apoptotic pathways via Beclin 1-Bcl-2 interplay to establish a successful infection in the host cell. Of note, this pathogen-host cell model has allowed uncovering a novel function of Beclin 1 as a regulator of the anti-apoptotic activity of Bcl-2. We have also established that a proper interplay between Beclin 1 and Bcl-2 is required for both autophagy and apoptosis modulation.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Apoptose/genética , Infecções Bacterianas/patologia , Proteínas de Membrana/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Proteínas Reguladoras de Apoptose/genética , Autofagia/genética , Autofagia/fisiologia , Infecções Bacterianas/genética , Proteína Beclina-1 , Coxiella burnetii/fisiologia , Células HeLa , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Febre Q/genética , Febre Q/patologiaRESUMO
During rat hepatocarcinogenesis preneoplastic lesions (PNL) emerge which may persist (pPNL) and be sites of progress to cancer or suffer remodeling (rPNL) tending to disappear. Cellular and molecular mechanisms involved in both phenotypes are not sufficiently elucidated. pPNL and rPNL cellular proliferation and apoptosis were evaluated in rats submitted to the resistant hepatocyte (RH) model, and an adjusted growth index (AGI) was established. p53, Bcl-2, and NF-kappaB p65 subunit expression was evaluated by immunohistochemistry in pPNL and rPNL. p65 expression and NF-kappaB activation was evaluated by Western blot assays in whole livers. A lower number of BrdU-stained hepatocyte nuclei/mm(2) and higher number of apoptotic bodies (AB) per mm(2) were observed in remodeling compared to pPNL. Cytoplasmic p53 accumulation is related to increased hepatocarcinoma malignancy. We observed that 71.3% pPNL and 25.4% rPNL (P < 0.05) presented p53 staining in the cytoplasm. Similarly, 67.7% pPNL and 23.1 % rPNL (P < 0.05) presented increased Bcl-2 staining. Thirty-two percent pPNL and 15.6% rPNL (P < 0.05) presented p65 staining. Compared to normal rats, increase (P < 0.05) of hepatic p65 expression and NF-kappaB activation in rats submitted to the RH model was observed. In agreement to previous studies hepatic pPNL and rPNL differ regarding cell proliferation and apoptosis. Moreover, persistence and remodeling involve differences in p53, Bcl-2, and NF-kappaB pathways. These data point to molecular pathways that may direct preneoplastic lesions to spontaneously regress or to progress to cancer. J. Cell. Biochem. 103: 538-546, 2008. (c) 2007 Wiley-Liss, Inc.
Assuntos
Hepatopatias/patologia , Neoplasias Hepáticas Experimentais/patologia , NF-kappa B/fisiologia , Lesões Pré-Cancerosas/patologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Fator de Transcrição RelA/fisiologia , Proteína Supressora de Tumor p53/fisiologia , 2-Acetilaminofluoreno/toxicidade , Animais , Apoptose , Doença Hepática Induzida por Substâncias e Drogas , Cocarcinogênese , Replicação do DNA , Dietilnitrosamina/toxicidade , Progressão da Doença , Regulação da Expressão Gênica , Hepatectomia/métodos , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepatopatias/genética , Hepatopatias/metabolismo , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/genética , Regeneração Hepática , Masculino , Lesões Pré-Cancerosas/induzido quimicamente , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismo , Distribuição Aleatória , Ratos , Ratos Endogâmicos F344RESUMO
Interferons alpha (IFNsalpha) are a family of related proteins exhibiting antiviral, antiproliferative and immunoregulatory activities. Although IFNsalpha have been widely employed for the pharmacological treatment of different types of cancer, the therapeutic efficacy occasionally can be diminished by the appearance of side effects, neutralizing antibodies or tumor resistance. In the search of mimetic peptides of the IFN-alpha2b molecule, we have recently synthesized a chimeric cyclic peptide that inhibits IFN-alpha2b binding to its receptor and exerts an IFN-like antiproliferative activity. In order to study the mechanism of growth inhibition of the cyclic chimera, we evaluated its ability to induce cell cycle arrest or apoptosis in WISH cells. We found that the chimeric peptide did not cause a cell cycle arrest, although the entire IFN-alpha2b molecule did modify cell cycle by increasing the number of S-phase cells. In spite of this difference, both molecules were able to induce apoptosis through the activation of caspases 8 and 9, indicating the involvement of death receptor and mitochondrial pathways. In addition, both peptidic derivative and IFN-alpha2b altered the expression of Bcl-2 family proteins and induced the release of cytochrome C to cytosol, supporting the participation of mitochondrial pathway in the induction of apoptosis. In conclusion, we demonstrated that the chimeric cyclic peptide behaved as a potent inducer of apoptosis and it could be a potentially useful agent for the treatment of certain malignancies.
Assuntos
Apoptose/efeitos dos fármacos , Interferon-alfa/farmacologia , Neoplasias/tratamento farmacológico , Fragmentos de Peptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Caspases/fisiologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocromos c/fisiologia , Humanos , Interferon alfa-2 , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Proteínas RecombinantesRESUMO
Reduction in corticosterone by acute adrenalectomy (5 d) promotes apoptosis in dentate gyrus (DG) granular neurons, an effect concomitant with variations in the expression of the Bcl-2 gene family implicated in apoptotic regulation. However, no studies exist correlating the effect of long-term adrenalectomy (30 d) on the hippocampus in terms of extent of apoptosis and the levels of proteins related to an apoptotic cascade. After 5 d of adrenalectomy, we found an increase in apoptosis of the DG granular region, correlated with an increase in the processing of caspase-9. The magnitude of apoptosis 30 d after adrenalectomy was reduced in the DG granular layer compared with 5 d after adrenalectomy, in close relation to a reduction in the level of processed caspase-9. To understand how the increase in cell survival long after adrenalectomy occurs, we analyzed changes in the expression of genes and proteins related to apoptosis. Long-term adrenalectomy did not change hippocampal pro-apoptotic Bax or antiapoptotic Bcl-2 mRNA levels or protein content with respect to control. However, we found an increase in mRNA levels of the GD's Bcl-x gene, in parallel with the increase in anti-apoptotic BCL-XL protein levels. These results suggest the reduction in apoptosis observed after long-term adrenalectomy occurs through mechanisms that repress proapoptotic genes previously found to be increased at shorter times of adrenalectomy.
Assuntos
Adrenalectomia/efeitos adversos , Apoptose , Hipocampo/citologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Animais , Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 9 , Caspases/metabolismo , Sobrevivência Celular , Hipocampo/fisiologia , Masculino , Modelos Biológicos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
In this study, the mechanisms involved in the inhibitory effect of histamine (HA) on PANC-1 cell proliferation were investigated. The action of HA on cell growth was evaluated by determining the cell doubling time from experimental growth curves and analysing the cell cycle using a flow cytometer. The expression of proteins related to cell death and proliferation (PCNA, p53, c-Fos and Bcl-2 family proteins) was studied using Western blot, immunocytochemistry and flow cytometric analysis. The results indicated that HA produced an accumulation of PANC-1 cells in GO/Gl-phase and increased the doubling time via H2HA (H2R) stimulation. Expression of p53, c-Fos and Bcl-2 were not modulated by HA. However, HA decreased PCNA and Bax expression, while it increased the Bcl-x level. In summary, the antiproliferative effect exerted by HA was associated with a G0/G1-phase arrest and a modulation of the Bcl-2 family proteins.