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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.
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
3.
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
4.
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
5.
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
6.
J Cell Physiol ; 234(1): 521-536, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-30071126

RESUMO

Cell death and differentiation appear to share similar cellular features. In this study, we aimed to investigate whether differentiation and mitochondrial cell death use a common pathway. We assessed the hallmarks of apoptosis during cardiomyocyte differentiation of human embryonic stem cells and found remarkable changes in P53, reactive oxygen species, apoptotic protease-activating factor 1, poly[ADP-ribose]polymerase 1, cellular adenosine triphosphate, and mitochondrial complex I activity. Furthermore, we observed reversible mitochondrial membrane permeabilization during cardiomyocyte differentiation accompanied by reversible loss of mitochondrial membrane potential, and these changes coincided with the fluctuating patterns of cytosolic cytochrome c accumulation and subsequent caspase-9 and -3/7 activation. Moreover, the use of apoptosis inhibitors (BCL2-associated X protein [BAX] inhibitor and caspase-3/7 inhibitor) during differentiation impaired cardiomyocyte development, resulting in substantial downregulation of T, MESP1, NKX2.5, and α-MHC. Additionally, although the expression of specific differentiation markers (T, MESP1, NKX2.5, MEF2C, GATA4, and SOX17) was enhanced in doxorubicin-induced human embryonic stem cells, the stemness-specific markers (OCT4 and NANOG) showed significant downregulation. With increasing doxorubicin concentration (0.03-0.6 µM; IC50 = 0.5 µM), we observed a marked increase in the expression of mesoderm and endoderm markers. In summary, we suggest that reversible mitochondrial outer membrane permeabilization promotes cardiomyocyte differentiation through an attenuated mitochondria-mediated apoptosis-like pathway.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/genética , Mitocôndrias/genética , Miócitos Cardíacos/citologia , Trifosfato de Adenosina/genética , Apoptose/genética , Fator Apoptótico 1 Ativador de Proteases/genética , Caspase 9/genética , Doxorrubicina/farmacologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Potencial da Membrana Mitocondrial/genética , Mitocôndrias/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética , Proteína X Associada a bcl-2/genética
7.
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
8.
J Comp Neurol ; 526(13): 2099-2114, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30004576

RESUMO

The orchestrated events required during brain development, as well as the maintenance of adult neuronal plasticity, highly depend on the accurate responses of neuronal cells to various cellular stress or environmental stimuli. Recent studies have defined a previously unrecognized, broad class of multidomain proteins, designated as signal transduction ATPases with numerous domains (STAND), which comprises a large number of proteins, including the apoptotic peptidase activating factor 1 (Apaf1) and nucleotide-binding oligomerization domain-like receptors (NLRs), central players in cell death and innate immune responses, respectively. Although the involvement of STANDs in the central nervous system (CNS) has been postulated in terms of neuronal development and function, it remains largely unclear. Here, we identified Nwd1 (NACHT and WD repeat domain-containing protein 1), as a novel STAND protein, expressed in neural stem/progenitor cells (NSPCs). Structurally, Nwd1 was most analogous to the apoptosis regulator Apaf1, also involved in mitosis and axonal outgrowth regulation in the CNS. Using a specific antibody, we show that, during the embryonic and postnatal period, Nwd1 is expressed in nestin-positive NSPCs in vivo and in vitro, while postnatally it is found in terminally differentiated neurons and blood vessels. At the subcellular level, we demonstrate that Nwd1 is preferentially located in the cytosolic compartment of cultured NSPCs, partially overlapping with cytochrome c. These observations imply that Nwd1 might be involved in the neuronal lineage as a new STAND gene, including having a pro-apoptotic or nonapoptotic role, similar to Apaf1.


Assuntos
Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Animais Recém-Nascidos , Fator Apoptótico 1 Ativador de Proteases/biossíntese , Fator Apoptótico 1 Ativador de Proteases/genética , Sistema Nervoso Central/embriologia , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Camundongos Endogâmicos ICR , Mitose/genética , Células-Tronco Neurais/metabolismo , Distribuição Tecidual
9.
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
10.
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
11.
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
12.
Biochem Biophys Res Commun ; 497(2): 513-520, 2018 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-29452092

RESUMO

MicroRNA-630 (miR-630) has been implicated in the development and progression of multiple cancers. The current study aimed to investigate the role of miR-630 in chemoresistant epithelial ovarian cancer. MiR-630 expression levels were detected in ovarian cancer cell line SKOV3 and paclitaxel-resistant SKOV3 (SKOV3-TR) via microarray and qRT-PCR. MiR-630 inhibitors and negative controls were transfected into SKOV3 and SKOV3-TR cells. Wound healing, invasion, chemosensitivity, and cell apoptosis assays were performed to determine proliferation and migration rates. Chemoresistant patient-derived xenograft (PDX) models were established and utilized to verify the effect of miR-630 on chemoresistant ovarian cancer. Inhibition of miR-630 decreased cell proliferation and enhanced the sensitivity of SKOV3-TR and SKOV3 cells to paclitaxel. In the chemosensitivity assay, we observed that the miR-630 inhibitor exhibited a synergistic effect with paclitaxel on SKOV3-TR cells. Inhibition was correlated with enhanced expression of apoptosis-related proteins. APAF-1 was predicted to be a potential target of miR-630. An in vivo PDX study showed that the miR-630 inhibitor sensitized chemoresistant ovarian cancer to paclitaxel. Thus, miR-630 inhibitor sensitizes chemoresistant epithelial ovarian cancer to chemotherapy by enhancing apoptosis. Our findings suggest that miR-630 might be a potential therapeutic target for chemotherapy-resistant ovarian cancer.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , MicroRNAs/antagonistas & inibidores , Neoplasias Ovarianas/tratamento farmacológico , Paclitaxel/farmacologia , Animais , Fator Apoptótico 1 Ativador de Proteases/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , MicroRNAs/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Ovário/efeitos dos fármacos , Ovário/metabolismo , Ovário/patologia
13.
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
14.
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
15.
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
17.
Reprod Domest Anim ; 53(1): 137-142, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29076565

RESUMO

Some of the highest genetic merit sires have been shown to harbour recessive mutations affecting fertility, which may spread rapidly in the population through AI. These disorders may result in abortion and decline in pregnancy per insemination in cows. This study was carried out on 240 Holstein-Friesian cows and 15 mummified foetuses. Blood and tissue samples were collected from the cows and mummified foetuses, respectively, for DNA extraction. Allele-specific PCR was designed for the detection of the cows and foetuses carrying the nonsense mutation (C/T) in apoptosis peptide activating factor 1 gene (APAF1). The mutant allele frequency of the APAF1 in carrier cows and mummified foetuses was calculated. Milk samples were taken from the carrier and non-carrier cows for progesterone assay. The allele-specific PCR reaction efficiently distinguished the C/T mutation in APAF1. Of 240 cows, seven cows (2.9%) were diagnosed to carry one copy of the mutant allele of APAF1. However, the carrier frequency was 33.3% in mummified foetuses (five of 15). The mutant allele frequency was 0.02 and 0.17 in the cows and mummified foetuses, respectively. Concentrations of progesterone did not differ between cows with APAF1 mutation and non-carrier cows during 45 days post-insemination. This study provided allele-specific PCR for the detection of APAF1 mutation in cows. Moreover, it reports the carrier and mutant allele frequencies of APAF1 in dairy cows and mummified foetuses in Japan.


Assuntos
Aborto Animal/genética , Fator Apoptótico 1 Ativador de Proteases/genética , Bovinos/genética , Morte Fetal , Mutação , Alelos , Animais , Doenças dos Bovinos/genética , Indústria de Laticínios , Feminino , Japão , Leite/química , Reação em Cadeia da Polimerase/veterinária , Gravidez , Progesterona/análise
18.
Am J Pathol ; 188(2): 404-416, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29154960

RESUMO

Porphyromonas gingivalis is able to invade and modulate host-immune response to promote its survival. This bacterium modulates the cell cycle and programed cell death, contributing to periodontal lesion worsening. Several molecular pathways have been identified as key triggers of apoptosis, including apoptosome apoptotic peptidase activating factor 1 (APAF-1). Apaf-1 and X-linked inhibitor of apoptosis protein (Xiap) mRNA were differentially expressed between gingival samples harvested from human healthy and chronic periodontitis tissues (Apaf-1, 19.2-fold; caspase-9, 14.5-fold; caspase-3, 6.8-fold; Xiap: 2.5-fold in chronic periodontitis) (P < 0.05), highlighting their potential role in periodontitis. An increased proteic expression of APAF-1 was also observed in a murine experimental periodontitis model induced by P. gingivalis-soaked ligatures. In vitro, it was observed that P. gingivalis targets APAF-1, XIAP, caspase-3, and caspase-9, to inhibit epithelial cell death at both mRNA and protein levels. Opposite effect was observed in fibroblasts in which P. gingivalis increased cell death and apoptosis. To assess if the observed effects were associated to APAF-1, epithelial cells and fibroblasts were transfected with siRNA targeting Apaf-1. Herein, we confirmed that APAF-1 is targeted by P. gingivalis in both cell types. This study identified APAF-1 apoptosome and XIAP as intracellular targets of P. gingivalis, contributing to the deterioration of periodontal lesion through an increased persistence of the bacteria within tissues and the subversion of host-immune response.


Assuntos
Apoptossomas/metabolismo , Fator Apoptótico 1 Ativador de Proteases/biossíntese , Infecções por Bacteroidaceae/metabolismo , Periodontite Crônica/microbiologia , Porphyromonas gingivalis/patogenicidade , Idoso , Animais , Fator Apoptótico 1 Ativador de Proteases/genética , Infecções por Bacteroidaceae/patologia , Células Cultivadas , Periodontite Crônica/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica/fisiologia , Gengiva/metabolismo , Gengiva/microbiologia , Gengiva/patologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , RNA Mensageiro/genética
19.
Sci Rep ; 7(1): 15278, 2017 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-29127384

RESUMO

Dasatinib and radotinib are oral BCR-ABL tyrosine kinase inhibitors that were developed as drugs for the treatment of chronic myeloid leukemia. We report here that the c-KIT (CD117) targeting with dasatinib and radotinib promotes acute myeloid leukemia (AML) cell death, and c-KIT endocytosis is essential for triggering c-KIT-positive AML cell death by dasatinib and radotinib during the early stages. In addition, dasatinib and radotinib reduce heat shock protein 90ß (HSP90ß) expression and release Apaf-1 in c-KIT-positive AML cells. Finally, this activates a caspase-dependent apoptotic pathway in c-KIT-positive AML cells. Moreover, the inhibition of c-KIT endocytosis by dynamin inhibitor (DY) reversed cell viability and c-KIT expression by dasatinib and radotinib. HSP90ß expression was recovered by DY in c-KIT-positive AML cells as well. Furthermore, the effect of radotinib on c-KIT and HSP90ß showed the same pattern in a xenograft animal model using HEL92.1.7 cells. Therefore, dasatinib and radotinib promote AML cell death by targeting c-KIT. Taken together, these results indicate that dasatinib and radotinib treatment have a potential role in anti-leukemic therapy on c-KIT-positive AML cells.


Assuntos
Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Dasatinibe/farmacologia , Sistemas de Liberação de Medicamentos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Proteínas Proto-Oncogênicas c-kit/análise , Pirazinas/farmacologia , Animais , Fator Apoptótico 1 Ativador de Proteases/genética , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Linhagem Celular Tumoral , Feminino , Proteínas de Choque Térmico HSP90/biossíntese , Proteínas de Choque Térmico HSP90/genética , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Int J Mol Med ; 40(6): 1971-1982, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29039468

RESUMO

Amyloid-ß (Aß), a main pathogenic factor of Alzheimer's disease (AD), induces apoptosis accompanied by caspase activation. However, limited caspase activation and the suppression of the intrinsic apoptotic pathway (IAPW) are frequently observed upon Aß treatment. In this study, we investigated whether these suppressive effects of Aß can be overcome; we also examined the death-related pathways. Single treatments of cells with Aß42 for up to 48 h barely induced caspase activation. In cells treated with Aß42 twice for 2 h followed by 22 h (2+22 h) or for longer durations, the apoptotic protease activating factor-1 (Apaf-1) apoptosome was formed and caspases-3 and -9 were activated to a certain extent, suggesting the activation of the IAPW. However, the Aß42-induced activation of the IAPW differed from that induced by treatment with other agents, such as staurosporine (STS) in that lower amounts of cytochrome c were released from the mitochondria, the majority of procaspase-9 in the Apaf-1 complex was not processed and caspase-3 was activated to a lesser extent in the peptide-treated cells. Thus, it seemed that the IAPW was not fully activated by Aß42. The 30- and 41/43-kDa fragments derived from procaspase-8 were detected, which appear to be produced through the IAPW without death-inducing signaling-complex (DISC) formation, a key feature of the extrinsic apoptotic pathway (EAPW). Bid cleavage was observed only after caspase-3 activity reached its maximal levels, suggesting that the cleavage may contribute in a limited capacity to the amplification process of the IAPW in the Aß-treated cells. Taken together, our data suggest that the IAPW, albeit functional only to a limited extent, plays a major role in Aß42-induced apoptosis without the EAPW.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/genética , Apoptose/genética , Estaurosporina/administração & dosagem , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Fator Apoptótico 1 Ativador de Proteases/genética , Caspase 3/genética , Caspase 9/genética , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Transdução de Sinais/efeitos dos fármacos
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