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1.
Gene ; 732: 144370, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31954860

RESUMO

Apoptosis plays a significant role in the cellular immune responses against infections, especially those related to viruses. Across various species, Caspase 3 is a prominent mediator of apoptosis and participates in the cell death signaling cascade. However, its role remains relatively unknown in cod fish. In this study, we aimed to reveal the role of Pacific cod Caspase-3 (GmCasp3) in apoptosis and its evolutionary position. Our results showed that the GmCasp3 cDNA contains an open reading frame of 864 nucleotides; that codes for 287 amino acids long protein with a molecular weight of 32.03 kDa. The sequence alignments and 3-D model indicated that GmCasp3 contained highly conserved domains, such as "QACRG", "GSWFI" and "HG" active sites, however, the phylogenetic tree analysis revealed that both GmCasp3 and Atlantic cod caspase-3 clustered together are far from the high vertebrate branch, indicating they are at a lower position in vertebrate evolution. Red fluorescent labeling vector pDsRed2-C1-GmCasp3 was constructed and it was transfected into EPC cell lines. The result showed that GmCasp3 protein was distribute in the protoplasm and expressed in apoptotic cell debris. Moreover, the GmCasp3 enzyme activity increased with the increased post-transfection analysis time, while the genome DNA was visibly fragmented at 36 h post transfection. Flow cytometry analysis showed that the proportion of apoptosis cells increased from 12 h to 24 h post transfection. In conclusion, the conserved functions of GmCasp3 in apoptosis indicated that Pacific cod has the similar apoptotic characteristics as other animals.


Assuntos
Apoptose/fisiologia , Caspase 3/fisiologia , Proteínas de Peixes/fisiologia , Sequência de Aminoácidos , Animais , Caspase 3/química , Caspase 3/genética , Proteínas de Peixes/química , Proteínas de Peixes/genética , Gadiformes , Filogenia , Alinhamento de Sequência , Frações Subcelulares/metabolismo
2.
Fish Shellfish Immunol ; 94: 792-799, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31585244

RESUMO

The caspase is an essential module in the Drosophila immune deficiency (IMD) pathway, which plays a crucial role in countering pathogen infection. In this study, a gene named PcCaspase-3C was found in Procambarus clarkia with a full-length of 4684 bp, including a 1572 bp opening reading frame, which encoded a putative protein of 523 amino acids. PcCaspase-3C contained a CASc domain constituted of 237 amino acids. The PcCaspase-3C gene was primarily expressed in heart, stomach, and intestine, while less in gonad, hepatopancreas, gills, and hemocytes, with the least expression in muscle. Infection with Staphyloccocus aureus, Vibrio parahaemolyticus or white spot syndrome virus (WSSV) induced an up-regulated expression of PcCaspase-3C in intestine or stomach to varying degrees. When PcCaspase-3C was silenced by double-stranded RNA, the expression of some antimicrobial peptides such as ALF2, ALF5, ALF6, Cru3, Cru4, and Lys was significantly inhibited. In addition, silencing of PcCaspase-3C accelerated infection with WSSV in vivo. According to these results, we suggest that PcCaspase-3C might play a crucial role in the immune response of P. clarkia against pathogenic bacterial and viral infections.


Assuntos
Astacoidea/genética , Astacoidea/imunologia , Caspase 3/genética , Caspase 3/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Caspase 3/química , Perfilação da Expressão Gênica , Filogenia , Alinhamento de Sequência
3.
Analyst ; 144(22): 6751-6759, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31612876

RESUMO

There is a great need to develop sensitive and specific methods for quantitative analysis of caspase-3 activities in cell apoptosis. Herein, we report a new method for sensitive detection of caspase-3 enzyme activities and inhibitor screening based on dual maleimide (DuMal) labeling quantitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Evaluation of caspase-3 activities is performed using MS analysis of the enzymatic product of the peptide probe, which fuses a caspase-3 cleavable peptide segment (DEVD) and a quantifiable "ID tag" (a peptide segment of FRGLRGFKC labeled by maleimide). The DuMal labeling technique features non-isotopic tagging, rapid reactions, and reproducible quantitation. We have achieved quantitative analysis of the enzyme activities with a limit of detection (LOD) and limit of quantitation (LOQ) of caspase-3 down to 0.11 nM and 0.29 nM respectively and a proof-of-concept demonstration of its inhibitor screening. Our method has further been tested for caspase-3 activities in a Parkinson's disease cellular model, suggesting a useful tool for protease activity research.


Assuntos
Caspase 3/análise , Ensaios Enzimáticos/métodos , Maleimidas/química , Peptídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , 1-Metil-4-fenilpiridínio/farmacologia , Sequência de Aminoácidos , Animais , Caspase 3/química , Inibidores de Caspase/química , Linhagem Celular Tumoral , Humanos , Limite de Detecção , Camundongos , Oligopeptídeos/química , Doença de Parkinson/enzimologia , Ácidos Pentanoicos/química , Ratos
4.
Analyst ; 144(13): 3959-3966, 2019 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-31134974

RESUMO

MDM2 can mediate the degradation of tumor suppressor p53 through an autoregulatory feedback loop, in which MDM2 abolishes wild-type p53 function and accelerates malignant transformation. However, the incorporation of MDM2 antagonist Nutlin-3 could reactivate the transcriptional activity of p53, up-regulate caspase-3, and induce apoptosis. In this work, the simultaneous and label-free monitoring of p53-MDM2 complex and caspase-3 levels in cancer cells before and after Nutlin-3 treatment was conducted using dual-channel surface plasmon resonance (SPR). The p53-MDM2 complex was captured in one fluidic channel covered with consensus double-stranded (ds)-DNA, while the other channel was pre-immobilized with caspase-3-specific biotinylated DEVD-containing peptides. To amplify the SPR signals, the attachment of streptavidin (SA)-conjugated anti-MDM2 antibody in both channels was achieved. The signal diversity before and after Nutlin-3 treatment is indicative of the difference in the levels of the intracellular p53-MDM2 complex and caspase-3. The limit of detection for p53-MDM2 and caspase-3 down to 4.54 pM and 0.03 ng mL-1, respectively, was attained. Upon treatment with Nutlin-3, MCF-7 cancer cells with wild-type p53 showed decreased expression of the p53-MDM2 complex and an increased caspase-3 level, while MDA-MB-231 cancer cells with mutant p53 exhibited an elevated caspase-3 level and unchanged p53-MDM2 complex expression. The apoptosis of MCF-7 and MDA-MB-231 cancer cells upon Nutlin-3 treatment follows a p53-dependent and a p53-independent pathway, respectively. The proposed method is sensitive, selective and label-free, holding great promise for assaying intracellular p53-MDM2 complex and caspase-3 levels and differentiating Nutlin-3-mediated p53-dependent or p53-independent apoptotic pathways.


Assuntos
Caspase 3/análise , Imidazóis/farmacologia , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/análise , Ressonância de Plasmônio de Superfície/métodos , Proteína Supressora de Tumor p53/análise , Apoptose/efeitos dos fármacos , Biotina/química , Caspase 3/química , Caspase 3/metabolismo , Linhagem Celular Tumoral , DNA/química , Relação Dose-Resposta a Droga , Humanos , Limite de Detecção , Ligação Proteica , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estreptavidina/química , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo
5.
Molecules ; 24(10)2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31117169

RESUMO

The engineering of enzymes for the purpose of controlling their activity represents a valuable approach to address challenges in both fundamental and applied research. Here, we describe and compare different design strategies for the generation of a human rhinovirus-14 (HRV14) 3C protease-inducible caspase-3 (CASP3). We exemplify the application potential of the resulting protease by controlling the activity of a synthetic enzyme cascade, which represents an important motif for the design of artificial signal transduction networks. In addition, we use our engineered CASP3 to characterize the effect of aspartate mutations on enzymatic activity. Besides the identification of mutations that render the enzyme inactive, we find the CASP3-D192E mutant (aspartate-to-glutamate exchange at position 192) to be inaccessible for 3C protease-mediated cleavage. This indicates a structural change of CASP3 that goes beyond a slight misalignment of the catalytic triad. This study could inspire the design of additional engineered proteases that could be used to unravel fundamental research questions or to expand the collection of biological parts for the design of synthetic signaling pathways.


Assuntos
Caspase 3/genética , Cisteína Endopeptidases/genética , Engenharia de Proteínas , Rhinovirus/enzimologia , Proteínas Virais/genética , Ácido Aspártico/metabolismo , Caspase 3/química , Caspase 3/metabolismo , Domínio Catalítico/genética , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Mutação , Proteínas Virais/química , Proteínas Virais/metabolismo
6.
Environ Sci Pollut Res Int ; 26(13): 13441-13452, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30911963

RESUMO

Radiation-induced brain injury is common and mainly occurs in patients receiving radiotherapy for malignant head and neck tumors. The brain is oversensitive to oxidant injury induced by radiation. Biotin is a member of the vitamin B complex family and its deficiency has been associated with neurogenesis impairment in animals and humans. The present study was undertaken to investigate the mitigating effect of biotin on the cerebral cortical and hippocampal damage induced by radiation exposure. Animals were exposed to radiation in the presence or absence of biotin and sacrificed on day 10. The results demonstrated that the administration of biotin 2 mg to irradiated rats had no significant effect on the radiation-induced damage of the cerebral cortex and the hippocampus, while the administration of biotin 6 mg has significantly attenuated oxidative stress in the hippocampus, manifested by a reduction of 4-hydroxynonenal (4HNE), total nitrate/nitrite (NOx), and xanthine oxidase (XO) levels associated with an elevation of glutathione (GSH) content as well as superoxide dismutase (SOD) and catalase (CAT) activities. In addition, biotin decreased the pro-inflammatory cytokines (interleukin-1 beta (IL-1ß), interleukin-6 (IL-6), and tumor necrotic factor alpha (TNF-α)), caspase-3, poly(ADP-ribose) polymerase 1 (PARP1) level, and PARP1 gene expression. Moreover, biotin 6 mg treatment diminished serum S100 protein (S100B) and neuron-specific enolase (NSE) levels. In conclusion, biotin treatment at high dose post-irradiation has efficiently neutralized the effect of free radicals in the hippocampal region of rats. Thus, it could be applicable as a radio-mitigator for reducing or delayed radiation-induced brain injury in patients post-radiotherapy.


Assuntos
Biotina/química , Encéfalo/efeitos dos fármacos , Caspase 3/metabolismo , Córtex Cerebral/fisiologia , Citocinas/metabolismo , Glutationa/metabolismo , Hipocampo/efeitos dos fármacos , Interleucina-6/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Xantina Oxidase/metabolismo , Animais , Caspase 3/química , Interleucina-6/química , Masculino , Ratos , Fator de Necrose Tumoral alfa/química
7.
Environ Sci Pollut Res Int ; 26(15): 15209-15217, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30924043

RESUMO

Breast cancer is a global public health problem where it is the second most prevalent cancer. Historical cancer treatment with graviola has been reported. This study aimed to investigate the protective effects of graviola on 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat breast cancer. Fifty female Wistar rats were allocated into four groups: control group (gastro-gavaged by sesame oil), DMBA-treated group (gastro-gavaged a single dose of DMBA [50 mg/kg body mass, diluted in 1 ml sesame oil]) at the age 57 days, DMBA+G37-treated group (gastro-gavaged a single dose of DMBA [50 mg/kg body mass, diluted in 1 ml sesame oil]) at the age of 57 days plus graviola (200 mg/kg body mass) two times weekly (p.o.) at the age of 37 days till the end of the experiment, and DMBA+G57-treated group (received a single dose of DMBA [50 mg/kg body mass, diluted in 1 ml sesame oil]) plus graviola (200 mg/kg body mass) two times weekly at the age of 57 days until the end of the experiment. After the 30-week experimental period, blood samples were collected. Then, animals were sacrificed to determine the apoptotic indices, antioxidant status, and mammary gland tumor marker (CA 15-3). The DMBA upregulated the expression of one of the main anti-apoptotic genes: B-cell lymphoma protein 2 (BCL2) and estrogen receptor alpha (ER-α) gene. Moreover, it significantly increased breast lipid peroxidation and serum CA 15-3 but decreased breast antioxidant enzymatic activities (glutathione peroxidase, glutathione S-transferase, catalase, and superoxide dismutase). Nevertheless, administration of DMBA and graviola especially DMBA+G37 induced apoptosis through at least 1.5-fold in gene expression levels of pro-apoptotic genes: BCL2-associated X protein (BAX), tumor suppressor gene (P53), and cysteinyl-aspartic acid-protease-3 (caspase-3). A critical role of P53 in the regulation of the BCL2 and BAX has been reported. These proteins can determine if the cell undergoes apoptosis or cancels the process. Once the BAX gene activates caspase-3, there is no irreversible way toward cell death. Also, graviola ameliorated the DMBA effects on antioxidant enzymatic activities and tumor marker CA 15-3. This study concludes that graviola ameliorated DMBA-induced breast cancer potentially through upregulating apoptotic genes, downregulating the ER-α gene, increasing antioxidants, and decreasing lipid peroxidation levels.


Assuntos
9,10-Dimetil-1,2-benzantraceno/toxicidade , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Neoplasias da Mama/induzido quimicamente , Caspase 3/metabolismo , Catalase/metabolismo , Regulação para Baixo/efeitos dos fármacos , Glutationa Peroxidase/metabolismo , Receptores Estrogênicos/metabolismo , Superóxido Dismutase/metabolismo , Proteína X Associada a bcl-2/genética , 9,10-Dimetil-1,2-benzantraceno/química , 9,10-Dimetil-1,2-benzantraceno/metabolismo , Animais , Antioxidantes/química , Caspase 3/química , Catalase/química , Feminino , Glutationa Peroxidase/química , Peroxidação de Lipídeos , Ratos , Ratos Wistar , Superóxido Dismutase/química , Proteína X Associada a bcl-2/química
8.
Molecules ; 24(1)2019 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-30626051

RESUMO

Chemical probes that covalently interact with proteases have found increasing use for the study of protease function and localization. The design and synthesis of such probes is still a bottleneck, as the strategies to target different families are highly diverse. We set out to design and synthesize chemical probes based on protease substrate specificity with inclusion of an uncleavable peptide bond mimic and a photocrosslinker for covalent modification of the protease target. With caspase-3 as a model target protease, we designed reduced amide and triazolo peptides as substrate mimetics, whose sequences can be conveniently constructed by modified solid phase peptide synthesis. We found that these probes inhibited the caspase-3 activity, but did not form a covalent bond. It turned out that the reduced amide mimics, upon irradiation with a benzophenone as photosensitizer, are oxidized and form low concentrations of peptide aldehydes, which then act as inhibitors of caspase-3. This type of photoactivation may be utilized in future photopharmacology experiments to form protease inhibitors at a precise time and location.


Assuntos
Caspase 3/química , Inibidores de Caspase/química , Inibidores de Caspase/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Biomimética , Inibidores de Caspase/síntese química , Química Click , Ativação Enzimática , Estrutura Molecular , Peptídeos/síntese química
9.
Cell Death Differ ; 26(2): 229-244, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-29748600

RESUMO

Apical caspases initiate and effector caspases execute apoptosis. Reagents that can distinguish between caspases, particularly apical caspases-8, 9, and 10 are scarce and generally nonspecific. Based upon a previously described large-scale screen of peptide-based caspase substrates termed HyCoSuL, we sought to develop reagents to distinguish between apical caspases in order to reveal their function in apoptotic cell death paradigms. To this end, we selected tetrapeptide-based sequences that deliver optimal substrate selectivity and converted them to inhibitors equipped with a detectable tag (activity-based probes-ABPs). We demonstrate a strong relationship between substrate kinetics and ABP kinetics. To evaluate the utility of selective substrates and ABPs, we examined distinct apoptosis pathways in Jurkat T lymphocyte and MDA-MB-231 breast cancer lines triggered to undergo cell death via extrinsic or intrinsic apoptosis. We report the first highly selective substrate appropriate for quantitation of caspase-8 activity during apoptosis. Converting substrates to ABPs promoted loss-of-activity and selectivity, thus we could not define a single ABP capable of detecting individual apical caspases in complex mixtures. To overcome this, we developed a panel strategy utilizing several caspase-selective ABPs to interrogate apoptosis, revealing the first chemistry-based approach to uncover the participation of caspase-8, but not caspase-9 or -10 in TRAIL-induced extrinsic apoptosis. We propose that using select panels of ABPs can provide information regarding caspase-8 apoptotic signaling more faithfully than can single, generally nonspecific reagents.


Assuntos
Caspase 10/isolamento & purificação , Caspase 8/isolamento & purificação , Caspase 9/isolamento & purificação , Peptídeos/química , Apoptose/genética , Caspase 10/química , Caspase 10/genética , Caspase 3/química , Caspase 3/genética , Caspase 3/isolamento & purificação , Caspase 8/química , Caspase 8/genética , Caspase 9/química , Caspase 9/genética , Inibidores de Caspase/química , Inibidores de Caspase/farmacologia , Humanos , Células Jurkat , Cinética , Transdução de Sinais , Especificidade por Substrato
10.
Biomolecules ; 8(4)2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30518159

RESUMO

Prostate apoptosis response-4 (Par-4) is a 38 kDa largely intrinsically disordered tumor suppressor protein that functions in cancer cell apoptosis. Par-4 down-regulation is often observed in cancer while up-regulation is characteristic of neurodegenerative conditions such as Alzheimer's disease. Cleavage of Par-4 by caspase-3 activates tumor suppression via formation of an approximately 25 kDa fragment (cl-Par-4) that enters the nucleus and inhibits Bcl-2 and NF-ƙB, which function in pro-survival pathways. Here, we have investigated the structure of cl-Par-4 using biophysical techniques including circular dichroism (CD) spectroscopy, dynamic light scattering (DLS), and intrinsic tyrosine fluorescence. The results demonstrate pH-dependent folding of cl-Par-4, with high disorder and aggregation at neutral pH, but a largely folded, non-aggregated conformation at acidic pH.


Assuntos
Doença de Alzheimer/genética , Proteínas Reguladoras de Apoptose/química , Núcleo Celular/química , Agregação Patológica de Proteínas/genética , Doença de Alzheimer/patologia , Proteínas Reguladoras de Apoptose/genética , Fenômenos Biofísicos , Caspase 3/química , Caspase 3/genética , Núcleo Celular/genética , Dicroísmo Circular , Difusão Dinâmica da Luz , Fluorescência , Genes Supressores de Tumor , Humanos , Concentração de Íons de Hidrogênio , NF-kappa B/genética , Domínios Proteicos/genética , Dobramento de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/genética , Tirosina/química
11.
J Cancer Res Ther ; 14(Supplement): S594-S599, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30249874

RESUMO

Introduction: Scutellaria baicalensis is commonly used in Asia as an herbal medicine to treat a variety of ailments, including cancer. Wogonoside, one major constituent of S. baicalensis, can be primarily converted to wogonin through deglycosylation via enteric microbiome metabolism. Materials and Methods: The antiproliferative effects of the glycoside (wogonoside) and its deglycosylated compound (wogonin) on a panel of human cancer cell lines from the most common solid tumors were evaluated using the MTS colorimetric assay. Cell cycle and apoptosis were determined using flow cytometry. Enzymatic activities of caspases were measured, and the interactions of wogonin and caspases were explored by a docking analysis. Results: Wogonoside did not have obvious antiproliferative effects on the cancer cells. In contrast, wogonin showed significant antiproliferative activities on all the tested cancer cells. Wogonin arrested the cells in the G1 phase and significantly induced cell apoptosis. The compound also activated the expression of caspases 3 and 9. The docking results suggest that the compound forms hydrogen bonds with Phe250 and Ser251, and π-π interactions with Phe256 in caspase 3, and with Asp228 in caspase 9. Conclusions: After wogonoside deglycosylation, wogonin significantly enhanced its anticancer potential as a potent anticancer compound derived from S. baicalensis.


Assuntos
Flavanonas/química , Flavanonas/farmacologia , Glucosídeos/química , Neoplasias/tratamento farmacológico , Extratos Vegetais/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/química , Caspase 3/genética , Caspase 9/química , Caspase 9/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Glucosídeos/farmacologia , Glicosilação/efeitos dos fármacos , Humanos , Ligação de Hidrogênio/efeitos dos fármacos , Células MCF-7 , Microbiota/efeitos dos fármacos , Simulação de Acoplamento Molecular , Neoplasias/patologia , Fitoterapia , Extratos Vegetais/química
12.
Proteins ; 86(11): 1202-1210, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30194780

RESUMO

The regulation of apoptosis is a tightly coordinated process and caspases are its chief regulators. Of special importance are the executioner caspases, caspase-3/7, the activation of which irreversibly sets the cell on the path of death. Dysregulation of apoptosis, particularly an increased rate of cell death lies at the root of numerous human diseases. Although several peptide-based inhibitors targeting the homologous active site region of caspases have been developed, owing to their non-specific activity and poor pharmacological properties their use has largely been restricted. Thus, we sought to identify FDA-approved drugs that could be repurposed as novel allosteric inhibitors of caspase-3/7. In this study, we virtually screened a catalog of FDA-approved drugs targeting an allosteric pocket located at the dimerization interface of caspase-3/7. From among the top-scoring hits we short-listed 5 compounds for experimental validation. Our enzymatic assays using recombinant caspase-3 suggested that 4 out of the 5 drugs effectively inhibited caspase-3 enzymatic activity in vitro with IC50 values ranging ~10-55 µM. Structural analysis of the docking poses show the 4 compounds forming specific non-covalent interactions at the allosteric pocket suggesting that these molecules could disrupt the adjacently-located active site. In summary, we report the identification of 4 novel non-peptide allosteric inhibitors of caspase-3/7 from among FDA-approved drugs.


Assuntos
Regulação Alostérica/efeitos dos fármacos , Anti-Inflamatórios não Esteroides/farmacologia , Caspase 3/metabolismo , Caspase 7/metabolismo , Inibidores de Caspase/farmacologia , Reposicionamento de Medicamentos , Sítio Alostérico/efeitos dos fármacos , Anti-Inflamatórios não Esteroides/química , Caspase 3/química , Caspase 7/química , Inibidores de Caspase/química , Aprovação de Drogas , Reposicionamento de Medicamentos/métodos , Células HEK293 , Humanos , Simulação de Acoplamento Molecular
13.
Cell Physiol Biochem ; 49(6): 2443-2462, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30261501

RESUMO

BACKGROUND/AIMS: Herbal materials derived from Juniperus communis (JCo) possess anticancer activity. In this study, we evaluated the efficacy of a JCo berry extract in suppressing glioblastoma growth. METHODS: The effects of JCo extract on the viability of normal and glioma cell lines was analyzed using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The synergistic therapeutic effect of JCo extract and temozolomide (TMZ) on glioma cells was examined by MTT analysis. Flow cytometry analysis, the terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) test, and western blotting were performed to identify the apoptotic pathway. To determine the in vivo efficacy of the JCo extract, rats were injected with 5 × 104 rat glioma RG2 cells in the back skin and brain hemisphere and then received a subcutaneous injection in the back skin that contained either JCo extract or vehicle. Finally, blood and histologic examinations were performed to evaluate JCo toxicity. RESULTS: The IC50 values of JCo extract were 57-69 µg/mL and 49-67 µg/mL in the glioblastoma cell lines after 24 and 48 h, respectively. However, in non-tumor cell lines, the respective IC50 values of JCo extract were 76-105 µg/mL and 77-108 µg/mL. The JCo extract had a stronger cytotoxicity and a larger range of IC50 values in glioma than in normal cells as compared to those effects caused by temozolomide (TMZ). In addition, the results of flow cytometry analysis, TUNEL test, and western blotting revealed that the JCo extract induced glioma cell cycle arrest through intrinsic and extrinsic apoptotic pathways. In the in vivo studies, a significant reduction of tumor size in JCo-treated rats, as measured by animal MRI, demonstrated that the JCo extract effectively inhibited glioma cell growth and successfully penetrated the blood-brain barrier. The immunohistochemical (IHC) staining detected positive signals of PCNA, VEGFR-1, and VEGFR -2 in 44.49%, 5.88%, and 5.85% of JCo-treated glioma cells, respectively. However, positive signals of PCNA, VEGFR-1, and VEGFR-2 were detected in 73.08%, 9.67%, and 11.70% of vehicle-treated glioma cells, respectively. The IHC examination of PCNA and VEGFR-1 and -2 indicated that JCo extract significantly decreased the degree of neovascularization. However, no significant differences in serum levels of blood cell count and hepatic enzymes, renal function index, and the histologic appearance of vital organs were detected between the JCo and vehicle-treated rats. CONCLUSION: The JCo extract penetrated the blood-brain barrier and significantly induced glioma cell apoptosis by reducing neovascularization via suppression of the PI3K/AKT/mTOR pathway. Furthermore, JCo extract was less cytotoxic to non-neoplastic vital organs than TMZ.


Assuntos
Apoptose/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Juniperus/química , Extratos Vegetais/farmacologia , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Caspase 3/química , Caspase 3/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Dacarbazina/uso terapêutico , Sinergismo Farmacológico , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Humanos , Juniperus/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Extratos Vegetais/química , Extratos Vegetais/uso terapêutico , Antígeno Nuclear de Célula em Proliferação/metabolismo , Temozolomida , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
14.
Food Funct ; 9(9): 4771-4780, 2018 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-30117517

RESUMO

Agaricus bisporus is one of the most important edible and medicinal mushrooms in the world. It has been well known that Agaricus bisporus has an immunoregulatory role, but its active ingredients have not been completely identified. In this study, a glucogalactomannan named TJ3 was isolated and purified from Agaricus bisporus. TJ3 (827 kDa) is composed of mannose, galactose, glucose and xylose in the ratio 28.26 : 27.82 : 20.88 : 9.87 mainly joined by ß-linkages. Functional analysis of TJ3 revealed that it effectively induced apoptosis in RAW 264.7 cells, a mouse macrophage cell line. Cell apoptosis was determined by an Annexin V/PI staining assay. After treatment with TJ3 (2 µg mL-1) for 16 h, apoptosis was observed in 34% of the Raw cells (9% in the non-treated control cells). TJ3 treatment remarkably increased the production of cleaved caspase-3, PARP and Bim, and decreased the level of Bcl-2 although no obvious change in the level of Bax was observed. Interestingly, further elucidation of the molecular mechanism underlying the role of TJ3 in the induction of apoptosis showed that TJ3 activated the JNK signaling pathway through TLR4 and subsequently promoted the expression of Bim and activation of caspase-3. Our results demonstrate that TJ3 may be a novel active component in Agaricus bisporus responsible for its immunoregulatory role by the induction of macrophage apoptosis.


Assuntos
Agaricus/química , Apoptose , Proteína 11 Semelhante a Bcl-2/agonistas , Carpóforos/química , Sistema de Sinalização das MAP Quinases , Macrófagos/metabolismo , Mananas/metabolismo , Animais , Anti-Inflamatórios não Esteroides/efeitos adversos , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/isolamento & purificação , Anti-Inflamatórios não Esteroides/metabolismo , Proteína 11 Semelhante a Bcl-2/metabolismo , Caspase 3/química , Caspase 3/metabolismo , Proliferação de Células , Sobrevivência Celular , Suplementos Nutricionais/efeitos adversos , Suplementos Nutricionais/análise , Ativação Enzimática , Etnofarmacologia , Macrófagos/citologia , Macrófagos/imunologia , Mananas/efeitos adversos , Mananas/química , Mananas/isolamento & purificação , Medicina Tradicional Chinesa , Camundongos , Estrutura Molecular , Peso Molecular , Poli(ADP-Ribose) Polimerases/metabolismo , Proteólise , Células RAW 264.7
15.
Proc Natl Acad Sci U S A ; 115(26): 6792-6797, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29891674

RESUMO

The inflammasomes are signaling platforms that promote the activation of inflammatory caspases such as caspases-1, -4, -5, and -11. Recent studies identified gasdermin D (GSDMD) as an effector for pyroptosis downstream of the inflammasome signaling pathways. Cleavage of GSDMD by inflammatory caspases allows its N-terminal domain to associate with membrane lipids and form pores that induce pyroptotic cell death. Despite the important role of GSDMD in pyroptosis, the molecular mechanisms of GSDMD recognition and cleavage by inflammatory caspases that trigger pyroptosis are poorly understood. Here, we demonstrate that the catalytic domains of inflammatory caspases can directly bind to both the full-length GSDMD and its cleavage site peptide, FLTD. A GSDMD-derived inhibitor, N-acetyl-Phe-Leu-Thr-Asp-chloromethylketone (Ac-FLTD-CMK), inhibits GSDMD cleavage by caspases-1, -4, -5, and -11 in vitro, suppresses pyroptosis downstream of both canonical and noncanonical inflammasomes, as well as reduces IL-1ß release following activation of the NLRP3 inflammasome in macrophages. By contrast, the inhibitor does not target caspase-3 or apoptotic cell death, suggesting that Ac-FLTD-CMK is a specific inhibitor for inflammatory caspases. Crystal structure of caspase-1 in complex with Ac-FLTD-CMK reveals extensive enzyme-inhibitor interactions involving both hydrogen bonds and hydrophobic contacts. Comparison with other caspase-1 structures demonstrates drastic conformational changes at the four active-site loops that assemble the catalytic groove. The present study not only contributes to our understanding of GSDMD recognition by inflammatory caspases but also reports a specific inhibitor for these caspases that can serve as a tool for investigating inflammasome signaling.


Assuntos
Proteínas Reguladoras de Apoptose/química , Inibidores de Caspase/química , Proteínas de Neoplasias/química , Peptídeos/química , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 3/química , Caspase 3/metabolismo , Inibidores de Caspase/metabolismo , Domínio Catalítico , Células HEK293 , Humanos , Células Jurkat , Camundongos , Proteínas de Neoplasias/metabolismo , Peptídeos/metabolismo , Estrutura Secundária de Proteína , Células RAW 264.7 , Células THP-1
16.
Toxicol Lett ; 294: 156-165, 2018 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-29763685

RESUMO

Methamphetamine (METH) is a commonly abused psychostimulant that can induce severe neurotoxicity. Cardiovascular injury caused by METH has recently gained increasing attention; however, the underlying mechanisms remain unclear. As autophagy has been shown to be associated with cell injury, the association between autophagy and METH-mediated cell apoptosis was investigated in the present study. METH treatment significantly increased the expression of two key autophagy proteins, i.e., Beclin-1 and LC3-II, in the cardiomyocyte cell line H9C2. Furthermore, according to western blot and flow cytometry analyses, METH contributed to cell injury and markedly enhanced cleaved-caspase 3 and PARP expression. In addition, the corresponding AKT-mTOR survival pathway axis was appeared deactivated. The autophagic activity was closely associated with METH-mediated cell injury because rapamycin, which is an autophagy inducer, markedly attenuated METH-induced cell injury, while 3-Methyladenine (3-MA), which is an autophagy inhibitor, and bafilomycinA1 (Baf-A1), which is a blocker of autophagosome-lysosome fusion, markedly exacerbated METH-induced cell injury. Notably, defective autophagosome-lysosome fusion might be partially involved in the METH-induced enhancement of LC3-II expression and cell injury. However, the underlying mechanisms require further investigation.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Estimulantes do Sistema Nervoso Central/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Metanfetamina/toxicidade , Miócitos Cardíacos/efeitos dos fármacos , Animais , Antibióticos Antineoplásicos/farmacologia , Autofagossomos/efeitos dos fármacos , Autofagossomos/enzimologia , Autofagossomos/metabolismo , Proteína Beclina-1/agonistas , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Caspase 3/química , Caspase 3/genética , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Estimulantes do Sistema Nervoso Central/agonistas , Estimulantes do Sistema Nervoso Central/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/enzimologia , Lisossomos/metabolismo , Macrolídeos/farmacologia , Fusão de Membrana/efeitos dos fármacos , Metanfetamina/agonistas , Metanfetamina/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/agonistas , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Poli(ADP-Ribose) Polimerase-1/química , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Sirolimo/farmacologia
17.
J Control Release ; 280: 1-10, 2018 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-29723615

RESUMO

Despite the extremely high substrate specificity and catalytically amplified activity of enzymes, the lack of efficient cellular internalization limits their application as therapeutics. To overcome this limitation and to harness enzymes as practical biologics for targeting intracellular functions, we developed the streptavidin-mirror DNA tetrahedron hybrid as a platform for intracellular delivery of various enzymes. The hybrid consists of streptavidin, which provides a stoichiometrically controlled loading site for the enzyme cargo and an L-DNA (mirror DNA) tetrahedron, which provides the intracellular delivery potential. Due to the cell-penetrating ability of the mirror DNA tetrahedron of this hybrid, enzymes loaded on streptavidin can be efficiently delivered into the cells, intracellularly expressing their activity. In addition, we demonstrate tumor delivery of enzymes in an animal model by utilizing the potential of the hybrid to accumulate in tumors. Strikingly, the hybrid is able to transfer the apoptotic enzyme specifically into tumor cells, leading to strong suppression of tumor growth without causing significant damage to other tissues. These results suggest that the hybrid may allow anti-proliferative enzymes and proteins to be utilized as anticancer drugs.


Assuntos
Caspase 3/química , DNA/química , Portadores de Fármacos/química , Neoplasias/tratamento farmacológico , Estreptavidina/química , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Transporte Biológico , Caspase 3/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Preparações de Ação Retardada/química , Preparações de Ação Retardada/uso terapêutico , Liberação Controlada de Fármacos , Humanos , Camundongos Endogâmicos BALB C , Distribuição Tecidual/efeitos dos fármacos
18.
Acta Histochem ; 120(4): 385-394, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29685720

RESUMO

Programmed cell death is a fundamental event that takes place during organ development and plays an important role in cellular homeostasis. Since various body organs of the camel are under high ecological and physiological stress during food and water deprivation, desiccation, and the long exposure to solar radiation in these desert nomads, we aimed to examine the immunohistochemical expression of apoptosis-related biomarkers in some of its normal body organs to illustrate a basic track for further pathological investigation. Regarding apoptosis, the present study has revealed that the higher expression of cleaved caspase-9 (CC9) [initiator of the intrinsic pathway] and CC3 (effector caspase), and the scanty expression of CC8 (initiator of the extrinsic pathway), highlight the role of the caspase-dependent, intrinsic apoptotic pathway particularly in the intestines and lymphoid organs. The apoptosis- inducing factor (AIF)-immunoexpression was completely missing in the cell nuclei of the examined tissues, indicating the absence of the caspase-independent pathway. The nuclear overexpression of the phospho-histone H2AX (γ H2AX) and the occasional expression of single-stranded DNA, particularly among the CNS neurons, suggest an efficient, protective DNA-repair mechanism in such cells. Thus, despite efficient anti-apoptotic mechanisms intrinsic apoptotic pathways exists in brain, intestine and lymph organs of adult desert camels.


Assuntos
Fator de Indução de Apoptose/química , Apoptose , Biomarcadores/química , Animais , Fator de Indução de Apoptose/metabolismo , Camelus , Caspase 3/química , Imuno-Histoquímica , Modelos Animais
19.
J Biol Chem ; 293(15): 5462-5463, 2018 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-29654071

RESUMO

Caspase-3 is well known as the "executioner" whose activation commits the cell to an apoptotic fate, but low levels of caspase-3 activity also play key roles in development. A new study explains how cells can balance these functions, using biophysical, structural, and computational approaches to demonstrate the mechanism by which phosphorylation of conserved sites on a distal surface loop reduces or abolishes catalytic activity. These results provide new insights into allosteric regulation mechanisms and offer new opportunities for development of caspase-3 modulators.


Assuntos
Caspase 3/química , Caspase 3/metabolismo , Regulação Alostérica , Animais , Caspase 3/genética , Catálise , Humanos , Fosforilação , Estrutura Secundária de Proteína , Relação Estrutura-Atividade
20.
Mol Nutr Food Res ; 62(8): e1700890, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29446867

RESUMO

SCOPE: We investigated the role of endoplasmic reticulum (ER) stress in the protective effects of EGCG against the neuronal apoptosis in Aß1-42 -induced SH-SY5Y cells and APP/PS1 transgenic mice. METHODS AND RESULTS: Cell viability (CCK8 assay), flow cytometry, Hoechst 33258 staining, immunohistochemistry, transmission electron microscopy (TEM), and western blotting were used. EGCG prevented Aß1-42-induced toxicity in SH-SY5Y cells, increased cell viability, and decreased apoptosis in a dose-dependent manner. In a subsequent mechanism study, it was found that this effect contributed to the down-regulation of GRP78, CHOP, cleaved-caspase-12 and -3. Moreover, EGCG also reduced the cytotoxicity induced by tunicamycin (TM) and thapsigargin (TG), two ER stress activators. Consistent with the in vitro study, EGCG inhibited neuronal apoptosis in the cortex of APP/PS1 transgenic mice, with the mitigation of ER abnormal ultrastructural swelling and the downregulation of ER-stress-associated proteins. CONCLUSION: These results indicate that EGCG attenuates the neurotoxicity in Alzheimer's disease (AD) via a novel mechanism that involves inhibition of ER-stress-associated neuronal apoptosis in vitro and in vivo, suggesting the tremendous potential of EGCG for use in a nutritional preventive strategy against AD.


Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Apoptose , Catequina/análogos & derivados , Suplementos Nutricionais , Estresse do Retículo Endoplasmático , Neurônios/metabolismo , Fármacos Neuroprotetores/metabolismo , Fragmentos de Peptídeos/antagonistas & inibidores , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/prevenção & controle , Peptídeos beta-Amiloides/metabolismo , Animais , Caspase 12/química , Caspase 12/genética , Caspase 12/metabolismo , Caspase 3/química , Caspase 3/genética , Caspase 3/metabolismo , Catequina/metabolismo , Catequina/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Córtex Cerebral/ultraestrutura , Proteínas de Choque Térmico/agonistas , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/patologia , Neurônios/ultraestrutura , Fármacos Neuroprotetores/uso terapêutico , Nootrópicos/metabolismo , Nootrópicos/uso terapêutico , Fragmentos de Peptídeos/metabolismo , Distribuição Aleatória , Fator de Transcrição CHOP/agonistas , Fator de Transcrição CHOP/antagonistas & inibidores , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo
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