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1.
Zhonghua Yan Ke Za Zhi ; 56(1): 32-40, 2020 Jan 11.
Artigo em Chinês | MEDLINE | ID: mdl-31937061

RESUMO

Objective: To investigate the role and mechanism of microglial activation in the process of retinal ganglion cell (RGC) death in the oxygen-glucose deprivation/reperfusion (OGD/R) model which mimicked retinal ischemia/reperfusion injury in vitro. Methods: Experimental study. Primary RGCs from C57BL/6 mice and BV2 microglia were co-cultured or cultured alone. The OGD/R model was established in vitro (reoxygenation time was set to 6 h, 24 h, 36 h, 48 h). BV2 microglial activation was assessed by immunofluorescence staining of ionized calcium binding adapter molecule 1 (iba1), and the survival rate of RGCs was detected by the Cell Counting Kit-8. The apoptosis rate of RGC was detected by using apoptosis detection kit. The levels of Toll-like receptor-4 (TLR4) and Nod-like receptor family pyrin domain containing protein 3 (NLRP3) in BV2 cells were detected by PCR, Western-blot and immunofluorescence staining. The activity of caspase-8 in BV2 cells was detected by the CaspGLOW Kit, and the content of interleukine-1ß (IL-1ß) in the supernatant was detected by enzyme linked immunosorbent assay. After the corresponding pathways were blocked by TLR4 small interfering RNA (siRNA) transfection or caspase-8 inhibitor, the expression changes of TLR4 and NLRP3, the activity of caspase-8, and the difference of IL-1ß content could be observed as well as the activity of RGCs co-cultured with BV2. Statistical analysis was performed using analysis of variance. Results: Under co-culture of RGC and BV2 cells, cellular immunofluorescence detection showed that the expression of iba1 in BV2 cells increased, which indicated BV2 cells were activated significantly in the OGD/R model. In the OGD/R model, the apoptosis rate of RGC co-cultured with BV2 cells (71.1%±3.2%) was significantly higher than that of RGC cultured alone (35.1%±1.8%) (t=10.10, P<0.01). Cellular immunofluorescence detection showed that the expression of TLR4 and NLRP3 in BV2 cells in the OGD/R model increased significantly when BV2 cells were cultured alone, and their mRNA levels increased significantly with prolongation of reoxygenation time (F=64.45, 72.74; P<0.01), and peaked at OGD/R 24 h (TLR4 mRNA, relative ratio to control was 2.83±0.23; NLRP3 mRNA, relative ratio to control was 3.12±0.27). Caspase-8 activity also increased with prolonged reoxygenation time, the difference was statistically significant (F=93.57, P<0.01), and peaked at OGD/R 24 h (relative ratio to control was 2.92±0.31). After transfection of BV2 cells with TLR4 siRNA, its caspase-8 activity was significantly inhibited, but using caspase-8 inhibitor did not affect the up-regulation of TLR4 expression in BV2 cells. However, the mature IL-1ß secreted by BV2 cells exposed to OGD/R was significantly reduced by using caspase-8 inhibitor (from 3.52±0.55 to 1.39±0.37, t=7.19, P<0.01), meanwhile, the expression of NLRP3 was also significantly decreased after caspase-8 inhibitor pretreatment (from 2.79±0.23 to 1.37±0.19, t=9.37, P<0.01). In the OGD/R model, the activity of RGC cells co-cultured with TLR4 siRNA-transfected BV2 cells was 74.5%±1.2%, and the activity of RGC cells co-cultured with BV2 cells treated with caspase-8 inhibitor was 62.8%±1.5%, those were both higher than that of RGC cells co-cultured with untreated BV2 cells (36.7%±0.3%), and the difference was statistically significant (t=11.60, 6.83; both P<0.01). Conclusion: TLR4-caspase-8-NLRP3 inflammasome pathway is activated in microglia exposed to OGD/R, resulting in the production of IL-1ß, thereby contributing to the death of RGCs. (Chin J Ophthalmol, 2020, 56: 32-40).


Assuntos
Caspase 8/metabolismo , Morte Celular , Inflamassomos/metabolismo , Microglia/metabolismo , Células Ganglionares da Retina/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Caspase 8/genética , Linhagem Celular , Inflamassomos/genética , Camundongos , Camundongos Endogâmicos C57BL , Hipertensão Ocular , Traumatismo por Reperfusão , Receptor 4 Toll-Like/genética
3.
Toxicol Lett ; 319: 102-110, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31706006

RESUMO

Crizotinib is a multi-target receptor tyrosine kinase inhibitor which is of great importance for the management of ALK-rearranged non-small cell lung cancer (NSCLC) patients. Serious erythroderma and toxic epidermal necrolysis have been reported associated with crizotinib treatment. The underlying mechanisms have not been examined. In this study, we tested the toxicity of crizotinib on immortal human keratinocytes (HaCaT) and human primary keratinocytes. We found that crizotinib directly cause cytotoxic on these two cells, which could be the explanation of the clinical characteristic of pathology. Apoptosis was observed and Z-VAD-FMK, a pan-caspase inhibitor can almost totally reverse the apoptosis induction effect of crizotinib. However, mitochondrial dysfunction and DNA damage were not involved in crizotinib-induced apoptosis, indicating the intrinsic apoptosis pathway have no connection with this cutaneous toxicity. Further studies showed that crizotinib significantly increased cleaved-caspase-8, a signaling protein of extrinsic apoptosis pathway, in a concentration and time-dependent manner. Moreover, we found the targets of crizotinib were not involved in HaCaT cells apoptosis. Collectively, our findings first report keratinocytes apoptosis is the key cause of crizotinib-induced cutaneous toxicity. We also reveal crizotinib induce apoptosis through the extrinsic apoptosis pathway due to detected up-regulated cleaved-caspase-8. Meanwhile, the apoptosis is independent of mitochondrial dysfunction, DNA damage and related drug targets inhibition.


Assuntos
Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Crizotinibe/toxicidade , Dermatite Esfoliativa/induzido quimicamente , Queratinócitos/efeitos dos fármacos , Clorometilcetonas de Aminoácidos/farmacologia , Caspase 8/metabolismo , Inibidores de Caspase/farmacologia , Linhagem Celular , Dano ao DNA , Dermatite Esfoliativa/patologia , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Cultura Primária de Células , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
5.
BMC Complement Altern Med ; 19(1): 312, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31729992

RESUMO

BACKGROUND: Cervical cancer is the second-leading cause of cancer-related mortality in females. Coix lacryma-jobi L. var. ma-yuen (Rom.Caill.) Stapf ex Hook. f. is the most widely recognized medicinal herb for its remedial effects against inflammation, endocrine system dysfunctions, warts, chapped skin, rheumatism, and neuralgia and is also a nourishing food. METHODS: To investigate the activity of Coix lacryma-jobi sprout extract (CLSE) on cell proliferation in human cervical cancer HeLa cells, we conducted a Cell Counting Kit-8 (CCK-8) assay. Flow-cytometric analysis and western blot analysis were performed to verify the effect of CLSE on the regulation of the cell cycle and apoptosis in HeLa cells. RESULTS: We observed that CLSE significantly inhibited cell proliferation. Furthermore, CLSE dose-dependently promoted cell cycle arrest at the sub-G1/ S phase in HeLa cells, as detected by bromodeoxyuridine (BrdU) staining. The cell-cycle-arrest effects of CLSE in HeLa cells were associated with downregulation of cyclin D1 and cyclin-dependent kinases (CDKs) 2, 4, and 6. Moreover, CLSE induced apoptosis, as determined by flow-cytometric analysis and nuclear DNA fragmentation with Annexin V/propidium iodide (PI) and 4'6'-diamidino-2-phenylindole (DAPI) staining. Induction of apoptosis by CLSE was involved in inhibition of the antiapoptotic protein B-cell lymphoma 2 (Bcl-2) and upregulation of the apoptotic proteins p53, cleaved poly (ADP-ribose) polymerase (PARP), cleaved caspase-3, and cleaved caspase-8. Finally, we observed that CLSE inactivated the phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) pathways. CONCLUSIONS: CLSE causes cell cycle arrest and apoptotic cell death through inactivation of the PI3K/AKT pathway in HeLa cells, suggesting it is a viable therapeutic agent for cervical cancer owing to its anticancer effects.


Assuntos
Apoptose/efeitos dos fármacos , Carcinoma/fisiopatologia , Coix/química , Extratos Vegetais/farmacologia , Neoplasias do Colo do Útero/fisiopatologia , Carcinoma/tratamento farmacológico , Carcinoma/genética , Carcinoma/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Coix/crescimento & desenvolvimento , Feminino , Humanos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo
6.
Mol Biol (Mosk) ; 53(5): 830-837, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31661481

RESUMO

Caspase-8 performs initiatory functions during the induction of apoptosis through the extrinsic pathway. Apoptosis is a type of programmed cell death that plays an important role in regulating embryogenesis and maintaining homeostasis in the tissue of an adult organism, as well as differentiating and removing damaged cells. Dysregulation of the apoptosis mechanisms is associated with the pathogenesis and progression of a number of oncological and neurodegenerative diseases. Caspase-8 (also called СAP4, FLICE, MACH, MCH5) is one of two members of the death effector domain (DED)-containing caspases. Despite the fact that the role of caspase-8 in apoptosis has been well known since the mid 1990s, we are only now beginning to understand the subtle mechanisms of its activation and regulation in response to the activation of death receptors (DRs). In particular, it was demonstrated that the activation of caspase-8 requires the formation of specific oligomeric structures, which are named DED filaments. In this review, the recent data on the mechanisms of activating initiator caspase-8 in DED filaments are considered that allow us to better understand the subtle mechanisms of the initiation of the programmed cell death.


Assuntos
Apoptose , Caspase 8/metabolismo , Precursores Enzimáticos/metabolismo , Citoesqueleto/metabolismo , Ativação Enzimática , Humanos
7.
Int J Nanomedicine ; 14: 7123-7139, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31564869

RESUMO

Background: Poly(amidoamine) (PAMAM) dendrimers are of considerable interest when used as a carrier for topical drugs for the skin, although little is known about their possible side effects. Therefore, our study was about the impact of 2nd and 3rd generation PAMAM dendrimers on human keratinocytes and fibroblasts cells. Methods: The effect of the tested compounds on collagen biosynthesis was determined using 5[3H]-proline incorporation bioassay. Morphological changes accompanying cell growth inhibition were observed using a confocal microscope. To evaluate the percentage of apoptotic/necrotic cells and the cell growth dynamic of apoptotic features, we performed Annexin V/PI double staining assay, assessed caspase activity, and performed cell cycle analysis by flow cytometry. The flow cytometry method was also used to determine the effect of dendrimers on pro-inflammatory cytokines (IL-6, IL-8 IL-1ß). Results: The obtained results showed that as the concentration and the generation of dendrimers increased, collagen biosynthesis decreased. We also observed abnormalities in cell differentiation, which may have caused disturbed secretion of pro-inflammatory cytokines. We found that dendrimers cause chronic inflammation which may cause adverse changes in the skin, ultimately- leading to apoptosis in the case of dendrimers in lower concentrations or necrosis at higher concentrations (especially 3rd generation dendrimers). In addition, the inflammatory path induced by the tested compounds was caused by damage in the mitochondria, which we observed as a significant decrease in the mitochondrial membrane potential. Conclusion: The results of our study showed that PAMAM dendrimers can cause disorders of cell proliferation and differentiation and may be the cause of cell cycle deregulation and chronic adverse inflammation.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Citocinas/metabolismo , Dendrímeros/farmacologia , Fibroblastos/metabolismo , Mediadores da Inflamação/metabolismo , Queratinócitos/metabolismo , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 8/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Colágeno/biossíntese , Dendrímeros/química , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fluorescência , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Necrose
8.
PLoS Comput Biol ; 15(9): e1007374, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31553717

RESUMO

Ligand binding to death receptors activates apoptosis in cancer cells. Stimulation of death receptors results in the formation of intracellular multiprotein platforms that either activate the apoptotic initiator Caspase-8 to trigger cell death, or signal through kinases to initiate inflammatory and cell survival signalling. Two of these platforms, the Death-Inducing Signalling Complex (DISC) and the RIPoptosome, also initiate necroptosis by building filamentous scaffolds that lead to the activation of mixed lineage kinase domain-like pseudokinase. To explain cell decision making downstream of death receptor activation, we developed a semi-stochastic model of DISC/RIPoptosome formation. The model is a hybrid of a direct Gillespie stochastic simulation algorithm for slow assembly of the RIPoptosome and a deterministic model of downstream caspase activation. The model explains how alterations in the level of death receptor-ligand complexes, their clustering properties and intrinsic molecular fluctuations in RIPoptosome assembly drive heterogeneous dynamics of Caspase-8 activation. The model highlights how kinetic proofreading leads to heterogeneous cell responses and results in fractional cell killing at low levels of receptor stimulation. It reveals that the noise in Caspase-8 activation-exclusively caused by the stochastic molecular assembly of the DISC/RIPoptosome platform-has a key function in extrinsic apoptotic stimuli recognition.


Assuntos
Apoptose/fisiologia , Caspase 8 , Modelos Biológicos , Receptores de Morte Celular , Caspase 8/química , Caspase 8/metabolismo , Sobrevivência Celular/fisiologia , Biologia Computacional , Humanos , Neoplasias/metabolismo , Receptores de Morte Celular/química , Receptores de Morte Celular/metabolismo
9.
Int J Mol Sci ; 20(17)2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31480289

RESUMO

Modern molecular medicine demands techniques to efficiently deliver molecules directly into mammalian cells. As proteins are the final mediators of most cellular pathways, efficient intracellular protein delivery techniques are highly desired. In this respect, photoporation is a promising recent technique for the delivery of proteins directly into living cells. Here, we show the possibility to deliver a model saccharide (FD70) and a model protein (FITC-BSA) into murine B16 melanoma cells by using the vapor nanobubble photoporation technique with an efficiency of 62% and 38%, respectively. Next, we delivered the mixed-lineage kinase domain-like (MLKL) protein, the most terminal mediator of necroptosis currently known, and caspase-8 and -3 protein, which are important proteins in the initiation and execution of apoptosis. A significant drop in cell viability with 62%, 71% and 64% cell survival for MLKL, caspase-8 and caspase-3, respectively, was observed. Remarkably, maximal cell death induction was already observed within 1 h after protein delivery. Transduction of purified recombinant MLKL by photoporation resulted in rapid cell death characterized by cell swelling and cell membrane rupture, both hallmarks of necroptosis. As necroptosis has been identified as a type of cell death with immunogenic properties, this is of interest to anti-cancer immunotherapy. On the other hand, transduction of purified recombinant active caspase-3 or -8 into the tumor cells resulted in rapid cell death preceded by membrane blebbing, which is typical for apoptosis. Our results suggest that the type of cell death of tumor cells can be controlled by direct transduction of effector proteins that are involved in the executioner phase of apoptosis or necroptosis.


Assuntos
Apoptose , Sistemas de Liberação de Medicamentos , Luz , Melanoma Experimental/terapia , Nanopartículas/química , Proteínas Quinases/metabolismo , Animais , Caspase 3/metabolismo , Caspase 8/metabolismo , Linhagem Celular Tumoral , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Peso Molecular , Necrose , Volatilização
10.
Molecules ; 24(17)2019 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-31454945

RESUMO

Tartary buckwheat (Fagopyrum tataricum (L.) Gaertn) is rich in functional compounds such as rutin, quercetin, d-chiro-inositol, dietary fiber, and essential amino acids. Electric field (EF) treatment before sprout germination results in physiological and chemical changes, and some alterations might lead to positive applications in plant seeds. MTT assay showed that the effect of total flavonoids on human gastric cancer cell line MGC80-3 was significantly changed after EF treatment for different germination days (3-7 days). Among them, the total flavonoids of tartary buckwheat (BWTF) on the third day had the most obvious inhibitory effect on MGC80-3 (p < 0.01). In addition, flow cytometry evidenced that different ratios of quercetin and rutin had effects on the proliferation of MGC80-3. The same content of quercetin and rutin had the best effect, reaching 6.18 ± 0.82%. The anti-cancer mechanism was mainly promoted by promoting the expression of apoptotic proteins. The expression of Bax/Bcl-2 and caspase-8 in MGC80-3 cells was mediated by BWTFs. This study has good research value for improving the biological and economic value of tartary buckwheat.


Assuntos
Fagopyrum/fisiologia , Quercetina/farmacologia , Rutina/farmacologia , Neoplasias Gástricas/metabolismo , Caspase 8/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Fagopyrum/química , Flavonoides/isolamento & purificação , Flavonoides/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Germinação , Humanos , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Quercetina/isolamento & purificação , Rutina/isolamento & purificação , Neoplasias Gástricas/tratamento farmacológico , Proteína X Associada a bcl-2/metabolismo
11.
BMC Genomics ; 20(1): 682, 2019 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-31464583

RESUMO

BACKGROUND: The brown plant hopper (BPH), Nilaparvata lugens, is one of the major pest of rice (Oryza sativa). Plant defenses against insect herbivores have been extensively studied, but our understanding of insect responses to host plants' resistance mechanisms is still limited. The purpose of this study is to characterize transcripts of BPH and reveal the responses of BPH insects to resistant rice at transcription level by using the advanced molecular techniques, the next-generation sequencing (NGS) and the single-molecule, real-time (SMRT) sequencing. RESULTS: The current study obtained 24,891 collapsed isoforms of full-length transcripts, and 20,662 were mapped to known annotated genes, including 17,175 novel transcripts. The current study also identified 915 fusion genes, 1794 novel genes, 2435 long non-coding RNAs (lncRNAs), and 20,356 alternative splicing events. Moreover, analysis of differentially expressed genes (DEGs) revealed that genes involved in metabolic and cell proliferation processes were significantly enriched in up-regulated and down-regulated sets, respectively, in BPH fed on resistant rice relative to BPH fed on susceptible wild type rice. Furthermore, the FoxO signaling pathway was involved and genes related to BPH starvation response (Nlbmm), apoptosis and autophagy (caspase 8, ATG13, BNIP3 and IAP), active oxygen elimination (catalase, MSR, ferritin) and detoxification (GST, CarE) were up-regulated in BPH responses to resistant rice. CONCLUSIONS: The current study provides the first demonstrations of the full diversity and complexity of the BPH transcriptome, and indicates that BPH responses to rice resistance, might be related to starvation stress responses, nutrient transformation, oxidative decomposition, and detoxification. The current result findings will facilitate further exploration of molecular mechanisms of interaction between BPH insects and host rice.


Assuntos
Hemípteros/genética , Oryza/genética , Animais , Proteínas Relacionadas à Autofagia/metabolismo , Caspase 8/metabolismo , Catalase/metabolismo , Feminino , Ferritinas/metabolismo , Proteína Forkhead Box O1/metabolismo , Regulação da Expressão Gênica , Genótipo , Glutationa Transferase/metabolismo , Hemípteros/metabolismo , Herbivoria , Oryza/metabolismo , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Transaminases/metabolismo , Transcriptoma
12.
Food Chem Toxicol ; 133: 110750, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31390533

RESUMO

Human peripheral blood mononuclear cells (PBMCs) are one of the main cell models used in studies concerning the exposure of humans (in vitro) to various chemical substances. Changes in PBMCs may reflect the general reaction of the organism regarding the effect of xenobiotics. The aim of this work was to evaluate the effect of di-n-butyl phthalate (DBP), butylbenzyl phthalate (BBP) and their metabolites: mono-n-butylphthalate (MBP), mono-benzylphthalate (MBzP) upon the induction of apoptosis in human peripheral blood mononuclear cells in vitro. PBMCs were incubated with the studied compounds at concentrations from 1 to 100 µg/mL for 12 h and/or 24 h. In order to clarify the mechanism of phthalates-induced programmed cell death, the changes in the calcium ions (Ca2+) level, alterations in the transmembrane mitochondrial potential (ΔÑ°m) and caspase-8, -9, -3 activity as well as externalization of phosphatidylserine have been determined. An increased Ca2+ level and a reduction of the ΔÑ°m were observed in PBMCs incubated with all of the studied compounds, and particularly with DBP and BBP. Phthalates caused an increase of caspases activity. The most pronounced increase was observed for caspase -9. The most pronounced pro-apoptotic changes were caused by DBP followed by BBP and then by their metabolites.


Assuntos
Apoptose/efeitos dos fármacos , Dibutilftalato/toxicidade , Linfócitos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Ácidos Ftálicos/toxicidade , Cálcio/metabolismo , Caspase 3/metabolismo , Caspase 8/metabolismo , Caspase 9/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Necrose/induzido quimicamente , Plastificantes/toxicidade
13.
Indian J Pharmacol ; 51(3): 181-207, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31391686

RESUMO

AIM: Caspases-3 and 8 are key mediators of intrinsic and extrinsic pathway of apoptosis, respectively. Triterpenoids of natural and synthetic origin reported as anticancer agents with apoptotic potential and hence may prove to be good candidates for in silico testing against caspases-3 and 8. MATERIALS AND METHODS: Various naturally-occurring and synthetic triterpenoids were subjected to activity prediction using PASS Online software, and among them, 67 compounds were selected for further processing. Protein structure of caspase-3 (3DEI) and caspase-8 (3KJQ) was obtained from the protein data bank and docked with selected triterpenoids using AutoDock Tools and AutoDock Vina. Toxicological profile was predicted based on clinical manifestations using PASS online software. RESULTS: The high docking score of -10.0, -9.9, -9.8, and -9.5 were shown by friedelin, tingenone, albiziasaponin A, and albiziasaponin C, respectively, for caspase-3, and -11.0, -9.6, -9.6, and -9.4 by ß-boswellic acid, bryonolic acid, canophyllic acid, and CDDO, respectively, for caspase-8. Possible adverse events were predicted with varying degree of probability and major relevant effects were reported. Hydrostatic interactions along with formation of hydrogen bonds with specific amino acids in the binding pocket were identified with each triterpenoid. CONCLUSION: Lead molecules identified through this in silico study such as friedelin, tingenone, albiziasaponin, bryonolic acid, and canophyllic acid may be utilized for further in vitro/in vivo studies as apoptotic agents targeting caspases-3 and 8.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Triterpenos/farmacologia , Caspase 3/metabolismo , Caspase 8/metabolismo , Simulação de Acoplamento Molecular
14.
Gastroenterology ; 157(5): 1310-1322.e13, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31352002

RESUMO

BACKGROUND & AIMS: Interferon lambda (IFNL) is expressed at high levels by intestinal epithelial cells (IECs) and mucosal immune cells in response to infection and inflammation. We investigated whether IFNL might contribute to pathogenesis of Crohn's disease (CD). METHODS: We obtained serum samples and terminal ileum biopsies from 47 patients with CD and 16 healthy individuals (controls). We measured levels of IFNL by enzyme-linked immunosorbent assay and immunohistochemistry and location of expression by confocal microscopy. Activation of IFNL signaling via STAT1 was measured in areas of no, mild, moderate, and severe inflammation and correlated with Paneth cell homeostasis and inflammation. IFNL expression and function were studied in wild-type mice and mice with intestinal epithelial cell-specific (ΔIEC) disruption or full-body disruption of specific genes (Mlkl-/-, Stat1ΔIEC, Casp8ΔIEC, Casp8ΔIECRipk3-/-, Casp8ΔIECTnfr-/-, Casp8ΔIECMlkl-/-, and Nod2-/- mice). Some mice were given tail vein injections of a vector encoding a secreted form of IFNL. Intestinal tissues were collected from mice and analyzed by immunohistochemistry and immunoblots. We generated 3-dimensional small intestinal organoids from mice and studied the effects of IFNL and inhibitors of STAT-signaling pathway. RESULTS: Patients with CD had significant increases in serum and ileal levels of IFNL compared with controls. Levels of IFNL were highest in ileum tissues with severe inflammation. High levels of IFNL associated with a reduced number of Paneth cells and increased cell death at the crypt bottom in inflamed ileum samples. Intestinal tissues from the ileum of wild-type mice injected with a vector expressing IFNL had reduced numbers of Paneth cells. IFNL-induced death of Paneth cells in mice did not occur via apoptosis, but required Mixed Lineage Kinase Domain Like (MLKL) and activation of Signal transducer and activator of transcription 1 (STAT1). In organoids, inhibitors of Janus kinase (JAK) signaling via STAT1 (glucocorticoids, tofacitinib, or filgotinib) reduced expression of proteins that mediate cell death and prevented Paneth cell death. CONCLUSIONS: Levels of IFNL are increased in serum and inflamed ileal tissues from patients with CD and associated with a loss of Paneth cells. Expression of a secreted form of IFNL in mice results in loss of Paneth cells from intestinal tissues, via STAT1 and MLKL, controlled by caspase 8. Strategies to reduce IFNL or block its effects might be developed for treatment of patients with CD affecting the terminal ileum.


Assuntos
Doença de Crohn/metabolismo , Íleo/metabolismo , Interferons/metabolismo , Interleucinas/metabolismo , Celulas de Paneth/metabolismo , Fator de Transcrição STAT1/metabolismo , Animais , Caspase 8/genética , Caspase 8/metabolismo , Morte Celular , Doença de Crohn/imunologia , Doença de Crohn/patologia , Modelos Animais de Doenças , Humanos , Íleo/imunologia , Íleo/patologia , Interferons/genética , Interleucinas/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Celulas de Paneth/imunologia , Celulas de Paneth/patologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Fator de Transcrição STAT1/deficiência , Fator de Transcrição STAT1/genética , Transdução de Sinais , Técnicas de Cultura de Tecidos , Regulação para Cima
15.
Vet Microbiol ; 235: 151-163, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31282373

RESUMO

This study demonstrates that the Muscovy duck reovirus (MDRV) p10.8 protein is one of many viral non-structural proteins that induces both cell cycle arrest and apoptosis. The p10.8 but not σC is a nuclear targeting protein that shuttles between the nucleus and the cytoplasm. Our results reveal that p10.8-induced apoptosis in cultured cells occurs by the nucleoporin Tpr/p53-dependent and Fas/caspase 8-mediated pathways. Furthermore, a compelling finding from this study is that the p10.8 and σC proteins of MDRV facilitate CDK2 and CDK4 degradation via the ubiquitin-proteasome pathway. We found that depletion of Cdc20 reversed the p10.8- and σC- mediated CDK4 degradation and p10.8-induced apoptosis, suggesting that Cdc20 plays a critical role in modulating p10.8-mediated cell cycle and apoptosis. Furthermore, we found that depletion of chaperonin-containing tailless complex polypeptide 1 (CCT) 2 and CCT5 reduced the level of Cdc20 and reversed the p10.8- and σC-mediated CDK4 degradation and p10.8-induced apoptosis, indicating that molecular chaperone CCT2 and CCT5 are required for stabilization of Ccd20 for mediating both cell cycle arrest and apoptosis. This study provides mechanistic insights into how p10.8 induces both cell cycle arrest and apoptosis.


Assuntos
Proteínas Cdc20/metabolismo , Chaperonina com TCP-1/metabolismo , Orthoreovirus/genética , Doenças das Aves Domésticas/virologia , Infecções por Reoviridae/veterinária , Proteínas não Estruturais Virais/metabolismo , Animais , Apoptose , Caspase 8/genética , Caspase 8/metabolismo , Proteínas Cdc20/genética , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Chaperonina com TCP-1/genética , Patos/virologia , Fibroblastos/virologia , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , RNA Interferente Pequeno , Células Vero , Proteínas não Estruturais Virais/genética
16.
BMC Cancer ; 19(1): 670, 2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31286874

RESUMO

BACKGROUND: In epithelial cells, tyrosine kinases induce tyrosine phosphorylation and ubiquitination of the E-cadherin complex, which is responsible for the epithelial-mesenchymal transition (EMT). However, the precise mechanisms remain unclear. METHODS: Protein antibody microarray analysis and E3 ligase profiling were performed to detect the unique E3 ligase underlying E-cadherin downregulation in lung adenocarcinoma tissues. Gene knockdown was performed using viral shRNA. Immunoblotting, immunofluorescence, immunoprecipitation, and xenograft models in vivo were integratively applied to explore RNF43-induced EMT in lung adenocarcinoma cell lines. RESULTS: Protein antibody microarray analysis and E3 ligase profiling revealed that the RING finger protein 43 (RNF43) was linked to E-cadherin downregulation within the context of c-Src activation in lung adenocarcinoma tissues. In addition, the c-Src-Caspase-8 interaction markedly increased c-Src activity. Activated c-Src phosphorylated E-cadherin at the tyrosine 797 site to initiate RNF43-mediated E-cadherin ubiquitination at lysine 816 and subsequent degradation, thus allowing the nuclear translocation of ß-catenin and upregulation of Vimentin and RNF43 expression in lung adenocarcinoma cells. Decreased E-cadherin expression and increased Vimentin expression induced the EMT phenotype and promoted tumor metastasis. The Frizzled 8 (Frz8)-RNF43-induced ubiquitination of phosphorylated E-cadherin was blocked by a monoclonal antibody against the cysteine-rich domain (CRD) of Frz8 but not by antibodies against the protease domain (PA) of RNF43. CONCLUSIONS: Our data suggest that RNF43 participates in the regulation of EMT in the metastasis of lung adenocarcinoma through the ubiquitination and degradation of phosphorylated E-cadherin by activated c-Src.


Assuntos
Adenocarcinoma de Pulmão/secundário , Antígenos CD/metabolismo , Caderinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Transição Epitelial-Mesenquimal , Neoplasias Pulmonares/patologia , Proteínas Oncogênicas/metabolismo , Células A549 , Adenocarcinoma de Pulmão/metabolismo , Animais , Anticorpos Monoclonais , Caspase 8/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/imunologia , Seguimentos , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Oncogênicas/imunologia , Fosforilação , Proteólise , Receptores de Superfície Celular/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Vimentina/metabolismo , beta Catenina/metabolismo
17.
Toxicol Lett ; 314: 18-26, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31299270

RESUMO

Epidemiological investigations indicate that effects related to prenatal adverse environments on the organs of the offspring could continue to adulthood. This study intends to confirm that prenatal nicotine exposure (PNE) increases the susceptibility of osteoarthritis (OA) in the male offspring, and to explore the potential intrauterine programming mechanism. During pregnancy, rats were divided into a PNE group and a control group. After birth, rats were given a high-fat diet for 6 months and long-distance running for 6 weeks. The rats were euthanized at 18 months after birth (PM18) and on gestational day 20 (GD20), respectively. Knee joints were collected for histochemistry, immunohistochemistry, and quantitative polymerase chain reaction (qPCR) assays. Histological analyses and the Mankin's score showed increased cartilage destruction and accelerated OA progression in adult offspring from the PNE group. Immunohistochemistry results showed decreased expression of transforming growth factor beta (TGFß) signaling pathway. Furthermore, the expression of apoptosis factors (caspase-3 and caspase-8), inflammatory factors [interleukin (IL)-1, IL-6] and matrix degradation enzymes [matrix metalloproteinase (MMP)-3, MMP-13] were also significantly increased. Traced back to the intrauterine period, it was found that the number of chondrocytes and the contents of Col2A1 and aggrecan in the matrix in the PNE group were decreased. And, the expression of the TGFß signaling pathway was inhibited. These results suggested that PNE enhanced the susceptibility of OA in male elderly offspring rats by down-regulating TGFß signaling, which increased articular cartilage local inflammation, matrix degradation, and cell apoptosis. This study confirmed the developmental origin of OA, and clarified the congenital and the living environment impact on the occurrence and development of OA. Our findings provide a theoretical and experimental basis for OA early prevention.


Assuntos
Articulações/efeitos dos fármacos , Nicotina/toxicidade , Agonistas Nicotínicos/toxicidade , Osteoartrite/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Fatores Etários , Agrecanas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 8/metabolismo , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Condrócitos/patologia , Condrogênese/efeitos dos fármacos , Colágeno Tipo II/metabolismo , Feminino , Idade Gestacional , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Articulações/metabolismo , Articulações/patologia , Masculino , Exposição Materna , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , Gravidez , Ratos Wistar , Fatores de Risco , Fatores Sexuais
18.
Ren Fail ; 41(1): 455-466, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31163002

RESUMO

Purpose: To investigate whether Niban protein plays a role in renal interstitial fibrosis by regulating renal tubular epithelial cell apoptosis and explore the underlying mechanism. Methods: Unilateral ureteral obstruction (UUO) model was performed in C57B/6J mice, and divided into sham operation group and groups of days 3, days 7, and days 14. Niban expression was detected by immunohistochemistry and Western blot. TUNEL assays were used to detected apoptosis. Niban siRNA and overexpression Niban plasmid were transfected in HK-2 cells respectively to explore apoptosis related mechanisms of Niban during angiotensin II (AngII) - and endoplasmic reticulum (ER) stress-induced injury. Results: With the development of obstruction, Niban's expression decreased gradually while apoptosis increased. Silencing of Niban not only increased the AngII- and ER stress-induced apoptosis, but also promoted the expression of caspase 8, caspase 9, Bip, and Chop. Overexpression of Niban reduced AngII-induced apoptosis and the expression of caspase 8 and caspase 9. Conclusions: Niban protein is involved in apoptosis regulation in HK-2 cells, and most likely via caspase-dependent pathway.


Assuntos
Apoptose , Biomarcadores Tumorais/metabolismo , Nefropatias/patologia , Túbulos Renais/patologia , Proteínas de Neoplasias/metabolismo , Animais , Biomarcadores Tumorais/genética , Caspase 8/metabolismo , Caspase 9/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático , Células Epiteliais , Fibrose , Humanos , Nefropatias/etiologia , Túbulos Renais/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/genética , RNA Interferente Pequeno , Obstrução Ureteral/complicações
19.
J Toxicol Sci ; 44(6): 435-440, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31168030

RESUMO

Fas/CD95 plays a pivotal role in T cell-mediated cytotoxicity. Accumulating evidence has suggested that resistance to Fas-mediated apoptosis contributes to the escape of cancer cells from immune destruction, and allows to undergo proliferation and outgrowth of cancer cells. In this study, we found that the anti-cancer drug gefitinib, a tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR), has an ability to enhance Fas-mediated cytotoxicity. In the presence of nontoxic concentrations of gefitinib, Fas-induced activation of caspase-8 and subsequent apoptosis was dramatically promoted, suggesting that gefitinib increases the sensitivity to Fas-mediated apoptosis. Interestingly, the effects of gefitinib were observed in EGFR or p53 knockout (KO) cells. These observations indicate that both EGFR and p53 are dispensable for the enhancement. On the other hand, gefitinib clearly downregulated heat shock protein 70 (HSP70) as previously reported. Considering that HSP70 contributes to protection of cells against Fas-mediated apoptosis, gefitinib may increase the sensitivity to Fas-mediated apoptosis by downregulating HSP70. Thus, our findings reveal novel properties of gefitinib, which may provide insight into the alternative therapeutic approaches of gefitinib for Fas-resistant tumors.


Assuntos
Antineoplásicos/farmacologia , Caspase 8/metabolismo , Proteína Ligante Fas/fisiologia , Gefitinibe/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Proteínas de Choque Térmico HSP72/metabolismo , Humanos , Camundongos
20.
Food Funct ; 10(6): 3626-3636, 2019 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-31162493

RESUMO

In this study, the apoptosis induction and antitumor activity of a novel complex, seleno-ß-lactoglobulin (Se-ß-Lg), on H22 cells were explored. In in vitro experiments, the MTT assay showed that Se-ß-Lg was cytotoxic to H22 cells in a concentration- and time-dependent manner and displayed few proliferation inhibition effects on normal liver L02 cells. Annexin V-FITC/PI and PI staining assays showed that Se-ß-Lg induced apoptosis changes of H22 cells from early to late apoptosis and led to S phase cell cycle arrest. Western blot and Z-VAD-FMK inhibitor assays showed that Se-ß-Lg triggered the Fas/FasL-mediated caspase 8-dependent extrinsic death receptor pathway in H22 cells. In in vivo experiments, Se-ß-Lg effectively repressed the growth of transplanted H22 solid tumors in a dose-dependent manner and exhibited few toxic effects on the host animals. H&E and PI staining of tumor tissues showed that Se-ß-Lg caused the occurrence of typical apoptosis morphology features and dose-dependently increased the proportion of apoptosis peaks (Sub-G1 peak) in H22 solid tumors. These results suggest that Se-ß-Lg has the capacity to induce H22 tumor cell apoptosis in vitro and in vivo and support that Se-ß-Lg can be applied as a functional complex in food.


Assuntos
Lactoglobulinas/farmacologia , Leite/química , Selênio/farmacologia , Animais , Apoptose/efeitos dos fármacos , Caspase 8/genética , Caspase 8/metabolismo , Bovinos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Lactoglobulinas/química , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Selênio/química
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