RESUMEN
Protein-protein interaction studies using proximity labeling techniques, such as biotin ligase-based BioID, have become integral in understanding cellular processes. Most studies utilize conventional 2D cell culture systems, potentially missing important differences in protein behavior found in 3D tissues. In this study, we investigated the protein-protein interactions of a protein, Bcl-2 Agonist of cell death (BAD), and compared conventional 2D culture conditions to a 3D system, wherein cells were embedded within a 3D extracellular matrix (ECM) mimic. Using BAD fused to the engineered biotin ligase miniTurbo (BirA*), we identified both overlapping and distinct BAD interactomes under 2D and 3D conditions. The known BAD binding proteins 14-3-3 isoforms and Bcl-XL interacted with BAD in both 2D and 3D. Of the 131 BAD-interactors identified, 56% were specific to 2D, 14% were specific to 3D, and 30% were common to both conditions. Interaction network analysis demonstrated differential associations between 2D and 3D interactomes, emphasizing the impact of the culture conditions on protein interactions. The 2D-3D overlap interactome encapsulated the apoptotic program, which is a well-known role of BAD. The 3D unique pathways were enriched in ECM signaling, suggestive of hitherto unknown functions for BAD. Thus, exploring protein-protein interactions in 3D provides novel clues into cell behavior. This exciting approach has the potential to bridge the knowledge gap between tractable 2D cell culture and organoid-like 3D systems.
Asunto(s)
Técnicas de Cultivo de Célula , Proteína Letal Asociada a bcl , Humanos , Proteína Letal Asociada a bcl/metabolismo , Técnicas de Cultivo de Célula/métodos , Mapas de Interacción de Proteínas , Matriz Extracelular/metabolismo , Mapeo de Interacción de Proteínas/métodos , Proteínas 14-3-3/metabolismo , Ligasas de Carbono-Nitrógeno/metabolismo , Ligasas de Carbono-Nitrógeno/genética , Unión Proteica , Proteína bcl-X/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas RepresorasRESUMEN
Ischemic stroke causes a lack of oxygen and glucose supply to brain, eventually leads to severe neurological disorders. Retinoic acid is a major metabolic product of vitamin A and has various biological effects. The PI3K-Akt signaling pathway is an important survival pathway in brain. Phosphorylated Akt is important in regulating survival and apoptosis. We examined whether retinoic acid has neuroprotective effects in stroke model by regulating Akt and its downstream protein, Bad. Moreover, we investigated the relationship between retinoic acid and Bcl-2 family protein interactions. Animals were intraperitoneally administered vehicle or retinoic acid (5 mg/kg) for four days before surgery and ischemic stroke was induced by middle cerebral artery occlusion (MCAO) surgery. Neurobehavioral tests were performed 24 h after MCAO and cerebral cortical tissues were collected. Cresyl violet staining and TUNEL histochemistry were performed, Western blot and immunoprecipitation analysis were performed to elucidate the expression of various proteins. Retinoic acid reduced neurological deficits and histopathological changes, decreased the number of TUNEL-positive cells, and alleviated reduction of phospho-PDK1, phospho-Akt, and phospho-Bad expression caused by MCAO damage. Immunoprecipitation analysis showed that MCAO damage reduced the interaction between phospho-Bad and 14-3-3, which was attenuated by retinoic acid. Furthermore, retinoic acid mitigated the increase in Bcl-2/Bad and Bcl-xL/Bad binding levels and the reduction in Bcl-2/Bax and Bcl-xL/Bax binding levels caused by MCAO damage. Retinoic acid alleviated MCAO-induced increase of caspase-3 and cleaved caspase-3 expression. We demonstrate that retinoic acid prevented apoptosis against cerebral ischemia through phosphorylation of Akt and Bad, maintenance of phospho-Bad and 14-3-3 binding, and regulation of Bcl-2 family protein interactions. .
Asunto(s)
Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-bcl-2 , Tretinoina , Proteína Letal Asociada a bcl , Animales , Masculino , Ratas , Apoptosis/efectos de los fármacos , Proteína Letal Asociada a bcl/metabolismo , Modelos Animales de Enfermedad , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/metabolismo , Accidente Cerebrovascular Isquémico/metabolismo , Accidente Cerebrovascular Isquémico/tratamiento farmacológico , Accidente Cerebrovascular Isquémico/patología , Fármacos Neuroprotectores/farmacología , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Tretinoina/farmacologíaRESUMEN
BH3 mimetics, including the BCL2/BCLXL/BCLw inhibitor navitoclax and MCL1 inhibitors S64315 and tapotoclax, have undergone clinical testing for a variety of neoplasms. Because of toxicities, including thrombocytopenia after BCLXL inhibition as well as hematopoietic, hepatic and possible cardiac toxicities after MCL1 inhibition, there is substantial interest in finding agents that can safely sensitize neoplastic cells to these BH3 mimetics. Building on the observation that BH3 mimetic monotherapy induces AMP kinase (AMPK) activation in multiple acute leukemia cell lines, we report that the AMPK inhibitors (AMPKis) dorsomorphin and BAY-3827 sensitize these cells to navitoclax or MCL1 inhibitors. Cell fractionation and phosphoproteomic analyses suggest that sensitization by dorsomorphin involves dephosphorylation of the proapoptotic BCL2 family member BAD at Ser75 and Ser99, leading BAD to translocate to mitochondria and inhibit BCLXL. Consistent with these results, BAD knockout or mutation to BAD S75E/S99E abolishes the sensitizing effects of dorsomorphin. Conversely, dorsomorphin synergizes with navitoclax or the MCL1 inhibitor S63845 to induce cell death in primary acute leukemia samples ex vivo and increases the antitumor effects of navitoclax or S63845 in several xenograft models in vivo with little or no increase in toxicity in normal tissues. These results suggest that AMPK inhibition can sensitize acute leukemia to multiple BH3 mimetics, potentially allowing administration of lower doses while inducing similar antineoplastic effects.
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Proteínas Quinasas Activadas por AMP , Compuestos de Anilina , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Pirimidinas , Sulfonamidas , Proteína bcl-X , Humanos , Animales , Compuestos de Anilina/farmacología , Sulfonamidas/farmacología , Proteínas Quinasas Activadas por AMP/metabolismo , Ratones , Proteína bcl-X/metabolismo , Proteína bcl-X/antagonistas & inhibidores , Línea Celular Tumoral , Pirimidinas/farmacología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Pirazoles/farmacología , Proteína Letal Asociada a bcl/metabolismo , Apoptosis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Leucemia/tratamiento farmacológico , Leucemia/patología , Leucemia/metabolismo , Fosforilación/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Sinergismo FarmacológicoRESUMEN
RNA-binding proteins (RBP) regulate several aspects of co- and post-transcriptional gene expression in cancer cells. CSTF2 is involved in the expression of many cellular mRNAs and involved in the 3'-end cleavage and polyadenylation of pre-mRNAs to terminate transcription. However, the role of CSTF2 in human glioblastoma (GBM) and the underlying mechanisms remain unclear. In the present study, CSTF2 was found to be upregulated in GBM, and its high expression predicted poor prognosis. Knockdown CSTF2 induced GBM cell apoptosis both in vitro and in vivo. Specific mechanism studies showed that CSTF2 unstabilized the mRNA of the BAD protein by shortening its 3' UTR. Additionally, an increase in the expression level of CSTF2 decreased the expression level of BAD. In conclusion, CSTF2 binds to the mRNA of the BAD protein to shorten its 3'UTR, which negatively affects the BAD mediated apoptosis and promotes GBM cell survival.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Humanos , Glioblastoma/genética , Glioblastoma/metabolismo , Proteína Letal Asociada a bcl/genética , Proteína Letal Asociada a bcl/metabolismo , Apoptosis/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulación Neoplásica de la Expresión Génica , Línea Celular Tumoral , Proliferación Celular/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismoRESUMEN
BACKGROUND: Stroke is caused by disruption of blood supply and results in permanent disabilities as well as death. Chlorogenic acid is a phenolic compound found in various fruits and coffee and exerts antioxidant, anti-inflammatory, and anti-apoptotic effects. OBJECTIVES: The purpose of this study was to investigate whether chlorogenic acid regulates the PI3K-Akt-Bad signaling pathway in middle cerebral artery occlusion (MCAO)-induced damage. METHODS: Chlorogenic acid (30 mg/kg) or vehicle was administered peritoneally to adult male rats 2 h after MCAO surgery, and animals were sacrificed 24 h after MCAO surgery. Neurobehavioral tests were performed, and brain tissues were isolated. The cerebral cortex was collected for Western blot and immunoprecipitation analyses. RESULTS: MCAO damage caused severe neurobehavioral disorders and chlorogenic acid improved the neurological disorders. Chlorogenic acid alleviated the MCAO-induced histopathological changes and decreased the number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells. Furthermore, MCAO-induced damage reduced the expression of phospho-PDK1, phospho-Akt, and phospho-Bad, which was alleviated with administration of chlorogenic acid. The interaction between phospho-Bad and 14-3-3 levels was reduced in MCAO animals, which was attenuated by chlorogenic acid treatment. In addition, chlorogenic acid alleviated the increase of cytochrome c and caspase-3 expression caused by MCAO damage. CONCLUSIONS: The results of the present study showed that chlorogenic acid activates phospho-Akt and phospho-Bad and promotes the interaction between phospho-Bad and 14-3-3 during MCAO damage. In conclusion, chlorogenic acid exerts neuroprotective effects by activating the Akt-Bad signaling pathway and maintaining the interaction between phospho-Bad and 14-3-3 in ischemic stroke model.
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Isquemia Encefálica , Ácido Clorogénico , Accidente Cerebrovascular , Animales , Masculino , Ratas , Apoptosis , Proteína Letal Asociada a bcl/metabolismo , Isquemia Encefálica/veterinaria , Ácido Clorogénico/farmacología , Ácido Clorogénico/uso terapéutico , Modelos Animales de Enfermedad , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/veterinaria , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas Sprague-Dawley , Accidente Cerebrovascular/tratamiento farmacológico , Accidente Cerebrovascular/veterinaria , Proteínas 14-3-3/metabolismoRESUMEN
Objective: To investigate the effect of arsenic and its main metabolites on the apoptosis of human lung adenocarcinoma cell line A549 and the expression of pro-apoptotic genes Bad and Bik. Methods: In October 2020, A549 cells were recovered and cultured, and the cell viability was detected by the cell counting reagent CCK-8 to determine the concentration and time of sodium arsenite exposure to A549. The study was divided into NaAsO(2) exposure groups and metobol: le expoure groups: the metabolite comparison groups were subdivided into the control group, the monomethylarsinic acid exposure group (60 µmol/L) , and the dimethylarsinic acid exposure group (60 µmol/L) ; sodium arsenite dose groups were subdivided into 4 groups: control group (0) , 20, 40, 60 µmol/L sodium arsenite NaAsO(2). Hoechst 33342/propidium iodide double staining (Ho/PI) was used to observe cell apoptosis and real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression levels of Bad and Bik mRNA in cells after exposure. Western blotting was used to detect the protein expressions of Bad, P-Bad-S112, Bik, cleaved Bik and downstream proteins poly ADP-ribose polymerase PARP1 and cytochrome C (Cyt-C) , using spectrophotometry to detect the activity changes of caspase 3, 6, 8, 9. Results: Compared with the control group, the proportion of apoptotic cells in the 20, 40, and 60 µmol/L NaAsO(2) dose groups increased significantly (P<0.01) , and the expression levels of Bad, Bik mRNA, the protein expression levels of Bad, P-Bad-S112, Bik, cleaved Bik, PARP1, Cyt-C were increased (all P<0.05) , and the activities of Caspase 3, 6, 8, and 9 were significantly increased with significantly differences (P<0.05) . Compared with the control group, the expression level of Bad mRNA in the DMA exposure group (1.439±0.173) was increased with a significant difference (P=0.024) , but there was no significant difference in the expression level of Bik mRNA (P=0.788) . There was no significant differences in the expression levels of Bad and Bik mRNA in the poison groups (P=0.085, 0.063) . Compared with the control group, the gray values of proteins Bad, Bik, PARP1 and Cyt-C exposed to MMA were 0.696±0.023, 0.707±0.014, 0.907±0.031, 1.032±0.016, and there was no significant difference between the two groups (P=0.469, 0.669, 0.859, 0.771) ; the gray values of proteins Bad, Bik, PARP1 and Cyt-C exposed to DMA were 0.698±0.030, 0.705±0.022, 0.908±0.015, 1.029±0.010, and there was no difference between the two groups (P=0.479, 0.636, 0.803, 0.984) . Conclusion: Sodium arsenite induces the overexpression of Bad and Bik proteins, initiates the negative feedback regulation of phosphorylated Bad and the degradation of Bik, activates the downstream proteins PARP1, Cyt-C and Caspase pathways, and mediates the apoptosis of A549 cells.
Asunto(s)
Arsénico , Venenos , Células A549 , Adenosina Difosfato Ribosa/farmacología , Apoptosis , Proteínas Reguladoras de la Apoptosis , Arsenitos , Ácido Cacodílico/farmacología , Caspasa 3 , Caspasas/farmacología , Citocromos c/farmacología , Humanos , Proteínas Mitocondriales/farmacología , Propidio/farmacología , ARN Mensajero , Sincalida/farmacología , Compuestos de Sodio , Proteína Letal Asociada a bcl/metabolismoRESUMEN
Platyhelminthes can perhaps rightly be described as a phylum of the good, the bad, and the ugly: remarkable free-living worms that colonize land, river, and sea, which are often rife with color and can display extraordinary regenerative ability; parasitic worms like schistosomes that cause devastating disease and suffering; and monstrous tapeworms that are the stuff of nightmares. In this chapter, we will explore how our research expanded beyond free-living planarians to their gruesome parasitic cousins. We start with Schistosoma mansoni, which is not a new model; however, approaching these parasites from a developmental perspective required a reinvention that may hold generalizable lessons to basic biologists interested in pivoting to disease models. We then turn to our (re)establishment of the rat tapeworm Hymenolepis diminuta, a once-favorite model that had been largely forgotten by the molecular biology revolution. Here we tell our stories in three, first-person narratives in order to convey personal views of our experiences. Welcome to the dark side.
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Parásitos , Planarias , Animales , Humanos , Ratas , Proteína Letal Asociada a bclRESUMEN
A 68-year-old male presents with generalized lymphadenopathy and fever of short duration. Axillary lymph node excision was performed and was sent for histopathological evaluation. Microscopic evaluation of the submitted lymph node revealed diffuse proliferation of intermediate-sized atypical lymphoid cells with round nuclei, irregular membranes, finely dispersed chromatin, and inconspicuous nucleoli. Mitotic figures were frequently seen. Immunohistochemical evaluation revealed diffuse expression of CD20, CD5, CD10, B-cell lymphoma 2 (Bcl2), and B-cell lymphoma 6 (Bcl6). Atypical lymphoid cells were negative for cyclin D1; however, showed diffuse and strong nuclear expression of SOX11. MIB1 proliferation index was high (Ki67: 90%-95%). Based on morphological features and immunohistochemical findings a diagnosis of "cyclin D1 negative aggressive blastoid variant of mantle cell lymphoma (MCL)" was offered. The classic morphology of MCL is seen in 90% of cases, while the remaining (â¼10%) are considered as variants. A blastoid variant is an aggressive subtype that can lack expression of CD5 as well as cyclin D1, but instead expresses CD10, Bcl6, and CD23. SOX11 expression is seen in 90% cases of MCL and in almost 100% cases of cyclin D1 negative MCL. The current case highlights the unusual morphologic and aggressive variant of MCL and a significant role of SOX11 in its diagnosis.
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Linfoma de Células del Manto , Adulto , Anciano , Ciclina D1 , Humanos , Ganglios Linfáticos/patología , Linfoma de Células del Manto/diagnóstico , Linfoma de Células del Manto/patología , Masculino , Neprilisina , Proteínas Proto-Oncogénicas , Proteínas Represoras , Factores de Transcripción SOXC , Proteína Letal Asociada a bclRESUMEN
Studies have shown that the BH3-only domain Bad regulates brain development via the control of programmed cell death (PCD), but very few studies have addressed its effect on the molecular signaling of brain development in the system. In this work, we examined the novel role of zebrafish Bad in initial programmed cell death for brain morphogenesis through the priming of p53-mediated stress signaling. In a biological function study on the knockdown of Bad by morpholino oligonucleotides, at 24 h post-fertilization (hpf) Bad defects induced abnormal hindbrain development, as determined in a tissue section by means of HE staining which traced the damaged hindbrain. Then, genome-wide approaches for monitoring either the upregulation of apoptotic-related genes (11.8%) or the downregulation of brain development-related genes (29%) at the 24 hpf stage were implemented. The p53/caspase-8-mediated apoptotic death pathway was strongly involved, with the pathway being strongly reversed in a p53 mutant (p53M214K) line during Bad knockdown. Furthermore, we propose the involvement of a p53-mediated stress signal which is correlated with regulating Bad loss-mediated brain defects. We found that some major genes in brain development, such as crybb1, pva1b5, irx4a, pax7a, and fabp7a, were dramatically restored in the p53M214K line, and brain development recovered to return movement behavior to normal. Our findings suggest that Bad is required for (PCD) control, exerting a p53 stress signal on caspase-8/tBid-mediated death signaling and brain development-related gene regulation.
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Apoptosis/genética , Encéfalo/embriología , Encéfalo/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Pez Cebra/genética , Proteína Letal Asociada a bcl/genética , Animales , Animales Modificados Genéticamente , Caspasa 8/metabolismo , Regulación hacia Abajo/genética , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genoma , Mutación con Pérdida de Función/genética , Morfogénesis/genética , Rombencéfalo/embriología , Rombencéfalo/metabolismo , Natación , Proteínas de Pez Cebra/metabolismo , Proteína Letal Asociada a bcl/metabolismoRESUMEN
The design and development of a small molecule named NPB [3-{(4(2,3-dichlorophenyl)piperazin-1-yl}{2-hydroxyphenyl)methyl}-N-cyclopentylbenzamide], which specifically inhibited the phosphorylation of BAD at Ser99 in human carcinoma cells has been previously reported. Herein, the synthesis, characterization, and effect on cancer cell viability of NPB analogs, and the single-crystal X-ray crystallographic studies of an example compound (4r), which was grown via slow-solvent evaporation technique is reported. Screening for loss of viability in mammary carcinoma cells revealed that compounds such as 2[(4(2,3-dichlorophenyl)piperazin-1-yl][naphthalen-1-yl]methyl)phenol (4e), 5[(4(2,3-dichlorophenyl)piperazin-1-yl][2-hydroxyphenyl)methyl)uran-2-carbaldehyde (4f), 3[(2-hydroxyphenyl][4(p-tolyl)piperazin-1-yl)methyl)benzaldehyde (4i), and NPB inhibited the viability of MCF-7 cells with IC50 values of 5.90, 3.11, 7.68, and 6.5 µM, respectively. The loss of cell viability was enhanced by the NPB analogs synthesized by adding newer rings such as naphthalene and furan-2-carbaldehyde in place of N-cyclopentyl-benzamide of NPB. Furthermore, these compounds decreased Ser99 phosphorylation of hBAD. Additional in silico density functional theory calculations suggested possibilities for other analogs of NPB that may be more suitable for further development.
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Nitrobencenos/química , Proteína Letal Asociada a bcl/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cristalografía por Rayos X , Teoría Funcional de la Densidad , Femenino , Humanos , Células MCF-7 , Conformación Molecular , Nitrobencenos/farmacología , Fosforilación/efectos de los fármacos , Serina/metabolismoRESUMEN
The widespread use of chlorothalonil (CTL) has caused environmental residues and food contamination. Although the intestinal epithelial barrier (IEB) is directly involved in the metabolism and transportation of various exogenous compounds, there are few studies on the toxic effects of these compounds on the structure and function of IEB. The disassembly of tight junction (TJ) is a major cause of intestinal barrier dysfunction under exogenous compounds intake, but the precise mechanisms are not well understood. Here, we used Caco-2 cell monolayers as an in vitro model of human IEB to evaluate the toxicity of CTL exposure on the structure and function of IEB. Results showed that CTL exposure increased the paracellular permeability of the monolayers and downregulated mRNA levels of the TJ genes (ZO-1, OCLN, and CLDN1), polarity marker gene (SI), and anti-apoptosis gene (BCL-2) but upregulated the mRNA levels of apoptosis-related genes, including BAD, BAX, CASP3, and CASP8. Western blot analysis and immunofluorescence assay results showed the decreased levels and disrupted distribution of TJ protein network, including ZO-1 and CLDN1 in CTL-exposed IEB. In addition, the accumulation of intracellular reactive oxygen species, decreased mitochondrial membrane potential, and increased active CASP3 expression were observed in treated IEB. The result of TUNEL assay further confirmed the occurrence of cell apoptosis after CTL exposure. In addition, the phosphorylation of mitogen-activated protein kinases, including ERK, JNK and p38, was increased in CTL-exposed IEB. In summary, our results demonstrated that CTL exposure induced IEB dysfunction in Caco-2 cell monolayers by activating the mitogen-activated protein kinase pathway.
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Quinasas MAP Reguladas por Señal Extracelular/genética , Fungicidas Industriales/toxicidad , Mucosa Intestinal/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Nitrilos/toxicidad , Uniones Estrechas/efectos de los fármacos , Células CACO-2 , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 8/genética , Caspasa 8/metabolismo , Claudina-1/genética , Claudina-1/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación de la Expresión Génica , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Modelos Biológicos , Ocludina/genética , Ocludina/metabolismo , Permeabilidad/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Uniones Estrechas/metabolismo , Proteína de la Zonula Occludens-1/genética , Proteína de la Zonula Occludens-1/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismo , Proteína Letal Asociada a bcl/genética , Proteína Letal Asociada a bcl/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The novel anti-neoplastic glycopeptide T11TS retards glioma both in in-vitro clinical samples and in-vivo models. This study investigates the correlation between altering the glioma microenvironment with glioma arrest and death. Flow cytometry, immunoblotting, ELISA, and co-immunoprecipitation were employed to investigate glioma cell arrest and death. Results include a decline in phosphorylation of Akt and attenuation of p21 phosphorylation (Thr145,Ser146) and disassociation of p-Akt-Mdm2 and p-Akt-BAD facilitating death by Akt>BAD. T11TS influence phosphorylation patterns in two focal axes Akt>p21 and Akt>Mdm2>p53. The current article provides crucial insight in deciphering the mechanism of T11TS induced glioma cell arrest and death.
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Neoplasias Encefálicas/tratamiento farmacológico , Antígenos CD58/farmacología , Glioma/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Antígenos CD58/uso terapéutico , Puntos de Control del Ciclo Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Femenino , Glioma/metabolismo , Glioma/patología , Masculino , Fosfohidrolasa PTEN/análisis , Fosforilación , Proteínas Proto-Oncogénicas c-mdm2/análisis , Ratas , Ratas Wistar , Microambiente Tumoral , Proteína p53 Supresora de Tumor/análisis , Proteína Letal Asociada a bcl/metabolismoRESUMEN
We studied the effect of technogenic radiation on the degree of promoter methylation in genes involved in apoptosis in blood lymphocytes of workers exposed to long-term γ-radiation during their professional activities. Blood samples for the analysis were obtained from 11 conventionally healthy men aged from 54 to 71 years (mean 66 years), workers of the Siberian Group of Chemical Enterprises working experience from 27 to 40 years (mean 30 years); the external exposure dose was 175.88 mSv (158.20-207.81 mSv). In all examined subjects, the degree of methylation of the promoters of apoptosis-related genes ranged from 0.22 to 50.00%. A correlation was found between the degree of methylation of BCLAF1 promoters (p=0.035) with the age of workers, BAX promoters (p=0.0289) with high content of aberrant cells, and APAF1 promoters (p=0.0152) with increased number of dicentric chromosomes. A relationship was found between the dose of external irradiation and the degree of methylation of gene promoters of BAD (p=0.0388), BID (Ñ=0.0426), and HRK (Ñ=0.0101) genes.
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Aberraciones Cromosómicas/efectos de la radiación , Metilación de ADN , Epigénesis Genética , Linfocitos/efectos de la radiación , Exposición Profesional/efectos adversos , Regiones Promotoras Genéticas , Exposición a la Radiación/efectos adversos , Anciano , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Factor Apoptótico 1 Activador de Proteasas/genética , Factor Apoptótico 1 Activador de Proteasas/metabolismo , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/genética , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Aberraciones Cromosómicas/clasificación , Rayos gamma/efectos adversos , Humanos , Linfocitos/metabolismo , Linfocitos/patología , Masculino , Persona de Mediana Edad , Radiometría , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Siberia , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismo , Proteína Letal Asociada a bcl/genética , Proteína Letal Asociada a bcl/metabolismoRESUMEN
Activation of the apoptotic pathway is a major cause of progressive loss of function in chronic diseases such as neurodegenerative and diabetic kidney diseases. There is an unmet need for an anti-apoptotic drug that acts in the early stage of the apoptotic process. The multifunctional protein Na+,K+-ATPase has, in addition to its role as a transporter, a signaling function that is activated by its ligand, the cardiotonic steroid ouabain. Several lines of evidence suggest that sub-saturating concentrations of ouabain protect against apoptosis of renal epithelial cells, a common complication and major cause of death in diabetic patients. Here, we induced apoptosis in primary rat renal epithelial cells by exposing them to an elevated glucose concentration (20 mM) and visualized the early steps in the apoptotic process using super-resolution microscopy. Treatment with 10 nM ouabain interfered with the onset of the apoptotic process by inhibiting the activation of the BH3-only protein Bad and its translocation to mitochondria. This occurred before the pro-apoptotic protein Bax had been recruited to mitochondria. Two ouabain regulated and Akt activating Ca2+/calmodulin-dependent kinases were found to play an essential role in the ouabain anti-apoptotic effect. Our results set the stage for further exploration of ouabain as an anti-apoptotic drug in diabetic kidney disease as well as in other chronic diseases associated with excessive apoptosis.
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Apoptosis , Citoprotección , Glucosa/toxicidad , Microscopía , Transducción de Señal , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Proteína Letal Asociada a bcl/metabolismo , Animales , Apoptosis/efectos de los fármacos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Citoprotección/efectos de los fármacos , Citosol/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Riñón/patología , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Modelos Biológicos , Ouabaína/farmacología , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Proteína X Asociada a bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMEN
The BH3-only molecule Bad regulates cell death via its differential protein phosphorylation, but very few studies address its effect on early embryonic development in vertebrate systems. In this work, we examined the novel role of zebrafish Bad in the initial programmed cell death (PCD) for brain morphogenesis through reducing environmental stress and cell death signaling. Bad was considered to be a material factor that because of the knockdown of Bad by morpholino oligonucleotides, PCD was increased and the reactive oxygen species (ROS) level was enhanced, which correlated to trigger a p53/caspase-8 involving cell death signaling. This Bad knockdown-mediated environmental stress and enhanced cell dying can delay normal cell migration in the formation of the three germ layers, especially the ectoderm, for further brain development. Furthermore, Bad defects involved in three-germ-layers development at 8 hpf were identified by in situ hybridization approach on cyp26, rtla, and Sox17 pattern expression markers. Finally, the Bad knockdown-induced severely defected brain was examined by tissue section from 24 to 48 h postfertilization (hpf), which correlated to induce dramatic malformation in the hindbrain. Our data suggest that the BH3-only molecule Bad regulates brain development via controlling programmed cell death on overcoming environmental stress for reducing secondary cell death signaling, which suggests that correlates to brain developmental and neurological disorders in this model system.
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Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Desarrollo Embrionario , Pez Cebra/embriología , Pez Cebra/metabolismo , Proteína Letal Asociada a bcl/metabolismo , Animales , Apoptosis , Encéfalo/patología , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Genes p53 , Morfolinos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Proteína Letal Asociada a bcl/genéticaRESUMEN
Melanoma represents one of the most aggressive and drug resistant skin cancers with poor prognosis in its advanced stages. Despite the increasing number of targeted therapies, novel approaches are needed to counteract both therapeutic resistance and the side effects of classic therapy. Betulinic acid (BA) is a bioactive phytocompound that has been reported to induce apoptosis in several types of cancers including melanomas; however, its effects on mitochondrial bioenergetics are less investigated. The present study performed in A375 human melanoma cells was aimed to characterize the effects of BA on mitochondrial bioenergetics and cellular behavior. BA demonstrated a dose-dependent inhibitory effect in both mitochondrial respiration and glycolysis in A375 melanoma cells and at sub-toxic concentrations (10 µM) induced mitochondrial dysfunction by eliciting a decrease in the mitochondrial membrane potential and changes in mitochondria morphology and localization. In addition, BA triggered a dose-dependent cytotoxic effect characterized by apoptotic features: morphological alterations (nuclear fragmentation, apoptotic bodies) and the upregulation of pro-apoptotic markers mRNA expression (Bax, Bad and Bak). BA represents a viable therapeutic option via a complex modulatory effect on mitochondrial metabolism that might be useful in advanced melanoma or as reliable strategy to counteract resistance to standard therapy.
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Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Melanocitos/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Triterpenos Pentacíclicos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Apoptosis/genética , Línea Celular Tumoral , Regulación de la Expresión Génica , Glucólisis/efectos de los fármacos , Glucólisis/genética , Humanos , Concentración 50 Inhibidora , Melanocitos/metabolismo , Melanocitos/patología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/genética , Mitocondrias/metabolismo , Fosforilación Oxidativa/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/agonistas , Transducción de Señal , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismo , Proteína Letal Asociada a bcl/genética , Proteína Letal Asociada a bcl/metabolismo , Ácido BetulínicoRESUMEN
BACKGROUND: Hypertension is a leading cause of chronic kidney disease worldwide. Early studies demonstrated the short-term effects of hypertension on kidney function and morphology in ablative nephropathy. The aim of this study was to investigate the long-term consequences of hypertension in 5/6 nephrectomy (5/6NE) model. METHODS: Reduction of the kidney mass by 5/6NE was created in spontaneous hypertensive rats (SHR) and genetically similar normotensive Wistar Kyoto (WKY) rats. Blood pressure, serum creatinine (SCr), hematuria, and proteinuria were monitored weekly for 23 weeks. Kidney morphology was assessed at the end of the study. Sham-operated rats from both strains were used as controls. RESULTS: Rats with 5/6NE had increased SCr, blood pressure, hematuria, and proteinuria in both SHR and WKY. Even though the SCr levels and blood pressure were greater in 5/6NE SHR as compared with 5/6NE WKY rats, absolute changes from sham-operated rats were not statistically significant between these 2 groups. 5/6NE SHR had earlier onset and higher proteinuria than 5/6NE WKY rats. Hematuria was similar in 5/6NE SHR and 5/6NE WKY rats. However, 5/6NE SHR had enlarged glomeruli, increased interstitial fibrosis, and prominent intimal thickening in the small arteries/arterioles as compared with 5/6NE WKY rats. CONCLUSIONS: The long-term severity of kidney injury correlated with higher blood pressure. Reduction of the kidney mass increases SCr, hematuria, proteinuria, and blood pressure in both normotensive and hypertensive rats. Histological assessment provides better information about underlying chronic kidney injury than actual changes in SCr and urinalysis.
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Hematuria , Hipertensión , Riñón , Animales , Ratas , Proteína Letal Asociada a bcl/farmacología , Presión Sanguínea , Creatinina , Hematuria/patología , Hipertensión/patología , Riñón/fisiopatología , Proteinuria/etiología , Ratas Endogámicas SHR , Ratas Endogámicas WKYRESUMEN
The BCL2-associated agonist of cell death protein is a key participant in apoptosis dependent on mitochondria and in disease progression that involves the regulation of cell death, such as tumorigenesis, diabetes, sepsis shock, and epilepsy. Nevertheless, the mechanisms underlying the immune responses to teleost BAD bacterial infection and mitochondrial-dependent apoptosis remains unclear. In order to elucidate the mechanisms involved, in this study, a Ctenopharyngodon idella (grass carp) BAD gene named GcBAD1 was firstly cloned and characterized. The results indicated that the ORF (open reading frame) of GcBAD1 was 438 bp in length, encoding a 145-amino acid putative protein of 16.66 kDa. This deduced amino acid sequence has a better identity than another teleost species according to a phylogenetic analysis, and contains a Bcl2-BAD-1 domain. In healthy grass carp fish, the mRNA transcripts of GcBAD1 were widely present in the studied tissues, which could be ranked as follows; spleen > brain > middle-kidney > head-kidney > liver > gills > intestines > heart and muscle. In addition, during infection by Aeromonas hydrophila and Staphylococcus aureus, the mRNA transcription and protein levels expression of GcBAD1 in the head-kidney, spleen, and liver tissues of the fish were significantly up-regulated. Moreover, when the C. idellus kidney cell line (CIK) cells were incubated with Lipopolysaccharide (LPS) and lipoteichoic acid (LTA), the GcBAD1 expression transcripts were also significantly up-regulated. Additionally, overexpression of GcBAD1 in CIK cells was able to activate apoptosis-related genes, including those encoding p53, Cytochrome C (CytoC), caspase-3, and caspase-9. Besides, in the TUNEL assays, when pEGFP-BAD1 was over-expressed, the number of red signals associated with apoptosis were significantly increased in the CIK cells infected with LPS or LTA at 12 h. This study demonstrates that GcBAD1 has a significant role in the mitochondrial apoptosis pathway of grass carp's innate immunity. Our findings provide new insight into the potential mechanisms of teleost antibacterial immunity.
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Aeromonas hydrophila/fisiología , Carpas/inmunología , Proteínas de Peces/metabolismo , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/fisiología , Proteína Letal Asociada a bcl/metabolismo , Secuencia de Aminoácidos , Animales , Apoptosis , Línea Celular , Clonación Molecular , Proteínas de Peces/genética , Inmunidad Innata , Lipopolisacáridos/inmunología , Filogenia , Bazo , Transcriptoma , Regulación hacia Arriba , Proteína Letal Asociada a bcl/genéticaRESUMEN
OBJECTIVES: The aim of the current study was to identify the long noncoding RNAs (lncRNAs) ANRIL function and molecular pathways underlying hepatocellular carcinoma progression. METHODS: ANRIL knockdown with specific siRNA, and transfected into HepG2 cells according to the protocol of Lipofectamine 2000. Cell proliferation, apoptosis, migration and metastasis were assessed with MTT assay, flow cytometry and wound healing assay, respectively. Moreover, the expression level of ANRIL, apoptosis-related genes, and the Wnt pathway-associated genes were assessed by real time-PCR and Western blot assay. KEY FINDINGS: Knocking down of ANRIL led to alleviated cell growth and increased cell apoptosis of HepG2 cells through markedly increased expression levels of Bax and Bad. In contrast, dramatically diminished the expressions of anti-apoptotic factors including Bid and Bcl-2 in comparison to the scrambled control group (si-NC). Furthermore, ANRIL silencing resulted in an inactivated Wnt/ß-catenin pathway by suppressing key genes associated with this pathway. CONCLUSIONS: Taken together, these findings imply new insights into the regulatory network of the Wnt pathway through lncRNA ANRIL that indicate ANRIL may be a therapeutic factor potential for hepatocellular carcinoma.
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Apoptosis , Carcinoma Hepatocelular , Silenciador del Gen , Neoplasias Hepáticas , ARN Largo no Codificante , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Ensayos de Migración Celular/métodos , Proliferación Celular , Supervivencia Celular , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen/métodos , Genes bcl-2/genética , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Análisis de Secuencia de ARN , Vía de Señalización Wnt/genética , Proteína Letal Asociada a bcl/genéticaRESUMEN
Elucidation of non-canonical protein functions can identify novel tissue homeostasis pathways. Herein, we describe a role for the Bcl-2 family member BAD in postnatal mammary gland morphogenesis. In Bad3SA knock-in mice, where BAD cannot undergo phosphorylation at 3 key serine residues, pubertal gland development is delayed due to aberrant tubulogenesis of the ductal epithelium. Proteomic and RPPA analyses identify that BAD regulates focal adhesions and the mRNA translation repressor, 4E-BP1. These results suggest that BAD modulates localized translation that drives focal adhesion maturation and cell motility. Consistent with this, cells within Bad3SA organoids contain unstable protrusions with decreased compartmentalized mRNA translation and focal adhesions, and exhibit reduced cell migration and tubulogenesis. Critically, protrusion stability is rescued by 4E-BP1 depletion. Together our results confirm an unexpected role of BAD in controlling localized translation and cell migration during mammary gland development.