RESUMEN
Thimerosal (THIM) induces neurotoxic changes including neuronal death and releases apoptosis inducing factors from mitochondria to cytosol. THIM alters the expression level of factors involved in apoptosis. On the other hand, the anti-apoptotic effects of exercise have been reported. In this study, we aimed to discover the effect of three protocols of treadmill exercise on the expression level of mitochondrial transcription factor A (TFAM), peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), BCL-2-associated death (BAD), BCL-2-associated X (BAX), BCL-XL, and BCL-2 (a pro-survival BCL-2 protein) in the hippocampus of control and THIM-exposed rats. Male Wistar rats were used in this research. Real-time PCR was applied to assess genes expression. The results showed that THIM increased the expression of pro-apoptotic factors (BAD and BAX), decreased the expression of anti-apoptotic factors (BCL-2 and BCL-XL), and decreased the expression of factors involved in mitochondrial biogenesis (TFAM and PGC-1α). Treadmill exercise protocols reversed the effect of THIM on all genes. In addition, treadmill exercise protocols decreased the expression of BAD and BAX, increased the expression of BCL-2, and increased the expression of TFAM and PGC-1α in control rats. In conclusion, THIM induced a pro-apoptotic effect and disturbed mitochondrial biogenesis and stability, whereas treadmill exercise reversed these effects.
Asunto(s)
Prueba de Esfuerzo/métodos , Hipocampo/efectos de los fármacos , Condicionamiento Físico Animal/fisiología , Timerosal/toxicidad , Animales , Expresión Génica , Hipocampo/metabolismo , Masculino , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/biosíntesis , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Condicionamiento Físico Animal/métodos , Conservadores Farmacéuticos/toxicidad , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Ratas , Ratas Wistar , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Proteína X Asociada a bcl-2/biosíntesis , Proteína X Asociada a bcl-2/genética , Proteína Letal Asociada a bcl/biosíntesis , Proteína Letal Asociada a bcl/genética , Proteína bcl-X/biosíntesis , Proteína bcl-X/genéticaRESUMEN
BACKGROUND: Non-small cell lung cancer (NSCLC) is one of the most widespread cancer with increasing morbidity and mortality. FAS-associated protein with death domain (FADD) is considered as an essential instrument in cell death, whereas Bcl-XS promotes apoptosis through inhibiting the activity of Bcl-2 and Bcl-XL. OBJECTIVE AND METHODS: We detected the expression of FADD and Bcl-XS in resected NSCLC tissues by immunohistochemistry, and investigated their association with clinicopathological characteristics and prognostic significance of NSCLC patients. RESULTS: Bcl-XS expression was significantly increased in well and moderate differentiated lung SCC (P= 0.004). Lung ADC patients with overexpression of FADD and lung SCC patients with low expression of Bcl-XS had importantly lower overall survival rates by Kaplan-Meier analysis (P= 0.033, P= 0.02, respectively). Multivariate analysis confirmed that elevated expression of FADD was an independent poor prognostic factor for patients with surgically resected lung ADC (P= 0.027) and increased expression of Bcl-XS was an independent good prognostic factor for patients with surgically resected lung SCC (P= 0.016)CONCLUSION: Elevated expression of FADD was identified as independent poor prognostic factor for patients with surgically resected lung ADC, however, increased expression of Bcl-XS was an independent good prognostic biomarker for patients with surgically resected lung SCC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/biosíntesis , Neoplasias Pulmonares/metabolismo , Proteína bcl-X/biosíntesis , Adulto , Anciano , Biomarcadores de Tumor/biosíntesis , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/cirugía , Femenino , Humanos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/cirugía , Masculino , Persona de Mediana Edad , PronósticoRESUMEN
Imatinib mesylate (IM) resistance has become a major clinical problem for chronic myeloid leukaemia (CML). It is known that Bcl-x splicing is deregulated and is involved in multiple malignant cancer initiation and chemotherapy resistance, including CML. The aim of the present study was to correct the abnormal splicing of Bcl-x in CML and investigate the subsequent malignant phenotype changes, especially response to IM. The aberrant Bcl-x splicing in CML cells was effectively restored using vivo-Morpholino Antisense Oligomer (vMO). CCK-8 cell viability assay and flow cytometry showed that restoring of Bcl-x splicing increases IM-induced growth inhibition and apoptosis of K562 cells. Moreover, a more significant similar phenomenon was observed in imatinib-resistant CML cell lines K562/G01. Finally, establishment of CML xenograft model had also proved that correcting Bcl-x splicing in vivo can also enhance the anti-tumor effect of IM. Our findings suggest that vMO co-operating with IM can effectively increase the sensitivity of CML cells to IM both in vitro and in vivo, and Bcl-x splicing could become good candidates for chemotherapy-sensitized target in IM-resistant CML.
Asunto(s)
Resistencia a Antineoplásicos , Mesilato de Imatinib/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva , Morfolinos/farmacología , Neoplasias Experimentales , Empalme del ARN/efectos de los fármacos , Proteína bcl-X , Animales , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Humanos , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína bcl-X/biosíntesis , Proteína bcl-X/genéticaRESUMEN
OBJECTIVE: The aim of this study was to investigate the role of interleukin-1ß (IL-1ß) in the apoptosis of synovial cells in rheumatoid arthritis (RA) rats, and to explore the underlying mechanism. MATERIALS AND METHODS: The apoptosis of the synovial cells in RA rats in the IL-1ß group and the control group was analyzed by scoring under an electron microscope. The expressions of cleaved-poly (ADP-ribose) polymerase (PARP), PARP and anti-apoptosis gene products in synovial cells of IL-1ß treated RA rats were explored as well. Meanwhile, the expressions of B-cell lymphoma 2 (Bcl-2), Bcl-xL, and Active-Caspase3 in the synovial cells of RA rats with IL-1ß treatment were evaluated by the Western blotting. To further clarify the relationship between IL-1ß and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway in the synovial cells of RA rats, the expressions of NF-κB regulated the gene products of matrix metalloproteinase-3 (MMP-3), MMP-9, cyclooxygenase-2 (Cox-2), and vascular endothelial growth factor (VEGF) in synovial cells of RA rats after that we investigated the treatment with IL-1ß (was investigated). In addition, the expression of NF-κB in the synovial cells of RA rats treated with IL-1ß was determined. RESULTS: The results showed that, compared with the control group, IL-1ß treatment significantly increased the number of apoptotic cells. This meant that IL-1ß treatment could promote the apoptosis of the synovial cells (p<0.05). IL-1ß treatment significantly promoted the expression level of cleaved-PARP (p<0.05). However, it remarkably reduced the expressions of Bcl-2 and Bcl-xL (p<0.05). Meanwhile, the level of the active-Caspase3 in the synovial cells of RA rats treated with IL-1ß was significantly enhanced (p<0.01). In comparison with the control group, the IL-1ß group exhibited significantly elevated expressions of NF-κB-regulated gene products in the synovial cells of RA rats (p<0.01). Besides, the positive markers of the activated NF-κB were detected in the synovial cells of RA rats in the IL-1ß group and the control group. The results demonstrated that they were mainly located in the nucleus of the IL-1ß group. CONCLUSIONS: IL-1ß can promote the apoptosis of the synovial cells in RA rats via the NF-κB pathway.
Asunto(s)
Apoptosis/fisiología , Artritis Reumatoide/fisiopatología , Interleucina-1beta/fisiología , FN-kappa B/fisiología , Sinoviocitos/fisiología , Animales , Apoptosis/efectos de los fármacos , Artritis Reumatoide/inducido químicamente , Artritis Reumatoide/metabolismo , Caspasa 3/biosíntesis , Células Cultivadas , Ciclooxigenasa 2/biosíntesis , Adyuvante de Freund , Interleucina-1beta/farmacología , Masculino , Metaloproteinasas de la Matriz/biosíntesis , FN-kappa B/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Ratas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Sinoviocitos/metabolismo , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Proteína bcl-X/biosíntesisRESUMEN
Physiological stress conditions attenuate global mRNA translation via modifications of key eukaryotic initiation factors. However, non-canonical translation initiation mechanisms allow cap-independent translation of certain mRNAs. We have previously demonstrated that eIF5B promotes cap-independent translation of the mRNA encoding the antiapoptotic factor, XIAP, during cellular stress. Here, we show that depletion of eIF5B sensitizes glioblastoma multiforme cells to TRAIL-induced apoptosis by a pathway involving caspases-8, -9, and -7, with no significant effect on cell cycle progression. eIF5B promotes evasion of apoptosis by promoting the translation of several IRES-containing mRNAs, encoding the antiapoptotic proteins XIAP, Bcl-xL, cIAP1, and c-FLIPS. We also show that eIF5B promotes translation of nuclear factor erythroid 2-related factor 2 and suggest that reactive oxygen species contribute to increased apoptosis under conditions of eIF5B depletion. Finally, eIF5B depletion leads to decreased activation of the canonical NF-κB pathway. Taken together, our data suggest that eIF5B represents a regulatory node, allowing cancer cells to evade apoptosis by promoting the translation of pro-survival proteins from IRES-containing mRNAs.
Asunto(s)
Factores Eucarióticos de Iniciación/genética , Glioblastoma/genética , Glioblastoma/patología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Línea Celular Tumoral , Supervivencia Celular/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Resistencia a Antineoplásicos , Factores Eucarióticos de Iniciación/deficiencia , Factores Eucarióticos de Iniciación/metabolismo , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Células HEK293 , Humanos , Proteínas Inhibidoras de la Apoptosis/biosíntesis , Proteínas Inhibidoras de la Apoptosis/genética , Factor 2 Relacionado con NF-E2/biosíntesis , Factor 2 Relacionado con NF-E2/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Proteínas Recombinantes/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Transfección , Regulación hacia Arriba , Proteína Inhibidora de la Apoptosis Ligada a X/biosíntesis , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína bcl-X/biosíntesis , Proteína bcl-X/genéticaRESUMEN
miR-214 has been recently found to be significantly downregulated in calcified human aortic valves (AVs). ER stress, especially the ATF4-mediated pathway, has also been shown to be significantly upregulated in calcific AV disease. Since elevated cyclic stretch is one of the major mechanical stimuli for AV calcification and ATF4 is a validated target of miR-214, we investigated the effect of cyclic stretch on miR-214 expression as well as those of ATF4 and two downstream genes (CHOP and BCL2L1). Porcine aortic valve (PAV) leaflets were cyclically stretched at 15% for 48 h in regular medium and for 1 week in osteogenic medium to simulate the early remodeling and late calcification stages of stretch-induced AV disease, respectively. For both stages, 10% cyclic stretch served as the physiological counterpart. RT-qPCR revealed that miR-214 expression was significantly downregulated during the late calcification stage, whereas the mRNA expression of ATF4 and BCL2L1 was upregulated and downregulated, respectively, during both early remodeling and late calcification stages. When PAV leaflets were statically transfected with miR-214 mimic in osteogenic medium for 2 weeks, calcification was significantly reduced compared to the control mimic case. This implies that miR-214 may have a protective role in stretch-induced calcific AV disease.
Asunto(s)
Estenosis de la Válvula Aórtica/metabolismo , Válvula Aórtica/metabolismo , Válvula Aórtica/patología , Calcinosis/metabolismo , Regulación de la Expresión Génica , MicroARNs/metabolismo , Regulación hacia Arriba , Factor de Transcripción Activador 4/biosíntesis , Animales , Estenosis de la Válvula Aórtica/patología , Calcinosis/patología , Porcinos , Factor de Transcripción CHOP/biosíntesis , Proteína bcl-X/biosíntesisRESUMEN
PURPOSE: Cleft palate transmembrane protein 1 (Clptm1) and its paralog protein, Cisplatin resistance-related protein 9 (CRR9) constitute a highly conserved protein family, from Caenorhabditis elegans to Homo sapiens. In the present study, we examined the clinicopathological and biological significance of Clptm1 and CRR9 expression in oral squamous cell carcinoma (OSCC). METHODS: Ninety-eight OSCC tissue specimens were immunohistochemically stained with specific antibodies to Clptm1 and CRR9. The immunoreactivity of Clptm1 and CRR9 was then correlated with clinicopathological factors, including the prognosis of patients. siRNA-mediated gene silencing of CRR9 followed by cell proliferation, Matrigel invasion, anoikis assay, and gelatin zymography were performed using cultured OSCC cells. Subsequently, immunohistochemical examination including double staining was performed to determine the correlation between CRR9 and Bcl-xL expression in OSCC cells. RESULTS: Non-tumorous oral squamous cells exhibited vague, weak, or little cytoplasmic staining with anti-Clptm1 and CRR9 antibodies. By contrast, robust Clptm1 and CRR9 immunoreactivity was found at the cancer invasion front in 55 and 54 of the 98 OSCC tissue specimens, respectively. Notably, CRR9 immunoreactivity was associated with more than 5 mm of depth of invasion, poor prognosis of the patients, and smoking habits (P < 0.05). siRNA-mediated gene silencing of CRR9 did not alter the cell proliferation but decreased Matrigel invasion and impaired anoikis resistance in cultured Ca9-22 and SAS cells. CRR9 and anti-apoptotic Bcl-xL expression levels were correlated in pT1 OSCC tissue specimens. CONCLUSIONS: Clptm1 and CRR9 were overexpressed in many OSCC tissues. In particular, CRR9 expression may promote tumor development and have a significant poor prognostic value in OSCC, possibly through conferring invasion ability and resistance to apoptotic stimuli possibly related to Bcl-xL expression. CRR9 could be a novel molecular target for patients with OSCC.
Asunto(s)
Proteínas de la Membrana/biosíntesis , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Línea Celular Tumoral , Femenino , Silenciador del Gen , Humanos , Inmunohistoquímica , Masculino , Proteínas de la Membrana/genética , Persona de Mediana Edad , Neoplasias de la Boca/genética , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Estadificación de Neoplasias , Pronóstico , Estudios Retrospectivos , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Proteína bcl-X/biosíntesis , Proteína bcl-X/genéticaRESUMEN
AIMS: As a fifth most common cancer type, Hepatocellularcarcinoma (HCC) ranked third leading cause of cancer deaths worldwide. Arsenic trioxide (As2O3) is known as chemotherapeutic agent against few cancer including Acute promyelocyticleukemia and solid tumors. But its effect and possible associated mechanism in HCC is meager. Present study aimed to assess As2O3 modulatory effect on liver cancer by assessing cell growth and viability. METHODS: Liver normal (Chang liver) and cancerous cells (Hep3B) were exposed to different concentration's (0, 1, 5, 10 & 15⯵M) of As2O3 at different intervals (24, 48 & 72â¯h). Cell growth was assessed microscopically, and Cytotoxicity assays were done through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Water-soluble tetrazolium salt (WST) growth inhibition assays. Cell viability was studied by trypan blue staining. Apoptosis was analyzed by Annexin V/PI assay, and expression of genes (Notch and anti-apoptotic) were determined through western blotting and Q-PCR method. KEY FINDINGS: A significant reduction in cell growth and viability was reported in liver cancerous cells as compare to normal cells at 5⯵M As2O3. Consistently, As2O3 induced apoptosis along with down-regulation of anti-apoptotic protein Bcl-xL, and up regulates expression of Notch that leads towards apoptosis. SIGNIFICANCE: Results clearly suggest that As2O3 restricted growth and induces apoptosis more in liver cancer cells as compared to normal cells. This finding suggests that it could be a promising potential therapeutic agent against liver cancer which need further testing by in-vivo investigations.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Arsenicales/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Óxidos/farmacología , Trióxido de Arsénico , Carcinoma Hepatocelular/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Humanos , Neoplasias Hepáticas/patología , Receptores Notch/biosíntesis , Receptores Notch/efectos de los fármacos , Proteína bcl-X/antagonistas & inhibidores , Proteína bcl-X/biosíntesisRESUMEN
Protein phosphatase 2A (PP2A) is an important enzyme within various signal transduction pathways. The present study was investigated PP2A mediates JS-K-induced apoptosis by affecting Bcl-2 family protein. JS-K showed diverse inhibitory effects in five HCC cell lines, especially HepG2 cells. JS-K caused a dose- and time-dependent reduction in cell viability and increased in levels of LDH release. Meanwhile, JS-K- induced apoptosis was characterized by mitochondrial membrane potential reduction, Hoechst 33342+ /PI+ dual staining, release of cytochrome c (Cyt c), and activation of cleaved caspase-9/3. Moreover, JS-K-treatment could lead to the activation of protein phosphatase 2A-C (PP2A-C), decrease of anti-apoptotic Bcl-2 family-protein expression including p-Bcl-2 (Ser70), Bcl-2, Bcl-xL, and Mcl-1 as well as the increase of pro-apoptosis Bcl-2 family-protein including Bim, Bad, Bax, and Bak. Furthermore, JS-K caused a marked increase of intracellular NO levels while pre-treatment with Carboxy-PTIO (a NO scavenger) reduced the cytotoxicity effects and the apoptosis rate. Meanwhile, pre-treatment with Carboxy-PTIO attenuated the JS-K-induced up-regulation of PP2A, Cyt c, and cleaved-caspase-9/3 activation. The silencing PP2A-C by siRNA could abolish the activation of PP2A-C, down-regulation of anti-apoptotic Bcl-2 family-protein (p-Bcl-2, Bcl-2, Bcl-xL, and Mcl-1), increase of pro-apoptosis Bcl-2 family-protein (Bim, Bad, Bax, and Bak) and apoptotic-related protein (Cyt c, cleaved caspase-9/3) that were caused by JS-K in HepG2 cells. In addition, pre-treatment with OA (a PP2A inhibitor) also attenuated the above effects induced by JS-K. In summary, NO release from JS-K induces apoptosis through PP2A activation, which contributed to the regulation of Bcl-2 family proteins.
Asunto(s)
Apoptosis/efectos de los fármacos , Compuestos Azo/farmacología , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Piperazinas/farmacología , Proteína Fosfatasa 2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Carcinoma Hepatocelular/patología , Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/biosíntesis , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína Fosfatasa 2/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteína bcl-X/biosíntesis , Proteína bcl-X/genéticaRESUMEN
Several miRNAs have been demonstrated to be involved in endothelial dysfunction during atherosclerosis (AS). However, the detailed roles and underlying mechanisms of miR-34a in AS-associated endothelial cell apoptosis are far from being addressed. Apolipoprotein E-deficient (ApoE-/-) mice fed with high-fat diet (HFD) were used as in vivo model of AS. Oxidized low-density lipoprotein (ox-LDL)-treated human aortic endothelial cells (HAECs) were applied as in vitro model of AS. The effects of miR-34a on atherosclerotic lesions were evaluated by hematoxylin-eosin (HE) and Oil Red O staining. Pecam-1+ endothelial cells were isolated from the aortic arch with flow cytometry. qRT-PCR and western blot were employed to measure gene and protein expression. The effects of miR-34a on cell viability, cell cycle distribution, and apoptosis were assessed by Cell counting kit (CCK)-8 and flow cytometry analysis. The relationship between miR-34a and Bcl-2 was confirmed by online softwares, luciferase reporter assay, and RNA immunoprecipitation (RIP). miR-34a was upregulated in HFD-induced ApoE-/- mice and ox-LDL-treated HAECs. Anti-miR-34a decreased atherosclerotic lesions and inhibited Pecam-1+ endothelial cells apoptosis in HFD-induced ApoE-/- mice. Moreover, anti-miR-34a significantly promoted cell viability, alleviated cell cycle arrest, and restrained apoptosis in ox-LDL-treated HAECs. Furthermore, Bcl-2 was identified as a target of miR-34a, and miR-34a inhibited Bcl-2 expression via binding to its 3'UTR. Rescue experiments demonstrated that Bcl-2 overexpression dramatically reversed miR-34a-mediated inhibition of cell growth and promotion of apoptosis in ox-LDL-exposed HAECs. Depletion of miR-34a facilitated endothelial cell growth and blocked apoptosis in AS by upregulating Bcl-2, offering a promising avenue for AS therapy.
Asunto(s)
Apoptosis , Aterosclerosis/genética , Regulación hacia Abajo , Células Endoteliales/metabolismo , Regulación de la Expresión Génica , MicroARNs/genética , Proteína bcl-X/genética , Animales , Apolipoproteínas E/deficiencia , Aterosclerosis/metabolismo , Aterosclerosis/patología , Western Blotting , Supervivencia Celular , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/patología , Citometría de Flujo , Inmunoprecipitación , Masculino , Ratones , MicroARNs/biosíntesis , ARN/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteína bcl-X/biosíntesisRESUMEN
Little is known about how RNA binding proteins cooperate to control splicing, and how stress pathways reconfigure these assemblies to alter splice site selection. We have shown previously that SRSF10 plays an important role in the Bcl-x splicing response to DNA damage elicited by oxaliplatin in 293 cells. Here, RNA affinity assays using a portion of the Bcl-x transcript required for this response led to the recovery of the SRSF10-interacting protein 14-3-3ε and the Sam68-interacting protein hnRNP A1. Although SRSF10, 14-3-3ε, hnRNP A1/A2 and Sam68 do not make major contributions to the regulation of Bcl-x splicing under normal growth conditions, upon DNA damage they become important to activate the 5' splice site of pro-apoptotic Bcl-xS. Our results indicate that DNA damage reconfigures the binding and activity of several regulatory RNA binding proteins on the Bcl-x pre-mRNA. Moreover, SRSF10, hnRNP A1/A2 and Sam68 collaborate to drive the DNA damage-induced splicing response of several transcripts that produce components implicated in apoptosis, cell-cycle control and DNA repair. Our study reveals how the circuitry of splicing factors is rewired to produce partnerships that coordinate alternative splicing across processes crucial for cell fate.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Empalme Alternativo , Proteínas de Ciclo Celular/metabolismo , Daño del ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Ribonucleoproteína Nuclear Heterogénea A1/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Oxaliplatino/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/metabolismo , Factores de Empalme Serina-Arginina/metabolismo , Proteínas 14-3-3/metabolismo , Reparación del ADN , Células HEK293 , Humanos , Mutágenos/metabolismo , Precursores del ARN/metabolismo , Proteína bcl-X/biosíntesis , Proteína bcl-X/genéticaRESUMEN
BACKGROUND: Ameloblastoma (AMB), odontogenic keratocyst (OKC) and adenomatoid odontogenic tumor (AOT) are epithelial odontogenic lesions with diverse biologic profiles. Defects in regulation of apoptosis and cell cycle may be involved in the development and progression of those lesions, therefore we aimed to investigate the expression of Bcl-2, Bax and p53 to better understand the possible role of these proteins in AMBs, OKCs and AOTs. MATERIAL AND METHODS: The studied sample consisted of 20 AMBs, 20 OKCs and 20 AOTs. Immunohistochemistry technique was performed for the antibodies p53, Bcl-2 and Bax. Immunoreactivity was observed in the epithelial component and positive cells were counted in five fields (100x magnification). Statistical analysis was performed with Kruskal-Wallis and Spearman tests (p <0.05). RESULTS: All lesions exhibited staining for the three studied proteins. There was no statistically significant associations between the expression of proteins and the lesions, however we identified a positive correlation between the expression of p53 and Bcl-2 (r = 0.200) and a negative correlation between p53 and Bax expressions (r = -0.100). In addition, p53 and Bax were similarly expressed between AMBs and OKCs. Bcl-2 was similarly expressed in AMBs and AOTs. CONCLUSIONS: Apoptosis regulatory proteins, as well as cell cycle proteins, are differently expressed in epithelial odontogenic lesions and their expression is possibly related to the biological behavior of AMB, OKC and AOT.
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Ameloblastoma/metabolismo , Ameloblastoma/patología , Apoptosis , Ciclo Celular , Neoplasias Maxilomandibulares/metabolismo , Neoplasias Maxilomandibulares/patología , Quistes Odontogénicos/metabolismo , Quistes Odontogénicos/patología , Proteína p53 Supresora de Tumor/biosíntesis , Proteína X Asociada a bcl-2/biosíntesis , Proteína bcl-X/biosíntesis , Humanos , Inmunohistoquímica , OdontogénesisRESUMEN
Apoptosis has a negative impact on the cell survival state during cell cultivation. To optimize mammalian cell culture for production of biopharmaceuticals, one of the important approaches is to extend cell life through over-expression of anti-apoptotic genes. Here, we reported a cost-effective process to enhance cell survival and production of an antibody through transient co-transfection with anti-apoptotic genes Bcl-x L or Mcl-1 in Chinese hamster ovary (CHO) cells with polyethylenimine (PEI). Under the optimal conditions, it showed reduced levels of apoptosis and improved cell viability after co-transfected with Bcl-x L or Mcl-1. The overall production yield of the antibody anti-PD1 increased approximately 82% in CHO cells co-transfected with Bcl-x L , and 34% in CHO cells co-transfected with Mcl-1. This work provides an effective way to increase viability of host cells through delaying apoptosis onset, thus, raise production yield of biopharmaceuticals without the process of generating stable cell lines and subsequent screening.
Asunto(s)
Proteína 1 de la Secuencia de Leucemia de Células Mieloides/biosíntesis , Receptor de Muerte Celular Programada 1 , Anticuerpos de Cadena Única/biosíntesis , Transfección , Proteína bcl-X/biosíntesis , Animales , Células CHO , Cricetulus , Humanos , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Anticuerpos de Cadena Única/genética , Proteína bcl-X/genéticaRESUMEN
Doxycycline (Dox)-inducible transgenic approach is used to examine the neural mechanisms of anxiety and depression; however, its own effects on related behaviors are not clear. To address this, in the present study, we tested the anxiety- and depression-like behaviors in rats treated with Dox in drinking water (2 mg/ml) in the elevated plus-maze (EPM; on day 5) and forced swim (FST; on day 8) tests, respectively. In addition, the levels of mRNAs and proteins of brain-derived neurotrophic factor (BDNF) and anti-apoptotic protein Bcl-xL in the hippocampus (HIPP) and frontal cortex (FC) were also analyzed. Consumption of Dox for 4 days induced an anxiogenic-like phenotype that was manifested by the decreased percentages of open arm entries and time spent on the open arms of the EPM. After Dox for 7 days, animals demonstrated more active behavior in the FST than control rats as evidenced by the increase in climbing time. When assessed after the FST, expression of Bcl-xL was increased in the hippocampus of Dox-treated animals. Furthermore, hippocampal Bcl-xL content correlated positively with the duration of climbing in the test. This study is the first to find that Dox in treatment regime used to control transgene expression can affect anxiety- and depression-like behaviors in rats. Dox-induced increase in Bcl-xL expression in the hippocampus may be involved in the moderate activation of FST behavior.
Asunto(s)
Ansiedad/metabolismo , Depresión/metabolismo , Doxiciclina/farmacología , Hipocampo/metabolismo , Transgenes/fisiología , Proteína bcl-X/biosíntesis , Animales , Antibacterianos/farmacología , Antibacterianos/toxicidad , Ansiedad/inducido químicamente , Ansiedad/genética , Depresión/inducido químicamente , Depresión/genética , Doxiciclina/toxicidad , Expresión Génica , Hipocampo/efectos de los fármacos , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Aprendizaje por Laberinto/fisiología , Ratas , Ratas Wistar , Estrés Psicológico/inducido químicamente , Estrés Psicológico/genética , Estrés Psicológico/metabolismo , Natación/fisiología , Transgenes/efectos de los fármacos , Proteína bcl-X/genéticaRESUMEN
The protein bcl-xL is able to enhance the secretion of the proinflammatory chemokine interleukin 8 (CXCL8) in human melanoma lines. In this study, we investigate whether the bcl-xL/CXCL8 axis is important for promoting melanoma angiogenesis and aggressiveness in vivo, using angiogenesis and xenotransplantation assays in zebrafish embryos. When injected into wild-type embryos, bcl-xL-overexpressing melanoma cells showed enhanced dissemination and angiogenic activity compared with control cells. Human CXCL8 protein elicited a strong proangiogenic activity in zebrafish embryos and zebrafish Cxcr2 receptor was identified as the mediator of CXCL8 proangiogenic activity using a morpholino-mediated gene knockdown. However, human CXCL8 failed to induce neutrophil recruitment in contrast to its zebrafish homolog. Interestingly, the greater aggressiveness of bcl-xL-overexpressing melanoma cells was mediated by an autocrine effect of CXCL8 on its CXCR2 receptor, as confirmed by an shRNA approach. Finally, correlation studies of gene expression and survival analyses using microarray and RNA-seq public databases of human melanoma biopsies revealed that bcl-xL expression significantly correlated with the expression of CXCL8 and other markers of melanoma progression. More importantly, a high level of co-expression of bcl-xL and CXCL8 was associated with poor prognosis in melanoma patients. In conclusion, these data demonstrate the existence of an autocrine CXCL8/CXCR2 signaling pathway in the bcl-xL-induced melanoma aggressiveness, encouraging the development of novel therapeutic approaches for high bcl-xL-expressing melanoma.
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Interleucina-8/metabolismo , Melanoma/irrigación sanguínea , Proteína bcl-X/metabolismo , Animales , Animales Modificados Genéticamente , Línea Celular Tumoral , Xenoinjertos , Humanos , Interleucina-8/biosíntesis , Interleucina-8/genética , Interleucina-8/farmacología , Melanoma/genética , Melanoma/metabolismo , Neovascularización Patológica/metabolismo , Proteínas Recombinantes/farmacología , Microambiente Tumoral , Pez Cebra , Proteína bcl-X/biosíntesis , Proteína bcl-X/genéticaRESUMEN
Culture media modifications, including the addition of various factors, are important for the in vitro production of oocytes and embryos. In this study, we investigated the effects of lysophosphatidic acid (LPA) on porcine embryo development. Porcine parthenogenetic embryos were cultured with 0, 0.1, 1, and 10 µM LPA for 7 days, or cultured in basic medium until Day 4 and then treated with LPA from Days 4 to 7. No difference in the in vitro development of embryos cultured with LPA for 7 days was observed. Conversely, rates of blastocyst and over-expanded blastocyst formation were higher in the 0.1 and 1 µM LPA-treated versus the other groups of embryos treated from Days 4 to 7. Moreover, formation of early blastocysts occurred earlier and embryo size was larger in LPA-treated compared to control embryos. Expression of Connexin 43 and gap junction and cell adhesion-related genes (GJC1 and CDH1, respectively) was also higher in LPA-treated compared to control embryos. Despite no difference in the blastocyst total cell number between groups, the apoptotic index was lower in the LPA-treated group than in the control group; indeed, BCL2L1 (B-cell lymphoma 2-like protein 1) expression increased while BAK (Bcl-2 homologous antagonist killer) decreased in the LPA-treated group. Thus, addition of LPA to the medium from Days 4 to 7 of culture improves blastocyst formation and aids the development of preimplantation embryos.
Asunto(s)
Blastocisto/citología , Embrión de Mamíferos/embriología , Desarrollo Embrionario/efectos de los fármacos , Lisofosfolípidos/farmacología , Animales , Proteínas Cdh1/biosíntesis , Conexina 43/biosíntesis , Técnicas de Cultivo de Embriones , Partenogénesis , Porcinos , Proteína Destructora del Antagonista Homólogo bcl-2/biosíntesis , Proteína bcl-X/biosíntesisRESUMEN
Granulosa cells (GCs) play an important role in ovarian follicle growth, development, and follicular atresia. In the present study, we investigated the effects of Melatonin on bovine GCs, and asked if MTNR1A was involved in their response to this indole hormone. Our results indicated that Melatonin inhibited GC apoptosis by up-regulating the expression of BCL2, BCL-XL, GPX4, and SOD1, and down-regulating the expression of BAX, CASP3, and TP53. Moreover, Melatonin modulated bovine GC function by decreasing the expression of INHA, INHBB, FSHR, and TGFBR3, and the abundance of Inhibin ß and Activin B, while increasing the expression of LHR, INHBA, and secretion of progesterone by GCs. In contrast, knockdown of MTNR1A significantly increased the expression of BAX, CASP3, TP53, INHA, FSHR, and TGFBR3, as well as Inhibin ß abundance, while decreasing the expression of BCL2, GPX4, SOD1, and LHR, and production of progesterone and estradiol; no effect was observed on the expression of BCL-XL, INHBA, or INHBB. These results suggest that Melatonin and MTNR1A play an important role in modulating bovine GC function by regulating cellular progression, apoptosis, hormones secretion, and reproduction-related genes. Furthermore, altered expression of MTNR1A could affect how bovine GCs respond to Melatonin.
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Apoptosis/efectos de los fármacos , Células de la Granulosa/metabolismo , Melatonina/farmacología , Receptor de Melatonina MT1/metabolismo , Animales , Bovinos , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Glutatión Peroxidasa/biosíntesis , Células de la Granulosa/citología , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Receptores de HL/biosíntesis , Superóxido Dismutasa-1/biosíntesis , Proteína bcl-X/biosíntesisRESUMEN
The antiapoptotic protein Bcl-xL is associated with several neuroplastic processes such as formation of synapses, regulation of spontaneous and evoked synaptic responses, and release of neurotransmitters. Dependence of expression on activity of neurons is characteristic for many proteins participating in regulation of neuroplasticity. Whether such property is exhibited by the Bcl-xL protein was analyzed using in vivo optogenetic stimulation of hippocampal glutamatergic neurons expressing channelrhodopsin ChR2H134 under CAMKIIa promoter in the adeno-associated viral vector, followed by immunohistochemical determination of the level of Bcl-xL protein in these neurons and surrounding cells. Increase in the level of early response c-Fos protein following illumination with blue light was indicative of activation of these hippocampal neurons. The optogenetic activation of hippocampus resulted in a significant increase in the level of antiapoptotic protein Bcl-xL in the photosensitive neurons as well as in the surrounding cells. The dependence of the level of expression of Bcl-xL protein on the activity of neurons indicates that this protein possesses one more important property that is essential for participation in neuroplastic processes in the brain.
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Hipocampo/metabolismo , Plasticidad Neuronal/fisiología , Neuronas/metabolismo , Proteína bcl-X/biosíntesis , Animales , Channelrhodopsins , Optogenética , Ratas , Proteína bcl-X/genéticaRESUMEN
The antiapoptotic protein Bcl-xL is involved in development of neurobiological resilience to stress; hence, the possibility of use of psychotropic drugs to increase its expression in brain in response to stress is of considerable interest. Lithium is a neurotropic drug widely used in psychiatry. In work, we studied effects of lithium administration (for 2 or 7 days) on the expression of Bcl-xL mRNA and protein in the hippocampi and cortices of rats subjected to stress that induced depression-like behavior in the animals. In contrast to the brain-derived neurotrophic factor (BDNF), whose expression decreased in the hippocampus in response to acute stress, stress increased the level of Bcl-xL mRNA in the hippocampus, but decreased it in the frontal cortex. Treatment of stressed animals with lithium for 2 or 7 days increased Bcl-xL protein levels 1.5-fold in the hippocampus, but it decreased them in the cortex. Therefore, Bcl-xL expression in the brain can be modulated by both stress and psychotropic drugs, and the effects of these factors are brain region-specific: both stress exposure and lithium administration activated Bcl-xL expression in the hippocampus and suppressed it in the frontal cortex. The activation of Bcl-xL expression in the hippocampus by lithium, demonstrated for the first time in this study, suggests an important role of this protein in the therapeutic effects of lithium in the treatment of stress-induced psychoemotional disorders.
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Lóbulo Frontal/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/metabolismo , Cloruro de Litio/farmacología , Estrés Psicológico/metabolismo , Proteína bcl-X/biosíntesis , Enfermedad Aguda , Animales , Lóbulo Frontal/patología , Hipocampo/patología , Litio/farmacología , Masculino , Ratas , Ratas Wistar , Estrés Psicológico/tratamiento farmacológico , Estrés Psicológico/patologíaRESUMEN
An estimated 40,000 deaths will be attributed to breast cancer in 2016, underscoring the need for improved therapies. Evading cell death is a major hallmark of cancer, driving tumor progression and therapeutic resistance. To evade apoptosis, cancers use antiapoptotic Bcl-2 proteins to bind to and neutralize apoptotic activators, such as Bim. Investigation of antiapoptotic Bcl-2 family members in clinical breast cancer datasets revealed greater expression and more frequent gene amplification of MCL1 as compared with BCL2 or BCL2L1 (Bcl-xL) across three major molecular breast cancer subtypes, Luminal (A and B), HER2-enriched, and Basal-like. While Mcl-1 protein expression was elevated in estrogen receptor α (ERα)-positive and ERα-negative tumors as compared with normal breast, Mcl-1 staining was higher in ERα+ tumors. Targeted Mcl-1 blockade using RNAi increased caspase-mediated cell death in ERα+ breast cancer cells, resulting in sustained growth inhibition. In contrast, combined blockade of Bcl-2 and Bcl-xL only transiently induced apoptosis, as cells rapidly acclimated through Mcl-1 upregulation and enhanced Mcl-1 activity, as measured in situ using Mcl-1/Bim proximity ligation assays. Importantly, MCL1 gene expression levels correlated inversely with sensitivity to pharmacologic Bcl-2/Bcl-xL inhibition in luminal breast cancer cells, whereas no relationship was seen between the gene expression of BCL2 or BCL2L1 and sensitivity to Bcl-2/Bcl-xL inhibition. These results demonstrate that breast cancers rapidly deploy Mcl-1 to promote cell survival, particularly when challenged with blockade of other Bcl-2 family members, warranting the continued development of Mcl-1-selective inhibitors for targeted tumor cell killing.Implications: Mcl-1 levels predict breast cancer response to inhibitors targeting other Bcl-2 family members, and demonstrate the key role played by Mcl-1 in resistance to this drug class. Mol Cancer Res; 15(3); 259-68. ©2016 AACR.