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1.
Chemotherapy ; 64(3): 119-128, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31661694

RESUMO

OBJECTIVE: To investigate whether TRIAP1inhibition affects the ovarian cancer cell resistance to cisplatin (DDP) via the Cyt c/Apaf-1/caspase-9 pathway by in vitro and in vivo experiments. METHODS: CCK8 assay was performed to find out how treatment with both TRIAP1 siRNA and DDP affects the cell viability of SKOV3 cells and DDP-resistant human ovarian carcinoma cell line SKOV3/DDP. SKOV3/DDP cells were transfected with control siRNA or TRIAP1 siRNA before 24 h of treatment with DDP (5 µg/mL). Flow cytometry was employed to detect cell apoptosis and Western blot to examine the expressions of Cyt c/Apaf-1/caspase-9 pathway-related proteins. SKOV3/DDP cells transfected with control siRNA or TRIAP1 siRNA were subcutaneously injected into BALB/c-nu/nu nude mice followed by the intraperitoneal injection of DDP (4 mg/kg). Cyt c/Apaf-1/caspase-9 pathway in transplanted tumors was detected by immunohistochemistry. RESULTS: TRIAP1 expression declined in SKOV3 cells when compared with SKOV3/DDP cells. The proliferation rate was lower in SKOV3/DDP cells transfected with TRIAP1 siRNA combined with treatment of DDP (1, 2, 4, 6, 8, 16, 32 µg/mL) than in those transfected with control siRNA. Moreover, the TRIAP1 siRNA group had an increased SKOV3/DDP cell apoptosis rate with the activation of the Cyt c/Apaf-1/caspase-9 pathway. During DDP treatment, nude mice in TRIAP1 siRNA group had slower growth and smaller size of transplanted tumor than those in control siRNA group, with increased expression of Cyt c, Apaf-1, and caspase-9. CONCLUSION: TRIAP1 inhibition may enhance the sensitivity of SKOV3/DDP cells to cisplatin via activation of the Cyt c/Apaf-1/caspase-9 pathway.


Assuntos
Cisplatino/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Fator Apoptótico 1 Ativador de Proteases/genética , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Caspase 9/genética , Caspase 9/metabolismo , Linhagem Celular Tumoral , Cisplatino/uso terapêutico , Citocromos c/genética , Citocromos c/metabolismo , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Interferência de RNA , RNA Interferente Pequeno/uso terapêutico , Transplante Heterólogo
2.
Braz J Med Biol Res ; 52(11): e8772, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31664306

RESUMO

This study aimed to investigate the mechanism of fluorofenidone (AKF-PD) in treating renal interstitial fibrosis in rats with unilateral urinary obstruction (UUO). Thirty-two male Sprague-Dawley rats were randomly divided into sham, UUO, UUO + enalapril, and UUO + AKF-PD groups. All rats, except sham, underwent left urethral obstruction surgery to establish the animal model. Rats were sacrificed 14 days after surgery, and serum was collected for renal function examination. Kidneys were collected to observe pathological changes. Immunohistochemistry was performed to assess collagen I (Col I) protein expression, and terminal deoxynucleotidyl transferase-mediated nick end-labeling staining to observe the apoptosis of renal tubular epithelial cells. The expression of Fas-associated death domain (FADD), apoptotic protease activating factor-1 (Apaf-1), and C/EBP homologous protein (CHOP) proteins was evaluated by immunohistochemistry and western blot analysis. AKF-PD showed no significant effect on renal function in UUO rats. The pathological changes were alleviated significantly after enalapril or AKF-PD treatment, but with no significant differences between the two groups. Col I protein was overexpressed in the UUO group, which was inhibited by both enalapril and AKF-PD. The number of apoptotic renal tubular epithelial cells was much higher in the UUO group, and AKF-PD significantly inhibited epithelial cells apoptosis. The expression of FADD, Apaf-1, and CHOP proteins was significantly upregulated in the UUO group and downregulated by enalapril and AKF-PD. In conclusion, AKF-PD improved renal interstitial fibrosis by inhibiting apoptosis of renal tubular epithelial cells in rats with UUO.


Assuntos
Apoptose/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Nefropatias/patologia , Piridonas/farmacologia , Obstrução Ureteral/patologia , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Fator Apoptótico 1 Ativador de Proteases/efeitos dos fármacos , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Nitrogênio da Ureia Sanguínea , Colágeno Tipo I/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Creatinina/sangue , Modelos Animais de Doenças , Enalapril/metabolismo , Enalapril/farmacologia , Proteína de Domínio de Morte Associada a Fas/efeitos dos fármacos , Proteína de Domínio de Morte Associada a Fas/metabolismo , Fibrose , Masculino , Piridonas/metabolismo , Distribuição Aleatória , Ratos Sprague-Dawley , Fator de Transcrição CHOP/efeitos dos fármacos , Fator de Transcrição CHOP/metabolismo
3.
Gene ; 711: 143949, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31255735

RESUMO

As a transcriptional repressor, Chromobox 8 (CBX8) overexpression is found to be associated with tumorigenesis in several cancers. However, its role in radiotherapy resistance remains poorly characterized. Our study is the first to explore the correlation between CBX8 and radioresistance. We report here that CBX8 is upregulated in Esophageal Squamous Cell Carcinoma (ESCC) tissues and cells and serves as an indicator of poor prognosis for ESCC patients. CBX8 knockdown inhibits cell proliferation, colony formation capability, DNA repair and promotes cell apoptosis. Moreover, the transcriptome sequencing analysis demonstrates that CBX8 downregulates the expression of Apoptotic protease activating factor 1 (APAF1), which is the core protein that mediates mitochondrial apoptotic pathways. APAF1 depletion could abrogate apoptosis induced by CBX8 knockdown in irradiated ESCC cells. Our results provide novel insight into CBX8 as a therapeutic target to improve the radiosensitivity of ESCC.


Assuntos
Fator Apoptótico 1 Ativador de Proteases/genética , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/patologia , Complexo Repressor Polycomb 1/metabolismo , Tolerância a Radiação , Animais , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Transplante de Neoplasias , Complexo Repressor Polycomb 1/genética , Prognóstico , Regulação para Cima
4.
J Nat Med ; 73(2): 339-352, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30523551

RESUMO

The aim of this study was to elucidate the gastroprotective activity and possible mechanism of involvement of araloside A (ARA) against ethanol- and aspirin-induced gastric ulcer in mice. The experimental mice were randomly divided into control, model, omeprazole (20 mg/kg, orally) and ARA (10, 20 and 40 mg/kg, orally). Gastric ulcer in mice was induced by intragastric administration of 80% ethanol (10 mL/kg) containing 15 mg/mL aspirin 4 h after drug administration on day 7. The results indicated that ARA could significantly raise gastric juice volume and acidity; ameliorate gastric mucosal blood flow, gastric binding mucus volume, ulcer index and ulcer inhibition rate; suppress H+/K+-ATPase activity, which was confirmed by computer-aided docking simulations; inhibit the release of mitochondrial cytochrome c into the cytoplasm; inhibit caspase-9 and caspase-3 activities and down-regulate mRNA expression levels; down-regulate the mRNA and protein expressions of apoptosis protease-activating factor-1 and protein expression of cleaved poly(ADP ribose) polymerase-1; and up-regulate Bcl-2 mRNA and protein expressions and down-regulate Bax mRNA and protein expressions, thus elevating the Bcl-2/Bax ratio in a dose-dependent manner. Histopathological observations further provided supportive evidence for the aforementioned results. The results demonstrated that ARA exerted beneficial gastroprotective effects on alcohol- and aspirin-induced gastric ulcer in mice, which was related to suppressing H+/K+-ATPase activity as well as pro-apoptotic protein expression, and promoting anti-apoptotic protein expression, thus alleviating gastric mucosal injury and cell death.


Assuntos
Antiulcerosos/uso terapêutico , Mucosa Gástrica/efeitos dos fármacos , ATPase Trocadora de Hidrogênio-Potássio/efeitos dos fármacos , Ácido Oleanólico/análogos & derivados , Saponinas/uso terapêutico , Úlcera Gástrica/prevenção & controle , Animais , Antiulcerosos/isolamento & purificação , Antiulcerosos/farmacologia , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Aralia/química , Aspirina/efeitos adversos , Caspase 3/metabolismo , Caspase 9/metabolismo , Citocromos c/metabolismo , Avaliação Pré-Clínica de Medicamentos , Etanol/efeitos adversos , Suco Gástrico/efeitos dos fármacos , Mucosa Gástrica/irrigação sanguínea , Mucosa Gástrica/metabolismo , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Masculino , Camundongos , Mitocôndrias/metabolismo , Simulação de Acoplamento Molecular , Ácido Oleanólico/isolamento & purificação , Ácido Oleanólico/farmacologia , Ácido Oleanólico/uso terapêutico , Fitoterapia , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Distribuição Aleatória , Fluxo Sanguíneo Regional/efeitos dos fármacos , Saponinas/isolamento & purificação , Saponinas/farmacologia , Úlcera Gástrica/induzido quimicamente
5.
Toxicol Lett ; 303: 55-66, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30579903

RESUMO

Cigarette smoke is responsible for many fatal pulmonary diseases, however, the toxic mechanism is still unclear. In this study, we first confirmed that whole cigarette smoke condensates (WCSC) contain hydrophilic elements, lipophilic and gaseous components. Then, we treated BEAS-2B cells, a normal human bronchial epithelial cell line, at dosages of 0.25, 0.5, and 1% for 24 h and explored the toxic mechanism. Cell viability decreased in a dose-dependent manner, and fission and fusion of mitochondria, damage of endoplasmic reticulume (ER) structures, and formation of autophagosome-like vacuoles were found in cells treated with 1% WCSC. Mitochondrial and ER volumes, lysosomal fluorescence intensity, LDH release, and intracellular ROS levels notably decreased at the highest doses compared with the control, whereas intracellular calcium ion and NO levels were significantly elevated accompanying G2/M phase arrest. Expression of an iron-binding nuclear protein-related gene (pirin) was the most up-regulated in the WCSC-treated cells with enhanced expression of antioxidant-related genes, whereas expression of carbonic anhydrase IX gene, a marker of tumor hypoxia, was the most down-regulated. Additionally, levels of apoptosis (BAX, Apaf-1, and cleavage of caspase-3 and PARP), autophagy (p62 and LC3B-II), ER stress (PERK, IRE-1a, Bip, and CHOP), antioxidant (SOD-1 and SOD-2), and MAPkinase activation (p-ERK, p-p38, and p-JNK)-related proteins were clearly enhanced following exposure to WCSC, whereas expression of several mitochondrial dynamics-related proteins was reduced with dose. Interestingly, expression of ferritin protein (light chain) was dramatically enhanced near the ER along with that of p62 protein. More importantly, the hypoxia inducible factor-1 pathway and ferroptosis were proposed among the 20 terms in KEGG pathway analysis, and secretion of IL-6 and IL-8, which are involved in hypoxia-induced inflammation, were clearly elevated with dose. Taken together, we suggest that WCSC may induce ferroptosis in bronchial epithelial cells via ER stress and disturbed homeostasis in mitochondrial dynamics caused by induction of hypoxia conditions.


Assuntos
Apoptose/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Fumaça/efeitos adversos , Tabaco/efeitos adversos , Fator Apoptótico 1 Ativador de Proteases/genética , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Autofagossomos/efeitos dos fármacos , Brônquios/citologia , Brônquios/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
6.
Mol Vis ; 24: 746-758, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30581281

RESUMO

Objective: To investigate whether echinacoside (ECH) protects the retina against ischemia/reperfusion (I/R) injury and the underlying mechanisms. Methods: Adult male Wistar rats were randomly divided into four groups: sham, sham plus ECH, I/R plus vehicle, and I/R plus ECH. Before the retinal I/R injury produced by high intraocular pressure (HOP), ECH was administered (20 mg/kg daily) for 7 days. The level of retinal cell damage was evaluated using Fluoro-Gold (FG) retrograde labeling and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) analysis 7 days after I/R. Optic nerve histology was analyzed with transmission electron microscopy. Levels of retinal malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were determined. The expression of apoptosis-associated factors (Apaf-1, Parp, and Bad) were analyzed with western blotting and quantitative real-time PCR (qPCR). The production of proinflammatory cytokines (tumor necrosis factor-α [TNFα], interleukin-1 beta [IL-1ß], and IL-6) was analyzed with enzyme-linked immunosorbent assay (ELISA) 7 days after the I/R injury as well. Results: The administration of ECH not only preserved retinal morphology but also attenuated retinal inflammation and apoptosis at 7 days after the I/R injury and decreased I/R-induced oxidative stress in the retina statistically significantly. Conclusions: ECH protected against I/R-induced retinal injury, via activation of antioxidant enzymes and suppression of inflammation. Therefore, ECH could be a potential therapeutic candidate for the treatment and management of I/R retinal diseases.


Assuntos
Antioxidantes/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glicosídeos/farmacologia , Nervo Óptico/efeitos dos fármacos , Traumatismo por Reperfusão/prevenção & controle , Retina/efeitos dos fármacos , Animais , Fator Apoptótico 1 Ativador de Proteases/genética , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Catalase/genética , Catalase/metabolismo , Modelos Animais de Doenças , Esquema de Medicação , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Masculino , Malondialdeído/antagonistas & inibidores , Malondialdeído/metabolismo , Nervo Óptico/metabolismo , Nervo Óptico/patologia , Estresse Oxidativo/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Ratos , Ratos Wistar , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Retina/metabolismo , Retina/patologia , Transdução de Sinais , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteína de Morte Celular Associada a bcl/genética , Proteína de Morte Celular Associada a bcl/metabolismo
7.
Food Funct ; 9(11): 5891-5902, 2018 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-30375606

RESUMO

Acetaminophen (APAP) is commonly used to relieve pain and fever in a clinical setting, but its excessive use can lead to serious hepatotoxicity. Our previous study demonstrated that polydatin (PD) can effectively attenuate d-galactose- and alcohol-induced hepatotoxicity, however, its effect on APAP-induced hepatotoxicity is still unknown. In this study, we explore the protective effect and potential mechanism of PD against APAP-induced hepatotoxicity in mice. The results indicate that PD effectively improves the survival of mice with APAP-induced hepatotoxicity, significantly alleviating histopathologic alterations in the liver, and decreasing the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). PD significantly and dose-dependently reduces oxidative stress by lowering the content of oxidized glutathione (GSSG), reactive oxygen species (ROS), nitric oxide (NO) and malonaldehyde (MDA), while enhancing the hepatic activities of glutathione (GSH), glutathione peroxidase (GSH-Px) and the GSH/GSSG ratio. Meanwhile, PD also substantially inhibits the levels and mRNA expressions of inducible nitric oxide synthase (iNOS) and NADPH oxidase 2 (NOX2). Additionally, PD markedly arrests apoptosis by assuaging TUNEL-positive hepatocytes and the apoptotic index, decreasing the levels and expression of cytochrome c (CytC), cleaved-caspase-9, apoptotic protease activating factor 1 (Apaf-1), cleaved-caspase-3, and Bax and increasing the level and expression of Bcl-2. Overall, PD pretreatment shows a potent protective effect against APAP-induced hepatotoxicity by relieving oxidative stress and inhibiting apoptosis.


Assuntos
Acetaminofen/toxicidade , Apoptose/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Glucosídeos/farmacologia , Substâncias Protetoras/farmacologia , Estilbenos/farmacologia , Alanina Transaminase/sangue , Animais , Antioxidantes/metabolismo , Fator Apoptótico 1 Ativador de Proteases/genética , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Aspartato Aminotransferases/sangue , Caspase 3/genética , Caspase 3/metabolismo , Caspase 9/genética , Caspase 9/metabolismo , Glutationa/sangue , Dissulfeto de Glutationa/sangue , Dissulfeto de Glutationa/metabolismo , Glutationa Peroxidase/sangue , Glutationa Peroxidase/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Malondialdeído/sangue , Camundongos , Camundongos Endogâmicos ICR , NADPH Oxidase 2/genética , NADPH Oxidase 2/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
8.
Cell Death Dis ; 9(10): 1012, 2018 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-30262881

RESUMO

Apoptosis ensures removal of damaged cells and helps shape organs during development by removing excessive cells. To prevent the intracellular content of the apoptotic cells causing damage to surrounding cells, apoptotic cells are quickly cleared by engulfment. Tight regulation of apoptosis and engulfment is needed to prevent several pathologies such as cancer, neurodegenerative and autoimmune diseases. There is increasing evidence that the engulfment machinery can regulate the execution of apoptosis. However, the underlying molecular mechanisms are poorly understood. We show that dynein mediates cell non-autonomous cross-talk between the engulfment and apoptotic programs in the Caenorhabditis elegans germline. Dynein is an ATP-powered microtubule-based molecular motor, built from several subunits. Dynein has many diverse functions including transport of cargo around the cell. We show that both dynein light chain 1 (DLC-1) and dynein heavy chain 1 (DHC-1) localize to the nuclear membrane inside apoptotic germ cells in C. elegans. Strikingly, lack of either DLC-1 or DHC-1 at the nuclear membrane inhibits physiological apoptosis specifically in mutants defective in engulfment. This suggests that a cell fate determining dialogue takes place between engulfing somatic sheath cells and apoptotic germ cells. The underlying mechanism involves the core apoptotic protein CED-4/Apaf1, as we find that DLC-1 and the engulfment protein CED-6/GULP are required for the localization of CED-4 to the nuclear membrane of germ cells. A better understanding of the communication between the engulfment machinery and the apoptotic program is essential for identifying novel therapeutic targets in diseases caused by inappropriate engulfment or apoptosis.


Assuntos
Apoptose/fisiologia , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Dineínas/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Dineínas do Citoplasma/metabolismo , Células Germinativas/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Transdução de Sinais/fisiologia
9.
Int J Oncol ; 53(4): 1787-1799, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30066861

RESUMO

MicroRNAs (miRNAs) are a class of small non­coding RNAs involved in post­transcriptional gene regulation. Furthermore, dysregulation of miRNA expression is an important factor in the pathogenesis of neuroblastoma. Our previous study identified that overexpression of monocyte chemoattractant protein­induced protein 1 protein led to a significant downregulation of a novel miRNA molecule, miRNA­3613­3p. In the present study, the potential involvement of miRNA­3613­3p in the cell biology of neuroblastoma was investigated. It was identified that the expression of miRNA­3613­3p varies among a range of human neuroblastoma cell lines. As the delineation of the functions of a miRNA requires the identification of its target genes, seven putative mRNAs that may be regulated by miRNA­3613­3p were selected. Furthermore, it was identified that overexpression of miRNA­3613­3p causes significant downregulation of several genes exhibiting tumor suppressive potential [encoding apoptotic protease­activating factor 1 (APAF1), Dicer, DNA fragmentation factor subunit ß, von Hippel­Lindau protein and neurofibromin 1] in BE(2)­C human neuroblastoma cells. APAF1 mRNA was the most significantly decreased transcript in the cells with miRNA­3613­3p overexpression. In accordance with the aforementioned results, the downregulation of cleaved caspase-9 and lack of activation of executive caspases in BE(2)­C cells following miRNA­3613­3p overexpression was observed. The results of the present study suggest a potential underlying molecular mechanism of apoptosis inhibition via APAF1 downregulation in human neuroblastoma BE(2)­C cells with miRNA­3613­3p overexpression.


Assuntos
Apoptose/genética , Fator Apoptótico 1 Ativador de Proteases/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Neuroblastoma/genética , Regiões 3' não Traduzidas/genética , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , MicroRNAs/genética , RNA Mensageiro/genética
10.
Ann Diagn Pathol ; 35: 27-37, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30072015

RESUMO

Chemoresistance is the major obstacle to effective treatment in patients with serous ovarian carcinoma (SOC), which frequently related to the failure of chemotherapeutic agents to induce apoptosis. In this study, the immunohistochemical expression of Apaf-1, Cyclin D1, and Aquaporin-5 (AQP-5) was studied in 50 paraffin blocks of SOC. Data on overall survival (OS), disease-free survival (DFS) and response to the first-line chemotherapy were collected and then statistically analyzed. Apaf-1 expression was observed in 84% of the SOC cases with a significant down-regulation with higher tumor grade, lymph node metastasis, and advanced FIGO stage. Cyclin D1 expression was found in 70% of the cases with a significant up-regulation with higher tumor grade, lymph node metastasis, and advanced FIGO stage. Positive AQP-5 expression was noted in 84% of the cases with a significant positive association with higher tumor grade, lymph node metastasis, and advanced FIGO stage. During the follow-up period, the Apaf-1 expression had a significant negative association with OS and DFS (p < 0.001 for each), while both Cyclin D1 and AQP-5 expression had a significant positive association with unfavorable OS and DFS. The cases of SOC treated with suboptimal surgery revealed a significant association of low Apaf-1, high Cyclin D1, and strong AQPs with the poor response to the first-line chemotherapy (p = 0.047, p < 0.001, and 0.006 respectively). CONCLUSIONS: Down-regulation of Apaf-1 protein and the overexpression of Cyclin D1 and AQP-5 proteins possibly contribute to an aggressive SOC with a high risk of recurrence and poor response to the first-line chemotherapy.


Assuntos
Fator Apoptótico 1 Ativador de Proteases/metabolismo , Aquaporina 5/metabolismo , Biomarcadores Tumorais/metabolismo , Ciclina D1/metabolismo , Cistadenocarcinoma Seroso/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Ovarianas/metabolismo , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carboplatina/uso terapêutico , Cistadenocarcinoma Seroso/tratamento farmacológico , Cistadenocarcinoma Seroso/mortalidade , Cistadenocarcinoma Seroso/patologia , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Gradação de Tumores , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Paclitaxel/uso terapêutico , Prognóstico , Taxa de Sobrevida , Resultado do Tratamento
11.
Cell Death Differ ; 25(7): 1194-1208, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29765111

RESUMO

The apoptosome is a platform that activates apical procaspases in response to intrinsic cell death signals. Biochemical and structural studies in the past two decades have extended our understanding of apoptosome composition and structure, while illuminating the requirements for initiator procaspase activation. A number of studies have now provided high-resolution structures for apoptosomes from C. elegans (CED-4), D. melanogaster (Dark), and H. sapiens (Apaf-1), which define critical protein interfaces, including intra and interdomain interactions. This work also reveals interactions of apoptosomes with their respective initiator caspases, CED-3, Dronc and procaspase-9. Structures of the human apoptosome have defined the requirements for cytochrome c binding, which triggers the conversion of inactive Apaf-1 molecules to an extended, assembly competent state. While recent data have provided a detailed understanding of apoptosome formation and procaspase activation, they also highlight important evolutionary differences with functional implications for caspase activation. Comparison of the CARD/CARD disks and apoptosomes formed by CED-4, Dark and Apaf-1. Cartoons of the active states of the CARD-CARD disks, illustrating the two CED-4 CARD tetrameric ring layers (CED4a and CED4b; top row) and the binding of 8 Dronc CARDs and between 3-4 pc-9 CARDs, to the Dark and Apaf-1 CARD disk respectively (middle and lower rows). Ribbon diagrams of the active CED-4, Dark and Apaf-1 apoptosomes are shown (right column).


Assuntos
Apoptossomas/metabolismo , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Caspase 9/metabolismo , Proteínas de Drosophila/metabolismo , Animais , Fator Apoptótico 1 Ativador de Proteases/genética , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Caspase 9/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Humanos
12.
Am J Reprod Immunol ; 80(3): e12978, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29774968

RESUMO

PROBLEM: Granulysin (GNLY) is a cytotoxic molecule mostly present in decidual natural killer (NK) cells. Blighted ovum (BO) and missed abortion (MA) represent the early pathological pregnancies with hindered development of the embryoblast or a dead embryo. We investigated the GNLY-mediated apoptotic mechanism potentially responsible for delayed termination of pregnancy. METHOD OF STUDY: We performed immunohistological and immunofluorescence labeling of decidual tissues (GNLY, Apaf-1, NF-κB). NKG2A expression was analyzed by flow cytometry and GNLY mRNA by RT-qPCR. RESULTS: The GNLY labeling intensity (H score) was lower in the nuclei of trophoblast cells in BO and MA. GNLY gene levels were inversely detected in BO and MA. A decreased decidual NK cell percentage was found in MA. NK cells from pathological pregnancies expressed lower NKG2A levels. The highest frequency of Apaf-1 was found in trophoblast cells of MA. NF-kB was highly expressed in decidual cells of BO. CONCLUSION: The reduced activation of GNLY-mediated killing might be implicated in the slower rejection of trophoblast cells in BO and MA. A decreased authentic decidual NK cell number could be responsible for low cytotoxicity against trophoblast cells in MA. In BO, trophoblast cells have a higher survival potential due to increased NF-kB expression.


Assuntos
Aborto Retido/imunologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Decídua/patologia , Células Matadoras Naturais/imunologia , Trofoblastos/imunologia , Apoptose , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Células Cultivadas , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , NF-kappa B/metabolismo , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismo , Gravidez
13.
Cell Death Dis ; 9(3): 365, 2018 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-29511177

RESUMO

Apoptosis is a highly regulated form of programmed cell death, essential to the development and homeostasis of multicellular organisms. Cytochrome c is a central figure in the activation of the apoptotic intrinsic pathway, thereby activating the caspase cascade through its interaction with Apaf-1. Our recent studies have revealed 14-3-3ε (a direct inhibitor of Apaf-1) as a cytosolic cytochrome c target. Here we explore the cytochrome c / 14-3-3ε interaction and show the ability of cytochrome c to block 14-3-3ε-mediated Apaf-1 inhibition, thereby unveiling a novel function for cytochrome c as an indirect activator of caspase-9/3. We have used calorimetry, NMR spectroscopy, site mutagenesis and computational calculations to provide an insight into the structural features of the cytochrome c / 14-3-3ε complex. Overall, these findings suggest an additional cytochrome c-mediated mechanism to modulate apoptosome formation, shedding light onto the rigorous apoptotic regulation network.


Assuntos
Proteínas 14-3-3/metabolismo , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Citocromos c/metabolismo , Proteínas 14-3-3/química , Proteínas 14-3-3/genética , Motivos de Aminoácidos , Fator Apoptótico 1 Ativador de Proteases/genética , Caspase 3/genética , Caspase 9/genética , Citosol/enzimologia , Citosol/metabolismo , Ativação Enzimática , Humanos , Ligação Proteica
14.
Mol Biol Cell ; 29(9): 1125-1136, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29514931

RESUMO

Accumulating evidence has demonstrated that voltage-gated potassium channels (Kv channels) were associated with regulating cell proliferation and apoptosis in tumor cells. Our previous study proved that the Kv channel blocker 4-aminopyridine (4-AP) could inhibit cell proliferation and induce apoptosis in glioma. However, the precise mechanisms were not clear yet. MicroRNAs (miRNAs) are small noncoding RNAs that act as key mediators in the progression of tumor, so the aim of this study was to investigate the role of miRNAs in the apoptosis-promoting effect of 4-AP in glioma cells. Using a microRNA array, we found that 4-AP altered the miRNA expression in glioma cells, and the down-regulation of miR-10b-5p induced by 4-AP was verified by real-time PCR. Transfection of miR-10b-5p mimic significantly inhibited 4-AP-induced caspases activation and apoptosis. Moreover, we verified that apoptosis-related molecule Apaf-1 was the direct target of miR-10b-5p. Furthermore, miR-10b-5p mimic significantly inhibited 4-AP-induced up-regulation of Apaf-1 and its downstream apoptosis-related proteins, such as cleaved caspase-3. In conclusion, Kv channel blocker 4-AP may exert its anti-tumor effect by down-regulating the expression of miR-10b-5p and then raised expression of Apaf-1 and its downstream apoptosis-related proteins. Current data provide evidence that miRNAs play important roles in Kv channels-mediated cell proliferation and apoptosis.


Assuntos
4-Aminopiridina/metabolismo , Apoptose/genética , MicroRNAs/efeitos dos fármacos , 4-Aminopiridina/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Fator Apoptótico 1 Ativador de Proteases/genética , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Glioma/genética , Glioma/metabolismo , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores
15.
Eur J Cell Biol ; 97(2): 126-135, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29395479

RESUMO

Recent evidence suggests that mitochondrial apoptosis regulators and executioners may regulate differentiation, without being involved in cell death. However, the involved factors and their roles in differentiation and apoptosis are still not fully determined. In the present study, we compared mitochondrial pathway of cell death during early neural differentiation from human embryonic stem cells (hESCs). Our results demonstrated that ROS generation, cytosolic cytochrome c release, caspases activation and rise in p53 protein level occurred upon either neural or apoptosis induction in hESCs. However, unlike apoptosis, no remarkable increase in apoptotic protease activating factor-1 (Apaf-1) level at early stages of differentiation was observed. Also the caspase-like activity of caspase-9 and caspase-3/7 were seen less than apoptosis. The results suggest that low levels of Apaf-1 as an adaptor protein might be considered as a possible regulatory barrier by which differentiating cells control cell death upon rise in ROS production and cytochrome c release from mitochondria. Better understanding of mechanisms via which mitochondria-mediated apoptotic pathway promote neural differentiation can result in development of novel therapeutic approaches.


Assuntos
Fator Apoptótico 1 Ativador de Proteases/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Apoptose/fisiologia , Diferenciação Celular/fisiologia , Citocromos c/metabolismo , Humanos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo
16.
Arch Biochem Biophys ; 642: 46-51, 2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29410086

RESUMO

Apaf-1 is a cytosolic multi-domain protein in the apoptosis regulatory network. When cytochrome c releases from mitochondria; it binds to WD-40 repeats of Apaf-1 molecule and induces oligomerization of Apaf-1. Here in, a split luciferase assay was used to compare apoptosome formation in cell-free and cell-based systems. This assay uses Apaf-1 tagged with either N-terminal fragment or C-terminal fragment of P. pyralis luciferase. In cell based-system, the apoptosome formation is induced inside the cells which express Apaf-1 tagged with complementary fragments of luciferase while in cell-free system, the apoptosome formation is induced in extracts of the cells. In cell-free system, cytochrome c dependent luciferase activity was observed with full length Apaf-1. However, luciferase activity due to apoptosome formation was much higher in cell based system compared to cell-free system. The truncated Apaf-1 which lacks WD-40 repeats (ΔApaf-1) interacted with endogenous Apaf-1 in a different fashion compared to native form as confirmed by different retention time of eluate in gel filtration and binding to affinity column. The interactions between endogenous Apaf-1 and ΔApaf-1 is stronger than its interaction with native exogenous Apaf-1 as indicated by dominant negative effect of ΔApaf-1 on caspase-3 processing.


Assuntos
Apoptossomas/metabolismo , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Apoptose , Fator Apoptótico 1 Ativador de Proteases/química , Biopolímeros/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Sistema Livre de Células , Cromatografia de Afinidade , Cromatografia em Gel , Ativação Enzimática , Células HEK293 , Humanos , Luciferases/metabolismo , Ligação Proteica , Proteólise , Repetições WD40
17.
Cell Death Dis ; 9(2): 244, 2018 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-29445170

RESUMO

Chemotherapy is a standard treatment for non-small-cell lung cancer (NSCLC). However, the dose-limiting toxicity of drugs and the development of chemoresistance are major clinical challenges to successful management of NSCLC. Asian traditional medicine is gaining global attention as a non-toxic alternative to chemotherapy. BRM270 is an extract formulated from seven Asian medicinal plants that has been shown to inhibit tumor cell proliferation in diverse cancer types. We previously demonstrated that BRM270 suppresses tumorigenesis by negatively regulating nuclear factor-κB signaling in multidrug-resistant cancer stem cells (CSCs). In this study we report that the growth, migration, and invasion of normal human lung adenocarcinoma cells and their chemoresistant derivatives was inhibited by BRM270 treatment. Notably, BRM270 was found to modulate CSC self-renewal and tumor-initiating capacity via positive regulation of the miRNA-128. Thus, combination therapy with miRNA-128 and BRM270 may be an effective treatment strategy for chemoresistant NSCLC.


Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Antineoplásicos Fitogênicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/tratamento farmacológico , MicroRNAs/genética , Células A549 , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Animais , Fator Apoptótico 1 Ativador de Proteases/genética , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , MicroRNAs/agonistas , MicroRNAs/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Sci Rep ; 8(1): 1611, 2018 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-29371610

RESUMO

Caspase-3-related DEVDase activity is initiated upon apoptosis in unfertilized starfish eggs. In this study, we cloned a starfish procaspase-3 corresponding to mammalian effector caspase containing a CARD that is similar to the amino terminal CARD of mammalian capsase-9, and we named it procaspase-3/9. Recombinant procaspase-3/9 expressed at 15 °C was cleaved to form active caspase-3/9 which has DEVDase activity. Microinjection of the active caspase-3/9 into starfish oocytes/eggs induced apoptosis. An antibody against the recombinant protein recognized endogenous procaspase-3/9 in starfish oocytes, which was cleaved upon apoptosis in aged unfertilized eggs. These results indicate that caspase-3/9 is an effector caspase in starfish. To verify the mechanism of caspase-3/9 activation, we cloned starfish Apaf-1 containing a CARD, a NOD, and 11 WD40 repeat regions, and we named it sfApaf-1. Recombinant sfApaf-1 CARD interacts with recombinant caspase-3/9 CARD and with endogenous procaspase-3/9 in cell-free preparations made from starfish oocytes, causing the formation of active caspase-3/9. When the cell-free preparation without mitochondria was incubated with inactive recombinant procaspase-3/9 expressed at 37 °C, DEVDase activity increased and apoptosome-like complexes were formed in the high molecular weight fractions containing both sfApaf-1 and cleaved caspase-3/9. These results suggest that sfApaf-1 activation is not dependent on cytochrome c.


Assuntos
Apoptose , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Asterina , Caspase 3/metabolismo , Caspase 9/metabolismo , Peptídeo Hidrolases/metabolismo , Zigoto , Animais , Caspase 3/genética , Caspase 9/genética , Caspases Efetoras/metabolismo , Clonagem Molecular , Regulação Enzimológica da Expressão Gênica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
19.
Eur J Hum Genet ; 26(3): 420-427, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29358613

RESUMO

Neural tube defects (NTDs) remain one of the most serious birth defects, and although genes in several pathways have been implicated as risk factors for neural tube defects via knockout mouse models, very few molecular causes in humans have been identified. Whole exome sequencing identified deleterious variants in key apoptotic genes in two families with recurrent neural tube defects. Functional studies in fibroblasts indicate that these variants are loss-of-function, as apoptosis is significantly reduced. This is the first report of variants in apoptotic genes contributing to neural tube defect risk in humans.


Assuntos
Fator Apoptótico 1 Ativador de Proteases/genética , Caspase 9/genética , Defeitos do Tubo Neural/genética , Adulto , Apoptose , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Caspase 9/metabolismo , Células Cultivadas , Resistência a Medicamentos , Feminino , Morte Fetal , Fibroblastos/metabolismo , Fibroblastos/patologia , Ácido Fólico/administração & dosagem , Ácido Fólico/uso terapêutico , Humanos , Mutação com Perda de Função , Masculino , Defeitos do Tubo Neural/tratamento farmacológico , Defeitos do Tubo Neural/patologia , Gravidez
20.
Sci Rep ; 8(1): 992, 2018 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-29343765

RESUMO

The compound 29-(4-methylpiperazine)-luepol (M22), a novel derivative of lupeol has shown anti-proliferative effects against the human non-small cell lung cancer A549 cell line. M22 showed significant anti-proliferative activity at 6.80 µM and increased accumulation of G1 cells and effectively suppressed expression of the G1 arrest-related genes cyclins D1 and E1, CDK2 and CDC25A. This was further confirmed by Western blotting demonstrating decreased cyclin D1 and CDC25A protein levels. Furthermore, M22 caused induction of apoptosis that downregulated the anti-apoptotic BCL-2 gene and increased expression of BAX, CASP3 and CASP9 as well as the APAF1 gene. The effect of caspase-induced apoptosis was confirmed by an increase in reactive oxygen species (ROS), loss of mitochondrial membrane potential (MMP). Taken together, our findings indicated that M22 possessed potent anti-proliferative and apoptotic activities.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Triterpenos Pentacíclicos/farmacologia , Piperazinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Células A549 , Antineoplásicos Fitogênicos/síntese química , Apoptose/efeitos dos fármacos , Fator Apoptótico 1 Ativador de Proteases/agonistas , Fator Apoptótico 1 Ativador de Proteases/genética , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Caspase 9/genética , Caspase 9/metabolismo , Linhagem Celular Tumoral , Ciclina D1/antagonistas & inibidores , Ciclina D1/genética , Ciclina D1/metabolismo , Ciclina E/antagonistas & inibidores , Ciclina E/genética , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Células HeLa , Células Hep G2 , Humanos , Concentração Inibidora 50 , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Oncogênicas/antagonistas & inibidores , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Triterpenos Pentacíclicos/síntese química , Piperazinas/síntese química , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética , Proteína X Associada a bcl-2/agonistas , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Fosfatases cdc25/antagonistas & inibidores , Fosfatases cdc25/genética , Fosfatases cdc25/metabolismo
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