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1.
Molecules ; 26(16)2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34443374

RESUMO

The activation of NFAT (nuclear factor of activated T cells) transcription factors by calcium-dependent phosphatase calcineurin is a key step in controlling T cell activation and plays a vital role during carcinogenesis. NFATs are overexpressed in many cancers, including the most common primary brain tumor, gliomas. In the present study, we demonstrate the expression of NFATs and NFAT-driven transcription in several human glioma cells. We used a VIVIT peptide for interference in calcineurin binding to NFAT via a conserved PxIxIT motif. VIVIT was expressed as a fusion protein with a green fluorescent protein (VIVIT-GFP) or conjugated to cell-penetrating peptides (CPP), Sim-2 or 11R. We analyzed the NFAT expression, phosphorylation, subcellular localization and their transcriptional activity in cells treated with peptides. Overexpression of VIVIT-GFP decreased the NFAT-driven activity and inhibited the transcription of endogenous NFAT-target genes. These effects were not reproduced with synthetic peptides: Sim2-VIVIT did not show any activity, and 11R-VIVIT did not inhibit NFAT signaling in glioma cells. The presence of two calcineurin docking sites in NFATc3 might require dual-specificity blocking peptides. The cell-penetrating peptides Sim-2 or 11R linked to VIVIT did not improve its action making it unsuitable for evaluating NFAT dependent events in glioma cells with high expression of NFATc3.


Assuntos
Neoplasias Encefálicas/patologia , Calcineurina/metabolismo , Glioma/patologia , Fatores de Transcrição NFATC/metabolismo , Oligopeptídeos/farmacologia , Transdução de Sinais , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Peptídeos Penetradores de Células/farmacologia , Glioma/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Fatores de Transcrição NFATC/química , Oligopeptídeos/química , Peptídeos/farmacologia , Transporte Proteico/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos
2.
Int J Mol Sci ; 22(15)2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34361059

RESUMO

In vertebrates, nucleostemin (NS) is an important marker of proliferation in several types of stem and cancer cells, and it can also interact with the tumor-suppressing transcription factor p53. In the present study, the intra-nuclear diffusional dynamics of native NS tagged with GFP and two GFP-tagged NS mutants with deleted guanosine triphosphate (GTP)-binding domains were analyzed by fluorescence correlation spectroscopy. Free and slow binding diffusion coefficients were evaluated, either under normal culture conditions or under treatment with specific cellular proliferation inhibitors actinomycin D (ActD), 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), or trichostatin A (TSA). When treated with ActD, the fractional ratio of the slow diffusion was significantly decreased in the nucleoplasm. The decrease was proportional to ActD treatment duration. In contrast, DRB or TSA treatment did not affect NS diffusion. Interestingly, it was also found that the rate of diffusion of two NS mutants increased significantly even under normal conditions. These results suggest that the mobility of NS in the nucleoplasm is related to the initiation of DNA or RNA replication, and that the GTP-binding motif is also related to the large change of mobility.


Assuntos
Núcleo Celular/metabolismo , Dactinomicina/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Nucleares/metabolismo , Inibidores da Síntese de Ácido Nucleico/farmacologia , Transcrição Genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ligação ao GTP/genética , Células HeLa , Humanos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética
3.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203768

RESUMO

Mesembryanthemum crystallinum (common ice plant) is a halophyte species that has adapted to extreme conditions. In this study, we cloned a McHB7 transcription factor gene from the ice plant. The expression of McHB7 was significantly induced by 500 mM NaCl and it reached the peak under salt treatment for 7 days. The McHB7 protein was targeted to the nucleus. McHB7-overexpressing in ice plant leaves through Agrobacterium-mediated transformation led to 25 times more McHB7 transcripts than the non-transformed wild type (WT). After 500 mM NaCl treatment for 7 days, the activities of superoxide dismutase (SOD) and peroxidase (POD) and water content of the transgenic plants were higher than the WT, while malondialdehyde (MDA) was decreased in the transgenic plants. A total of 1082 and 1072 proteins were profiled by proteomics under control and salt treatment, respectively, with 22 and 11 proteins uniquely identified under control and salt stress, respectively. Among the 11 proteins, 7 were increased and 4 were decreased after salt treatment. Most of the proteins whose expression increased in the McHB7 overexpression (OE) ice plants under high salinity were involved in transport regulation, catalytic activities, biosynthesis of secondary metabolites, and response to stimulus. The results demonstrate that the McHB7 transcription factor plays a positive role in improving plant salt tolerance.


Assuntos
Mesembryanthemum/metabolismo , Proteínas de Plantas/metabolismo , Proteômica , Tolerância ao Sal/fisiologia , Sequência de Aminoácidos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Biologia Computacional , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Ontologia Genética , Mesembryanthemum/efeitos dos fármacos , Mesembryanthemum/genética , Filogenia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Transporte Proteico/efeitos dos fármacos , Salinidade , Tolerância ao Sal/efeitos dos fármacos , Tolerância ao Sal/genética , Cloreto de Sódio/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Frações Subcelulares/metabolismo , Fatores de Transcrição/metabolismo
4.
Commun Biol ; 4(1): 677, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083702

RESUMO

Immortalized erythroid cell lines are expected to be a promising source of ex vivo manufactured red blood cells (RBCs), however the induction of enucleation in these cell lines is inefficient at present. We utilized an imaging-based high-throughput system to identify chemical compounds that trigger enucleation of human erythroid cell lines. Among >3,300 compounds, we identified multiple histone deacetylase inhibitors (HDACi) inducing enucleated cells from the cell line, although an increase in membrane fragility of enucleated cells was observed. Gene expression profiling revealed that HDACi treatment increased the expression of cytoskeletal genes, while an erythroid-specific cell membrane protein, SPTA1, was significantly down-regulated. Restoration of SPTA1 expression using CRISPR-activation partially rescued the fragility of cells and thereby improved the enucleation efficiency. Our observations provide a potential solution for the generation of mature cells from erythroid cell lines, contributing to the future realization of the use of immortalized cell lines for transfusion therapies.


Assuntos
Núcleo Celular/efeitos dos fármacos , Eritrócitos/metabolismo , Células Eritroides/efeitos dos fármacos , Perfilação da Expressão Gênica/métodos , Inibidores de Histona Desacetilases/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Eritrócitos/citologia , Células Eritroides/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Inibidores de Histona Desacetilases/isolamento & purificação , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
5.
Int J Mol Sci ; 22(9)2021 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-34067160

RESUMO

Puccinia striiformis f. sp. tritici (Pst) is an important pathogen of wheat (Triticum aestivum L.) stripe rust, and the effector protein secreted by haustoria is a very important component involved in the pathogenic process. Although the candidate effector proteins secreted by Pst haustoria have been predicted to be abundant, few have been functionally validated. Our study confirmed that chitin and flg22 could be used as elicitors of the pathogenic-associated molecular pattern-triggered immune (PTI) reaction in wheat leaves and that TaPr-1-14 could be used as a marker gene to detect the PTI reaction. In addition, the experimental results were consistent in wheat protoplasts. A rapid and efficient method for screening and identifying the effector proteins of Pst was established by using the wheat protoplast transient expression system. Thirty-nine Pst haustorial effector genes were successfully cloned and screened for expression in the protoplast. We identified three haustorial effector proteins, PSEC2, PSEC17, and PSEC45, that may inhibit the response of wheat to PTI. These proteins are localized in the somatic cytoplasm and nucleus of wheat protoplasts and are highly expressed during the infection and parasitism of wheat.


Assuntos
Proteínas Fúngicas/metabolismo , Imunidade , Padrões Moleculares Associados a Patógenos/metabolismo , Protoplastos/microbiologia , Puccinia/fisiologia , Triticum/imunologia , Triticum/microbiologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Quitina/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Imunidade/efeitos dos fármacos , Doenças das Plantas/microbiologia , Imunidade Vegetal/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/imunologia , Folhas de Planta/microbiologia , Protoplastos/efeitos dos fármacos , Puccinia/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Reprodutibilidade dos Testes , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Transcrição Genética/efeitos dos fármacos , Triticum/efeitos dos fármacos , Triticum/genética
6.
Nat Commun ; 12(1): 3356, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099663

RESUMO

Since their discovery as drivers of proliferation, cyclin-dependent kinases (CDKs) have been considered therapeutic targets. Small molecule inhibitors of CDK4/6 are used and tested in clinical trials to treat multiple cancer types. Despite their clinical importance, little is known about how CDK4/6 inhibitors affect the stability of CDK4/6 complexes, which bind cyclins and inhibitory proteins such as p21. We develop an assay to monitor CDK complex stability inside the nucleus. Unexpectedly, treatment with CDK4/6 inhibitors-palbociclib, ribociclib, or abemaciclib-immediately dissociates p21 selectively from CDK4 but not CDK6 complexes. This effect mediates indirect inhibition of CDK2 activity by p21 but not p27 redistribution. Our work shows that CDK4/6 inhibitors have two roles: non-catalytic inhibition of CDK2 via p21 displacement from CDK4 complexes, and catalytic inhibition of CDK4/6 independent of p21. By broadening the non-catalytic displacement to p27 and CDK6 containing complexes, next-generation CDK4/6 inhibitors may have improved efficacy and overcome resistance mechanisms.


Assuntos
Ciclina D/metabolismo , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Células MCF-7 , Camundongos , Microscopia de Fluorescência , Piperazinas/farmacologia , Ligação Proteica , Piridinas/farmacologia , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo
7.
Molecules ; 26(9)2021 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068647

RESUMO

The anticancer activity of terretonin N (1) and butyrolactone I (2), obtained from the thermophilic fungus Aspergillus terreus TM8, was intensively studied against prostate adenocarcinoma (PC-3) and ovary adenocarcinoma (SKOV3) human cell lines. According to this study, both compounds showed potent cytotoxicity towards ovarian adenocarcinoma cells (SKOV3) with IC50 1.2 and 0.6 µg/mL, respectively. With respect to metastatic prostate cells (PC-3), the two compounds 1 and 2 showed a significantly promising cytotoxicity effect with IC50 of 7.4 and 4.5 µg/mL, respectively. The tested fungal metabolites showed higher rates of early and late apoptosis with little or no necrotic apoptotic pathway in all treated prostate adenocarcinoma (PC-3) and ovary adenocarcinoma (SKOV3) human cell lines, respectively. The results reported in this study confirmed the promising biological properties of terretonin N (1) and butyrolactone I (2) as anticancer agents via the induction of cellular apoptosis. However, further studies are needed to elucidate the molecular mechanism by which cellular apoptosis is induced in cancer cells.


Assuntos
4-Butirolactona/análogos & derivados , Apoptose/efeitos dos fármacos , Aspergillus/química , Neoplasias Ovarianas/patologia , Neoplasias da Próstata/patologia , Terpenos/farmacologia , 4-Butirolactona/química , 4-Butirolactona/farmacologia , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Forma Celular/efeitos dos fármacos , Feminino , Humanos , Concentração Inibidora 50 , Masculino , Terpenos/química
8.
Aging (Albany NY) ; 13(10): 13405-13420, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34038868

RESUMO

cDNA microarray data conducted by our group revealed overexpression of CXCL2 and CXCL8 in ovarian cancer (OC) microenvironment. Herein, we have proven that the chemokine receptor, CXCR2, is a pivotal molecule in re-sensitizing OC to cisplatin, and its inhibition decreases cell proliferation, viability, tumor size in cisplatin-resistant cells, as well as reversed the overexpression of mesenchymal epithelium transition markers. Altogether, our study indicates a central effect of CXCR2 in preventing tumor progression, due to acquisition of cisplatin chemoresistant phenotype by tumor cells, and patients' high lethality rate. We found that the overexpression of CXCR2 by OC cells is persistent and anomalously confined to the cellular nuclei, thus pointing to an urge in developing highly lipophilic molecules that promptly permeate cells, bind to and inhibit nuclear CXCR2 to fight OC, instead of relying on the high-cost genetic engineered cells.


Assuntos
Cisplatino/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Receptores de Interleucina-8B/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quimiocina CXCL2/metabolismo , Embrião de Galinha , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Invasividade Neoplásica , Proteínas de Neoplasias/metabolismo , Neovascularização Patológica/tratamento farmacológico , Neoplasias Ovarianas/irrigação sanguínea , Neoplasias Ovarianas/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Interleucina-8B/metabolismo , Análise de Sobrevida , Serina-Treonina Quinases TOR/metabolismo
9.
Cell Death Dis ; 12(4): 341, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33795649

RESUMO

The JAK2/STAT pathway is hyperactivated in many cancers, and such hyperactivation is associated with a poor clinical prognosis and drug resistance. The mechanism regulating JAK2 activity is complex. Although translocation of JAK2 between nucleus and cytoplasm is an important regulatory mechanism, how JAK2 translocation is regulated and what is the physiological function of this translocation remain largely unknown. Here, we found that protease SENP1 directly interacts with and deSUMOylates JAK2, and the deSUMOylation of JAK2 leads to its accumulation at cytoplasm, where JAK2 is activated. Significantly, this novel SENP1/JAK2 axis is activated in platinum-resistant ovarian cancer in a manner dependent on a transcription factor RUNX2 and activated RUNX2/SENP1/JAK2 is critical for platinum-resistance in ovarian cancer. To explore the application of anti-SENP1/JAK2 for treatment of platinum-resistant ovarian cancer, we found SENP1 deficiency or treatment by SENP1 inhibitor Momordin Ic significantly overcomes platinum-resistance of ovarian cancer. Thus, this study not only identifies a novel mechanism regulating JAK2 activity, but also provides with a potential approach to treat platinum-resistant ovarian cancer by targeting SENP1/JAK2 pathway.


Assuntos
Cisteína Endopeptidases/metabolismo , Resistência a Medicamentos/efeitos dos fármacos , Janus Quinase 2/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Platina/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Feminino , Humanos , Neoplasias Ovarianas/metabolismo , Transdução de Sinais/efeitos dos fármacos
10.
J Biol Chem ; 296: 100714, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33930463

RESUMO

Overconsumption of sucrose and other sugars has been associated with nonalcoholic fatty liver disease (NAFLD). Reports suggest hepatic de novo lipogenesis (DNL) as an important contributor to and regulator of carbohydrate-induced hepatic lipid accumulation in NAFLD. The mechanisms responsible for the increase in hepatic DNL due to overconsumption of carbohydrate diet are less than clear; however, literatures suggest high carbohydrate diet to activate the lipogenic transcription factor carbohydrate response element-binding protein (ChREBP), which further transcribes genes involved in DNL. Here, we provide an evidence of an unknown link between nuclear factor kappa-light chain enhancer of activated B cells (NF-κB) activation and increased DNL. Our data indicates high carbohydrate diet to enforce nuclear shuttling of hepatic NF-κB p65 and repress transcript levels of sorcin, a cytosolic interacting partner of ChREBP. Reduced sorcin levels, further prompted ChREBP nuclear translocation, leading to enhanced DNL and intrahepatic lipid accumulation both in vivo and in vitro. We further report that pharmacological inhibition of NF-κB abrogated high carbohydrate diet-mediated sorcin repression and thereby prevented ChREBP nuclear translocation and this, in turn, attenuated hepatic lipid accumulation both in in vitro and in vivo. Additionally, sorcin knockdown blunted the lipid-lowering ability of the NF-κB inhibitor in vitro. Together, these data suggest a heretofore unknown role for NF-κB in regulating ChREBP nuclear localization and activation, in response to high carbohydrate diet, for further explorations in lines of NAFLD therapeutics.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Núcleo Celular/efeitos dos fármacos , Carboidratos da Dieta/farmacologia , Lipogênese/efeitos dos fármacos , Fígado/metabolismo , Fator de Transcrição RelA/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Hep G2 , Humanos
11.
Int J Nanomedicine ; 16: 2833-2847, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33883894

RESUMO

Introduction: Peptides can be rationally designed as non-covalent inhibitors for molecularly targeted therapy. However, it remains challenging to efficiently deliver the peptides into the targeted cells, which often severely affects their therapeutic efficiency. Methods: Herein, we created a novel non-covalent peptide inhibitor against nuclear export factor CRM1 by a structure-guided drug design method and targetedly delivered the peptide into cancer cells by a nanoparticle-mediated gene expression system for use as a cancer therapy. Results: The nuclear export signal (NES)-optimized CRM1 peptide inhibitor colocalized with CRM1 to the nuclear envelope and inhibited nuclear export in cancer cell lines in vitro. The crystal structures of the inhibitors complexed with CRM1 were solved. In contrast to the covalent inhibitors, the peptides were similarly effective against cells harboring the CRM1 C528S mutation. Moreover, a plasmid encoding the peptides was delivered by a iRGD-modified nanoparticle to efficiently target and transfect the cancer cells in vivo after intravenous administration. The peptides could be selectively expressed in the tumor, resulting in the efficient inhibition of subcutaneous melanoma xenografts without obvious systemic toxicity. Discussion: This work provides an effective strategy to design peptide-based molecularly targeted therapeutics, which could lead to the development of future targeted therapy.


Assuntos
Espaço Intracelular/metabolismo , Carioferinas/antagonistas & inibidores , Melanoma Experimental/tratamento farmacológico , Nanopartículas/química , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Células A549 , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Sequência de Aminoácidos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Carioferinas/química , Carioferinas/metabolismo , Melanoma Experimental/patologia , Proteínas Mutantes/metabolismo , Mutação/genética , Nanopartículas/ultraestrutura , Sinais de Exportação Nuclear , Peptídeos/química , Ligação Proteica/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas não Estruturais Virais/química
12.
Molecules ; 26(7)2021 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-33917440

RESUMO

Periodontitis is a set of chronic inflammatory diseases caused by the accumulation of Gram-negative bacteria on teeth, resulting in gingivitis, pocket formation, alveolar bone loss, tissue destruction, and tooth loss. In this study, the contents of ginsenosides isolated from Panax ginseng fruit extract were quantitatively analyzed, and the anti-inflammatory effects were evaluated in human periodontal ligament cells. The major ginsenosides, Re, Ra8, and Rf, present in ginseng fruit were simultaneously analyzed by a validated method using high-performance liquid chromatography with a diode-array detector; Re, Ra8, and Rf content per 1 g of P. ginseng fruit extract was 1.01 ± 0.03, 0.33 ± 0.01, and 0.55 ± 0.04 mg, respectively. Ginsenosides-Re, -Ra8, and -Rf inhibited the production of pro-inflammatory factors and the expression of important cytokines in periodontitis by inducing the expression of heme oxygenase 1 (HO-1), promoting osteoblast differentiation of periodontal ligament cells, suppressing alveolar bone loss, and promoting the expression of osteoblast-specific genes, such as alp, opn, and runx2. An inhibitory effect of these ginsenosides on periodontitis and alveolar bone loss was observed via the regulation of HO-1 and subsequent epidermal growth factor receptor (EGFR) signaling. Silencing EGFR with EGFR siRNA confirmed that the effect of ginsenosides on HO-1 is mediated by EGFR. In conclusion, this study evaluated the contents of ginsenosides-Re, -Ra8, and -Rf isolated from P. ginseng fruit extract. Therefore, these results provide important basic data for future P. ginseng fruit component studies and suggest that ginsenosides Re, Ra8, and Rf have potential as future treatment options for periodontitis.


Assuntos
Anti-Inflamatórios/farmacologia , Receptores ErbB/metabolismo , Ginsenosídeos/isolamento & purificação , Ginsenosídeos/farmacologia , Heme Oxigenase-1/metabolismo , Osteogênese/efeitos dos fármacos , Panax/química , Ligamento Periodontal/citologia , Diferenciação Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Frutas/química , Regulação da Expressão Gênica/efeitos dos fármacos , Ginsenosídeos/química , Humanos , Mediadores da Inflamação/metabolismo , Limite de Detecção , Lipopolissacarídeos/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Extratos Vegetais/química , Porphyromonas gingivalis/química , Análise de Regressão , Transdução de Sinais/efeitos dos fármacos
13.
Int J Mol Sci ; 22(6)2021 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-33801927

RESUMO

BACKGROUND: Nuclear protein-1 (NUPR1, also known as p8/Com-1) is a transcription factor involved in the regulation of cellular stress responses, including serum starvation and drug stimulation. METHODS: We investigated the mechanism of NUPR1 nuclear translocation involving karyopherin ß1 (KPNB1), using a single-molecule binding assay and confocal microscopy. The cellular effects associated with NUPR1-KPNB1 inhibition were investigated by gene expression profiling and cell cycle analysis. RESULTS: The single-molecule binding assay revealed that KPNB1 bound to NUPR1 with a binding affinity of 0.75 nM and that this binding was blocked by the aminothiazole ATZ-502. Following doxorubicin-only treatment, NUPR1 was translocated to the nucleus in more than 90% and NUPR1 translocation was blocked by the ATZ-502 combination treatment in MDA-MB-231 with no change in NUPR1 expression, providing strong evidence that NUPR1 nuclear translocation was directly inhibited by the ATZ-502 treatment. Inhibition of KPNB1 and NUPR1 binding was associated with a synergistic anticancer effect (up to 19.6-fold) in various cancer cell lines. NUPR1-related genes were also downregulated following the doxorubicin-ATZ-502 combination treatment. CONCLUSION: Our current findings clearly demonstrate that NUPR1 translocation into the nucleus requires karyopherin ß1 binding. Inhibition of the KPNB1 and NUPR1 interaction may constitute a new cancer therapeutic approach that can increase the drug efficacy while reducing the side effects.


Assuntos
Acrilamidas/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Benzotiazóis/farmacologia , Doxorrubicina/farmacologia , Proteínas de Neoplasias/metabolismo , beta Carioferinas/metabolismo , Acrilamidas/química , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacologia , Benzotiazóis/química , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/química , Sinergismo Farmacológico , Humanos , Células MCF-7 , Microscopia Confocal , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos
14.
Nat Commun ; 12(1): 2398, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33893278

RESUMO

Arginine plays diverse roles in cellular physiology. As a semi-essential amino acid, arginine deprivation has been used to target cancers with arginine synthesis deficiency. Arginine-deprived cancer cells exhibit mitochondrial dysfunction, transcriptional reprogramming and eventual cell death. In this study, we show in prostate cancer cells that arginine acts as an epigenetic regulator to modulate histone acetylation, leading to global upregulation of nuclear-encoded oxidative phosphorylation (OXPHOS) genes. TEAD4 is retained in the nucleus by arginine, enhancing its recruitment to the promoter/enhancer regions of OXPHOS genes and mediating coordinated upregulation in a YAP1-independent but mTOR-dependent manner. Arginine also activates the expression of lysine acetyl-transferases and increases overall levels of acetylated histones and acetyl-CoA, facilitating TEAD4 recruitment. Silencing of TEAD4 suppresses OXPHOS functions and prostate cancer cell growth in vitro and in vivo. Given the strong correlation of TEAD4 expression and prostate carcinogenesis, targeting TEAD4 may be beneficially used to enhance arginine-deprivation therapy and prostate cancer therapy.


Assuntos
Arginina/farmacologia , Proteínas de Ligação a DNA/genética , Epigênese Genética/efeitos dos fármacos , Epigenômica/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Musculares/genética , Fosforilação Oxidativa/efeitos dos fármacos , Neoplasias da Próstata/genética , Fatores de Transcrição/genética , Animais , Arginina/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Masculino , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Musculares/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Transcrição/metabolismo
15.
Food Chem Toxicol ; 151: 112107, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33722596

RESUMO

Toxicant exposure can induce acute or chronic alterations in cellular numbers, morphology, and cell function. The quantification of these parameters can provide valuable information regarding a toxicant's effect and/or mechanism of action in organ-on-a-chip toxicity testing platforms. Unfortunately, manual quantification can be variable and time consuming. Additionally, the unique designs of Organ-Chips make automated imaging difficult as current microscopes were not specifically designed for Organ-Chip use. The development of semi-automated and automated imaging and quantification procedures greatly increases the quantity and quality of collected data. Using Emulate's transparent liver Organ-Chip (Liver-Chip) in combination with Keyence's bench-top BZ-X700 All-in-one fluorescence microscope we have developed semi-automated imaging and automated quantification methods for nuclei, mitochondrial viability, and apoptosis. The methods described herein provide alternative imaging options to more costly and space consuming microscopes while still providing necessary features for Organ-Chip evaluation. We were able to detect significant decreases in nuclear number and mitochondrial membrane potential, and significant increases in apoptosis with a model hepatotoxic compound, benzbromarone. These methods have greatly reduced the time and increased the quality of cell number/function data acquisition and demonstrated that these automated quantification methods can detect changes resulting from chemical exposure.


Assuntos
Fígado/diagnóstico por imagem , Microscopia de Fluorescência/métodos , Apoptose/efeitos dos fármacos , Automação , Benzobromarona/toxicidade , Núcleo Celular/efeitos dos fármacos , Células Cultivadas , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos , Fígado/citologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia de Fluorescência/instrumentação , Mitocôndrias Hepáticas/efeitos dos fármacos
16.
Biochem Biophys Res Commun ; 553: 154-159, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33773137

RESUMO

The glucocorticoid receptor (GR) plays an important role in steroid-dependent regulation of metabolism, development, and the immune response in humans. Although GR is known to be activated by the binding of glucocorticoid, the mechanism of action is poorly understood. We investigated dimerization of GR in the cytoplasm and nuclear trans-localization in response to treatment with the ligand dexamethasone. GFP-tagged GR and FLAG-tagged GR were co-expressed in COS-1 cells, and cell lysates were subjected to co-immunoprecipitation assay with anti-GFP antibody to determine their dimerization. FLAG-GR was co-precipitated with GFP-GR in the cytoplasmic fraction of COS-1 cells. Treatment with the GR agonist dexamethasone significantly decreased the cytoplasmic interaction between FLAG- and GFP-GR, and significantly increased interaction of the GRs in the nuclear fraction. The two amino acids, Pro625 and Ile628 known to be located in GR-GR dimer interface, were mutated to alanine and the influence of the mutation on dimerization, ligand-dependent nuclear localization, and transcriptional activities were determined. Mutant GR showed a dramatic decrease in interaction in the cytoplasmic fraction and no detectable nuclear translocation in the presence or absence of dexamethasone. Furthermore, luciferase assays showed that mutant GR showed no detectable transcriptional activation via the GR-responsive DNA element (GRE) compared to the wild-type. Our results suggest that GR exists as a dimer in the cytoplasm and this dimerization may be essential for GRE-mediated transcriptional activation following ligand binding.


Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Multimerização Proteica , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/metabolismo , Animais , Células COS , Núcleo Celular/efeitos dos fármacos , Chlorocebus aethiops , Citoplasma/efeitos dos fármacos , Dexametasona/metabolismo , Dexametasona/farmacologia , Humanos , Ligantes , Modelos Moleculares , Mutação , Multimerização Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Receptores de Glucocorticoides/genética
17.
FEBS Lett ; 595(10): 1438-1453, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33686684

RESUMO

The DEK oncoprotein regulates cellular chromatin function via a number of protein-protein interactions. However, the biological relevance of its unique pseudo-SAP/SAP-box domain, which transmits DNA modulating activities in vitro, remains largely speculative. As hypothesis-driven mutations failed to yield DNA-binding null (DBN) mutants, we combined random mutagenesis with the Bacterial Growth Inhibition Screen (BGIS) to overcome this bottleneck. Re-expression of a DEK-DBN mutant in newly established human DEK knockout cells failed to reduce the increase in nuclear size as compared to wild type, indicating roles for DEK-DNA interactions in cellular chromatin organization. Our results extend the functional roles of DEK in metazoan chromatin and highlight the predictive ability of recombinant protein toxicity in E. coli for unbiased studies of eukaryotic DNA modulating protein domains.


Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA/metabolismo , Escherichia coli/efeitos dos fármacos , Mutação com Perda de Função , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas Recombinantes/toxicidade , Viés , Núcleo Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Cromatina/química , Cromatina/genética , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/toxicidade , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genoma Bacteriano/efeitos dos fármacos , Genoma Bacteriano/genética , Humanos , Mutagênese , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Proteínas Oncogênicas/química , Proteínas Oncogênicas/toxicidade , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Proteínas de Ligação a Poli-ADP-Ribose/química , Proteínas de Ligação a Poli-ADP-Ribose/toxicidade , Domínios Proteicos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Testes de Toxicidade/métodos
18.
Toxins (Basel) ; 13(2)2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33673235

RESUMO

We are investigating plant species from the Canadian prairie ecological zone by phenotypic cell assays to discover toxins of biological interest. We provide the first report of the effects of extracts prepared from the shrub Symphoricarpos occidentalis in several human cell lines. S. occidentalis (Caprifoliaceae) extracts are cytotoxic, and, strikingly, treated cells undergo light-dependent vacuolation near the nucleus. The range of irradiation is present in standard ambient light and lies in the visible range (400-700 nm). Vacuolization in treated cells can be induced with specific wavelengths of 408 or 660 nm at 1 J/cm2 energies. Vacuolated cells show a striking phenotype of a large perinuclear vacuole (nuclear associated vacuole, NAV) that is distinct from vesicles observed by treatment with an autophagy-inducing agent. Treatment with S. occidentalis extracts and light induces an intense lamin A/C signal at the junction of a nuclear vacuole and the nucleus. Further study of S. occidentalis extracts and vacuolation provide chemical tools that may contribute to the understanding of nuclear envelope organization and human cell biology.


Assuntos
Núcleo Celular/efeitos dos fármacos , Extratos Vegetais/toxicidade , Plantas Tóxicas/toxicidade , Symphoricarpos/toxicidade , Toxinas Biológicas/toxicidade , Vacúolos/efeitos dos fármacos , Células A549 , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Núcleo Celular/efeitos da radiação , Células HT29 , Humanos , Lamina Tipo A/metabolismo , Luz , Extratos Vegetais/isolamento & purificação , Toxinas Biológicas/isolamento & purificação , Vacúolos/metabolismo , Vacúolos/patologia , Vacúolos/efeitos da radiação
19.
Oxid Med Cell Longev ; 2021: 8839479, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33747350

RESUMO

Black berry (Syzygium cumini) fruit is useful in curing diabetic complications; however, its role in diabetes-induced cardiomyopathy is not yet known. In this study, we investigated the regulation of gelatinase-B (MMP-9) by S. cumini methanol seed extract (MSE) in diabetic cardiomyopathy using real-time PCR, RT-PCR, immunocytochemistry, gel diffusion assay, and substrate zymography. The regulatory effects of MSE on NF-κB, TNF-α, and IL-6 were also examined. Identification and estimation of polyphenol constituents present in S. cumini extract were carried out using reverse-phase HPLC. Further, in silico docking studies of identified polyphenols with gelatinase-B were performed to elucidate molecular level interaction in the active site of gelatinase-B. Docking studies showed strong interaction of S. cumini polyphenols with gelatinase-B. Our findings indicate that MSE significantly suppresses gelatinase-B expression and activity in high-glucose- (HG-) stimulated cardiomyopathy. Further, HG-induced activation of NF-κB, TNF-α, and IL-6 was also remarkably reduced by MSE. Our results suggest that S. cumini MSE may be useful as an effective functional food and dietary supplement to regulate HG-induced cardiac stress through gelatinase.


Assuntos
Anti-Inflamatórios/farmacologia , Hiperglicemia/patologia , Metaloproteinase 9 da Matriz/metabolismo , Miócitos Cardíacos/patologia , Estresse Oxidativo , Extratos Vegetais/farmacologia , Sementes/química , Syzygium/química , Animais , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Cromatografia Líquida de Alta Pressão , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucose , Hiperglicemia/genética , Inflamação/patologia , Interleucina-6/metabolismo , Metaloproteinase 9 da Matriz/genética , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fenóis/farmacologia , Transporte Proteico/efeitos dos fármacos , Ratos , Especificidade por Substrato/efeitos dos fármacos , Termodinâmica , Fator de Necrose Tumoral alfa/metabolismo
20.
Arch Biochem Biophys ; 703: 108847, 2021 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-33766523

RESUMO

SIRT1 is a mammalian NAD+-dependent deacetylase, which is known to be involved in various physiological events, such as adaptive response to environmental stresses including caloric restriction, as well as in aging and cellular senescence. However, recent studies have revealed overexpression of SIRT1 in many different types of human malignancies, particularly colon cancer. Interleukin-1ß (IL-1ß) is a proinflammatory cytokine that plays a major role in invasiveness, stemness and progression of colon cancer. However, the interaction between IL-1ß and SIRT1 in the tumor development and progression remains elusive. In this study, we found that IL-1ß induces SIRT1 protein expression in human colon cancer HCT-116 cells. IL-1ß-induced SIRT1 upregulation led to enhanced expression of mRNA transcripts of pro-inflammatory cytokines, IL-6 and IL-8 as well as that of IL-1ß. Knockdown of SIRT1 prevented IL-1ß-induced phosphorylation and nuclear accumulation of c-Jun. Furthermore, pharmacologic inhibition of SIRT1 abrogated clonogenicity and migrative capability of human colon cancer cells stimulated with IL-1ß. In summary, IL-1ß-induced SIRT1 upregulation stimulates production of proinflammatory cytokines via a nuclear accumulation of c-Jun, leadng to colon cancer growth and progression.


Assuntos
Movimento Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , Citocinas/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Interleucina-1beta/farmacologia , Sirtuína 1/genética , Regulação para Cima/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células HCT116 , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Transcrição Genética/efeitos dos fármacos
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